130 PART ONE Fundamentáis of Microbiology

-•:FMc^l^AT>QN

FIGURE 5.1 ó Electron transport and the chemiosmotic generation of ATP.

In the mitochondrial membrana, electrón carriers are organizad into three complexes,
and protons (H"^) are pumped across the membrane at three points. In a eukaryotic
cell, they are pumped from the matrix side of the mitochondrial membrane to the op-
posite side. In a prokaryotic cell, protons are pumped across the plasma membrane
from the cytoplasmic side. The flow of electrons is shown in magenta.

• How does chemiosmosis differ in eukaryotes and prokaryotes?

for each molecule of glucose, the 34 from. chemiosmosis are c a n n o w summarize the o v e r a l l r e a c t i o n for aerobic respi-
added to those generated by o x i d a t i o n i n glycolysis and the r a t i o n i n prokaryotes as foUows: . •
Krebs cycle. I n aerobic respiration among prokaryotes, a
Q H i z O é + 6O2 + 38 A D P + 38 @, >
t o t a l of 38 molecules o f A T P can be generated f r o m one
Glucose Oxygen
molecule of glucose. N o t e t h a t four o f those A T P s come
f r o m substrate-level p h o s p h o r y l a t i o n i n glycolysis and the 6 CO2 + 6 H2O + 38 A T P

Krebs cycle. Table 5.3 provides a detailed a c c o u n t i n g of Carbón Water

dioxide
t h e A T P yield d u r i n g p r o k a r y o t i c aerobic respiration.
A e r o b i c respiration a m o n g eukaryotes produces a total A summary o f the various stages o f aerobic respiration i n
o f only 36 molecules of A T P . T h e r e are fewer ATPs t h a n i n prokaryotes is presented i n Figure 5.17.
prokaryotes because some energy is lost w h e n electrons are
shuttled across the m i t o c h o n d r i a l membranes t h a t sepá- Anaerobic Respiration

rate glycolysis ( i n the cytoplasm) f r o m the electrón trans- I n anaerobic respiration, the final electrón acceptor is an i n -
port c h a i n . N o such separation exists i n prokaryotes. W e organic substance other t h a n oxygen {Oi)- Some bacteria,
 CHAPTER 5' Microbial Metabolism 131

TABLE 5.3 ATP Y i e I d D u r i n g P r o k a r y o t i c Aerobíc R e s p i r o t i o n of O n e G l u c o s e M o l e c u i e

Glycolysis
1. Oxidation of glucose to pyruvic acid 2 ATP (substrate-level phosphorylation)

2. Production of 2 NADH ó ATP (oxidative phosphorylation in ""^CW

electrón transport chai
'^1 transport
Electron Chain
Preparatory Step and chemiosmosis

1. Formation of acetyl CoA produces Ó ATP (oxidative phosphorylation in

2 NADH electrón transport chain)

Krebs Cycle
1. Oxidation of succinyl CoA to succinic 2 GTP (equivalent of ATP; substrate-level phosphorylation)
acid

2. Production of ó NADH 1 8 ATP (oxidative phosphorylation in electrón transport chain)

3. Production of 2 FADH 4 ATP (oxidative phosphorylation in electrón transport chain)

Total: 38 ATP

FIGURE 5 . 1 7 A s u m m a r y of
aerobio respirotion in
p r o k a r y o t e s . Glucose is
broken d o w n completely to
carbón dioxide and water, a n d
ATP is generoted. This process
has three major phoses: glycol-
ysis, the Krebs cycle, and the
electrón transport chain. The
preparatory step is between
glycolysis a n d the Krebs cycle.
The key event in aerobio respi-
rotion is that electrons are
picked up from intermediates
of glycolysis a n d the Krebs
cycle by NAD"^ or FAD and
are carried by N A D H or
FADH2 to the electrón transport
chain. N A D H is olso produced
in the conversión of pyruvic
acid to acetyl C o A . Most of the
ATP generoted by aerobic
respiratión is mode by the
chemiosmotic mechanism dur-
ing the electrón transport chain
phase; this is called oxidative
phosphorylation.

• How do aerobic and anaero-

bio respirotion differ?

Electron
transport
chain and
chemiosmosis
88 SECCIÓN I Bases d e l a m i c r o b i o l o g í a

i Se sintetizan derivados ©
El compiejo NAM-pentapéptido se ) El UDP transfiere NAG al complejo bactoprenoI-NAM-
UDP de NAM y NAG transfiere al fosfato de bactoprenol. pentapéptido. Si se requiere la interposición de
(no se muestra). Se unen por medio de enlaces de pentaglicina, se crea utilizando moléculas especiales
pirofosfato. de glicil-tRNA, pero no ribosomas. La formación de
UDP—NAM puentes ocurre en la membrana.
L-Ala L.Aia
^ A d i c i ó n secuencia! de
aminoácidos a UDP-NAM para
formar el compiejo N A M - D-Glu
pentapéptido. Se utiliza ATP Cicloserina @ L o s transportadores de bactoprenol
para favorecer la reacción, pero transportan las unidades repetidas del
L-Lys (DAP)
no participan el RNA y los complejo NAG-NAM pentapéptido a
ribosomas en la formación través de la membrana.
de enlaces peptídicos que
r
D-Ala—D-Afa
unen los aminoácidos. Lípidol UDP—NAG Lípido I I

Citoplasma Pentapéptido Pentapéptido

UDP- NAM — Pentapéptido

UMP ©—NAM © — NAM—NAG

- > i Bactoprenol

Bacitracina

KttiviiíKit
Peripiasma ©
7
Peptidoglucano — N A M — N A G Peptidoglucano NAM—NAG

Vancomicina Pentapéptido
Pentapéptido

© L o s enlaces cruzados entre las cadenas
de peptidoglucano se forman por
transpeptidación (no se muestran).
© El bactoprenol transportador se desplaza
a través de la membrana. Conforme lo
tiace, pierde un fosfato. Atiora está
preparado para iniciar un nuevo ciclo.
© El complejo NAG-NAM pentapéptido se une
al extremo en crecimiento de la cadena
de peptidoglucano, incrementando la
longitud de la cadena en repeticiones de
una unidad.

Transpeptidación de Escherichia coli

- N A M •••
D-Aia

- *w

Transpeptidación de Staphyiococcus aureus Penicilinas

- NAM D-Ala
D-Ala
i I I
L-Ala D-Ala L-Ala D-Ala
I
D-GluNHg rL - Lys D-GluNHg
I ^ L-Lys
I
L-Lys D- GluNHa (Gly)5
L-Lys D-GluNHz
^ D - A I ¿ ^ N - < g Í ) 5 »--Ála
L-Ala
I D-Ala 1
D-Aía - NAM - NAM

F I G U R A 6-19 A: Síntesis de p e p t i d o g l u c a n o . NAM corresponde a ácido A/-acetiImurámico y NAG a A/-acet¡lglucosamina. Los pentapéptidos
contienen L-lisina en el peptidoglucano de Staphyiococcus aurews, y ácido diaminopimélico (DAP) en Escherichia coli. También se muestra la intiibición
con bacitracina, cicloserina y vancomicina. Los números corresponden a seis de las ocho etapas revisadas en el texto. Se ilustra la etapa ocho en la
figura 5-19B. B: Transpeptidación. Reacciones de transpeptidaci-ón en la formación de peptidoglucanos de Escherichia coli y Staphyiococcus aureus. .
 Appendix ni(a>.

LACTIC ACID FERMENTATIONS

(see entries: HOMOIACTIC FERI^ENTATION; HETEROLA.CTIC FERMENTATION; BIFIDOBACTERIUM)
Appendix ni(b) Appendix III(c)
MiXED ACID FERMENTATION BUTANEDIOL FERMENTATION
(see entries: MfXED ACID FERMENTATION; FORMATE HYDROGEN LYASE) (see entries: BUTANEDIOL FERMENTATION: FORMATE HYDROGEN
LYASE; VOGES-PROSKAUER TEST)
CH2OH
CH2OH
CH3
NAD NADHj CH2COOH
CHj MAO NADHj CH3
CH3COOH CHOH
I c=o • - *• CH2COOH
CHOH • CHjCOOH COOH lactate dehvdrogenase |
B dflhycJrogenase
SUCCINATE
I COOH COOH
LACTATE
COOH PYRUVATE
LACTATE
• HCOOH — • CO2 + H2
FORMATE
CH3
I
C=0
S—CoA
ACETYLCoA
0 - ® ;HO
ACETYL- ACETALDEHYDE
PHOSPHATE
CH3
COOH CH2OH
ACETATE ETHANOL
466 467
Appendix Ill(d) Appendix Ill(e)
BUTYRIC ACID FERMENTATION AND ACETONE-BUTANOL PROPIONIC ACID FERMENTATION
FERMENTATION (in Propionibacterium)
(see entries: BUTYRICACID FERMENTATION;ACETONE-BUTANOL (see entry: PROPIONIC ACID FERMENTATION)
FERMENTATION; HYDROGENASE; FERREDOXIN)
CH2OH
CH3 MAD NADHj
CH3
I
CHOH :0
e dehvdrogenase
I
COOH
COOH
PYRUVATE
LACTATE
:;OASH-^ ^ F E R R E D O X II2
hydrogenase
1 dehydfogenase
C02*^ ^ ^FÉRREDOX
JH3 MAD NADH2 JH3 T C^l3 p, CoASH I CH3
CH2OH •" • CHO •* ^ c=o COOH
' S —CoA O — ®
ACETALDEHYDE ACETYL-CoA ACETYLPHOSPHATE ACETATE
jlyl-CoA
CoASH
CH3 Ch3
CHOH
I
C=0
I
CH2
C=0
S—CoA S—CoA
fl-HYDROXYBUTYRYL-CoA ACETOACETYL-CoA
c=o
S—CoA
CROTONYL-CoA
NADH2
butvfv' CoA
dehvdrogenase
NAD •
CH3
CH3
CH2 CoASH
NADH2 NAÍ
CHj
H2
—.0
CHj
aldehyde dehydrr
S—CoA CHO COOH
BUTYRYLCoA BUTYRALDEHYDE ACETOACETATE
H2O NADH2 -v|
COOH
1 butyryl-C
hydrolase PROPIONIC ACID
3ASH «-^1
J.
CH3
CH3
CHj CH3
I
C=0
CH2OH CH3
COOH
iBUTANOL ACETONE
BUTYRIC AGID 469
 Appendix IV(a) Appendix IV(b)
BIOSYNTHESIS OF ARGÍNINE, GLUTAMATE, GLUTAMINE, PROLINE BIOSYNTHESIS OF ALANINE, LEUCINE, VAÜNE
CH3 glutamate a-ketoglu
• (J;HNH2
alaníne aminotraniferf
COOH COOH
PYRUVATE ALANINE
COOH
O-KETOGLUTARATE
CH3 CH3 CH3
NADPHj NAOP I I a-k«to-
CH3—C—OH. glutarate CH3-
HjO CH3 C H
CH3—ij;—COOH -
C 7 '"'1 ¿ = 0 ¿H NHj
OH COOH COOH
a-ACETO LACTATE a^DIHYDROXY- a-KETOISOVALERATE VALINE
ISOVALERATE
J
CH3—C—H
HO—¿—COOH
HO—CHCOOH CHCOOH
I
c/í-DIMETHYL- CH2COOH
rISOPROPYLMALATE
CITRACONATii Í-ISOPROPYLMALATE
isopropyl-
malate
tíebyd rogarme
COj .t^
CH3 ;H3
•keto
CH3
utaraie CH3—Ó — H
leucine aminotranifarase CHj
=0 6HNH2
CHjCOOH COOH ¿OOH
<KETOISÜCAPROATE LEUCINE
srginosuccinate lyaie
471
470
 Appendix IV(d)
BIOSYNTHESIS OF ASPARAGINE, ASPARTATE, ISOLEUCINE,
^'"""'"¿ToSYNTHESIS OF CYSTEINE. 6LYC1NE. SERINE
METHIONINE, THREONINE
COOH
EMBOEN - MEYERHOF - PARNAS ATP ADP
PATHWAY - Appendix Ka) V-^ ^ CHNH2
CHj
COOH
CH2O® OXALOACETATE
CHOH
ioOH
3-PHOSPHOGLYCERATE
L Y S I N E (see Appendix IV(e))
CH2O®
C = 0
3-PHOSPHOHYDROXY-
PYRUVATE
CH2O®
ÍHNH2
I
COOH
3-PHOSPHOSERINE
COOH
NH3 I
COOH " 2 ° ^ P V ^ " » ¿HNH2
CH20.CO.CH3 CHjOH CH2 CH2 CHNH2 CH2
CH2 0-cvst»
¿HNHj CH2NH2 I I
ÍHNH2 iHa CH2—O — C = 0 CH2—S—(I:H2 CH2SH
¿ 0 0 H
COOH a-KETOBUTYRATE 0-SUCCINYLHOMOSERINE CYSTATHIONE HOMOCYSTEINE
COOH
SERINE
0-ACETYLSERINE
N^-mtthyl-
TPP tetrahydrotolí
I
-C COCH3 NAOPH2 NADP H O CH3 H2O
CHCH3 COOH
I . I
9^2 «•tohydroxy-acid CH2 ÍHNH2
CHjSH
CH3 CH3
¿HNH2 ü-ACETO-a-HYDROXY- a.lí-DIHYDROXY-fí- a-KETO-(J-
BUTYRATE METHYLVALERATE METHYLVALERATE
¿OOH
CYSTEINE glutamote
CH3
METHIONINE
•Forffled bv ASSIMILATOBY SULPHATE BEDUCTION Ileo en.ryl. COOH
(1HNH2
I
:HCH3
*ln membariof thé Entsrobacteriaceat.
" In bflctflfia othar than memben ol
Hj the Enterobacteriaceae, and in fungí.
CH3
ISOLEUCINE
473
472
 AppemBx IV(e)

BIOSYNTHESIS OF LYSINE
(see entries: DIAMINOPIMEUC ACID PATHWAY: AMINOADIPIC ACID PATHWAY)

(i) D I A M I N O P I M E L I C ACID PATHWAY (¡i) A M t N O A D I P I C A C I D P A T H W A Y

Appendix IV(cl) COOH

I
I
I COOH

I
I
I acetyKCoA CoASH CH2
t,
COOH ^ I
homocitrate synthetase — * CHj
HNHj pvruvate 2H2O HO—C—CHj—COOH
COOH
CH2 dihvdrodtpicolinate COOH
a-KETO-
svnthetase
CHO HOOC GLUTARATE HOMOCITRATE
HOOC ^ "COOH
ASPARTATE 2,3-DIHYDRO DIPICOLINIC
SEMIALDEHYDE DIPICOLINATE ACID

dihydrodiptcotinate
reductase

COOH 1=
COOH I
CH2 NAD(P)H2 NAD(P)

CH2 homoisocitrate
dehvdrogenase H i ; — C H O H — cC O O H
COOH H—C—CO—COOH
I COOH
A^-PIPERIDEINE
2,6-DICARBOXYLATE COOH HOMOISOCITRATE
N-SUCCINYL-2-AMINO OXALOGLUTARATE
6-KETO-L-PIMELATE

homoisocitrate
glutamate > ^ CO2 dehvdrogenase

a-keto- J
glutarate

COOH COOH

(CH2)3
i-keto- I
glutara (CH2)3
COOH COOH (j:ooH
I C = 0 — " CHNH2
¿Hj H2O suco
H2N—C—H
CHNH2 COOH COOH
CH2 — (CH2)3
N-succínvIdtamtnopimelate a-KETOADIPATE a-AMINOADIPATE
(CH2)3
— C = 0 desuccinylase H C NH2
H—C—NH
I COOH
NADPHj ^
ATP >v
COOH a-aminoadipate
N-SUCCINYL-L,L- L,L-2,6-DIAMINO- semialdehyde
PIMELATE dehvdrogenase
2,6-DIAMINOPIMELATE
NADP y
A D P + Pi

diamíno-
pimelate CH2—NH—CH—COOH CHO
epimerase
!
(C
C:H2)3
H2)3 CH2 M ^ D H 2 glutamate '9^^2)3

CHNH2 CH2 HNH2

COOH COOH COOH

i
SACCHAROPINE a-AMINOADIPATE-
H—C—NH2
e-SEMIALDEHYDE
CH2NH2
(CH2)3 H2O-S
CO2
(CH2)3 NAD
I H—C—NH2
saccharopíne
meso-diaminopimelat(
CHNH2 decarboxylase NADH2V dehvdrogenase
COOH
I
/T>eso-2,6-DIAMINO- Q-keto- V
COOH PIMELATE glutarate
LYSINE
CH2NH2
I
(CH2)3

CHNH2
I
COOH
LYSINE
 Appendix lV(f)
Appendix IV(g)
BIOSYNTHESIS OF AROMATIC AMINO ACIDS
COOH
BIOSYNTHESIS OFHISTIDINE
®0H2C ®0H2C
ATP AMP ATP pp, ®0H2C
¿ - 0 ®
OH OH
CHj OH ÓH Ribose-5<P>®><P
RIB0SE-5PH0SPHATE 5-PHOSPHORIBOSYL-
PHOSPHOENOL- ERYTHROSE- PYROPHOSPHATE N-1(5'-PH0SPH0RIB0SYL)-ATP
PYRUVATE 4PH0SPHATE
HjO
PPi •u^
® 0 H : V" ©OHjC
'Ribose-5-® Ribosc-5-®
PH0SPHORIB0SYLFORMIMIN0-5AMIN0- NM5'PH0SPH0RIB0SYL)-AMP
IMIDAZOLE CARBOXAMIDE RIBONUCLEOTIDE
phosphoribosyl-
formimino-5-amino-
imidaiolc carboxamide
fibonucleotide i:
p-AMINOBENZOATE
Tiinoimidazole
O, NH2 ifboxamide
^CC ribonucleotide
Qlutamíne fliutamate ''bonucleotide «2^
C: H
H2-NH Y \ ^ ^ ( C H O H ) 2 C H 2 0 ® j ^_^CH2COCH20®
CH2O® c= 0 Sg>^N'^ aminotransferase cyclase im.dazoleglycero. * ^ ^ ^ ^ ^
\e dehvdratase
I Ribose-5-®
H—C—OH IMIDAZOLEGLYCEROL IMIDAZOLEACETOL
PHOSPHATE PHOSPHATE
I
OH H—C—OH
PREPHENATE ENOLl'loCARBOXYPHENYLAMINOj- N-B'-PHOSPHORIBOSYL- CH2O®
l'-DEOXYRISULOSE-B'-PHOSPHATE ANTHR AÑILATE
PHOSPHORIBULOSYLFORMIMINO-5-AMINO-
IMIDAZOLE CARBOXAMIDE RIBONUCLEOTIDE
CO2 - indoteglycerol -
COOH -pr.ph.n,,, NADH2 phosphatK
d«liydralnin lynlhiílaip
CHj H3O . » keto-
glull
1 -(CH0H)2CH20®
p-HYDROXYPHENYL
INDOLEGLYCEROL-
PYRUVATE
PHOSPHATE
^ tyrosine -CH2CHNH2COOH 2NADHj 2NA0
aminotrani- ^ / —\ CH2CHNH2CH20H
"2° ^^=Y-CH2CHNH2CH30®
a-tc«io- ^ ferase H N
HNN, ^ M «
glularat histidínol dehvdrogenaí.e
3-phospho-
Blyceraldehyd HISTIDINOL HISTIDINOL
COOH
PHOSPHATE
I
CHNH2
CHj
H
NH3 pytui
•CH2CHNH2COOH
OH
PHENYLALANINE TYROSINE TRYPTOPHAN
476 477
 Appendix V(a)

BIOSYNTHESIS OF PURINE NUCLEOTIDES

(see enfríes: NUCLEOS/DE; NUCLEOTIDE)

N
/ H2
CH2
®OH2C H ATP AMP
(D0H2C
glutamine glutamate
PPi (P)0H2C
^ T

OH
ríbose phosphate ' i - < P H p ) A . í
pyrophosphokinase P R P P aminotransferase
OH OH :¡namide ^ ^ " ^ ^ " l
OH OH
D-RIBOSE-5-PHOSPHATE 5-PH0SPH0-a,D-RIB0SYL 5-PHOSPHO-/3-D-
P Y R O P H G S P H A T E (PRPP) B'-PHOSPHORIBOSYL-
RIBOSYLAMINE
GLYCINAMIDE
OO

N^, i^^°-methenyl- .
tetrahydrofolate
nhosphoribosyi-
glvcmamide
formyltransfera$e
tetrahydrofolate

CO2 ADP ATP

HO-^ J H H

phosphoribosy lamino
azofe carboxylast
phosphoribosylamino- CH2 H'^ phosphoribosylformylglycin-
H2N' imidazole sythetase
amidtne synthetase
¡bose-5-phosphate O^-NH^
I 1
5'-PHOSPHORIBOSYL-5-AMINO- 5 ' - P H 0 SRP Hibose-5-phosphate
ORIBOSYL- Ribose-5-phosphate R ibose-5-phosphate
IMIDAZOLE-4-CARBOXYLATE 5-AMINOIMIDAZOLE 5'-PH0SPH0RlB0SYL-N- 5 ' - P H 0 S P H 0 R I B O S Y L-N-
FORMYLGLYCINAMIDINE FORMYLGLYCINAMIDE

aspartate .... 1
ATP ^ 1 phosphoribosyl-
succinocarboxamido-
aminoimidazole
A D P .»y\ synthetase
Pi

O NlO-formyl-

HN
II tetrahydrof oíate tetrahydrofofate

HÍ—|NÍH

H7N
adenyiosuccinate iya

H 2 ' ^ ^ V
phosphoribosylamtnoimidazole-
carboxamide formyttransferase 0=CH
COOH ¡bose-5-phosphate R ibose-5-phosphate Ribose-5-phosphate

P
5 H
-'A
C RO
BS
PH
O O
XA
VR
IL
IDB5
O-)O
-S
AYN
ML -N
I4 ;S
(-CM-U
IC
DCZ
AN
IO
OL-E 5'PHOSPHORIBOSYL-4-CARBOXAMIDE- 5'-PH0SPH0RIB0SYL-4-CARB0XAMIDE-
5-AMINOIMIDAZOLE 5-FORMAMIDOIMIDAZOLE

Vibose-5-phosphate

ADENYLOSUCCINATE X A N T H 0 S I N E - 5 'R
-M 0N0P
ibose- H0SPHATE
5-phosphate
(XMP)

^ 3 ^
ATP-vi Suanosine
^ monophosphate
I synthetase

H2O

H2N'

0 O H 2 C

OH
OH

ADENOSINE-5 - GUANOSINE-5'-
MONOPHOSPHATE MONOPHOSPHATE
(AMP) (GMP)
Appendix V(b)
Appendix V(c)
BIOSYNTHESIS OF PYRIMIDINE NUCLEOTIDES THE S T R U C T U R E S OF SOME NUCLEOTIDES
(see entries: NUCLEOSIDE: NUCLEOTIDE)
(see entry: NUCLEOTIDE)
m-2 NH2
NHj CH2
HjN-^^COOH 0-^ ^ 0 —(P)
ASPARTATE CARBAMOYL
PHOSPHATE N-CARBAMOYL DIHYDRO- II II
ASPARTATE OROTATE 3=S—O—P—O—CI
NAD. /
0=P, O
ADENOSINE-B'-PHOSPHOSULPHATE
NAnu _ dihydroorotale ADENOSINE-5'-TRIPHOSPHATE
NADH2 v-7 dehvdrogenaie (APS)
(ATP) (PAPS = 3 ' - P H 0 S P H 0 - A P S )
(see e n t r y : A T P I 3',5'-CYCLIC ADENOSINE (see e n t r y : A S S I M I L A T O R Y S U L P H A T E
MONOPHOSPHATE REDUCTION)
(cAMP)
(P)0H2C
0<=^1 •COOH NHj
H .CONH2
NH2
OROTATE
OROTIDINE-5'-
MONOPHOSPHATE O O
(OMP)
CH2—O—P—O—P—O—CH
NICOTINAMIDE ADENINE DINUCLEOTIDE (NAD)
(NADP = NAD-2'-PH0SPHATE¡
(see entries: N A D ; N A D P ; N I C O T I N I C A C I D )
®-(P>-OH
CH2—O—P—O—P—O—CH2—(CHOHla—CH2
O- 0-
adenosine-5 -phosphate F L A V I N MONONUCLEOTIDE (FMN)
© - © - © - O H z C
F L A V I N ADENINE DINUCLEOTIDE (FAD)
(see e n t r y : R I B O F L A V I N )
OH H
DEOXYURIDINE-5'
MONOPHOSPHATE
(dUMPl
0 0
ATP 11
11 CH30H0 J
CH2—0- JpLo- - PII —
1 1
ADP -/[ 1
0 0- CH3H
H
.Jl I
pantothenic acid 2-mercaptoethvlamtne
-p—0-
®-(P>-(P>-0H2 T 11
0 -
_ J L.
adenosine-3'-phosphate-5'-phosphaio pantotheino-4'-pliosphato
C O E N Z Y M E A (CoASH)
(see entries: C O E N Z Y M E A ;
CYTIDINE-B'- DEOXYTHYMIDINE- PANTOTHENIC ACID)
TRIPHOSPHATE 5'-MONOPHOSPHATE
(CTP) (dTMP)
480 481
 resultad > la inhibición del consumo de glucosa (fig. 4.12). La con
práctica del efecto Pasteur es que la producción de alcohol es dr~
inhibida por el O2 y que para obtener una producción rentable
o cerveza mediante la levadura el O , debe excluirse rigurosam-^
fermentación.
Existe u n mínimo de concentración de O2 que es necesario an
puedan tener lugar los procesos aeróbicos de generación de ene
concentración es aproximadamente de un 0 , 2 % , que es un 1
concentración de O2 en la atmósfera actual (20 % ) . Por debajo de;
de Oj nc tienen lugar los procesos aeróbicos y los organismos
o bien u ilizan la fermentación o no crecen en absoluto. La
de la Tierra primitiva era anaeróbica y la concentración de O
lentamente como resultado de la producción de O2 debida a la fo
de las algas (véase cap. 15). Sólo cuando la concentración de Q-
el 0,2 % existió probablemente O, suficiente para la evolución
procesos respiratorios.

4.7

Respiración anaerobia

Aunque el O, es el aceptor de electrones más común en la oxid-

NADH2 en los sistemas de transporte de electrones, algunos or
p'ieden usar en su lugar compuestos orgánicos o inorgánicos (ta'
La oxidación con estos otros aceptares de electrones se denomina res^
anaerobia. Uno de los más comunes de esos aceptores sustitutiv
nitrato, NO3". que se convierte en formas de nitrógeno más r '
NO,-, NjO y Nt, proceso denominado desnitrificación. El nitrato es
ramente re incido a nitrito por la nitrato-reductasa; este enzima es"

Tabla 4.3 Aceptores de electrones utilizados en la respiración

Aceptor de electrones Productos reducidos

Respiración
anaerobia Inorgánico: Nitrito ( N O r ) , óxido'
Nitrato (NO3-) (N2O), nitrógeno (N
Férrico (Fe'*j* Ferroso (Fe-*)
Sulfato (SO.--) Sulfuro de hidrógeno'
Dióxido de carbono (CO2) Metano (CH^)
Orgánico:
Fumarato Succinato
(HÜOC—CH = C H — (HÜOC—CHí—CHrí.
—COOH) —COOH)
Respiración
aerobia H:0

* No se ha demostrado t o d a v í a que la reducción del hierro férrico sea una rea"

ductora de e n e r g í a .
reducido por el citocromo b. Así, la cadena transportadca des electrones e;
más corta que la que incluye O j (véase fig. 4 . 1 8 ) y sclo se generan do;
moléculas de ATP, no tres, por la fosforilación oxidati^ a. Esto concuerdí
con el hecho de que el potencial redox para la reducñón del nitrato Í
nitrito es 4-0,4 V, en oposición al potencial redox de + 0 , 8 V para la reduc
ción del O2. El crecimiento en presencia de nitrato es menos eficaz qu€
el crecimiento en presencia de O2 y, en la mayoría de los organismos
la reducción del nitrato es fuertemente inhibida por el Oj.
La mayoría de las bacterias que reducen el nitrato son llamadas anaero-
bios facultativos porque transfieren electrones al O2 cuai do éste está pre-
sente o al NO3 cuando no hay O2. El nitrito formado t s luego reducido
a los gases N2O y N2, pero la bioquímica de estas reacciones no se conoce
todavía. El anión inorgánico clorato (CIO3-) es u n inhi j i d o r específico de
la reducción del nitrato: inhibirá el crecimiento de bacterias si se utiliza
nitrato como aceptor de electrones pero no si se utiliza 'O2. L o s productos
gaseosos de la desnitrificación, si se producen en el suelo, escapan a la
atmósfera. Por ello la desnitrificación se considera u n proceso perjudicial
para la agricultura puesto que determina una pérdida de nitrógeno del
suelo (véase cap. 14).
El ion férrico (Fe^+) puede ser utilizado por varias bacterias como
aceptor de electrones, reduciéndose a ion ferroso {¥é^+). Este proceso
se encuentra en muchos organismos que reducen el nitrato, y hay algún
indicio de que quizás el mismo sistema enzimático interviene en ambas
reducciones. E l i o n férrico está presente en suelos y rocas, a menudo en
forma del hidróxido férrico insoluble Fe(OH)3, y cuando las condiciones
son anaeróbicas puede ocurrir la reducción al estado ferroso. E l ion ferroso
es mucho más soluble que el ion férrico, y la reducción por los microorga-
nismos conduce pues a la solubilización del hierro, ¡o cual es u n primer
paso importante en la form.ación del tipo de depósito de m i n e r a l llamado
hierro de pantanos (véase lám. en color y cap. 14). Sin c iibargo, si bien
la reducción del hierro férrico tiene lugar en muchas bac::rias reductoras
de nitrato, no se ha demostrado todavía que esto sea un prDceso productor
de energía.
Otro aceptor de electrones utilizado por algunas bacter as es el sulfato
(S04^-), que es reducido a H^S. Las bacterias reductoras de sulfatos son
generalmente anaerobios estrictos (u obligados), incapaces de crecer en la
presencia de oxígeno, o de utilizarlo, siendo a menudo ncluso matadas
por el O2. E l potencial redox para la reducción de sulfato es incluso más
bajo que para la reducción de nitrato, y la eficacia de crecimiento es
también más baja. L a bioquímica de la reducción del sulfato se discute en
el capítulo 5, y en el 14 la importancia ecológica de las bacterias reductoras
de sulfatos.
Las bacterias productoras de m.etano son u n grupo de anaerobios estric-
tos que usan CO2 como aceptor de electrones, reduciéndolo a metano (CH4).
Algunos miembros de este grupo son habitantes importantes del rumen
(véase cap. 12) y de las plantas de tratamiento anaerobio de las aguas
residuales (véase cap. 14). Algunas bacterias pueden utilizar ciertos com-
puestos orgánicos como aceptores de electrones. Por ejemplu, Strepíccoccus
faecalis convierte el fumarato en succinato, un compuesto más oxidado
(tabla 4.3).
 Carbón D i o x i d e F i x a t i o n
6C02
6 00000C6

A characteristic o f i
RuBP 2 PGA a p o p u l a t i o n o f orga
l i s m is a n increase ir
6 OOOOOC5 1 2 OOOC3 successful growth in

CARBON-FfXING Growth RequireiTií

REACTION

12 ADP + (P)
Chemical requii
ganisms must have¿
12 NADPH
stances i n c l u d i n g rn:
oxygen. Virtually al
carbón i nsome f o n
12 NADP+
o r l i p i d s . P e r h a p s 5(
Carbón c a n b e obta:
or i t m a y be derive
and photoautotroph
duce their nutrients
bon dioxide. Chem
while photoauíotro
A m o n g the o t h
(P^OOOOOOCf
trogen and phospho
Sucrose teins, a m i n o acids,
directly f r o m the atn
Glucose include species ofR
Other organic phosphate Glucose
molecules Phosphorus i s a n ei
the construction o f
Starch
O x y g e n is used
r e s p i r a t i o n a s a fina
ll^fr^^^^^'-^^^^^^ of photosynthesis. The ATP and
NADPH used in this process are synthesized in the eneroy-fixino: gen is an absolute 1
phase. ^
Certain microorgan
M Figure 1 1 • described as a n a e r

CLIFFS QUICK REViEW MICROBIOLOGY

2t
 ta
C h a p t e r 7

The Control of
Microbial Growth

BACTERIA TRAPPED /N A MEMBRANE

FILTER. Filtration can be use'^ te remove
microorganisms from water and solutions.

The Terminology of
Microbial Control
Learning Objective
• Define the following key terms relatad fo microbial con-
trol: sterilization, disinfection, antisepsis, degerming,
sanitization, biocide, germicida, bacteriostasis, and
asepsis. ,
The scientific control of infections, or n o s o c o m i a l
microbial growth begon only inrections, were the cause of A word frequently used, a n d m i s u s e d , i n d i s c u s s i n g t h e
obout 100 years ago. Recaí! death in at ¡east 10% of c o n t r o l o f m i c r o b i a l g r o w t h is steñÜzation. S t e r i l i z a t i o D is
t h e r e m o v a l o r d e s t r u c t i o n o f aü forms o f m i c r o b i a l Ufe.
'rom Chapter / that Pasteur's surgicol cases, and deaths
H e a t i n g is t h e most c o m m o n m e t h o d used f o r k i l l i n g m i -
work on microorganisms led of delivering mothers were
crobes, i n c l u d i n g t h e m o s t r e s i s t a n t f o r m s s u c h as e n -
scientists to believe that as high os 25%. Ignorance
dospores. S t e r i l i z a t i o n b y r e m o v a l o f m i c r o b e s f r o m l i q u i d s
riicrobes were a possible of microbes was such that, o r gases c a n be d o n e b y filtration.
cause of di sea se. In the mid- during the American Civil O n e would t h i n k that canned food i n t h e supermarket
' SOOs, the Hungarian physi- War, a surgeon might have is c o m p l e t e l y sterile. I n reality, t h e h e a t t r e a t m e n t r e q u i r e d

zian Ignatz Semmeiweis and cleaned his scalpel on his t o ensure absolute s t e r i l i t y w o u l d u n n e c e s s a r i l y degrade
t h e q u a l i t y o f t h e f o o d . I n s t e a d , f o o d is s u b j e c t e d o n l y t o
zngüsh physician joseph bootsole between incisions.
e n o u g h heat to.destroy t h e endospores o f Cíóstndium botu-
. síer used this 'hinking to Over the last century,
linum, w h i c h can produce a deadly t o x i n . T h i s l i m i t e d
'jevelop som.e of the first scientists have continued to h e a t t r e a t m e n t is t e r m e d c o m m e r c i a l s t e r i l i z a t i o n . T h e
"üCíobíai control practices develop a variely of physica'l endospores o f a n u i n b e r o f t h e r m o p h i l i c b a c t e r i a , c a p a b l e
--JÍ medical prccedufes. methods and chemical o f causing food spoilage b u t n o t h u m a n disease, are c o n -
"-lese pfccfices ¡ncluded ogenis to control microbial siderably m o r e resistant t o h e a t t h a n C . botulinum. l í pres-
e n t , t h e y w i l l s u r v i v e , b u t t h e i r s u r v i v a l is usually of n o
-'•jnd vva5f¡í/"!t; .vífii microbe- growth. In Chapter 20 we
p r a c t i c a l consequer\ce; t h e y w i l l n o t g r o w a t n o r m a l t o o d
<Jling chionde cf lime and wiil discuss methods for the
storage t e m p e r a t u r e s . I f c a n n e d foods i n a s u p e r m a r k e t
•zseptic surgery iechniques to control of microbes once were i n c u b a t e d at t e m p e r a t u r e s i n t h e g r o w r h range o f
'jevent microc'ol contamina- infection has occurred, mainly these t h e r m o p h i l e s (above a b o u t 4 5 ° C ) , s i g n i f i c a n t food
' on of surgico: wounds. Until antibiotic chemolherapy spoilage w o u l d occur.
•-at tim.e. l'.osc'ol-ocquired Complete s t e r i l i z a t i o n is o f t e n n o t r e q u i r e d i n o t h e r
settings. For e x a m p l e , t h e body's n o r m a l defenses c a n c o p e
2.3.
186 PART O N E Fundamento! ^bioiogy

Actíons of Microbial A s early as t h e S t o n e A g e , i t is l i k e l y t h a t h u m a n s w e r e a l -

Control Agents ready u s i n g some p h y s i c a l m e t h o d s o f m i c r o b i a l c o n t r o l t o

preserve foods. D r y i n g ( d e s i c c a t i o n ) a n d s a l t i n g (osmotic
pressure) were p r o b a b l y a m o n g t h e earliest t e c h n i q u e s .
Learning Objective
W h e n selecting methods o f m i c r o b i a l c o n t r o l , consid-
• Describe f/ie effects of microbial control agents on ceilu-
^eration must be g i v e n t o effects o n t h i n g s besides t h e m i -
lar structures. '
crobes. For e x a m p l e , c e r t a i n v i t a m i n s o r a n t i b i o t i c s i n a
I n t h i s s e c t i o n , w e e x a m i n e t h e ways various agents a c t u - s o l u t i o n m i g h t be i n a c t i v a t e d b y h e a t . M a n y l a b o r a t o r y o r
aily k i l l o r i n h i b i t microbes. h o s p i t a l m a t e r i a l s , s u c h as r u b b e r a n d látex t u b i n g , are
d a m a g e d b y repeated h e a t i n g . T h e r e are also e c o n o m i c
Alteratíon of Membrane Permeabilify c o n s i d e r a t i o n s ; f o r e x a m p l e , i t m a y be less e x p e n s i v e t o use

A m i c r o o r g a n i s m ' s p l a s m a m e m b r a n e (see Figure 4 - 1 3 ) , l o - p r e s t e r i l i z e d , disposable plasticware t h a n t o repeatedly

c a t e d j u s t i n s i d e t h e c e l l w a l l , is t h e target o f m a n y m i c r o - w a s h a n d resterilize glassware.

b i a l c o n t r o l agents. T h i s m e m b r a n e a c t i v e l y regulares t h e
passage o f n u t r i e n t s i n t o t h e c e l l a n d t h e e l i m i n a t i o n o f
wastes from- t h e c e l l . D a m a g e t o t h e l i p i d s o r p r o t e i n s o f
t h e p l a s m a m e m b r a n e b y a n t i m i c r o b i a l agents causes c e i -
l u l a r c o n t e n t s t o l e a k i n t o t h e s u r r o u n d i n g médium a n d i n -
terferes w i t h t h e g r o w t h o f t h e c e l l .
Damage to Proteins and Nucleic Acids
Heat
A v i s i t t o a n y s u p e r m a r k e t w i l l demónstrate t h a t h e a t -
preserved c a n n e d goods represent o n e o f t h e m o s t c o m m o n
m e t h o d s o f f o o d preservation. L a b o r a t o r y m e d i a a n d glass-
ware, a n d h o s p i t a l i n s t r u m e n t s , are also usually sterilized b y
heat. H e a t appears co k i l l microorganisms by d e n a t u r i n g t h e i r
enzymes; t h e r e s u l t a n t changes t o t h e t h r e e - d i m e n s i o n a l

B a c t e r i a are s o m e t i m e s t h o u g h t o f as " l i t t l e bags o f e n - shapes o f these p r o t e i n s i n a c t i v a t e t h e m (see Figure 5.6 o n

zyTnes." Enzymes, w h i c h are p r i m a r i l y p r o t e i n , are v i t a l t o a l l page 1 1 8 ) .

c e i l u l a r a c t i v i t i e s . R e c a l l t h a t t h e íunctional properties o f H e a t resistance varies a m o n g d i f f e r e n t m i c r o b e s ; these

p r o t e i n s are t h e result o f t h e i r t h r e e - d i m e n s i o n a l shape (see
F i g u r e 2.15 o n page 4 6 ) . T h i s shape is m a i n t a i n e d b y c h e m -
ical bonds t h a t l i n k adjoining portions o f the a m i n o acid
c h a i n as i t folds b a c k a n d f o r t h u p e n itself. S o m e o f those
b o n d s are h y d r o g e n b o n d s , w h i c h are susceptible t o break-
age b y h e a t o r c e r t a i n c h e m i c a l s ; breakage results i n d e n a -
t u r a t i o n o f t h e p r o t e i n . C o v a l e n t bonds, w h i c h are stronger,
are also subject t o a t t a c k . For e x a m p l e , disulfide bridges,
w h i c h play a n i m p o r t a n t r o l e i n p r o t e i n structure b y j o i n i n g
a m i n o acids w i t h exposed sulfhydryl ( — S H ) groups, c a n be
b r o k e n b y c e r t a i n c h e m i c a l s or sufiíicient heat.
differences c a n be expressed t h r o u g h t h e c o n c e p t o f t h e r -
mal d e a t h p o i n t . T h e n n a l d e a t h p o i n t ( T D P ) is t h e
lowest t e m p e r a t u r e a t w h i c h a l l t h e m i c r o o r g a n i s m s i n a
p a r t i c u l a r l i q u i d suspensión w i l l b e k i l l e d i n 10 m i n u t e s .
A n o t h e r f a c t o r t o b e c o n s i d e r e d i n s t e r i l i z a t i o n is t h e
l e n g t h o f t i m e r e q u i r e d . T h i s is expressed as t h e r m a l
d e a t h t i m e ( T D T ) , the minimal length o f time for all
b a c t e r i a i n a p a r t i c u l a r l i q u i d c u l t u r e t o be k i l l e d a t a g i v e n
temperature. B o t h T D P a n d T D T are useful guidelines
t h a t i n d i c a r e t h e severity o f t r e a t m e n t r e q u i r e d t o k i l l a
given p o p u l a t i o n o f bacteria.

T n e n u c l e i c acids D N A a n d R N A are t h e carriers o f t h e D e c i m a l r e d u c t i o n t i m e ( D R T , o r D valué) is a t h i r d

cell's g e n e t i c I n f o r m a t i o n . Damage t o these n u c l e i c acids by c o n c e p t r e l a t e d t o b a c t e r i a l h e a t resistance. D R T is t h e

h e a t , r a d i a t i o n , o r c h e m i c a l s is f r e q u e n t l y l e t h a l t o t h e c e l l ; time, i n minutes, i n w h i c h 9 0 % o f a p o p u l a t i o n o f bacteria

t h e c e l l can n o l o n g e r replícate, ñor c a n i t carry o u t n o r m a l at a g i v e n t e m p e r a t u r e w i l l be k i l l e d ( i n T a b l e 7.2 a n d Fig-

m e t a b o l i c áinctions s u c h as t h e synthesis o f enzymes. ure 7.1a, D R T is 1 m i n u t e ) . I n C h a p t e r 28 y o u c a n f i n d a n

i m p o r t a n t a p p l i c a t i o n o f D R T i n t h e c a n n i n g i n d u s t r y ; see

Physical Methods of t h e dis^ussion o f t h e 1 2 D t r e a t m e n t o f . c a n n e d goods o n

Microbial Control page 794.

MoisfHeat
Learning Objectives
M o i s t heat k i l l s m i c r o o r g a n i s m s p r i m a r i l y by t h e coagula-
• Comzcre the eñectiveness of moisi heat (boiling, auto- t i o n o f p r o t e i n s ( d e n a t u r a t i o n ) , w h i c h is caused b y b r e a k -
V clavirc, pasteurization) and dry heat.
age o f t h e h y d r o g e n bonds t h a t h o l d t h e p r o t e i n s i n t h e i r
• Descr oe how filtration, low temperatures, high pressure, t h r e e - d i m e n s i o n a l s t r u c t u r e . T h i s c o a g u l a t i o n process is
desiczation, and osmotic pressure suppress microbial f a m i l i a r t o a n y o n e w h o has w a t c h e d a n egg w h i t e f r y i n g .
grov.~ -**
O n e type o f moist heat s t e r i l i z a t i o n is b o i l i n g , w h i c h
• Expíe ' how radiation kills cells. k i l l s v e g e t a t i v e fqrms o f b a c t e r i a l p a t h o g e n s , a l m o s t a l l
184 PART O N E Fundamentáis of Microbiology

TABLE 7 . 1 Terminology Relating to the Control of Microbial G r o w t h

Definition Commenfs

Sterilization Destruction or removal of al! forms of mi- Usually done b y steom under pressure or
crobial life, including endospores. o sterilizing gas such as efhyiene o x i d e .

Commercial Sterilization Sufficient heat treatment-to-'kill endo- More-resistonf endospores of thermophilic

spores of Clostrid'tum botulinum in bacterio moy survive, but they will not
canned food. germinóte a n d g r o w under normal
storage conditions.

Disinfection Destruction of vegetative potfiogens. M o y moke use of physical or chemical

methods.

Antisepsis Destruction o f vegetative pathogens o n Treatment is almost alwbys b y chemical

living tissue. antimicrobials.

Degerming Removal of microbes from o limited Mostly a mechanical removal b y a n

área, such as the skin around a n injec- olcohol-soaked swab.
tion site.

Sanitization Treatment ¡ntended to lower microbial M o y b e done with high-íemperoture

counts o n eating a n d drínking utensils washing o r b y d i p p i n g into o chemical
to safe public health levéis. disinfectant.

w i t h a few microbes e n t e r i n g a surgical w o u n d . A d r i n k i n g T a b l e 7 . 1 summarizes t h e t e r m i n o l o g y r e l a t i n g t o t h e

glass o r a f o r k i n a r e s t a u r a n t requires o n l y e n o u g h m i c r o - control o f microbial growth.
b i a l c o n t r o l t o p r e v e n t t h e transmissíon o f possibly p a t h o - N a m e s o f t r e a t m e n t s t h a t cause t h e o u t r i g h t d e a t h o f
g e n i c microbes f r o m o n e person t o another. m i c r o b e s h a v e t h e sufíix -<:ide, m e a n i n g k i l l . A b i o c i d e , o r
C o n t r o l directed a t destroying h a r m f u l microorganisms g e r m í c i d e , k i l l s m i c r o o r g a n i s m s ( u s u a l l y w i t h c e r t a i n ex-
is c a l l e d d i s i n f e c t i o n . I t usually refers t o t h e d e s t r u c t i o n o f c e p t i o n s , s u c h as endospores); Afungicide k i l l s f u n g i ; a viru-
vegetative (non-endospore-forming) p a t h o g e n s , w h i c h is dde i n a c t i v a t e s viruses; a n d so o n . O t h e r t r e a t m e n t s o n l y
n o t t h e s a m e t h i n g as c o m p l e t e sterility. Disinfection i n h i b i t t h e g r o w t h a n d m u l t i p l i c a t i o n o f bacteria; their
m i g h t m a k e use o f c h e m i c a l s , u l t r a v i o l e t r a d i a t i o n , b o i l i n g ñames h a v e t h e sufíix -stat o r stasis, m e a n i n g t o stop o r to
w a t e r , o r s t e a m . I n p r a c t i c e , t h e t e r m is m o s t c o m m o n l y steady, as i n b a c t e r i o s t a s i s . O n c e a b a c t e r i o s t a t i c a g e n t is
a p p l i e d t o t h e use o f a c h e m i c a l ( a disinfectant) t o treat a n r e m o v e d , g r o w t h m i g h t resume.
i n e r t surface o r substance. W h e n t h i s t r e a t m e n t is d i r e c t e d S e p s i s , f r o m t h e G r e e k f o r decay o r p u t r i d , i n d i c a r e s
a t l i v i n g tissue, i t is c a l l e d a n t i s e p s i s , a n d t h e c h e m i c a l is b a c t e r i a l c o n t a m i n a t i o n , as i n septic t a n k s f o r sewage
t h e n c a l l e d a n antiseptic. T h e r e f o r e , i n p r a c t i c e t h e same t r e a t m e n t . ( T h e t e r m is also used l o describe a disease c o n -
c h e m i c a l m i g h t be c a l l e d a d i s i n f e c t a n t f o r o n e use a n d a n d i t i o n ; see C h a p t e r 23 o n page 6 4 1 . ) Aseptic means t h a t a n
a n t i s e p t i c f o r a n o t h e r . O f course, m a n y c h e m i c a l s s u i t a b l e o b j e c t o r área is free o f p a t h o g e n s . R e c a l l firom C h a p t e r 1
f o r s w a h b i n g a t a b l e t o p w o u l d be t o o h a r s h t o use o n l i v i n g t h a t a s e p s i s is t h e absence o f s i g n i f i c a n t c o n t a m i n a t i o n .
tissue. A s e p t i c t e c h n i q u e s are i m p o r t a n t i n surgery t o m i n i m i z e
T h e r e are m o d i f i c a t i o n s o f d i s i n f e c t i o n a n d antisepsis. c o n t a m i n a t i o n from the instruments, operating personnel,
For e x a m p l e , w h e n s o m e o n e is a b o u t t o receive a n i n j e c - and the patient.
t i o n , t h e s k i n is s w a b b e d w i t h a l c o h o l — t h e process o f
degerming ( o r degermation), w h i c h m o s t l y results i n t h e The Rate of Microbial Death
mechanical removal, rather t h a n t h e k i l l i n g , o f most o f t h e
m i c r o h e i i n a l i m i t e d área. Restaurant glassware, c h i n a , a n d Learning Objective
:ableware are subjected t o s a n i t i z a t i o n , w h i c h is i n t e n d e d
• Describe the pa^erns of m Jirobial death caused by treat-
zo l o w : m i c r o b i a l c o u n t s t o safe p u b l i c h e a l t h levéis a n d
ments with microbial control agents.
m i n i m i i c t h e chances o f disease t r a n s m i s s i o n f r o m o n e user
: o anotbitr. T h i s is usually accomplished by h i g h - t e m p e r a t u r e W h e n bacterial p o p u l a t i o n s are heated o r treated w i t h
'A-ashing or, i n t h e case o f glassware i n a bar, w a s h i n g i n a a n t i m i c r o b i a l chemicals, t h e y usually d i e at a c o n s t a n t

>ínk f o l l o w e d b y a d i p i n a c h e m i c a l d i s i n f e c t a n t . rate. For e x a m p l e , suppose a population of 1 million

 CHAPTER 7 The Control of M i c r o b i a l G r o w t h

TABLE 7 . 2 Microbial Death Rate: 1.000,000

A n Example o
>

Deaths Numberof > O n e log d e c r e a s e =

750,000 ^
V t i m e (mm) • • per Minute Survhrors. 9 0 % of population killed g
o
0 0 1,000,0X)CL

^. • .1 - 900,000 ] 00,000 500,000 -5

5)
2 90,000' 10,000
•- •
3 9000 . 1000
250.000
4 900 100 a
E
5 90 10
100,000 i
ó 9 1 2 3 4 5 6 ;
Time (min.)

(a) The c u n / e is plotted logaritfimicaHy (solid line) a n d arlthmetically

(broken line). In this c a s e , the cells are d y i n g at a rate of 9 0 % e a c h
m i c r o b e s has b e e n t r e a t e d f o r 1 m i n u t e , a n d 9 0 % o f t h e minute.

p o p u l a t i o n has d i e d . W e are n o w l e f t w i t h 1 0 0 , 0 0 0 m i -
crobes. I f t h e p o p u l a t i o n is t r e a t e d f o r a n o t h e r m i n u t e , 10^
9 0 % o f those m i c r o b e s d i e , a n d w e are l e f t w i t h 10,000
survivors. I n o t h e r words, for each m i n u t e t h e t r e a t m e n t > 105
is a p p l i e d , 9 0 % o f t h e r e m a i n i n g p o p u l a t i o n is k i l l e d W

( T a b l e 7 . 2 ) . I f t h e d e a t h c u r v e is p l o t t e d l o g a r i t h m i c a l l y , o 10^
t h e d e a t h r a t e is c o n s t a n t , as s h o w n b y t h e s t r a i g h t U n e i n ü
E
population ; ;
F i g u r e 7.1a.
103 load ,
S e v e r a l factors i n f l u e n c e t h e effectiveness o f a n t i m i - s\ X
^ L o w
crobial treatments:
102 ^"^.^^ population
• The number of microbes. T h e m o r e m i c r o b e s t h e r e are \d ,..
\
\
t o b e g i n w i t h , t h e longer i t takes t o elimínate t h e 101 - • \
co \
e n t i r e p o p u l a t i o n (Figure 7 . 1 b ) . O)
o \
• Environmental influences. T h e presence o f o r g a n i c 10° N
O 2 3 4
matter often inhibits the action of chemical antimi-
Time (min.)
c r o b i a l s . I n h o s p i t a l s , t h e presence o f o r g a n i c m a t t e r
(b) The effect of high or low initial load of m i c r o b e s . If t h e rate of
i n b l o o d , v o m i t , o r feces influences t h e s e l e c t i o n o f
küli.ig is the same, it will take longer to kill al! m e m b e r s of a larger
d i s i n f e c t a n t s . M i c r o b e s i n surface b i o f i l m s , s h o w n i n population than a smaller one. This Is true for b o t h heat a n d c h e m i c a l
Figure 2 7 . 1 0 , are difñcult f o r biocides t o r e a c h effec- treatments. , . -

: i v e l y . Because t h e i r a c t i v i t y is due t o t e m p e r a t u r e -
FIGURE 7 . 1 A microbial death curve.
j e p e n d e n t c h e m i c a l reactions, d i s i n f e c t a n t s w o r k
:(3mewhat b e t t e r u n d e r w a r m c o n d i t i o n s . D i r e c t i o n s • If the g r a p h ¡n p a r t (a) reflects the experience w i t h a vege-
: n d i s i n f e c t a n t c o n t a i n e r s f r e q u e n t l y specify t h e use o f tative bacterium, w h a t w o u l d the logarithmic curve !ook
like for the endospores of the same bacterium? Less steep?
j warm solution.
Steeper?
T h e n a t u r e o f t h e suspending médium is also a
r a c t o r i n h e a t t r e a t m e n t . Facs a n d p r o t e i n s are espe-
: i a l l y p r o t e c n s ' e , a n d a médium r i c h i n these
longer exposure c a n compénsate f o r a l o w e r t e m p e r a -
lubstanccs p r o t e c t s microbes, w h i c h w i l l t h e n h a v e a
t u r e , a p h e n o m e n o n o f p a r t i c u l a r i m p o r t a n c e t o pas-
i g h e r s u r v i v a l rate. H e a t is also measurably m j r e
teurization o f dairy products.
-rrfecrivc u n d e r acidic c o n d i t i o n s .
Microbial characteristics. T h e concluding section of this
• Ttme o/cxposme. C h e m i c a l a n t i m i c r o b i a l s o f t e n re-
c h a p t e r discusses h o w m i c r o b i a l c h a r a c t e r i s t i c s affect
4uire e x t e n d e d exposure for more-resistant microbes
chemical a n d physical c o n t r o l methods.
;r e n d o s p o r e s co be atfected. I n heat t r e a t m e n t s , a
 CHAPTER 7 The Control of M i c r o b i a l G r o w t h . 187

Exhaust vaive Operating valve FIGURE 7 . 2 A n a u t o c l a v e . The

(to remove steam Steam to Saíety (controls steam from
entering sfecm forces the o i r out
aíter stenlizaíion) chamber valve Pressure gauge jacket to chamber)
of the bottom (blue o r r o w s ) . The
outomotic ejector v a l v e r e m o i n s
o p e n as long as o n oir-sfeam
mixture is possing o u t of the
waste line. W h e n a l l the ai.'- has
been ejected, the h i g h e r t e m p e r a -
ture of the puré steam closes the
valve, a n d the pressure in the
c h a m b e r increases.

• Autoclaving is the p r e f e r r e d
method of sterilization, p r o v i d e d
the material to be sterilized w i l l
not be d a m a g e d b y heat o r
moisture.

Sediment
screen

vThermometer
3

Automatic ejector vaive is 'Pressure regulator

thermostatically controiled for steam supply
and Gloses on contact with
puré steam v^/hen air is
To waste line exhausted. Steam supply

% viruses, a n d f u n g i a n d t h e i r spores w i t h i n a b o u t 10 m i n u t e s ,
TABLE 7 . 3 The Relationship Betvs^een
f_. u s u a l l y m u c h faster. F r e e - t l o w i n g (unpressurized) s t e a m is
the Pressure a n d
^ e q u i v a l e n t m t e m p e r a t u r e t o b o i l i n g water. Endospores a n d
Temperature of Steam
gi s o m e viruses, h o w e v e r , are n o t destroyed t h i s q u i c k l y . S o m e
at Sea Level*
I h e p a t i t i s viruses, for e x a m p l e , c a n s u r v i v e u p t o 3 0 m i n u t e s
0 o f b o i l i n g . a n d some b a c t e r i a l endospores c a n resist b o i l i n g Pressure (psi i n excess -
ó f d t j m o s p h e r í c pres s ure) T e m p e r a t u r a (°C)
1 for m o r e t h a n 2 0 h o u r s . B o i l i n g is therefore n o t always a re-
V Hable s t e n . i r a t i o n p r o c e d u r e . H o w e v e r , b r i e f b o i l i n g , e v e n 0 psi 100
- a t h i g h alr.rudes, w i l l k i l l most pathogens. T h e use o f b o i l -
5 psi n o
i n g co ¿an¡:::e baby boceles is a f a m i l i a r e x a m p l e .
10 psi 116
R e l i a b l r s t e r i l i z a t i o n w i t h moist h e a t requires t e m p e r a -
tures abo'.c r h a t o f b o i l i n g water. T h e s e h i g h temperatures 15 psi 121
are mosi: : r r i m o n l y a c h i e v e d by steam u n d e r pressure i n 12Ó
^ ^ 2 0 psi
a n a u t o c l a v e ( F i g u r e 7.2). A u t o c l a v i n g is the preferred
3 0 psi 135
method ::eriíizariün, unless t h e m a t e r i a l co be sterilized
c a n be J u : ; . :i;;cd by h e a t o r m o i s t u r e . ' *At higher altitudes the atmospheric pressure is less, which
T h e "r.:-ner t h e pressure i n che a u t o c l a v e , che h i g h e r must be token into account in operotion of on autoclave. For
example, in order to reach sterilizing temperatures (121°C) in
t h e r c i n r c : ; ' i j r e . For e x a m p l e , w h e n tree-flc^wing sceam at
Denver, Colorado, whose altitude is 528C feet (lóOO me-
'i c e m p e r ^ : l O O ^ ' C i-,placed u n d e r a pressure of l ac- ters), the pressure shown on the autoclave gauge would need
:no>pherc • ;ove sea level pressure—chac is, abouc 15 to be higher than the 15 psi shown in the tab'e.

••üuriJs o: r--::-.süre per square i n c h ( p s i ) — c h e cemperature

lie.-, co 11'/' J. I n c r c a s i n g che pressure co 20 psi raises t h e
en-ircracu:-: ' o 1 2 6 ° C T h e r e l a t i o n s h i p h e r w e c n cenipera-
¡rc a n d r : v nre is s l i o w n \n T i b i e 7.3.
188 PART O N E Fundamentáis of Microbiology

TABLE 7 . 4 The Effect of Container Size
o n Autoclave Sterilization
Times for Liquid Solutions*
liquid .
G o n t o i n e r Size Volume
- Sterilization
T i m e (min)
w h i c h is l i g h t e r t h a n air. T h e t r a p p e d air is t h e e q u i v a l e n t
o f a s m a l l h o t - a i r o v e n , w h i c h , as we w i l l see s h o r t l y , re-
quires a h i g h e r t e m p e r a t u r e a n d l o n g e r t i m e t o sterilize
materials. C o n t a i n e r s t h a t c a n t r a p a i r s h o u l d be p l a c e d i n
a t i p p e d p o s i t i o n so t h a t t h e steam w i l l forcé o u t t h e air.
Products t h a t d o n o t p e r m i t p e n e t r a t i o n by moi.sture, such

Test tube: 10 mi 15 as m i n e r a l o i l o r p e t r o l c u m j e l l y , are n o t sterilized b y the

1 8 X 1 5 0 mm
Erlenmeyer flask: * 9 5 mi 15
125 mi
Erlenmeyer flask: 1 5 0 0 mi 30
2 0 0 0 mi -
Fermentation bottie: 6 7 5 0 mi 70
9 0 0 0 mi '
•Sterilization times iñ tfie autoclave include the time for the
contents of the containers to reach sterilizotion temperatures.
For smaller containers, this is only 5 min or less; but for a
9000^nl bottie, it might be as much as 7 0 min. A container is
usually not filled post 7 5 % of ¡ts capacity.
S t e r i l i z a t i o n i n a n a u t o c l a v e is m o s t eífective w h e n t h e
organisms are e i t h e r c o n t a c t e d b y t h e s t e a m d i r e c t l y o r are
c o n t a i n e d i n a s m a l l v o l u m e o f aqueous ( p r i m a r i l y w a t e r )
l i q u i d . U n d e r t h e s e c o n d i t i o n s , s t e a m a t a pressure
a b o u t 15 psi ( 1 Z 1 ° C ) w i l l k i l l all organisms a n d t h e i r e n -
dospores i n a b o u t 15 m i n u t e s .
A u t o c l a v i n g is used t o sterilize c u l t u r e m e d i a , i n s t r u -
m e n t s , dressings, i n t r a v e n o u s e q u i p m e n t , a p p l i c a t o r s , so-
l u t i o n s , syringes, transfusión e q u i p m e n t , a n d n u m e r o u s
o t h e r Ítems t h a t c a n w i t h s t a n d h i g h t e m p e r a t u r e s a n d
pressures. Large i n d u s t r i a l autoclaves are c a l l e d retorts
F i g u r e 2 8 . 2 ) , b u t t h e same p r i n c i p i e applies f o r t h e c o m -
mon h o u s e h o l d pressure c o o k e r used i n t h e h o m e c a n n i n g
o f foods.
H e a t requires e x t r a t i m e t o r e a c h t h e c e n t e r o f s o l i d
m a t e r i a l s . s u c h as c a n n e d meats, because s u c h m a t e r i a l s d o
n o t deve.op t h e e f f i c i e n t h e a t - d i s t r i b u t i n g c o n v e c t i o n c u r -
r e n t s th¿: o c c u r i n l i q u i d s . H e a t i n g large c o n t a i n e r s also
same m e t h o d s t h a t w o u l d sterilize aqueous s o l u t i o n s .
Several c o m m e r c i a l l y availáble m e t h o d s c a n i n d i c a t e
w h e t h e r s t e r i l i z a t i o n has b e e n a c h i e v e d by h e a t t r e a t m e n t .
S o m e o f these are c h e m i c a l r e a c t i o n s i n w h i c h a n i n d i c a -
tor changes c o l o r w h e n t h e p r o p e r t i m e s a n d t e m p e r a t u r e s
h a v e been reached (Figure 7.3). I n some designs, t h e w o r d
" s t e r i l e " o r " a u t o c i a v e d " appears o n w r a p p i n g s o r tapes. I n
a n o t h e r m e t h o d , a p e l l e t c o n t a i n e d w i t h i n a glass v i a l
m e l t s . A w i d e l y used test consists o f p r e p a r a t i o n s o f speci-
fied species o f b a c t e r i a l endospores i m p r e g n a t e d i n t o paper
strips. A f t e r a u t o c l a v i n g , these c a n t h e n be a s e p t i c a l l y i n -
oculated i n t o culture media. G r o w t h i n the culture media
indicares s u r v i v a l o f t h e endospores a n d t h e r e f o r e inade-
q u a t e processing. O t h e r designs use e n d o s p o r e suspensions
t h a t c a n be released, after h e a t i n g , i n t o a s u r r o u n d i n g c u l -
t u r e médium w i t h i n t h e same v i a l .
S t e a m u n d e r pressure fails t o sterilize w h e n t h e a i r is
n o t completely exhausted. T h i s c a n h a p p e n w i t h t h e pre-
m a t u r e c l o s i n g o f t h e autoclave's a u t o m a t i c e j e c t o r v a l v e
of
(see Figure 7.2). T h e p r i n c i p i e s o f h e a t s t e r i l i z a t i o n h a v e a
direct bearing o n home c a n n i n g . A s anyone familiar w i t h
h o m e c a r m i n g knows, the steam must flow vigorously o u t
o f t h e v a l v e i n t h e l i d for several m i n u t e s t o carry w i t h i t
a l l t h e a i r before t h e pressure c o o k e r is sealed. I f t h e a i r is
n o t c o m p l e t e l y e x h a u s t e d , t h e c o n t a i n e r w i l l n o t r e a c u "^lle
t e m p e r a t u r e e x p e c t e d for a g i v e n pressure. Because o f th^.
(see
possibility o f b o t u l i s m , a k i n d o f food p o i s o n i n g resulting
f r o m i m p r o p e r c a n n i n g m e t h o d s (see C h a p t e r 2 2 , page
6 2 1 ) , people i n v o l v e d i n h o m e c a n n i n g s h o u l d o b t a i n r e l i -
able d i r e c t i o n s a n d f o l l o w t h e m exactly.
Pasteurization
R e c a l l f r o m C h a p t e r 1 t h a t i n t h e early days o f m i c r o b i o l -

requires t x t r a t i m e . T a b l e 7.4 shows t h e d i f f e r e n t t i m e re- ogy, Louis Pasteur f o u n d a p r a c t i c a l m e t h o d o f p r e v e n t i n g

quiremer.r^ for s t e r i l i z i n g l i q u i d s i n v a r i o u s c o n t a i n e r sizes. t h e sgpüage o f beer and w i n e . Pasteur^used m i l d h e a t i n g ,

U n l i k e s-.trilizing aqueous s o l u t i o n s , s t e r i l i z i n g t h e surface w h i c h was sufficient t o k i l l t h e organisms t h a t caused t h e

o t a solic requires t h a t s t e a m a c t u a l l y c o n t a c t i t . T o s t e r i l - p a r t i c u l a r spoilage p t o b l e m w i t h o u t seriously damaging

ize dr\ i-..assware, bandages, a n d t h e l i k e , care m u s t be t h e taste o f t h e p r o d u c t . T h e same p r i n c i p i e was later ap-

t a k e n te t n s u r e t h a t s t e a m c o n t a c t s a l l surfaces. p l i e d t o m i l k t o p r o d u c e w h a t w e n o w c a l i pasteurized

For c.-;.dmple, a l u m i n u m f o i l is i m p e r v i o u s t o s t e a m a n d m i l k . T h e i n t e n t o f p a s t e u r i z a t i o n o f m i l k was t o e l i m i n a t e

s h o u l d r. ••: be used t o w r a p dry m a t e r i a l s t h a t are t o be ster- pathogenií m i c r o b e s . I t also lowers m i c r o b i a l n u m b e r s ,

i l i z e d ; p¿:er s h o u l d be used instead. C a r e s h o u l d also be w h i c h prolongs milk's good quality under refrigeration.

t a k e n te ^ v o i d t r a p p i n g air i n t h e b o t t o m o f a d r y c o n - M a n y relatively heat-resistant ( t h e r m o d u r i c ) bacteria

t a i n e r b-eiause t r a p p e d a i r w i l l n o t be replaced by s t e a m , survive p a s t e u r i z a t i o n , b u t these are u n l i k e l y t o cause dis-

ease or cause refrigeFated m i l k t o s p o i l .
 CHAPTER 7 The Control of M i c r o b i a l G r o w t h 18^

T h i s is m o r e useful i n parts o f t h e w o r l d w h e r e r e f r i g e r a t i o n
f a c i l i t i e s are n o t always availáble. I n t h e U n i t e d States,
s t e r i l i z a t i o n is sometimes used o n t h e s m a l l c o n t a i n e r s o f
v.offee creamers f o u n d i n restaurants. T o a v o i d g i v i n g t h e
m i l k a c o o k e d taste, a U H T system is used i n w h i c h t h e l i q -
u i d m i l k n e v e r touches a surface h o t t e r t h a n t h e m i l k i t s e l f
w h i l e b e i n g heated by steam. T h e m i l k falls i n a t h i n film
through a chamber o f superheated steam and reaches
140°C i n less t h a n a second. I t is h e l d for 3 seconds i n a
h o l d i n g tube and t h e n cooled i n a v a c u u m chamber, where
t h e steam flashes off. W i t h t h i s process, i n less t h a n 5 sec-
o n d s t h e m i l k t e m p e r a t u r e rises f r o m 74°C t o 140°C a n d
drops b a c k t o 74°C.
T h e h e a t t r e a t m e n t s we h a v e j u s t discussed i l l u s t r a t e
the concept of e q u i v a l e n t t r e a t m e n t s : As the tempera-
t u r e is increased, m u c h less t i m e is n e e d e d t o k i l l t h e same
n u m b e r o f microbes. For e x a m p l e , the destruction of
h i g h l y r e s i s t a n t endospores m i g h t t a k e 7 0 m i n u t e s a t
115°C, whereas o n l y 7 m i n u t e s m i g h t be n e e d e d a t 125°C.
B o t h t r e a t m e n t s y i e l d t h e same r e s u l t . T h e c o n c e p t of
FIGURE 7 . 3 Examples of steríiizaHon índicotors. e q u i v a l e n t t r e a t m e n t s also e x p l a i n s w h y classic pasteuriza-
The strips i n d i c a t e if the item has been p r o p e r l y sterilized,
t i o n a t 6 3 ° C f o r 3 0 m i n u t e s , H T S T t r e a t m e n t a t 72*'C f o r
the w o r d NOT a p p e a r s if heating has been i n q d e q u a t e .
In the illustration, the indicator that w a s w r o p p e d w i t h 15 seconds, a n d U H T t r e a t m e n t a t 1 4 0 * 0 f o r less t h a n a
a l u m i n u m foil w a s not sterilized because steam c o u l d n ' t s e c o n d c a n h a v e s i m i l a r effects.
penétrate the foil.
Dry Heot Sterilization
• W h a t should have been used instead of aluminum foil to -
w r a p the ítems? D r y h e a t k i l l s b y o x i d a t i o n effects. A s i m p l e a n a l o g y is t h e
s l o w c h a r r i n g o f paper i n a h e a t e d o v e n , e v e n w h e n t h e
t e m p e r a t u r e r e m a i n s b e l o w t h e i g n i t i o n p o i n t o f paper.
P r o d u c t s o t h e r t h a n m i l k , s u c h as ice c r e a m , y o g u r t , O n e o f t h e s i m p l e s t m e t h o d s o f d r y héat s t e r i l i z a t i o n is d i -
a n d beer, a l l h a v e t h e i r o w n p a s t e u r i z a t i o n t i m e s a n d t e m - r e c t í l a m i n g . Y o u w i l l use t h i s p r o c e d u r e m a n y t i m e s i n
peratures, w h i c h o f t e n diífer considerably. T h e r e are sev- t h e m i c r o b i o l o g y l a b o r a t o r y w h e n y o u sterilize i n o c u l a t i n g
eral rcasons for these v a r i a t i o n s . For e x a m p l e , h e a t i n g is loops. T o e f f e c t i v e l y sterilize t h e i n o c u l a t i n g l o o p , y o u h e a t
less e f f i c i e n t i n foods t h a t are m o r e viscous, a n d fats i n f o o d t h e w i r e t o a r e d g l o w . A s i m i l a r p r i n c i p i e is used i n incin-
c a n h a v e a p r o t e c t i v e effect o n m i c r o o r g a n i s m s . T h e d a i r y eration, a n e f f e c t i v e w a y t o sterilize a n d dispose o f c o n t a m -
i n d u s t r y r o u t i n e l y uses a test t o d e t e r m i n e w h e t h e r p r o d - i n a t e d paper cups, bags, a n d dressings.
uces h a v e b e e n pasteurized: t h e phosphatase test (phos- A n o t h e r f o r m o f d r y h e a t s t e r i l i z a t i o n is h o t ^ a i r s t e r i l '
phat'ise is a n e n z y m e n a t u r a l l y present i n m i l k ) . I f t h e i z a t i o n . I t e m s t o be sterilized b y t h i s p r o c e d u r e are p l a c e d
p r o d u c t has b e e n pasteurized, phosphatase w i l l h a v e b e e n i n a n o v e n . G e n e r a l l y , a t e m p e r a t u r e o f a b o u t 170°C m a i n -
inacr.vaced. - t a i n e d for n e a r l y 2 h o u r s ensures s t e r i l i z a t i o n . T h e longer
Ir. the classic pasteurization treatment of m i l k , the p e r i o d a n d h i g h e r t e m p e r a t u r e ( r e l a t i v e t o m o i s t h e a t ) are
m i l k .vas e.xposed t o a t e m p e r a t u r e o f a b o u t 63°C for 3 0 r e q u i r e d because t h e h e a t i n w a t e r is m o r e r e a d i l y t r a n s -
m i n u " js. M o s t i n i l k p a s t e u r i z a t i o n today uses h i g h e r t e m - ferred t o a c o o l body t h a n is t h e h e a t i n air. For exam.ple,
pera:.res, at least 72°C, b u t for o n l y 15 seconds. T h i s i m a g i n e t h e d i f f e t e n t effects o f i m m e r s i n g y o u r h a n d i n
crearrcnc, known a s high-temperature short-time b o i l i n g w a t e r at 100°C ( 2 1 2 T ) a n J o f h o l d i n g i t i n a h o t -
( H T S T ) p a s t e u r i z a t i o n , is a p p l i e d as t h e m i l k flows c o n - air o v e n at t h e same t e m p e r a t u r e for t h e same a m o u n t o f
t i n u . ,>ly past a h e a t exchanger. I n addition to k i l l i n g cime. ••••
p a c h . - n s , HTST p a s t e u r i z a t i o n lowers total bacterial
c o u n : : so che m i l k keeps w e l l u n d e r r e f r i g e r a t i o n . Filtration
N ' . k c a n also be s t e r i l i z e d — s o m e t h i n g t^uite d i f f e r e n t
R e c a l l f r o m C h a p t e r 6 that/í!tratíon is t h e passage o f a l i q -
f r o m : :>ceuri-acion—by ultra-high-temperature (UHT)
u i d o r gas t h r o u g h a screenlike m a t e r i a l w i t h porcs s m a l l
treatments so t h a t ic c a n be scorcd wichouc refrigeración.
190 PART O N E Fundamentáis of Microbiology
FIGURE 7 . 4Filter sterilization with o disposable,
presterilized plástic unit. The sample ¡s p l a c e d into the
u p p e r c h a m b e r a n d forced through the membrane filter b y a
v a c u u m in the l o w e r chamber. Pores in the membrane filter
a r e smaller than the b a c t e r i a , so bacteria are retained o n íhe
filter. The sterilized s a m p l e c a n then be decanted from the
l o w e r chamber. Similar equipment w i t h removable filter disks
is used to count b a c t e r i a in samples (see Figure 6 . 1 7 a ) .
• Filters are availáble w i t h pores small enough to retain
vñruses.
e n o u g h t o r e t a i n m i c r o o r g a n i s m s ( o f t e n t h e same appara-
tus used for c o u n t i n g ; see Figure 6.17 o n page 1 7 6 ) . A vac-
u u m t h a t is c r e a t e d i n t h e r e c e i v i n g flask h e l p s g r a v i t y pulí
t h e l i q u i d t h r o u g h t h e filter. F i l t r a t i o n is used t o sterilize
h e a t - s e n s i t i v e m a t e r i a l s , s u c h as some c u l t u r e m e d i a , e n -
r v m e s , vaccines, a n d a n t i b i o t i c s o l u t i o n s .
S o m e o p e r a t i n g t h e a t e r s a n d r o o m s o c c u p i e d by b u m
patients receive filtered air to lower the numbers o f air-
b o m e microbes. H i g h - e f í i c i e n c y p a r t i c u l a t e a i r ( H E P A )
f i l t e r s r e m o v e a l m o s t a l l m i c r o o r g a n i s m s larger t h a n a b o u t
C.3 /xm •'. d i a m e t e r .
In Z7.t e a r l y days o f m i c r o b i o l o g y , h o l l o w candle-
s h a p e d r.>.ers o f unglazed p o r c e l a i n w e r e used t o filter l i q -
u i d s . T l . t ' o n g a n d i n d i r e c t passageways t h r o u g h t h e walls
or t h e T.r.tT adsorbed t h e bacteria. U n s e e n pathogens t h a t
p-iised -„'.: j u g h t h e filters ( c a u s i n g s u c h diseases as rabies)
w e r e Ctí/.td/ííterabíe vincses. ^ peratures or by a l t e m a t i n g pressure cycles t h a t cause sporc
I n : c : - n t years, m e m b r a n e filters, composed o f such
s u b s t a n c r i as c e l l u l o s e esters or plástic p o l y m e r s , have be-
c o m e r ^ - i - i l a r for i n d u s t r i a l a n d l a b o r a t o r y use (Figure 7.4).
Tne.se r..--:rs are o n l y C . l m m t h i c k . T h e pores o f m e m -
b.-ane r..:-rs i n c l u d e , for e x a m p l e , 0 . 2 2 - / i m a n d 0 . 4 5 - ^ t m
sizes, w h i c h are i n t e n d e d for b a c t e r i a . S o m e v e r y flexible
b a c t e r i a , such as spirochetes, o r t h e wall-less m y c o p l a s m a ,
w i l l sometimes pass t h r o u g h s u c h filters, h o w e v e r . F i l t e r s
are availáble w i t h pores as s m a l l as 0 . 0 1 /xm, a size t h a t w i l l
r e t a i n viruses a n d e v e n some large p r o t e i n m o l e c u l e s .
í o w Temperatures
T h e effect o f l o w temperatures o n m i c r o o r g a n i s m s d e p e n d s
o n the particular microbe and the intensity of the applica-
t i o n . For e x a m p l e , at temperatures o f o r d i n a r y r e f r i g e r a t o r s
( 0 - 7 ° C ) , t h e m e t a b o l i c rate o f m o s t m i c r o b e s is so r e d u c e d
t h a t t h e y c a n n o t reproduce o r synthesize t o x i n s . I n o t h e r
words, o r d i n a r y r e f r i g e r a t i o n has a b a c t e r i o s t a t i c effect. Yet
psychrotrophs d o grow slowly at refrigerator temperatures
a n d w i l l a l t e r t h e appearance a n d taste o f foods a f t e r a
t i m e . For example, a single m i c r o b e r e p r o d u c i n g o n l y
t h r e e t i m e s a day w o u l d r e a c h a p o p u l a t i o n o f m o r e t h a n 2
m i l l i o n w i t h i n a week. P a t h o g e n i c b a c t e r i a g e n e r a l l y w i l l
n o t g r o w a t refrigerator t e m p e r a t u r e s , b u t f o r a t least o n e
i m p o r t a n t e x c e p t i o n , see t h e d i s c u s s i o n o f l i s t e r i o s i s i n
C h a p t e r 22 o n page 6 1 9 .
Surprisingly, some bacteria c a n g r o w at temperatures sev-
eral degrees b e l o w fireezing. M o s t foods r e m a i n u n f r o z e n
u n t i l — 2°C o r lower. R a p i d l y a t t a i n e d subfreezing t e m p e r a -
tures t e n d t o render microbes d o r m a n t b u t d o n o t necessar-
ily k i l l t h e m . S l o w freezing is m o r e h a r m f u l t o b a c t e r i a ; t h e
ice crystals t h a t f o r m a n d g r o w d i s m p t t h e c e i l u l a r a n d m o -
lecular structure o f t h e bacteria. T h a w i n g , b e i n g i n h e r e n t l y
slower, is actually t h e m o r e d a m a g i n g p a r t o f a freeze-thaw
cycle. O n c e frozen, o n e - t h i r d o f t h e p o p u l a t i o n o f some veg-
e t a t i v e bacteria m i g h t survive a year, whereas o t h e r species
m i g h t have very few survivors after t h i s t i m e . M a n y e u k a r y -
o t i c parasites, s u c h as t h e r o u n d w o r m s t h a t cause t r i c h i -
nosis, are k i l l e d by several days o f freezing t e m p e r a t u r e s .
Some i m p o r t a n t temperatures associated w i t h m i c r o o r g a n -
isms a n d food spoilage are s h o w n i n Figure 6.2 o n page 157.
High Pressure
H i g h pressure applied t o l i q u i d suspensions is transferred i n -
stantly a n d e v e n l y t h r o u g h o u t the sample. I f t h e pressure is
h i g h epQUgh, t h e molecular structures p r o t e i n s a n d car-
bohydrates are altered, r e s u l t i n g i n t h e r a p i d i n a c t i v a t i o n u f
vegetative bacterial cells. Endospores are r e l a t i v e l y resistant
t o h i g h pressure. T h e y can, .owever, be k i l l e d by o t h e r t e c h -
niques, such as c o m b i n i n g ; igh pressure w i t h elevated t e m -
germination, followed by pressure-caused death of the
resulting vegetative cells. -"ruit juices preserved by h i g h -
pressure treatments have boen m a r k e t e d i n Japan a n d t h e
U n i t e d States. A n advantage is t h a t these t r e a t m e n t s pre-
serve the flavors, éolors, a n d n u t r i e n r valúes o f t h e p r o d u c t s .
 CHAPTER 7 The Control of M i c r o b i a l G r o w t h 19|Í

Desiccation e n v i r o n m e n t t h a t causes w a t e r t o leave t h e m i c r o b i a l c e l l

(see F i g u r e 6.4 o n page 1 5 9 ) . T h i s process resembles
I n t h e <ihsence o f water, a c o n d i t i o n k n o w n as d e s i c c a -
p r e s e r v a t i o n by d e s i c c a t i o n , i n t h a t b o t h m e t h o d s Jeny
t i o n , t n i c r o o r g a n i s m s c a n n o t g r o w or reproduce b u t c a n
t h e c e l l t h e m o i s t u r e i t needs f o r g r o w t h . T h e p r i n c i p i e o f
r e m a i n v i a b l e for years. T h e n , w h e n water is made a v a i l -
o s m o t i c pressure is used i n t h e p r e s e r v a t i o n o f foods. F o r
áble t o t h e m , r h e y c a n resume t h e i r g r o w t h a n d división.
example, concentrated salt s o l u t i o n s a r e used t o c u r e
T h i s a b i i i t y is u s e d ' i n t h e l a b o r a t o r y w h e n microb2s*'are
m e a t s , a n d t h i c k sugar s o l u t i o n s a r e used t o p r e s e r v e
preserved by l y o p h i l i z a t i o n , or freeze-drying, a process de-
fruits.
scribed m C h a p t e r 6 o n page 169. C e r t a i n foods are also
A s a general rule, m o l d s a n d yeasts are m u c h m o r e c a -
freeze-dried ( f o r e x a m p l e , coffee a n d some f r u i t a d d i t i v e s
pable t h a n bacteria o f g r o w i n g i n m a t e r i a l s w i t h l o w m o i s -
for d r y cereals).
t u r e o r h i g h o s m o t i c pressures. T h i s p r o p e r t y o f m o l d s ,
T h e resistance o f v e g e t a t i v e cells t o d e s i c c a t i o n varies
sometimes combiní-d w i t h t h e i r a b i l i t y t o g r o w under
w i t h t h e species a n d t h e organism's e n v i r o n m e n t . F o r ex-
a c i d i c c o n d i t i o n s , is t h e reason firuits a n d g r a i n s are s p o i l e d
a m p l e , t h e g o n o r r h e a b a c t e r i u m c a n w i t h s t a n d dryness for
b y m o l d s r a t h e r t h a n b y bacteria. I t is also p a r t o f t h e rea-
o n l y a b o u t a n h o u r , b u t t h e tuberculosis b a c t e r i u m c a n r e -
s o n m o l d s are able t o f o r m m i l d e w o n a d a m p w a l l o r a
m a i n v i a b l e f o r m o n t h s . Viruses are g e n e r a l l y resistant t o
shower c u r t a i n .
d e s i c c a t i o n , b u t t h e y are n o t as resistant as b a c t e r i a l e n -
dospores, some o f w h i c h h a v e s u r v i v e d f o r c e n t u r i e s . T h i s
a b i l i t y o f c e r t a i n d r i e d microbes a n d endospores t o r e m a i n
Radiation
v i a b l e is i m p o r t a n t i n a h o s p i t a l s e t t i n g . D u s t , c l o t h i n g , R a d i a t i o n has various effects o n cells, d e p e n d i n g o n i t s
b e d d i n g , a n d dressings m i g h t c o n t a i n i n f e c t i o u s m i c r o b e s wavelength, intensity, a n d d u r a t i o n . R a d i a t i o n t h a t k i l l s
t u >'ried m u c u s , uriñe, pus, a i i d feces. m i c r o o r g a n i s m s ( s t e r i l i z i n g r a d i a t i o n ) is o f t w o types: i o n -
izing and nonionizing.
Osmotic Pressure l o n i z i n g r a d i a t i o n — g a m m a rays, X rays, o r h i g h -
e n e r g y electrón b e a m s — h a s a wavelength shorter t h a n
T h e use o f h i g h c o n c e n t r a t i o n s o f salts a n d sugars t o p r e -
t h a t o f n o n i o n i z i n g r a d i a t i o n , less t h a n a b o u t 1 n m . T h e r e -
serve f o o d is based o n t h e effects o f osmotic pressure. High
f o r e , i t carries m u c h m o r e e n e r g y ( F i g u r e 7 . 5 ) . Gamma
c o n c e n t r a t i o n s o f these substances créate a h y p e r t o n i c

• • •' 1m
10-^nm ICr^nm 1 nm 10^nm lO^nm (10^nm) lO^m
1 I —
1 1
Gamma
X rays UV Infrared Microwaves Radio waves
rays

Ultraviolet (UV) light

700 nm 750 nm
Ijj nm 250 nrry/ ^ 3 0 0 nnn/350 nm 400 nm 450 nm 500 nm 550 nm 600 nm 650 nm

280 nm 295'nm 330 nm

Wavelengtii increases'
— Energy increases —

r 3URE 7 , 5 The radiant energy spectrum. Visible light o n d other forms of rodiont
e - e r g y radióte t h r o u g h space os waves of various lengths. l o n i z i n g r a d i a t i o n , s u ' h as
gz-r,ma rays a n d X rays, has a wavelength shorter than 1 nm. N o n i o n i z i n g r a d i a t i o n , such
C: j l t r o v i o l e t (UV) light, has o w a v e l e n g t h between 1 nm a n d a b o u t 3 8 0 n m , where the
V i.ole spectrum begins.

• 'Vhct effect might increased U V radiation (due to decrease in the ozone layer) have o n the
Earth's ecosystems?
192 PART O N E Fundamentáis of Microbiology
rays are e m i t t e d b y c e r t a i n r a d i o a c t i v e e l e m e n t s s u c h as
c o b a k , a n d electrón beams are p r o d u c e d b y a c c e l e r a t i n g
e l e c t r o n s t o h i g h e n e r g i e s i n special m a c h i n e s . X rays,
w h i c h are p r o d u c e d b y m a c h i n e s i n a m a n n e r s i n i i l a r t o
t h e p r o d u c t i o n o f electrón beams, are s i m i l a r i n n a t u r e t o
g a m m a rays. G a m m a rays penétrate deeply b u t m a y r e -
q u i r e h o u r s t o s t e r i l i z e large masses; high-energy electrón
beams h a v e m u c h l o w e r p e n e t r a t i n g p o w e r b u t usually r e -
q u i r e o n l y a f e w seconds o f exposure. T h e p r i n c i p a l effect
of i o n i z i n g r a d i a t i o n is t h e i o n i z a t i o n o f water, w h i c h
f o r m s h i g h l y r e a c t i v e h y d r o x y l radicáis (see t h e discus-
sion of toxic-forms o f oxygen i n Chapter 6, pages
1 6 1 - 1 6 3 ) . T h e s e radicáis r e a c t w i t h o r g a n i c c e i l u l a r
c o m p o n e n t s , especially D N A .
T h e so-called t a r g e t t h e o r y o f damage b y r a d i a t i o n sup-
poses t h a t i o n i z i n g partióles, o r p a c k e t s o f energy, pass
t h r o u g h o r cióse t o v i t a l p o r t i o n s o f t h e c e l l ; these c o n s t i -
t u t e " h i t s . " O n e , o r a few, h i t s m a y o n l y cause n o n l e t h a l
m u t a t i o n s , some o f t h e m c o n c e i v a b l y useful. M o r e h i t s are
h k e l y t o cause s u f f i c i e n t m u t a t i o n s t o k i l l t h e m i c r o b e .
T h e f o o d i n d u s t r y hcis r e c e n d y r e n e w e d its i n t e r e s t i n
t h e use o f r a d i a t i o n f o r f o o d p r e s e r v a t i o n (discussed m o r e
f u l l y i n C h a p t e r 2 8 o n page 7 9 5 ) . L o w - l e v e l i o n i z i n g r a d i -
a t i o n , used f o r years i n m a n y c o u n t r i e s , has b e e n a p p r o v e d
i n t h e U n i t e d S t a t e s f o r p r o c e s s i n g spices a n d c e r t a i n
m e a t s a n d vegetables. l o n i z i n g r a d i a t i o n , especially h i g h -
e n e i ^ electrón beams, is used f o r t h e s t e r i l i z a t i o n o f p h a r -
m a c e u t i c a l s a n d disposable d e n t a l a n d m e d i c a l supplies,
s u c h as plástic syringes, s u r g i c a l gloves, s u t u r i n g m a t e r i a l s ,
and c a t h e t e r s . A s a p r o t e c t i o n against b i o t e r r o r i s m , t h e
p o s t a l service o f t e n uses electrón b e a m r a d i a t i o n t o s t e r i l -
ize c e r t a i n classes o f m a i l .
N o n i o n i z i n g : r a d i a t i o n has a w a v e l e n g t h l o n g e r t h a n
t h a t o f i o n i i i n g r a d i a t i o n , u s u a l l y greater t h a n a b o u t 1 n m .
T h e best e x a m p l e o f n o n i o n i z i n g r a d i a t i o n is u l t r a v i o l e t
(U\ l i g h t . U V l i g h t damages t h e D N A o f exposed cells
by causing bonds t o f o r m between adjacent p y r i m i d i n e
bases (page 2 3 0 ) , u s u a l l y t h y m i n e s , i n D N A c h a i n s (see
F i g u r e 8.2C>. T h e s e Úiymme áin^s i n h i b i t correct r e p l i c a -
r i o n o f the D N A d u r i n g reproduction o f the cell. T h e U V
w a v e l e n g t h i m o s t effective f o r k i l l i n g m i c r o o r g a n i s m s are
a b o u t 26C r . m ; these w a v e l e n g t h s are specifically absorbed
by c e i l u l a r L.^^A. U V r a d i a t i o n is also used t o c o n t r o l m i -
crobes i n the air. A U V o r " g e r m i c i d a l " l a m p is c o m m o n l y
f o u n d i n hi-spital r o o m s , auiseries, o p e r a t i n g rooms, a n d
cafererias. L V l i g h t is also^jsed t o d i s i n f e c t vaccines a n d
o t h e r m e d i c i l p r o d u c t s . A m a j o r disadvantage o f U V l i g h t
aí> a d j s i n f c : : a n t is t h a t t h e r a d i a t i o n is n o t very p e n e t r a t -
i n g . ív'j t h e rganisms t o b e k i l l e d must be d i r e c t l y exposed
t o t h e rays. Organisms p r o t e c t e d by solids a n d such cover-
ings xs pape: glass, a n d t e x t i l e s are n o t affected. A n o t h e r
p o t e r . t i a l p r . h l e m is t h a t U V l i g h t c a n damage h u m a n
eyes, a n d p r o l o n g e d exposure c a n cause b u m s a n d s k i n
cáncer i n h u m a n s .
S u n l i g h t c o n t a i n s some U V r a d i a t i o n , b u t t h e s h o r t e r
w a v e l e n g t h s — t h o s e m o s t effective against b a c t e r i a — a r e
screened o u t b y t h e ozone layer o f t h e a t m o s p h e r e . T h e a n -
tiTnicrchial effect o f s u n l i g h t is d u e a l m o s t e n t i r e l y t o t h e
f o r m a t i o n o f s i n g l e t o x y g e n i n t h e c y t o p l a s m (see C h a p t e r
6 o n page 162). M a n y p i g m e n t s p r o d u c e d b y b a c t e r i a p r o -
vide p r o t e c t i o n f r o m s u n l i g h t .
M i c r o w a v e s d o n o t h a v e m u c h d i r e c t eñtcx. o n m i -
croorganisms, a n d b a c t e r i a c a n r e a d i l y be i s o l a t e d f r o m t h e
interior o f recently operated m i c r o w a v e ovens. M o i s t u r e -
c o n t a i n i n g foods are h e a t e d b y m i c r o w a v e a c t i o n , a n d t h e
h e a t w i l l k i l l m o s t v e g e t a t i v e p a t h o g e n s . S o l i d foods h e a t
u n e v e n l y because o f t h e u n e v e n d i s t r i b u t i o n o f m o i s t u r e .
For t h i s reason, p o r k c o o k e d i n a m i c r o w a v e o v e n has b e e n
responsible f o r o u t b r e a k s o f t r i c h i n o s i s .
* * *
T a b l e 7.5 summarizes t h e p h y s i c a l m e t h o d s o f m i c r o b i a l
control.
Chemical Methods of
Microbial Control
C h e m i c a l agents are used t o c o n t r o l t h e g r o w t h o f m i -
crobes o n b o t h l i v i n g tissue a n d i n a n i m a t e o b j e c t s . U n f o r -
t u n a t e l y , f e w c h e m i c a l agents a c h i e v e s t e r i l i t y ; m o s t o f
t h e m merely reduce m i c r o b i a l p o p u l a t i o n s t o safe levéis o r
remove vegetative forms o f pathogens f r o m objects. A
c o m m o n p r o b l e m i n d i s i n f e c t i o n is t h e s e l e c t i o n o f a n
agent. N o single d i s i n f e c t a n t is a p p r o p r i a t e f o r a l l c i r c u m -
stances.
Principies of Effective Disinfection
Learning Objective
• List tile factors related to effective disinfection.
By r e a d i n g t h e l a b e l , w e c a n l e a m a great d e a l a b o u t a d i s -
infectant's properties. T h e l a b e l w i l l usually i n d i c a t e w h a t
groups o f o r g a n i s m s t h e d i s i n f e c t a n t w i l l be effective
against. ^ ^ e m b e r that t h e concentratioji o f a disinfec-
t a n t affects its a c t i o n , so i t s h o u l d always be d i l u t e d e x a c t l y
as specified by t h e m a n u f a c t u r e n
A l s o consider t h e n a t u r e o f t h e m a t e r i a l b e i n g d i s i n -
fected. For e x a m p l e , are o r g a n i c m a t e r i a l s p r e s e n t that
m i g h t interfere w i t h t h e a c t i o n o f t h e d i s i n f e c t a n t ? S i m i -
larly, t h e p H o f t h e médium o f t e n has a great effect o n a
disinfectants a c t i v i t y
A n o t h e r very i m p o r t a n t c o n s i d e r a t i o n is w h e t h e r t h e
d i s i n f e c t a n t w i l l easily m a k e c o n t a c t w i t h t h e m i c r o b e s .
An área m i g h t need t o h e s c r u b b e d a n d r i n s e d before t h e
TABLE 7 . 5 Physical Methods Used to Control Microbial G r o w t h
Mechonism
Method o f A ct i o n . Commeht P r e f e r r e d Use

Heat
1. Moist heat
o. Boiling or ^ P.ot'^in denaturation Kills vegetative bacterial ond Dishes, basins, pitchers, various
flowing steam fungal pathogens ond almost equipment
all vir-üses within 10 m i n ; less
effective on endospores.
b. Autoclaving Protein denaturation Very effective method of steriliza- Microbiologicol medio, solu-
tion; at about 15 psi of pressure tions, linens, utensils, dress-
(121 °C), olí vegetative cells ings, equipment, ond other
and their endospores are killed Ítems that con withstand
in about 15 min. temperature o n d pressure
2. Pasteurization Protein denaturation Heat treatment for milk (72°C for Milk, creom, o n d certain
about 15 sec) that kills all patho- olcoholic beveroges (beer
gens ond most nonpathogens. ond wine)
3. Dry heat
a.. Direct floming Burning contaminants Very effective method of Inoculating loops *
to ashes sterilization.

b. Incineration Burning to ashes Very effective method of Paper cups, contaminoted dress-
sterilization. ings, animal corcasses, bags,
o n d wipes
c. Hot-oir Oxidation Very effective method of steriliza- Empty glassware, instruments,
sterilization tion, but requires temperature needles, a n d glass syringes
of 170°C for a b o u t 2 hr.

Filtration Separation of bacteria Removes microbes by passage of Useful for sterilizing liquids
from suspending o liquid or gas through a screen- (enzymes, vaccines) that a r e
liquid like material. Most filters in use destroyed by heat
consist of cellulose acetóte
or nitrocellulose.
Coid
1. Refrigeration Decreased chemical Has o bacteriostatic effect Food, drug, o n d culture
reactions and possible preservation
changes in proteins

2. Deep-freezing Decreased chemical A n effective method for preserv- Food, drug, a n d culture
(see Chapter ó, reactions ond possible ing microbial cultures, in which preservation
page 1ó9) changes in proteins cultures ore quick-frozen
between - 5 0 ° o n d - 9 5 ° C .

3. Lyophilization Decreased chemical Most effective method for long- Food, drug, o n d culture
•see Chapter ó, reactions ond possible term preservation of microbial preservation
cage 169) cfionges in proteins cultures; water removed by high
vacuum ot low temperature.

High pressure Aiteration of molec- Preservation of colors, flavors, Fruit juices

ular structure nutrient valúes.
of proteins and
carbohydrotes

Desiccation Disruption of Involves removing water from Food preservation

meíobolism microbes; primarily bacteriostatic.

Csmotic Pressure Plasmolysis Results. in loss of water from Food preservation

microbial cells.

Radiation
1, o n i z i n g Destruction of D N A Not widespreod in routine Used for sterilizing phormoceu-
sterilization. . • ticals and medical and dental
supplies

2. ' ionionizing Domoge to D N A Radiation not very penetrating. Control of closed environment
with UV (germicidol) lamp
194 PART O N E Fundamentáis of M i c r o b i o l o g y

d i s i n f e c t a n t is a p p l i e d . I n g e n e r a l , d i s i n f e c t i o n is a g r a d u a l Types of Disinfectants
process. T h u s , t o be effective, a d i s i n f e c t a n t m i g h t n e e d t o
Learning Objectives
be left o n a surface for several h o u r s .
• Identify the methods of action and preferred uses of v
chemical disinfectants.
Evaluating a Disinfectant
• Differenfiate between halogens used as antiseptics and
Learning Objective as disinfectants.
• Interpret tiie results of use-dilution tests and the disli-
• Identify the appropriate uses for surface-active agents.
diffusion method.
• List the advantages of glutaraldehyde over other chemi-
Use-Dilufion Tests cal disinfectants.

T h e r e is a n e e d t o evalúate t h e effectiveness o f d i s i n f e c - • Identify the method of sterilizing plástic labware.

t a n t s a n d a n t i s e p t i c s . F o r m a n y years t h e s t a n d a r d test was
Phenol and Phenolics
t h e phenol coefficient test, w h i c h c o m p a r e d t h e a c t i v i t y o f a
given disinfectant w i t h t h a t of phenol. However, t h e cur- L i s t e r was t h e first t o use p h e n o l ( c a r b o l i c a c i d ) t o c o n t r o l

rent standard is the American Official Analytical surgical i n f e c t i o n s i n t h e o p e r a t i n g r o o m . I t s use h a d b e e n

C h e m i s t ' s u s e - d i i u t i o n t e s t . F o r m o s t purposes, t h e t h r e e suggested by its effectiveness i n c o n t r o l l i n g o d o r i n sewage.

b a c t e r i a used i n t h i s test are Sdmonetia choleraesíús, Staphy- I t is n o w rarely used as a n a n t i s e p t i c o r d i s i n f e c t a n t be-

lococcus aureiis, a n d Pseudomonas aeru^nosa. M e t a l carrier cause i t i r r i t a t e s t h e s k i n a n d has a disagreeable o d o r , l i is

r i n g s are d i p p e d i n t o s t a n d a r d i z e d c u l t u r e s o f t h e test bac- o f t e n used i n t h r o a t lozenges f o r i t s l o c a l a n e s t h e t i c effect

teria grown i n liquid media, removed, a n d dried at 3TC b u t has l i t t l e a n t i m i c r o b i a l effect a t t h e l o w c o n c e n t r a -

f o r a s h o r t rime. T h e d r i e d c u l t u r e s are t h e n p l a c e d i n t o a t i o n s used. A t c o n c e n t r a t i o n s a b o v e 1 % ( s u c h as i n some

s o l u t i o n o f t h e d i s i n f e c t a n t a t t h e concentratíon r e c o m - t h r o a t sprays), h o w e v e r , p h e n o l has a s i g n i f i c a n t a n t i b a c -

m e n d e d by t h e m a n u f a c t u r e r a n d left t h e r e f o r 10 m i n u t e s t e r i a l effect. T h e s t r u c t u r e o f a p h e n o l m o l e c u l e is s h o w n

a t 2 0 ° C . F o l l o w i n g t h i s e x p o s u r e , t h e c a r r i e r rings are i n Figure 7.7a.

t r a n s f e r r e d t o a médium t h a t w i l l p e r m i t t h e g r o w t h o f a n y D e r i v a t i v e s o f p h e n o l , c a l l e d phene*'*->í, c o n t a i n a m o l -
s u r v i v i n g b a c t e r i a . T h e effectiveness o f t h e d i s i n f e c t a n t ecule o f p h e n o l t h a t has b e e n c h e m i c a l l y íiltered t o reduc e
c a n t h e n be d e t e r m i n e d b y t h e n u m b e r o f c u l t u r e s t h a t its i r r i t a t i n g q u a l i t i e s o r increase i t s a n t i b a c t e r i a l a c t i v i t y i n
grow. c o m b i n a t i o n w i t h a soap or, d e t e r g e n t . P h e n o l i c s e x e r t a n -

V a r i a t i o n s o f t h i s m e t h o d are used f o r t e s t i n g t h e effec- t i m i c r o b i a l a c t i v i t y by i n j u r i n g l i p i d - c o n t a i n i n g plasma

tiveness o f a n t i m i c r o b i a l agents against endospores, m y - membranes, w h i c h results i n leakage o f c e i l u l a r c o n t e n t s .

c o b a c t e r i a t h a t cause t u b e r c u l o s i s , a n d f u n g i , because t h e y T h e c e l l w a l l o f m y c o b a c t e r i a , t h e causes o f t u b e r c u l o s i s

are diíficult t o c o n t r o l w i t h c h e m i c a l s . A l s o , tests o f a n a n d leprosy, are r i c h i n lipids, w h i c h m a k e t h e m susceptible

l i m i c r o b i a l s i n t e n d e d f o r special purposes, s u c h as d a i r y t o p h e n o l d e r i v a t i v e s . A useful p r o p e r t y o f p h e n o l i c s as dis-

u t e n s i l d i s i n f e c t i o n , m a y s u b s t i t u t e o t h e r test b a c t e r i a . i n f e c t a n t s is t h a t t h e y r e m a i n a c t i v e i n t h e presence o f or-

\ * i r u c i d a l c h e m i c a l s are usually tested against c u l t u r e s o f ganic c o m p o u n d s , t h e y are stable, a n d t h e y persist f o r l o n g

K e w c a s t l e disease v i r u s ( a disease o f b i r d s a n d d o m e s t i c periods after a p p l i c a t i o n . For these reasons, p h e n o l i c s are

t o w l ) . A f t e r exposure t o t h e c h e m i c a l , t h e c u l t u r e s are i n - suitable agents f o r d i s i n f e c t i n g pus, saliva, a n d feces.

j e c t e d i n t o e m b r y o n a t e d chicleen eggs; i f any viruses sur- O n e o f t h e most f r e q u e n t l y used p h e n o l i c s is d e r i v e d

v i v e , thev w i l l k i l l t h e e m b r y o s . f r o m c o a l tat, a g r o u p o f c h e m i c a l s c a l l e d cresols. A very

i m p o r t a n t cresol is O-phenylphenol (see Figures 7.6 a n d
The Disk-Diffusion Method 7.7b), t h e m a i n i n g r e d i e n t i n m o s t f o r m u l a t i o n s o f Lysol.
Cresots are very g o o d surface d i s i n f e c t a n t s .
T h e áisk'iíffusion method is used i n t e a c h i n g laboratories t o
evalúate -.he efficacy o f a c h e m i c a l a g e n t . A disk o f filrer
Bisphenoís
p.-íper i5 ^oaked w i t h a c h e m i c a l a n d p l a c e d o n a n agar
p'.are th¿: has been p r e v i o u s l y i n o c u l a t e d a n d i n c u b a t e d B i s p h e n o l s are d e r i v a t i v e s o f p h e n o l t h a t c o n t a i n t w o

u ith th . -^r orgíinism. .After i n c u b a t i o n , i f the c h e m i c a l is p h e n o l i c groups c o n n e c t e d by a b r i d g e (bis i n d i c a t e s t w o ) .

e r t e c t i v e . ^ clear zone repr^isenting i n h i b i t i o n o f g r o w t h O n e b i s p h e n o l , hexachlorophene (Figures 7.6 a r . d 7.7c), is

c . m be i<rtn a r o u n d t h e d i s k (Figure 7 . 6 ) . . a n i n g r e d i e n t o f a p r e s c r i p t i o n l o t i o n , p H i s o H e x , used for

surgical a n d h o s p i t a l m i c r o b i a l c o n t r o l procedures. G r a m -
D i s k - l o n t a i n i n g a n t i b i o t i c s are c o m m e r c i a l l y a v a i l -
p o s i t i v e s t a p h y l o c o c c i a n d s t r e p t o c o c c i , w h i c h c a n cause
c'.rle a n c jsed t o d e t e r m i n e m i c n ^ b i a l s u s c e p t i b i l i t y t o a n -
s k i n i n f e c t i o n s i n n e w b o r n s , are p a r r i c i i l a r l y susceptible t o
t:'ri(>ncí '.-e Figure 20.1 7 o n page 5 7 9 ) .
 CHAPTER 7 The Control of M i c r o b i a l Grov.ih 195

I
phenylphenol, MHexachioroph

Staphyiococcus aureus ; Escherichia coH Pseudomonas aeruginosa

(gram-positive) V . "(gram-negative) . (gram-negative)

FIGURE 7 . 6 Evaluation of disinfectants by the disir-diffusion method. In this

experiment, p a p e r disks are soaked in a solution of disinfectant a n d p l a c e d o n the surface of
a nutrient médium o n w h i c h a culture of test bacteria has been spread to p r o d u c e uniform
growth.
A t the t o p o f e a c h píate, the tests show that chlorine (as sodium hypochlorite) w a s effec-
tive a g a i n s t all the test bacteria but w a s more effective against gram-positive bacteria.
A t the b o t t o m r o w of each píate, the tests show that the q u o t e r n a r y a m m o n i u m c o m p o u n d
( " q u a t ' l w a s also m o r e effective against the gram-positive bacteria, but it d i d not affect the
pseudomonads at all. "
A t the left side o f e a c h píate, the tests show that h e x a c h l o r o p h e n e w a s effective a g a i n s t
g r a n v p o s i t i v e b a c t e r i a only.
A i rhe r i g h t sides, O-phenylphenol w a s ineffecíive a g a i n s t pseudomonads but w a s almost
equolfy effective a g a i n s t the gram-positive bacteria a n d the gram-negaíive b a c t e r i a .
AJI four c h e m i c a l s w o r k e d against the gram-positive test b a c t e r i a , but o n l y o n e of the four
chemicals affected p s e u d o m o n a d s .

• W h c h g r o u p of bacteria is most resistant to the disinfectants tested?

h e x a c " i o r o p h e n e , so i t is o f t e n used t o c o n t r o l s u c h i n f e c - m a n y o t h e r a n t i b i o t i c s a n d d i s i n f e c t a n t s (see t h e discus-

t i o n s .n nurseries. H o w e v e r , excessive use p f t h i s b i s p h e - s i o n o n pages 3 1 2 , 4 2 3 , a n d 5 9 7 ) .
a o l , i _ c h a s b a t h i n g i n f a n t s w i t h i t several t i m e s a day, c a n
i e a d : : n e u r o l o g i c a l damage. Biguanides
t h e r widely used b i s p h e n o l is triclosan (Figure Chlorhexidine is a m e m b e r o f t h e b i g u a n i d e g r o u p w i t h a
7.7d V - a i n g r e d i e n t i n a n t i b a c t e r i a l soaps a n d at least o n e b r o a d s p e c t r u m o f a c t i v i t y . I t is f r e q u e n t l y used for m i c r o -
t o o t r . : ;.>te. T r i c l o s a n has e v e n been incorporated i n t o bial c o n t r o l o n skin and mucous membranes. Combined
k i c c h r : . c u t t i n g b o a r d s a n d the handles of k n i v e s a n d o t h e r w i t h a d e t e r g e n t or a l c o h o l , c h l o r h e x i d i n e is also used for
pla-r;: a c c h e n w a r e . Its use is n o w so widespread t h a t resis- surjiical h a n d scrubs a n d p r e o p e r a t i v e s k i n p r e p a r a t i o n i n
t a n r "r -creria h a v e b e e n reported, a n d c o i K e r n s a b o u t its p a t i e n t s . ín s u c h a p p l i c a t i o n s , its s t r o n g a f f i n i t y f o r b i n d i n g
eftec: n m i c r o b e s ' resistance t o certain antibiotics have t o t h e s k i n o r mucous m e m b r a n e s is a n a d v a n t a g e , as is its
b e e n ::;.>ed. ~"riclosan i n h i b i t s a n e n z y m e needed for t h e l o w t o x i c i t y . H o w e v e r , c o n t a c t w i t h t h e eyes c a n cause
hios'.T.'.'.esis o fattv acids ( l i p i d s ) , w h i c h affects t h e i n - damage. Its k i l l i n g effect is r e l a t e d t o t h e i n j u r y i t causes t o
r e g n : r t h e p l a s m a m e m b r a n e . It is e.specially e t t e c t i v e r h e plasma m e m b r a n e . I t is b i o c i d a l against m o s t vegeta-
.igair.r- i r a m - p o s i t i \ b a c t e r i a but also works w e l l against t i v e bacteria a n d f u n g i . M y c o b a c t e r i a are r e l a t i v e l y resis-
t u n g i : ' : \c b a c t e r i a . Tliere are certain excep- t a n t , a n d endospores a n d p r o t o z o a n cysts are n o t affected.
t i o n - . i c h as Pseudomonas acruginosa, a gram-negative The o n l y viruses affected are c e r t a i n e n v e l o p e d (lipo-
b a c ' c : . n i t h a t is v e r v rcsistani t o t r i c l o s a n , as w e l l as t o p h i k c ) types (see C h a p t e r 13).
196 PART O N E Fundamentáis of Microbiology

OH (1)
CI2 + H2O; H^ + c r + HOCl
Chlorine Water Hydrogen Chloride Hypochlorous
ion ion acid
(a) Ptienol (b) 0 - p h e n v l p t i e n o l
(2)
HOCl ^ H" + o c r
Hypochlorous Hydrogen Hypochlorite
acid ion ion

01 OH HO Cl
(c) H e x a c f i l o r o p h e n e (a bisptienol)
( d ) Triclosan (a bisptienol)
FIGURE 7 . 7 The structure of phenolics and
bisphenoíS.
• At i ifrotions a b o v e 10%^ phenol has a significant
anííi5c-ctrial effect.
T h e h s . ú g e n s , p a r t i c u l a r l y i o d i n e a n d c h l o r i n e , are effec-
t i v e a n : m i c r o b i a l a g e n t s , b o t h a l o n e a n d as c o n s t i t u e n t s
o f inor¿ m i c o r o r g a n i c c o m p o u n d s . Iodine (I2) is o n e o f t h e
plde'^t a n d m o s t effeccive antiseptics^ I t is effective against
a l l k i n d s o f b a c t e r i a , m a n y endospores, v a r i o u s f u n g i , a n d
s o m e viruses. O n e p r o p o s e d m e c h a n i s m for t h e a c t i v i t y o f
i o d i n e , i s t h a t i t c o m b i n e s w i t h c e r t a i n a m i n o acids o f e n -
zymes a n d o t h e r c e i l u l a r p r o t e i n s . T h e e x a c t m o d e o f ac-
t i o n is n o t k n o w n .
I o d i n e is availáble as a t i n c t u r e — t h a t is, i n s o l u t i o n
i n aquec'us a l c o h o l — a n d as a n i o d o p h o r . A n iodophor
is a c o m b i n a t i o n o f i o d i n e a n d a n o r g a n i c m o l e c u l e , f r o m
w h i c h t h e i o d i n e is released s l o w l y . l o d o p h o r s h a v e t h e
antimicrobial activity o f iodine, but they do n o t stain and
are less i r r i t a t i n g . T h e m o s t c o m m o n c o m m e r c i a l p r e p a -
r a t i o n s are B e t a d i n e a n d I s o d i n e . T h e s e are povídone-
lodines: povidone is a surface-active iodophor that
i m p r o v c s t h e w e t t i n g a c t i o n a n d serves as a r e s e r v o i r o f
tree i o c i n e . l o d i n e s are used m a i n l y for s k i n d i s i n f e c t i o n
a n d w c u n d t r e a t m e n t . M a n y c a m p e t s are f a m i l i a r w i t h
i o d i n e r'^r w a t e r t r e a t m e n t . T o t r e a t water, i o d i n e t a b l e t s
are a d i . d o r t h e w a t e r c a n be passed t h r o u g h i o d i n e -
treatec resin filters.
C L : - i n e ( C I 2 ) , as a gas or i n c o m b i n a t i o n w i t h o t h e r
chemic';i.s, is a n o t h e r w i d e l y used d i s i n f e c t a n t . Its g e r m i c i -
d a l a c t : : n is caused b y t h e h y p o c h l o r o u s acid ( H O C l ) t h a t
forms V . - e n c h l o r i n e i.< added t o w a t e r :
E x a c t l y h o w h y p o c h l o r o u s a c i d exerts its k i l l i n g p o w e r i ^
n o t k n o w n . I t is a s t r o n g o x i d i z i n g a g e n t t h a t p r e v e n ts
m u c h o f t h e c e i l u l a r e n z y m e system f r o m f u n c t i o n i n g .
H y p o c h l o r o u s a c i d is t h e m o s t effective f o r m o f c h l o r i n e
because i t is n e u t r a l i n e l e c t r i c a l c h a r g e a n d diffiises as
r a p i d l y as w a t e r t h r o u g h t h e c e l l w a l l . Because o f i t s nega-
t i v e charge, t h e h y p o c h l o r i t e i o n ( O C P ) c a n n o t enter the
c e l l fireely.
A l i q u i d f o r m o f compressed c h l o r i n e gas is used e x t e n -
sively for d i s i n f e c t i n g m u n i c i p a l d r i n k i t Í water, y^ater i n
s w i m m i n g p o o l s , a n d sewage. Several c o m p o i m d s o f c h l o -
r i n e are also effective d i s i n f e c t a n t s . F o r e x a m p l e , s o l u t i o n s
o f caldum hypochlorite [ C a ( O C l ) 2 ] are used t o d i s i n f e c t dair\
e q u i p m e n t a n d restaurant e a t i n g u t e n s i l s . T l i i s c o m p o u n d ,
o n c e c a l l e d c h l o r i d e o f l i m e , was used as e a r l y as 1 8 2 5 , l o n g
before t h e c o n c e p t o f a g e r m t h e o r y for disease, t o soak hos-
p i t a l dressings i n París hospitals. I t was also t h e d i s i n f e c t a n t
used i n t h e 1840s b y Senunelweis t o c o n t r o l h o s p i t a l i n f e c -
tior\ d u r i n g c h i l d b i r t h , as m e n t i o n e d i n C h a p t e r 1, page 9.
A n o t h e r c h l o r i n e c o m p o u n d , sodium hypochlorite (NaOCl;
see F i g u r e 7 . 6 ) , is used as a h o u s e h o l d d i s i n f e c t a n t a n d
b l e a c h ( C l o r o x ) , a n d as a d i s i n f e c t a n t i n d a i r i e s , f o o d -
processing establishments, and hemodialysis systems.
W h e n t h e q u a l i t y o f d r i n k i n g w a t e r is i n q u e s t i o n , h o u s e -
h o l d bleach can provide a rough equivalent o f m u n i c i p a l
c h l o r i n a t i o n . A f t e r t w o drops o f b l e a c h are a d d e d t o a l i t e r
o f w a t e r ( f o u r drops i f t h e w a t e r is c l o u d y ) a n d t h e m i x t u r e
has sat f o r 3 0 m i n u t e s , t h e w a t e r is c o n s i d e r e d safe for
d r i n k i n g u n d e r emergency c o n d i t i o n s . U . S . m i l i t a r y forces
i n t h e field are issued tablets ( C h l o r - F l o c ) that contain
sodium dichloroisocyanurctc, a form of chlorine combined
w i t h a n agent t h a t flocculates (coagulates) suspended m a -
terials i n a w a t e r sample, causing t h e m t o settle o u t , t h u s
clarifying it.
Chbrine dioxide (CIO2) is a gaseous f o r m o f c h l o r i n e ,
o c c a s i o n a l l y used for área d i s i n f e c t i o n , m o s t n o t a b l y , t o
k i l l endospores o f t h e a n t h r a x b a c t e r i u m .
A n o t h e r group of chlorine compounds, the chlora-
mines, consist o f c h l o r i n e a n d a m m o n i a . T h e y are used as
disinfectants, antiseptics, or s a n i t i z i n g agents. Chlo-
r a m i n e s are very stable c o m p o u n d s t h a t reléase c h l o r i n e
o v e r l o n g periods. T h e y are r e l a t i v e l y effective i n organic
m a t t e r , b u t t h e y h a v e t h e disadvantages o f a c t i n g m o r e
 CHAPTER 7 The Conhol of M i c r o b i a l G r o w l h 197

T w o o f t h e most c o m m o n l y used a l c o h o l s are e t h a n o i

TABLE 7.Ó Biocidai Action of Various
and isopropanol. T h e recommended o p t i m u m concentra-
Concentrations of Ethanoi in
t i o n o f ethanoi is 7 0 % , b u t c o n c e n t r a t i o n s b e t w e e n 60%
Aqueous Solution Against
and 9 5 % seem t o k i l l as w e l l ( T a b l e 7 - 6 ) . Puré e t h a n o i is
Streptococcus pyogenes
less effective t h a n aqueous s o l u t i o n s ( e t h a n o i m i x e d w i t h
T i m e (sec) w a t e r ) because d e n a t u r a t i o n r e q u i r e s w a t e r . Isopropanol,
_.<C<dnceiitrat¡fm/^
<
o f t e n sold as r u b b i n g a l c o h o l , is s l i g h t l y s u p e r i o r t o e t h a n o i
o f E t h a n o i i%} i ta,;: 20, 40 50
as a n a n t i s e p t i c a n d d i s i n f e c t a n t . M o r e o v e r , i t is less
100 - - - - - v o l a t i l e , less e x p e n s i v e , and m o r e easily o b t a i n e d than

95 + + + + ethanoi.
E t h a n o i a n d i s o p r o p a n o l are o f t e n used t o e n h a n c e t h e
90 + 4- + +
effectiveness o f o t h e r c h e m i c a l agents. F o r e x a m p l e , a n
80 + + + . + + aqueous s o l u t i o n o f Z e p h i r a n ( d e s c r i b e d o n page 1 9 8 ) k i l l s

70 + + a b o u t 4 0 % o f t h e p o p u l a t i o n o f a test o r g a n i s m i n t w o m i n -
'i' utes, whereas a t i n c t u r e o f Z e p h i r a n k i l l s a b o u t 8 5 % i n t h e
60 + + ' ' .' + +
same p e r i o d . T o c o m p a r e t h e effectiveness o f t i n c t u r e s a n d
50 - aqueous s o l u t i o n s , see F i g u r e 7.10 o n page 1 9 9 .

40 — — — — —

Heovy Metáis and Their Compounds

NOTE: A minus sign indicates no biocidal action (bacterial
S e v e r a l h e a v y metáis c a n be b i o c i d a l o r a n t i s e p t i c , i n c l u d -
growth); a plus sign indicates biocidal action (no bacterial
growth). The highiighfed área represents bacteria killed by i n g silver, mercury, a n d copper. T h e a b i l i t y o f v e r y s m a l l
biocidal action. a m o u n t s o f h e a v y metáis, especially s i l v e r a n d c o p p e r , t o
e x e r t a n t i m i c r o b i a l a c t i v i t y is r e f e r r e d t o as o i i g o d y n a m i c
a c t i o n {oligo means f e w ) . T h i s a c t i o n c a n b e s e e n w h e n w e
place a c o i n o r o t h e r c l e a n p i e c e o f m e t a l c o n t a i n i n g s i l v e r
s l o w l y a n d b e i n g less effective purifiers t h a n m a n y o t h e r o r c o p p e r o n a c u l t u r e o n a n i n o c u l a t e d P e t r i píate. E x -
c h l o r i n e c o m p o u n d s . C h l o r a m i n e s are used t o s a n i t i z e t r e m e l y s m a l l a m o u n t s o f m e t a l diffíise f r o m t h e c o i n a n d
glassware a n d e a t i n g utensils a n d t o t r e a t d a i r y a n d f o o d - i n h i b i t the g r o w t h o f bacteria for some distance a r o u n d t h e
manufacturing equipment. A m m o n i a is u s u a l l y m i x e d c o i n ( F i g u r e 7.8). T h i s effect is p r o d u c e d b y t h e a c t i o n o f
w i t h c h l o r i n e i n m u n i c i p a l w a t e r - t r e a t m e n t systems to h e a v y m e t a l ions o n m i c r o b e s . W h e n t h e m e t a l i o n s c o m *
f o r m c h l o r a m i n e s . T h e c h l o r a m i n e s c o n t r o l taste a n d o d o r b i n e w i t h t h e s u l f h y d r y l groups o n c e i l u l a r p r o t e i n s , d e n a -
p r o b l e m s caused b y t h e r e a c t i o n o f c h l o r i n e w i t h o t h e r n i - t u r a t i o n results.
t r o g e n o u s c o m p o u n d s i n t h e w a t e r . Because c h l o r a m i n e s S i l v e r is used as a n a n t i s e p t i c i n a 1 % silver nitrate solu-
are less e f f e c t i v e as germicides, s u f f i c i e n t c h l o r i n e m u s t be t i o n . A t o n e t i m e , m a n y states r e q u i r e d t h a t t h e eyes o f
added t o ensure a r e s i d u a l o f c h l o r i n e i n t h e f o r m o f H O C l . n e w b o r n s be t r e a t e d w i t h a few d r o p s o f s i l v e r n i t r a t e t o
( C h l o r a m i n e s are t o x i c t o a q u a r i u m fish, b u t p e t shops sell g u a r d against a n i n f e c t i o n o f t h e eyes c a l l e d g o n o r r h e a l
chemicals t o neutralize them.) , , .... ophthalmia neonatorum, w h i c h the infants m i g h t have
c o n t r a c t e d as t h e y passed t h r o u g h t h e b i r t h c a n a l . I n re-
Alcchols c e n t years, a n t i b i o t i c s h a v e r e p l a c e d s i l v e r n i t r a t e f o r t h i s
Alcohols effectively k i l l bacteria and fungi but n o t en- purpose.
d o i r - i t e s a n d n o n e n v e l o p e d viruses. T h e m e c h a n i s m o f ac- R e c e n t l y , t h e r e has b e e n r e n e w e d i n t e r e s t i n s i l v e r as
[lor. f a l c o h o l is usually p r o t e i n d e n a t u r a t i o n , b u t a l c o h o l aji..antimicrobial agent. Silver-impregnated dressings
c a n ; L s o d i s r u p t m e m b r a n e s a n d dissolve m a n y l i p i d s , i n - t h a t s l o w l y reléase s i l v e r i o n s h a v e p r o v e n e s p e c i a l l y use-
cluí.ng t h e lipid component of enveloped viruses. A l c o - ful w h e n a n t i b i o t i c - r e s i s t a n t b a c t e r i a are a p r o b l e m . A
h c i : n a v e d^e advantage of a c t i n g and t h e n evaporating c o i n b i n a t i o n o f silver and the d r u g sulfadiazine, silver-
rap-.j'V a n d leaving n o residue. W h e n t h e s k i n is s w a b b e d sulfadiazine, is t h e m o s t c o m m o n f o r m u l a t i o n . I t is a v a i l -
i d e ^ - r m e d ) before a n i n j e c t i o n , most of the m i c r o b i a l c o n - áble as a t o p i c a l c r e a m for use o n b u m s . S i l v e r c a n also
rro: cccivicy c o m e s t r o m s i m p l y w i p i n g away d i r t a n d m i - be i n c o r p o r a t e d i n t o i n d w e l l i n g c a t h e t e r s , w h i c h are a
c r c - . r i a n i s m s , a l o n g w i t h s k i n o i l s . H o w e v e r , a l c o h o l s are c o m m o n source o f h o s p i t a l i n f e c t i o n s , a n d i n w o u n d
unic:-¡sfactory antiseptics w h e n applied to wounds. T h e y dressings. Surfacine, a c o m b i n a t i o n of silver iodide and
cau- c o a g u l a t i o n o f a layer ot p r o t e i n u n d e r w h i c h b a c t e - b i g u a n i d e , is a p r o m i s i n g , v e r y p e r s i s t e n r a n t i m i c r o b i a l
ria : n t i n u e t o grow. agent t h a t ¿an h e used t o k i l l a w i d e v a r i e t y o f m i c r o b e s
198 PAi'-T O N c Fundamentáis of Microbiology

T h e r o o f is l i g h t e r - c o l o r e d w h e r e b i o l o g i c a l g r o w t h is i m -
peded. C o p p e r - a n d z i n c - t r e a t e d s h i n g l e s are availáble.
Zmc chloride is a c o m m o n i n g r e d i e n t i n m o u t h w a s h e s , a n d
zinc oxide is p r o b a b l y t h e most w i d e l y used a n t i f u n g a l agent
i n p a i n t s , m a i n l y because i t is o f t e n p a r t o f t h e p i g m e n t
Jormulation. ,

FIGURE 7 . 8 OligoJynamic action of heavy metáis.
O l e a r z o n e s w h e r e b a c t e r i a l g r o w t h has been ¡nhibited are
seen a r o u n d the s o m b r e r o c h a r m (pushed aside), the d i m e ,
a n d t h e penny. The c h a r m a n d the d i m e c o n t a i n silver; the
p e n n y contains c o p p e r .
• The coins used i n this demonstration w e r e minted m a n y
y e a r s a g o ; w h y w e r e c u r r e n t coins not used?
Surface-Active Agents
S u r f a c e - a c t i v e a g e n t s , or s u r f a c t a n t s , c a n decrease sur-
face tensión a m o n g molecules o f a l i q u i d . S u c h agents i n -
c l u d e soaps a n d d e t e r g e n t s . S o a p has l i t t l e valué as a n
a n t i s e p t i c , b u t i t does h a v e a n i m p o r t a n t f u n c t i o n i n t h e
mechanical removal o f microbes t h r o u g h scrubbing. T h e
s k i n n o r m a l l y c o n t a i n s dead cells, d u s t , d r i e d sweat, m i -
crobes, a n d o i l y secretions f r o m o i l glands. S o a p breaks t h e
o i l y film i n t o t i n y d r o p l e t s , a process c a l l e d emulsification,
a n d t h e w a t e r a n d soap t o g e t h e r l i f t u p t h e e m u l s i f i e d o i l
a n d debris a n d float t h e m away as t h e l a t h e r is w a s h e d off.
I n t h i s sense, soaps are g o o d d e g e r m i n g agents.
Acid-arúonic surface-active sanitizers are v e r y i m p o r -
t a n t i n t h e cleaning o f dairy utensils a n d e q u i p m e n t . T h e i r
s a n i t i z i n g a b i l i t y is r e l a t e d t o t h e n e g a t i v e l y c h a r g e d p o r -
t i o n ( a n i ó n ) o f t h e m o l e c u l e , w h i c h reacts w i t h t h e p l a s m a
m e m b r a n e . T h e s e sanitizers, w h i c h a c t o n a w i d e s p e c t r u m
o f microbes, i n c l u d i n g troublesome t h e r m o d u r i c bacteria,
are n o n t o x i c , n o n c o r r o s i v e , a n d fast a c t i n g .

Quaternary Ammonium Compounds (Quats]

The most widely used surface-active agents are the
t h a t c o n t a c t s k i n a n d e n v i r o n m e n t a l surfaces. W h e n a p - c a t i o n i c d e t e r g e n t s , especially t h e q u a t e r n a r y ammo-
p r o v e d by t h e F D A , i t is e x p e c t e d t o find w i d e use o n n i u m c o m p o u n d s ( q u a t s ) . T h e i r c l e a n s i n g a b i l i t y n re-
m e d i c a l d e v i c e s a n d as a h a n d a n t i s e p t i c . lated t o the positively charged p o r t i o n — t h e c a t i ó n — o i
I n o r g a n i c m e r c u r y comp(3unds, s u c h as mercuñc chlo- t h e m o l e c u l e . T h e i r ñame is d e r i v e d f r o m t h e fact t h a t
ride , h a v e a l o n g h i s t o r y o f use as d i s i n f e c t a n t s . T h e y h a v e t h e v are m o d i f i c a t i o n s o f t h e f o u r - v a l e n c e a m m o n i u m i o n ,
a v e r y b r o a d s p e c t r u m o f a c t i v i t y ; t h e i r effect is p r i m a r i l y NH4'*" ( F i g u r e 7.9). Q u a t e r n a r y a m m o n i u m c o m p o u n d s
b a c t e r i o s t a t i c . H o w e v e r , t h e i r use is n o w l i m i t e d because are s t r o n g l y b a c t e r i c i d a l against g r a m - p o s i t i v e b a c t e r i a a n d
o f t h e i r t o x i c i t y , c o r r o s i v e n e s s , a n d ineffectiveness in'or- less a c t i v e against g r a m - n e g a t i v e b a c t e r i a (see F i g u r e 7.6).
g a n i c m a t t e r . A t p r e s e n t , t h e p r i m a r y use o f m e r c u r i a l s is Q u a t s are also f u n g i c i d a l , a m o e b i c i d a l , a n d v i r u c i d a l
to control rr.ildew i n paints. I n addition, mercurochrome against e n v e l o p e d viruses. T h e y d o n o t k i l l endospores or
antiseptic, o r g a n i c m e r c u r y c o m p o u n d , is o f t e n f o u n d m y c o b a c t e r i a . (See t h e b o x o n page 2 0 0 . ) T h e i r c h e m i c a l
i n h o m e m e d i c i n e chests. m o d e o f a c t i o n is u n k n o w n , b u t t h e y p r o b a b l y affect t h e
Copper .n t h e f o r m oí copper sulfate or o t h e r copper- plasma m e m b r a n e . T h e y c h a n g e t h e cejl's p e r m e a b i l i t y
c o n t a i n m g ; d d i t i v e s is used c h i e f l y t o destroy green algae a n d cause t h e loss o f essential c y t o p l a s m i c c o n s t i t u e n t s ,
(algicide) g r o w i n reservoirs, s t o c k p o n d s , s w i m m i n g such as potassium.
p o o l s , a n d :.íh t a n k s . I f t h e w a t e r does n o t c o n t a i n exces- T w o p o p u l a r quats are Z e p h i r a n , a b r a n d ñame o f bem-
s i v e o r g a n . ; m a t t e r , c o p p e r c o m p o u n d s are e f f e c t i v e i n alkonium chloride (see Figure 7.9), a n d C e p a c o l , a b r a n d
c o n c e n t r a : , .ns o f o n e pare per m i l l i o n o f water. T o p r e v e n t nanie o f cetylpyridinium chloride. T h e y are s t r o n g l y a n t i m i -
miij.ew. c-pper c o m p o u n d s s u c h as c o p p e r 8-hydroxy- c r o b i a l , coloiless, odorless, tasteless, stable, easily d i l u t e d ,
q u m o l i n e a:t s o m e t i m e s i n c l u d e d i n p a i n t . and n o n t o x i c , except at h i g h c o n c e n t r a t i o n s . I f y o u r
Anoíht: m e t a l used as a n a n t i m i c r o b i a l is zinc. T h e ef- mouthwash bottie filis with foam when shaken, the
fec: <,( t r a c - - . m o u n t s o f z i n c c a n be seen o n roofs o f b u i l d - mouthwash probably contains a quat. However, organic
ini:> dov..T.- . o p c f r o m galvanized (zinc-coated) httings.
 CHAPTER 7 The Control of M i c r o b i a l G r o w t h 199

H H CHo
I I I
H-N^-H
i C-N*-Ci8H37
H
H CHt
Aiiimonium ion £ snzaikonium chiloride

FIGURE 7 . 9 The amiríonium ion and o quaternary-''

ammonium compound, benzaikonium chloride
(Zephiran). N o t i c e h o w other groups replace the hydro-
gens of the a m m o n i u m i o n .

• Q u a t e r n a r y a m m o n i u m compounds are strongly bacterici-

dal against gram-positive bacteria but less effective against
gram-negative bacterio. '

m a t t e r interferes w i t h t h e i r a c t i v i t y , a n d t h e y are r a p i d l y
n e u t r a l i z e d by soaps a n d a n i o n i c detergents.
40 60 80 100 í
A n y o n e associated w i t h m e d i c a l a p p l i c a t i o n s o f quats
Time (sec)
should remember t h a t c e r t a i n b a c t e r i a , s u c h as some
FIGURE 7 . 1 0 A comparison of the effectiveness of
species o f Pseudomonas, n o t o n l y survive i n quatemary am-
various antiseptics. The steeper the d o w n w a r d slope of
m o n i u m c o m p o u n d s b u t a c t i v e l y g r o w i n t h e m . T h i s resis- the killing c u r v e o f the antiseptic, the m o r e effective it is. A
t a n c e o c c u r s n o t o n l y t o t h e d i s i n f e c t a n t s o l u t i o n b u t also 1 % i o d i n e in 7 0 % ethanoi solution is the most effective;
t o m o i s t e n e d gauze a n d bandages, w h o s e fibers tend to soap a n d w a t e r are the least effective. N o t i c e that a tinc-
n e u t r a l i z e t h e q u a t s . (See t h e b o x o n page 2 0 0 . ) ture o f Z e p h i r a n is more effective than a n a q u e o u s solution
of the same antiseptic.
Before we m o v e o n t o t h e n e x t group o f chemical
agents, refer t o F i g u r e 7.10, w h i c h compares t h e effective- • W h y is'the tincture of Z e p h i r a n more effective than the
ness o f s o m e o f t h e a n t i s e p t i c s w e h a v e discussed so far. aqueous solution?

Chemical Food Preservatives

C h e m i c a l p r e s e r v a t i v e s are f r e q u e n t l y a d d e d t o foods t o a n a e r o b i o c o n d i t i o n s . T h e n i t r i t e has t w o m a i n f u n c t i o n s :
r e t a r d spoilage. Sulfur dioxide ( S O 2 ) has l o n g b e e n used as t o preserve t h e pleasing r e d c o l o r o f t h e m e a t b y r e a c t i n g
a disinfectant, especially i n w i n e - m a k i n g . H o m e r men- w i t h blood components i n the meat, a n d t o p r e v e n t t h e
t i o n e d its use i n The Odyssey, w r i t t e n n e a r l y 2 8 0 0 years g e r m i n a t i o n a n d g r o w t h o f a n y b o t u l i s m endospores that
ago. . A m o n g t h e m o r e c o m m o n a d d i t i v e s are s o d i u m b e n - m i g h t b e present. T h e r e has b e e n some c o n c e r n t h a t t h e
zoate, s o r b i c a c i d , a n d c a l c i u m p r o p i o n a t e . T h e s e c h e m i - r e a c t i o n o f n i t r i t e s w i t h a m i n o acids c a n f o r m c e r t a i n car-
cals are s i m p l e o r g a n i c acids, o r salts o f o r g a n i c acids, cinogenic p r o d u c t s k n o w n as n í t r o s a m i n e s , a n d t h e
vvhich t h e b o d y r e a d i l y metabolizes a n d w h i c h are gener- a m o u n t o f n i t r i t e s added t o foods has g e n e r a l l y b e e n r e -
a l l y j u d g e d t o be safe i n foods. Sorbic acid, o r its m o r e sol- d u c e d r e c e n t l y for t h i s reason. H o w e v e r , t h e use o f n i t r i t e s
u b l e salt potassium sórbate, a n d sodium benzoate prevent continúes because o f t h e i r established valué i n p r e v e n t i n g
m o l d ; f r o m g r o w i n g i n c e r t a i n a c i d i c foods, s u c h as cheese b o t u l i s i n . Because n i t r o s a m i n e s are f o r m e d i n t h e b o d y
a n d >At d r i n k s . S u c h foods, u s u a l l y w i t h a p H o f 5.5 o r f r o m o t h e r sources, t h e added risk posed b y a l i m i t e d use o f
l o w e r . are m o s t s u s c e p t i b l e t o m o l d - t y p e spoilage. Calcium n i t r a t e s a n d n i t r i t e s i n meats is l o w e r t h a n was o n c e
¡yro-.:-.ate, a n effective f u n g i s t a t used i n b r e a d , p r e v e n t s thoijght. ^
the L ' : ) w t h o f surface m o l d s a n d t h e Bacillus bacterium
t h a t : i u s e s r o p y b r e a d . T h e s e o r g a n i c acids i n h i b i t m o l d Antibiotics . , '
g r o u r n . n o t by a f f e c t i n g che p H b u t by i n t e r f e r i n g w i t h T h e a n t i m i c r o b i a l s discussed i n t h i s c h a p t e r are n o t usetul
the ::. ild's m e t a b o l i s m o r t h e i n t e g r i t y o f t h e p l a s m a for ingestión o r i n j e c t i o n t o t r e a t disease. A n t i b i o t i c s are
meiT.r rane. used t o r t h i s purpose. T h e use o f a n t i b i o t i c s is h i g h l y r e -
> .'imm nitrate a n d sodium nitrite are a d d e d t o m a n y s t r i c t e d ; h o w e v e r , at least t w o h a v e c o n s i d e r a b l e use i n
m e a : r r o d u c t s , s u c h as h a m , b a c o n , h o t dogs, a n d sausage. food p r e s e r v a t i o n . N e i t h e r is o f valué for c l i n i c a l purposes.
T i l e i i t t v e i n g r e d i e n t is s o d i u m n i t r i t e , w h i c h c e r t a i n bac- N i s i n , w h i c h is o f t e n added t o cheese t o i n h i b i t t h e g r o w t h
teria n t h e meats c a n also p r o d u c e frorn s o d i u m n i t r a t e . o f c e r t a i n e n d o s p o r e - f o r m i n g spoilage b a c t e r i a , is a n e x -
T n e - - b a c t e r i a use n i t r a t e as a s u b s t i t u t e for o x y g e n u n d e r a m p l e o f a b ^ c t e r i o c i n , a p r o t e i n t h a t is p r o d u c e d by o n e
200 PART O N E Fundamentáis of Microbiology

C L I N I C A L P R O B L E M S O L V I N G

A HospitaUAcquired Infection

A
s you read through this box you
will encounter a series of ques-
tions that clinicians ask them-
selves as they formúlate a diagnosis and
treatment. Try to answer each question
before going on to the next one.
1. During a 17-month period, nine
patients íh eight hospitals acquired
surgical'Site infections w i t h i n 2
months after liposuction. The pa-
tients' symptoms included fever,
local iriflammation, and infected
surgical wounds.
What woidd you do next?
2. Gram stains arul acid-fast stains of pus
showed gram-positive, acid-fest rods.
What are possible orffmisms?
3. Slow-growing mycobacteria, includ-
ing M . tuberculosis and M . kprae, are
human pathogens. These infections,
however, were caused by rapidly
growing mycobacteria ( R O M ) :
Mycobacterium chelonae, M . fartui-
tum, and M . abscessus.
Where are other species of mycobacteria
normally found?
4. R O M are found in soil and water.
The liposuction procedure involves
making a small surgical wound and
using a cannula (needle) for fat
suctioning. T h e cannulae were
cleaned w i t h tap water and soap
followed by a quat.
What was wrong uñth this procedure?
5. R G M can be found i n tap water and
soap used to remove dust and m i -
crobes. Quats do have some disin-
fecting properties but are considered
low-level disinfectants because they
are ineffectíve against endospores
and mycobacteria.
Why are mycobacteria resistant lo some
ái5infa:mnts?
6. T h e Upid-rich cell wall prevents
entry o f most biocides into the
cells.
How coiM you find the source of the
RGM?
7. Nosocomial infections associated
with contaminated quats that had
been used to disinfect patienc-care
supplies or equipment have been
reported. Nosocomial infections
have also been associated w i t h R G M
growing in a hospital's hot water
system. ín this case, cultures of the
quat and environmental cultures did
not yield bacteria or mycobacteria.
What changes in procedures woiúd you
recommend?
8. Surgical instruments used i n liposuc-
tion are intended to enter normally
sterile tissue and should be steriliied
between patient procedures. T h e
hospitals modified their procedures
by replacing the quat w i t h either a
high-level disinfectant using 2 %
glutaraldehyde or ethylene oxide gas
sterilization.
SOURCE: Adapted feSm MAWR 47(49):
1065-1067(12/18/98). '

b a c t e r i u m a n d i n h i b i t s a n o t h e r (see C h a p t e r 8, page 2 4 1 ) .
N i s i n is present n a t u r a l l y i n s m a l l a m o u n t s i n m a n y d a i r y
p r o d u c t s . I t is tasteless, r e a d i l y d i g e s t e d , a n d n o n t o x i c .
Natamycin (pimaricin) is a n a n t i f u n g a l a n t i b i o t i c a p -
p r o v e d for use i n foods, m o s t l y cheese.
AJdehydes
A l d e h y d e s are a m o n g t h e most effective a n t i m i c r o b i a l s .
TvVQ examples are f o r m a l d e h y d e a n d glutaraldehyde. T h e y
i n a c t i v a t e p r o t e i n s by f o r m i n g covalent cross-links w i t h sev-
era.! organic f u n c t i o n a l groups o n p r o t e i n s ( — N H 2 , — O H ,
— C O O H , a n d — S H ) . Formaldehyde gas is a n e x c e l l e n t dis-
i n r e c t a n t . H o w e v e r , i t is m o r e c o m m o n l y availáble as/o77n¿i-
íín. a 3 7 % aqueous s o l u t i o n o f f o r m a l d e h y d e gas. F o r m a l i n
vvs^ o n c e used e x t e n s i v e l y t o preser\'e b i o l o g i c a l specimens
a r . d i n a c t r . ate bacteria a n d viruses i n vaccines.
Gluuir:iídehyde is a c h e m i c a l r e l a t i v e o f f o r m a l d e h y d e
t h a t is les: i r r i t a t i n g a n d more effective t h a n formalde-
h y d e . G l u : a r a l d e h y d e is u s e d t o d i s i n f e c t h o s p i t a l i n s t r u -
m e n t s , i n c l u d i n g respiratory-therapy equipment. When
u>cd i n a 1 % s o l u t i o n ( C i d e x ) , i t is b a c t e r i c i d a l , t u b e r c u -
l o c i d a l , a n d v i r u c i d a l i n 10 m i n u t e s a n d s p o r i c i d a l i n 3 - 1 0
h o u r s . G l u t a r a l d e h y d e is o n e o f t h e f e w l i q u i d chemical
d i s i n f e c t a n t s t h a t c a n be c o n s i d e r e d a s t e r i l i z i n g a g e n t .
H o w e v e r , 3 0 m i n u t e s is o f t e n c o n s i d e r e d t h e máximum
t i m e a l l o w e d f o r a sporicide t o act, w h i c h is a c r i t e r i o n g l u -
taraldehyde c a n n o t meet. B o t h glutaraldehyde a n d forma-
l i n are used b y m o r t i c i a n s f o r e m b a l m i n g .
A possible r e p l a c e m e n t f o r g l u t a r a l d e h y d e f o r m a n y
uses is ortho'phthalaldehyde ( O P A ) , w h i c h is m o r e e f f e c t i v e
against m a n y m i c r o b e s a n d has fewer i r r i t a t i n g p r o p e r t i e s .
Gaseous Chemosterifizers
Gaseous chemosterilizers are c h e m i c a l s t h a t s t e r i l i z e i n a
closed c h a m b e r ( s i m i l a r t o m a u t o c l a v e ) . A gas s u i t a b l e
for t h i s m e t h o d is ethylene 03 ide:
H,C - C H - ,
Its a c t i v i t y d e p e n d s o n t h e d e n a t u r a t i o n o f p r o t e i n s : T h e
protein.^' l a b i l e h y d r o g e n s , such as — S H , — C O O H , or

— O H , are r e p l a c e d b y a l k y l groups (alkylation), such as
—CH2CH2OH. E t h y l e n e oxide kills a l l microbes a n d
endospores b u t requires a l e n g t h y exposure period o f
4 - 1 8 h o u r s . I t is t o x i c a n d e x p l o s i v e i n its puré f o r m , so i t
is u s u a l l y m i x e d w i t h a n o n t l a m m a b l e gas, s u c h as carbón
d i o x i d e o r n i t r o g e n . O n e o f i t s advantages is t h a t i t is
h i g h l y p e n e t r a t i n g , so m u c h so t h a t e t h y l e n e o x i d e was
chüsen t o sterilize spacecraft sent t o l a n d o n t h e m o o n
a n d M a r s . U s i n g h e a t t o sterilize t h e e l e c t r o n i c gear o n
these v e h i c l e s was n o t p r a c t i c a l .
Because o f t h e i r a b i l i t y t o sterilize w i t h o u t heat, gases
l i k e e t h y l e n e o x i d e are also w i d e l y used o n medie?' sup-
plies a n d e q u i p m e n t . M a n y large hospitals h a v e e t h y l e n e
o x i d e c h a m b e r s , some large e n o u g h t o sterilize mattresses,
as p a r t o f t h e i r s t e r i l i z i n g e q u i p m e n t . Propylene o x i d e a n d
b e t a - p t o p i o l a c t o n e are also used for gaseous s t e r i l i z a t i o n :
H3C—CH—CH2 H2C—CH2
O O — c = o
Propylene oxide Beta-propiolactone
A disadvantage o f a l l these gases is t h a t t h e y are sus-
p e c t e d c a r c i n o g e n s , especially b e t a - p r o p i o l a c t o n e . For t h i s
reason, t h e r e has b e e n c o n c e r n a b o u t t h e exposure o f h o s -
p i t a l w o r k e r s t o e t h y l e n e o x i d e f r o m such sterilizers. B e -
cause o f d i i s hazard, these gases m a y e v e n t u a l l y be r e p l a c e d
by plasma gas sterilization. T h i s m a k e s use o f vapors o f h y -
d r o g e n p e r o x i d e (discussed s h o r t l y ) subjected t o r a d i o fre-
quencies o r m i c r o w a v e r a d i a t i o n t o p r o d u c e reactive free
radicab. N o b y - p r o d u c t s t o x i c t o h u m a n s are p r o d u c e d ,
a n d i t is a n effective s t e r i l a n t .
Peroxygens (Oxidizing Agents)
P e r o x ^ - g e n s e x e r t a n t i m i c r o b i a l a c t i v i t > ' by o x i d i z i n g c e i -
l u l a r c o m p o n e n t s o f t h e t r e a t e d m i c r o b e s . Examples are
ozone, h v d r o g e n p e r o x i d e , a n d p e r a c e t i c a c i d . Ozone (O3)
is a h i z h l y r e a c t i v e f o r m o f o x y g e n t h a t is generated b y
passing o x y g e n t h r o u g h h i g h - v o l t a g e e l e c t t i c a l discharges.
It '5 r e s r o n s i b l e for t h e air's r a t h e r fresh o d o r after a l i g h t -
ning : m , i n the v i c i n i t y o f electrical sparking, or around
m u h r i v i o l e t l i g h t . O z o n e is o f t e n used t o s u p p l e m e n t
c h l o r : r . - m t h e d i s i n f e c t i o n o f water, because i t helps n e u -
tralize r.i.^ces a n d o d o r s . . A l t h o u g h ozone is a m o r e effective
k i l l i n z ;v_-ent, its r e s i d u a l a c t i v i t y is d i f f i c u l t t o m a i n t a i n i n
'.vacer. i:.¿ i t is m o r e e x p e n s i v e t h a n c h l o r i n e .
. H ; - - ' j o ; e n peroxicU is a n a n t i s e p t i c f o u n d i n m a n y
housc'r. :d m e d i c i n e c.ibinets a n d i n h o s p i t a l supply r o o m s .
It 15 r . : : 1 g o o d a n t i s e p t i c for o p e n w o u n d s ; i n fact, i t m a y
úow ••. ' j n d h e a l i n g . I t is q u i c k l y b r o k e n d o w n t o w a t e r
rmJ ' U S o x y g c n b v rtie a c t i o n o f t h e enzyme catalase,
.'.hic'r. . present i n h u m a n cells (see C h a p t e r 6, page 1 6 3 ) .
: í . v-.c hydrogen peroxide does effectively disinfect
CHAPTER 7 The Control of M i c r o b i a l G r o w t h 201
i n a n i m a t e objects, a n a p p l i c a t i o n i n w h i c h i t is e v e n spo-
r i c i d a l , especially at elevated t e m p e r a t u r e s . O n a n o n l i v -
i n g surface, t h e n o t m a l l y p r o t e c t i v e e n z y m e s o f a e r o b i c
b a c t e r i a a n d f a c u l t a t i v e anaerobes are o v e r w h e l m e d b y t h e
h i g h c o n c e n t r a t i o n s o f p e r o x i d e used. Because o f these fac-
tors t h e food industry is i n c r e a s i n g its use o f h y d r o g e n per-
o x i d e for aseptic packaging (see C h a p t e r 2 8 o n page 7 9 5 ) .
T h e p a c k a g i n g materials pass t h r o u g h a h o t s o l u t i o n o f t h e
c h e m i c a l before b e i n g assembled i n t o a c o n t a i n e r . I n a d d i -
t i o n , m a n y wearers o f c o n t a c t lenses are f a m i l i a r w i t h dis-
infection by hydrogen peroxide. A f t e r disinfection, a
p l a t i n u m c a t a l y s t i n t h e l e n s - d i s i n f e c t i n g k i t destroys
residual h y d r o g e n p e r o x i d e so t h a t i t does n o t persist o n
t h e lens, w h e r e i t m i g h t be a n i r r i t a n t .
O x i d i z i n g agents are useful f o r i r r i g a t i n g deep w o u n d s ,
w h e r e t h e o x y g e n released m a k e s a n e n v i r o n m e n t t h a t i n -
hibits the g r o w t h o f anaerobio bacteria.
Benzcryl peroxide is a n o t h e r c o m p o u n d useful f o r t r e a t i n g
w o u n d s i n f e c t e d b y anaerobio pathogenis, b u t i t is p r o b a b l y
m o r e f a m i l i a r as t h e m a i n i n g r e d i e n t i n o v e r - t h e - c o u n t e r
m e d i c a t i o n s f o r acné, w h i c h is caused b y a t y p e o f a n a e r o -
bio bacterium infecting h a i r follicles.
Peracetic add is o n e o f t h e m o s t e f f e c t i v e l i q u i d c h e m i -
c a l sporicides availáble a n d is c o n s i d e r e d a s t e r i l a n t . I t is
g e n e r a l l y effective o n endospores a n d viruses w i t h i n 3 0
m i n u t e s , a n d k i l l s v e g e t a t i v e b a c t e r i a a n d f u n g i i n less
t h a n 5 m i n u t e s . Peracetic a c i d has m a n y a p p l i c a t i o n s i n
t h e d i s i n f e c t i o n o f food-processing a n d m e d i c a l e q u i p m e n t
because i t leaves n o t o x i c residues a n d is m i n i m a l l y af-
fected b y t h e presence o f o r g a n i c m a t t e r .
Microbial Characteristics
and Microbial Control
Learning Objective
• Explain t)ow tt)e control of microbial growth is affected
by the type of microbe.
M a n y biocides t e n d t o b e m o r e e f f e c t i v e against g r a m -
p o s i t i v e bacteria, as a g r o u p , t h a n against gram-negative
bacteria. T h i s is i l l u s t r a t e d i n Figure 7 . 1 1 , w h i c h presents a
simpUfied h i e r a r c h y o f r e l a t i v e resistance o f m a j o r m i c r o -
b i a l groups t o biocides. A p r i n c i p a l f a c t o r i n t h i s r e l a t i v e
resistance t o biocides is t h e e x t e r n a l l i p o p o l y s a c c h a r i d e
layer o f g r a m - n e g a t i v e bacteria. W i t h i n gram-negative
bacteria, members o f t h e genera Pseudomonas a n d Buríc-
holderia are o f special i n t e r e s t . T h e s e c'osely relamed b a c t e -
ria are u n u s u a l l y resistant t o b i o c i d e s (see Figure 7.6) a n d
w i l l e v e n grow a c t i v e l y i n some d i s i n f e c t a n t s a n d a n t i s e p -
tics, most n o t a b l y t h e q u a t e m a r y a m m o n i u m c o m p o u n d s .
(See t h e b o x o n page 2 0 0 ) . I n C h a p t e r 2 0 , y o u w i l l see t h a t
these bacteria &re also resistant t o m a n y a n t i b i o t i c s . T h i s
202 PART O N E Fundamentáis of M i c r o b i o l o g y

Most Resistant v,-^'''''""'

• Knons y ¡f
TABLE 7 . 7 The Effectiveness of
::,,ieiíM^ *. . . . Chemical Antimicrobials
Against Endospores a n d
E n d c i p o r e s of b a c t e r i a ' Mycobaceria
Chemical A g e n t Endospores ; Mycobocteri^

Mercury N o activity No activity

Afyiycobacteria'.í
Pfienolics Poor Good

Bisphenols N o activity N o activity

Gysts'of p r o t o z ó á %
Quaternary ammonium N o activity No activity
compounds

Chiorines Foir Fair

Iodine Poor Good

Alcohols Poor Good

Glutaraldehyde - Fair Good

Chlorhexidine N o activity Foir

b e e n d e v e l o p e d t o evalúate t h e effectiveness o f biocides

against t h i s b a c t e r i a l g r o u p .
B a c t e r i a endospores are affected b y r e l a t i v e l y f e w b i o -
cides. ( T h e a c t i v i t y o f t h e m a j o r c h e m i c a l a n t i m i c r o b i a l
groups against m y c o b a c t e r i a a n d endospores is s u m m a r i z e d
i n T a b l e 7.7.) T h e cysts a n d oocysts o f p r o t o z o a are also
r e l a t i v e l y resistant t o c h e m i c a l d i s i n f e c t i o n .
Viruses are n o t especially resistant t o b i o c i d e s , b u t a
d i s t i n c t i o n m u s t be m a d e b e t w e e n t h o s e t h a t possess a
Least Resistant l i p i d - c o n t a i n i n g envelope and those t h a t d o n o t . A n t i m i -
c r o b i a l s t h a t are l i p i d - s o l u b l e are m o r e l i k e l y t o be effec-
Decreasing order of resistance of
FIGURE 7 . 1 1
microorganisms to chemical biocides. t i v e against e n v e l o p e d viruses. I f so, t h i s w i l l u s u a l l y be
i n d i c a t e d o n t h e label by a s t a t e m e n t t h a t t h e y are effec-
• ore viruses w i t h lipid-containing envelopes relatively t i v e against l i p o p h i l i c viruses. N o n e n v e l o p e d "iruses, w i t h
susceptible to certain biocides? o n l y a p r o t e i n coat, are more r e s i s t a n t — f e w e r b i o c i d e s a r e
a c t i v e against t h e m .
A special p r o b l e m , n o t yet c o m p l e t e l y s o l v e d , is t h e
resi5:¿rice t o c h e m i c a l a n t i m i c r o b i a l s is m o s t l y r e l a t e d t o
r e l i a b l e k i l l i n g o f p r i o n s (see C h a p t e r 13, page 4 0 1 ) . Pri-
t h e cr.aracteristics oí t h e i r porim (structural openings i n
ons are i n f e c t i o u s p r o t e i n s t h a t are t h e cause o f n e u r o l o g -
t h e '.Vill o f g r a m - n e g a t i v e b a c t e r i a ; see Figure 4.13c on
ical diseases, t h e s p o n g i f o r m e n c e p h a l o p a t h i e s , such a >
page : 4 ) . P o r i n s are h i g h l y selective o f molecules t h a t t h e y
th"e p o p u l a r l y n a m e d mad c o w disease (see C h a p t e r 22.
penr.:: to enter the cell.
page 6 3 4 ) . T o destroy p r i o n s , i n f e c t e d a n i m a l carcasses are
Tr-i mycobacteria are another group of non-
i n c i n e r a t e d . A major p r o b l e m is t h e d i s i n f e c t i o n o f surgi-
e n d ; - ' o r e - f o r m i n g bacteria t h a t e x h i b i t greater t h a n n o r -
cal i n s t r u m e n t s exposed t o p r i o n c o n t a m i n a t i o n . N o r m a l
m a l :r:;istance t o c h e m i c a l biocides. T h i s g r o u p i n c l u d e s
a u t t ) c l a v i n g has p n n - e n t o be inadequate. T h e World
r'icterzuni tuberculosis, t h e p a t h o g e n t h a t causes t u -
H e a l t h O r g a n i z a t i o n has r e c o m i u c n d e d t n e c o m b i n e d use
b-erc_. -sis. T h e c e l l w a l l o f t h i s o r g a n i s m a n d o t h e r m e m -
o f a s o l u t i o n o f s o d i u m h y d r o x i d e a n d a u t o c l a v i n g at
bers .- t h i s genus h a v e a waxy, l i p i d - r i c h component.
1 3 4 ° G . H o w e v e r , a recent r c p o r t d e s c r i b e d a n o t h e r t e c h -
I n s t r . - . n i o n labels o i i d i s i n f e c t a n t s o f t e n state whether
n i q u c , w h e r e b y i n s t r u m e n t s were S(X\ked i n a strcmg solu-
t h e v : - r e t u b e r c u l o c i d a l , i n d i c a t i n g i f t h e y are effective
t i o n o f s o d i u m hydrv>xidc for a n h o u r f(^liowcd by a n lu>ur
agair.;- m y c o b a c t e r i a . Special t u b e r c u l o c i d a l tests h a v e
 CHAPTER 7 The C o n t r o l o f M i c r o b i o l G r o w t h 203

TABLE 7 . 8 Chemical Agents Used to Control Microbial G r o w t h

Mechanism.
Chemical A g e n t . of Action / Prefen'ed Use Comment

Phenol a n d Phenolics
1. Phenol Disruption of p l a s m o Rarely u s e d , e x c e p t a s a Seldom used os o disinfectant
membrane, denaturation stondard of c o m p a r i s o n . or antiseptic because of its
of enzymes. irritoting qualities o n d
disagreeable odor.

2. Pfienolics Disruption of p l a s m a Environmental surfaces, Derivatives of phenol that ore

membrane, denaturation instruments, skin surfaces, reactive even in the pres-
of e n z y m e s . and mucous membranes. ence of organic material;
O-phenylphenol is on
example.
"9
3. Bisphenols Probably disruption of Disinfectant hand soaps ond Triclosan is on especially

I plasmo membrane. skin lotions. common exampie of a

bisphenol. Broad spectrum,
>4 but most effective against
grom-positives.

Biguanides
(Chlorhexidine) Disruption of plasmo Skin disinfection, especially Bactericidal to grom-positives
membrane. for surgical scrubs. ond gram-negatives;
nontoxic, persistent.

Halogens Iodine inhibits pjrotein func- Iodine is on effective anti- Iodine a n d chlorine may act
tion and is a strong oxidiz- septic availáble os a alone or os components of
ing agent; chlorine forms tincture ond on iodophor; inorganic o n d organic
the strong oxidizing agent chlorine gas is used to compounds.
hyp>ochlorous acid, which disinfect water; chlorine
olters ceilular components. compounds are used to
disinfect dairy equipment,
eating utensils, household
Ítems, and glassware.

Alcohols Protein denaturation o n d Thermometers ond other Bactericidal o n d fungicidal,

lipid dissolution. instruments; in s w o b b i n g
the skin with alcohol
before on injection, most
of the disinfecting action
probably comes from o
simple w i p i n g a w a y
(degerming) of dirt o n d
some microbes.
but not effective against en-
dospores or nonenveloped
viruses; commonly used
alcohols ore ethanoi a n d
isopropanol.

oí a u c c c . V v i n g at 1 3 6 " C . Nevertheless, t h e r e p o r t de- T a b l e 7.8 summarizes c h e m i c a l agents used t o c o n t r o l m i -

-^nbed :'r.:: t r e a t m e n t as o n l y " f a i r l y e f f e c t i v e . " Surgeons crobial g r o w t h .

5 o r n e t i i T . r í r e s o r t co t h e use o f disposable i n s t r u m e n t s . T l i e c o m p o u n d s discussed i n t h i s í h a p t e r are n o t g e n -

I n S U " :-nar\-, i t is i m p o r t a n t t o r e m e m b e r t h a t m i c r o b i a l erally useful i n t h e t r e a t m e n t o f diseases. Because antibi-

control rr.--rhods, e s p e c i a l l y b i o c i d e s , are n o t u n i f o r m l y et- otics are used i n chemocherapy, antibiotics and the

'ecti\-e ::^-.:ns!: all microbes. pathogens against w h i c h t h e y are a c t i v e w i l l be discussed

together, i n C h a p t e r 20.
204 PART O N E Fundamentáis of Microbiology

TABLE 7 . 8 Chemical Agents Used to Control Microbial Growth ¡continuedj

Mechanism
Chemical A g e n t of Action P r e f e r r e d Use Comqient

Heavy Metols and Denaturation of enzymes
Their Compounds a n d o t h e r essential
proteins.
Silver nitrate m o y b e used t o Heavy metáis such os silver
prevent gonorrheal oph- ond mercury ore b i o c i d a l .
t h a l m i a n e o n a t o r u m ; mer-
c u r o c h r o m e disinfects skin
a n d mucous membranes;
c o p p e r sulfate is on o i g i c i d e .

Surface-Active Agents
1. Soaps a n d acid- Mechanical r?moval of mi- Skin degerming a n d M a n y antibacterial soaps
anionic detergents* crobes through scrubbing. removal of debris. contain antimicrobials.

2. Acid-onionic N o t certain; moy involve Sanitizers in dairy a n d W i d e spectrum of activity;

detergents enzyme inactivation o r food-processing industries. nontoxic, noncorrosive,
disruption. fost-octing.

3. Cationic detergents Enzyme inhibition, protein Antiseptic for skin, instru- Bactericidal, bacteriostatic,
(quaternary denaturation, o n d disrup- ments, utensils, rubber fungicidal, o n d v i r u d d o l
ammonium tion of plasmo membranes. goods. against enveloped viruses;
compounds) examples of quats are
Zephiran a n d Cepacol.

Organic Acids Metabolic inhibition, mostly
affecting molds; action
not related to their acidity.
Sorbic a c i d a n d benzoic W i d e l y used to control molds
acid effective ot l o w p H ; ond some bacterio in foods
porabens much used in ond cosmetics.
cosmetics, shampoos;
calcium propionate used
in bread.

Aldehydes Protein denaturation. Glutaraldehyde (Cide)^ is Very effective antimicrobials.

less irritating than formalde-
hyde ond is used íbr disinfec-
tion of medical equipment.

Gaseous Sterilonts Protein denaturation. Excellent sterilizing agent, Ethylene oxide is the most
especially for objects that commonly used.
would be damaged by heat.

Peroxygens Oxidation. Contaminated surfaces; O z o n e is widely used os o

(Oxidizing Agents) some deep wounds, in supplement for chlorination;
which they ore very effec- hydrogen peroxide is o poor
tive against oxygen- antiseptic but a g o o d disin-
sensitive anaerobes. fectant. Peracetic acid is
especially effective.
 CHAPTER 7 The C o n t r o l of M i c r o b i a l G r o w t h 205

STUDY OUTLINE
The T e r m i n o l o g y of 2. Moist heat kills microbes by denaturing enzymes.
M i c r o b i a l C o n t r o l (pp. 183-184) i 3. Thermal death point (TDP) is the lowest temperature at
1. The c o n t r o l of microbial growth can prevent infections and vvhich all the microbes in a liquid culture will be killed i n
food spoilage. 10 minutes.

2. Sterilization is the process of removing or destroying all 4. Thermal death time ( T D T ) is the length of time required to
microbial life o n an object. k i l l all bacteria i n a liquid culture at a given temperature.

3. Commercial sterilization is heat treatment of canned foods
to destroy C . botulinum endospores.
4. Disinfection is the process o f reducing or i n h i b i t i n g micro-
bial g r o w t h o n a n o n l i v i n g surface.
5. Antisepsis is the process of reducing or i n h i b i t i n g microor-
ganisms o n l i v i n g tissue.
6. The sufifix -cide means to k i l l ; the suffix -stat means to inhibit.
T r e p s i s is bacterial c o n t a m i n a t i o n .
Thel^ate of Microbial Death (pp. i 8 4 - i 8 5 j
1. Bacterial populations subjected to heat or antimicrobial
chemicals usually die at a constant rate.
2. Such a death curve, when p l o t t e d logarithmically, shows
this coristant d e a t h rate as a straight line.
3 . T h e t i m e i t takes t o k i l l a microbial population is propor-
tional t o t h e n u m b e r o f microbes.
4. Microbial species and life cycle phases (e.g., endospores) have
different susceptibilities to physical and chemical controls.
5. Organic matter may interfere w i t h heat treatments and
chemical c o n t r o l agents.
6. Longer exposure t o lower heat can produce the same effect
as shorter t i m e at higher heat.
Actions o f M i c r o b i a l C o n t r o l A g e n t s (p I8Ó)
Alteratíon of Membrane Permeability (p.
5. Decimal reduction time (DRT) is the length of time in
w h i c h 9 0 % of a bacterial population w i l l be killed at a
given temperature.
6. Boiling (100°C) kills many vegetative cells and viruses
w i t h i n 10 minutes.
7. Autoclaving (steam under pressure) is the most effective
method of moist heat sterilization. T h e steam must directly
contact the material to be sterilized.
8. I n H T S T pasteurization, a h i g h temperature is used for a
short time (72'*C for 15 seconds) t o destroy pathogens w i t h -
out altering the flavor of the food. Ultra-high-temperature
( U H T ) treatment (140*'C for 3 seconds) is used to sterilize
dairy products.
9 . Methods of dry heat sterilization include direct flaming,
incineration, and hot-air sterilization. Dry heat kills by
oxidation.
10. Different methods that produce the same effect (reduction
in microbial growth) are called equivalent treatments.
Filtratíon (pp. 189-190)
1. Filtration is the passage of a liquid or gas through a filter
w i t h pores small enough to retain microbes.
2. Microbes can be removed from air by high-efficiency partic-
ulate air filters.
3. Membrane filters composed of nitrocellulose or cellulose
acétate are commonly used to filter out bacteria, viruses,
and even 'arge proteins.
I86)

1. Tt.t susceptibility o f the plasma membrane is due to its lipid Lov/ Temperatures (p. 190)

ar.i protein components. 1. The effectiveness of low temperatures depends on the par-
2. C e r i a i n chemical c o n t r o l agents damage the plasma mem- ticular microorganism and the intensity of the application.
br;r,e by altering it5 permeability. 2. Most microorganisms do not reproduce at ordinary refriger-
ator temperatures ( 0 - 7 ° C ) .
amage to Proteins and Nucleic Acids (p. 18Ó)
3T]vIany microbes survive (but do not grow) at the subzero
1. 5 : - e m i c r o b i a l c o n t r o l agents damage ceilular proteins by temperatures used to store foods.
b:ti.<ing hydrogen bonds and covalent bonds.
2. C : - tr agents interrere w i t h D N A and R N A replication and high Pressure (p 190) '

p : : - i : i n synthesis. l . Hign pressure áenatures proteins i n vegetative cells.

P h y s k a l M e t h o d s of Desiccation {p 191)

M icro b ia l C o n t r o l (pp 186-192) 1. In the absence of water, microorganisms cannot grow but
can remain viable.
rieat (pp. 1 8 6 - 1 8 9 )
2. Viruses angl endospores can resist desiccation.
1. Hf.:' is frequently used to elimínate microorganisms.
206 PART O N E Fundamentáis of Microbiology

Osmotic Pressure {p]9]] Bígfuan;c/es (pp. 1 9 5 - 1 9 6 )

1. Microorganisms in high concentrations of salts and sugars 1. Chlorhexidine damages plasma membranes of vegetative cells.
undergo plasmolysis.
Ho/ogens (pp. 1 9 6 - 1 9 7 )
2 . Molds <md yeasts are more capable than bacteria o f growing
1. Some halogens (iodine and chlorine) are used alone or as
in materials w i t h low moisture or high osmotic pressure.
components of inorganic or organic solutions.

Radiation (pp. ]9]-]92)
1. T h e ettects o f radiation depend on its wavelength, intensity,
and duration.
2 . lonizing radiation (gamma rays, X rays, and high-energy
electrón beams) has a high degree o f penetration and exerts
Its effect primarily by ionizing water and forming h.ghly
reactive hydroxyl radicáis.
3. U l t r a v i o l e t ( U V ) radiation, a form o f nonionizing radia-
t i o n , has a low degree o f penetration and causes cell damage
by making t h y m i n e dimers i n D N A that interfere w i t h
D N A replication; the most effective germicidal wavelength
is 260 n m .
4. Microwaves can k i l l microbes indirectly as materials get hot.
Chemical M e t h o d s of
2 . Iodine may combine with certain amino acids t o inactivate
enzymes and other ceilular proteins.
3. Iodine is availáble as a tincture ( i n solution w i t h alcohol)
or as an iodophor (combined w i t h an organic molecule).
4. T h e germicidal action of chlorine is based o n the formation
of hypochlorous acid when chlorine is added t o water.
5. Chlorine is used as a disinfectant i n gaseous form (Cl? or
C I O 2 ) or i n the form of a compound, such as calcium
hypochlorite, sodium hypochlorite, sodium dichloroiso-
cyanurare, and chloramines.
Alcohols (p. 197)
1. Alcohols exert their action by denaturing proteins a n d
dissolving lipids.
2 . I n tinctures, they enhance the effectiveness o f other a n t i m i -
crobial chemicals.

M i c r o b i a l C o n t r o l (pp. 1 9 2 - 2 0 1 ) 3. Aqueous ethanoi ( 6 0 - 9 5 % ) and isopropanol are used as

disinfectants.
1. Chemical agents are used o n living tissue (as antiseptics)
and o n inanimate objects (as disinfectants). Heavy Metals and Their Compounds (pp. 1 9 7 - 1 9 8 )

2 . Few chemical agents achieve sterility. 1. Silver, mercury, copper, and zinc are used as germicidals.

Principies of Effective Disinfection
1. Careful a t t e n t i o n should be paid t o the properties and c o n -
centratíon o f the disinfectant to be used.
2 . T h e presence o f organic matter, degree of contact w i t h
microcrganisms, and temperature should also be considered.
Evaluating a Disinfectant (p. ]94)
1. I n the use-dilution test, bacterial (S. choleraesuis, S. aureus,
and P. zeruginosa) survival i n the manufacturer's recom-
mended d i l u t i o n o f a disinfectant is determined.
2. Viruí<-Ñ endospore-forming bacteria, mycobacteria, and
(pp. 1 9 2 - 1 9 4 ) 2 . They exert their antimácrobial action through oiigodynamic
action. W h e n heavy metal ions combine w i t h sulfhydryl
( — S H ) groups, proteins are denatured.
Surfoce-Acf/Ve Agfenfs (p. 198)
1. Surface-active agents decrease the surface tensión among
molecules o f a liquid; soaps and detergents are examples.
2 . Soaps have limited germicidal action but assist i n the re-
moval o f microorganisms through scrubbing.
3. Acid-anionic detergents are used t o clean dairy equipm.ent.
Quaternary Ammonium Compounds (Quats) (pp. 1 9 8 - 1 9 9 )

fungi : 3 n also be used i n the use-dilution test. 1. Quats are cationic detergents attached t o NH4''".
3. In tht :¡sk-diffusion method, a disk o f filter paper is soaked 2 . By disrupting plasma membranes, they allow cytoplasmic
uuh ihernical and placed on an inoculated agar píate; a constituents to leak out of the cell.
:one • nhibition indicates effectiveness.
3. Quats are most effective against gram-positive bacteria.
Types of Disinfectants (pp. 1 9 4 - 2 0 1 )
Chemical Food Preservatives (p. 199)

Phenoi o"J Phenolics (p. 194) 1. S O i , sorbic acid, benzoic acid, and propionic acid i n h i b i t

l. i'her. :.s exert their action by injuring plasma membranes.

fungal metabolism and are used as food preservatives.
2 . Nitrate and nitrite salts prevent germination of Clostridium
Bispherz : (pp. 194-195)
botulinum endospores i n meats.
1. í^!-rr -. 'Is such ;is triclosan ((n-cr the counter) and hcxa-
Antibiotics (pp. 1 9 9 - 2 0 0 )
tb.: - :-lene (prcscripnon) are widely used in household
1. N i s i n and natamycin are anibiotics used to preserve foods,
especially cheese.
 CHAPTER 7 The C o n t r o l of M i c r o b i a l G r o w t h 207

Aldehydes (p. 200) 2 . They exert their effect by oxidizing molecules inside c t l ;

1. Aldehydes such as formaldehyde and glutaraldehyde exert

their a n t i m i c r o b i a l effect by inactivating proteins. M i c r o b i a l Characteristics a n d
2 . T h e y are among the most effective chemical disinfectants. M i c r o b i a l Control (pp 2 0 1 - 2 0 4 )
Gaseous Chemosferihzers (pp. 2 0 0 - 2 0 1 ) 1. Gram-negative bacteria are generally more resistaíit thar
gram-positive bacteria to disinfectants and antiseptics.
1. Ethylene oxide is the gas most frequently used for sterilization.
2 . Mycobacteria, endospores, and protozoan cysts and oocv
2. It penetrares most materials and kills all microorganisms by
are very resistant to disinfectants and antiseptics.
protein denaturation.
3. Nonenveloped viruses are generally more resistant than
Peroxygens (Oxidizing Agents) (p. 20]) enveloped viruses to disinfectants and antiseptics.
1. Ozone, peroxide, and peracetic acid are used as antimicro- 4 . Prions are resistant to disinfection and autoclaving.
,^ bial agents.

STUDY QUESTIONS
Review 7. Pili i n the following table:

1. Ñame the cause o f cell death resulting firom damage to each

of the following: Method of Type of P r e f ' d T ^ e s of
a. cell w a l l c. proteins Sterilization Temp. Time Heat Use Action
b. plasma membrane d . nucleic acids
Autoclaving
2 . T h e thermal death time for a suspensión of Badüus subtiUs Hot A i r
endospores is 30 minutes i n dry heat and less than 10 minutes Pasteurization
i n an autoclave. W h i c h type of heat is more efiPective? Why?
3 . I f pasteurization does r o t achieve sterilization, why is food
treated by pasteurization? 8. H o w do the examples i n question 7 illustrate the concept of
equivalent treatments?
4 . T h e n n a l death p o i n t is not considered an accurate measure
of the effectiveness of heat sterilization. List three factors 9. H o w do salts and sugars preserve foods? W h y are these
that can alter t h e r m a l death point. considered physical rather t h a n chemical methoc of micro-
bial control? Ñame one food that is preserved w i t h se ;íí
5. T h e a n t i m i c r o b i a l effect of gamma radiation is due to
and one preserved w i t h salt. H o w do you account for the
T h e antimicrobial effect of ultraviolet radia-
occasional growth of PeniciUium mold i n jelly, w h i c h is 50%
t i o n is due to
sucrose?
6. A bacterial culture was i n log phase i n the following figure.
10. List five factors to consider before selecting a disinfectant.
A t time X, an antibacterial compound was added to the
culture. W h i c h line indicates addition of a bactericidal 1 1 . Give the method of action and at least one standard use of
compound? A bacteriostatic compound? How can you tell? each of the following types of disinfectants:
Explain why the viable count does not immediately drop to a. phenolics e. heavy metáis
zero at X. b. iodine f. aldehydes
c. chlorine g. ethylene oxide
d. alcohol ' h . oxidizing agents
12^jrhe use-dilution valúes for two disiaíectants tested under
the .same conditions are: Disinfectant A — 1 : 2 ; Disinfectant
B—1:10,000. If both disinfectants are designed for the .same
purpose, which would you selcct? - .

13. A large hospital washes b u m patients in a stainiess stee!

tub. After each patient, the tub is cleaned w i t h a qi'iit.dt
was noticed that 14 of 20 hurn patients acquired
P.sc'iidomímfl.s infections after being bathcd. Provide an
Time
explanation for this high rate of infection.
208 PART O N E Fundamentáis of Microbiology

M ú l t i p l e Cholee Bacterial G r o w t h After E x p o s u r e t o

1. W h i c h ot the following does not k i l l endospores? Disinfec- Disinfec- Disinfec- Disinfec-

a. autocbvií-ig d . pas eurization Dilution tant A tant B tant C tant D
b. incineration e. ñor e of the above
c. hot-air sterilization 1:2
1:4
2. W h i c h of the following i s ^ o s t effec tive for sterilizing '.nat- -
1:8
tresses and plástic Petri dishes? 1:ló
a. chlorine d. autoclaving
b. ethylene oxide . e. nonionizing radiation
r. glutaraldehyde 9. W h i c h disinfectant is the most effective?
3. W h i c h of these disinfectants does not act by disrupting the 10. W h i c h disinfectant(s) is (are) bactericidal?
plasma membrane? . .< a. A , B, C, and D d . B only
a. phenolics b. A , C, and D e. none of the above
b. phenol c. A only
c. quaternary a m m o n i u m compounds
d. halogens
e. biguanides Crifical Thínkíng
4. W h i c h o f the following cannot be used t o sterilize a heat- 1. T h e disk-diffusion method was used t o evalúate three disin-
labile solution stored i n a plástic container? fectants. T h e results were as follows:
a. gamma radiation
b. ethylene oxide Disinfectant Zone of Inhibition
c. nonionizing radiation
d. autoclaving O mm
e. short-wavelength radiation 5 mm
1 0 mm
5. W h i c h o f the f o l l o w i n g is not a characteristic of quatemary
a m m o n i u m compounds?
a. bactericidal against gram-positive bacteria
a. W h i c h disinfectant was the most effective against t h e or-
b. sponcidal
ganism?
c. amoebicidal
b. C a n you determine whether compound Y was bacterici-
d. fungicidal
dal or bacteriostatic?
e. kills enveloped vimses
2. W h y is each o f the following bacteria often resistant t o
6. A classmate is trying t o determine how a disinfectant might
disinfectants?
k i l l cells. You observed that when he spilled the disinfectant
a. Mycobacterium
in your reduced litmus m i l k , the litmus t u m e d blue again.
b. Pseudomonas
You susgest t o your classmate that
c. Bacillus
a. the disinfectant m i g h t i n h i b i t cell wall synthesis.
b. the disinfectant m i g h t oxidize molecules. 3. A use-dilution test was used t o evalúate t w o disinfectants
c. the disinfectant m i g h t i n h i b i t protein synthesis. against Sabnonella choleraesuis. T h e results were as follows:
d. the disinfectant m i g h t denature proteins. >
e. he c¿ke his work away from yours. Bacterial G r o w t h After Exposures

7. Which of the following is most likely to be bactericidal? Time o f Disinfectant B Disinfectant B

a. merr.'-rane filtration Exposure Disinfec- Diluted w i t h Diluted w i t h
b. ior.::.ng radiation (min) tant A Distilled W a t e r Tap Water
c. rrc¿:--drying
10
d. jc=r-rreezing • --•
e. a!. • 'he above
30
S. \ h-.c'^ 'f the following is used to control microbial growth ,
in • ' • -
a. ,rj/'.c acids d. heavy metáis a. W h i c h disinfectant was the most effective?
h. a\c - ;ls e. all of the above b. W h i c h disinfectant should be used against
c. a!jr' -des Sra/)h)iíococcus?
'L'-<.- rhe : wing information to answer questions 9 and 10. 4. To determine the lethal action of microwave radiation, two
T:-.L dc::¿ . -re obtaiiied from a use-dilution test comparing four 10^ suspensions o f E. cali were prepared. O n e cell suspen-
d-.-.nrc'c:.^r • against Saíínor.cíííi cholerat'sui.s. sión was exposed to microwave radiation while wet,

whereas the other was lyophitized (treeze-dried) and then
exposed to radiation. T h e results are shown i n the following
hgure. Dashed lines indicate the temperature of the sam-
ples. W h a t is the most likely method of lethal action of
microwave radiation? H o w do you suppose these data might
differ for Cíü.sm'diuTn?
Clínicai A p p l i c a t i o n s
1. Entamoeba histolytica and Giardia lamblia were isolated from
the stool sample o f a 45-year-old man, and Shigella sonnei
was isolated firom the stool sample o f an 18-year-old woman.
B o t h patients experienced dianhea and severe abdominal
cramps, and prior t o onset of digestive symptoms b o t h had
been treated by the same chiropractor. T h e chiropractor
had administered colonic inigations (enemas) to these
patients. T h e device used for this treatment was a gravity-
depjendent apparatus using 12 liters of tap water. There were
no check valves to prevent backflow, so all parts of the
apparatus could have become contaminated w i t h feces
during each colonic treatment. T h e chiropractor provided
colonic treatment to four or five patients per day. Between
patients, the adaptor piece that is inserted i n t o the rectum
was placed i n a "hot-water sterilizer."
W h a t two errors were made by the chiropractor?
CHAPTER 7 The C o n t r o l o f M i c r o b i a l G r o w t h 209
2. B e t w e e n M a t c h 9 a n d A p r i l 12, five c h r o n i c p e r i t o n e a l
dialysis patients at one hospital became i n f e c t e d w i t h
Pseudomonas aeru^nosa. Four patients d e v e l o p e d p e r i t o n i t i s
(inílammation of the abdominal cavity), a n d o n e d e v e l o p e d
a skin infection at the catheter insertion site. A l l p a t i e n t s
w i t h peritonitis had l o w - g r a d e fever, c l o u d v p e r i t o n e a l t l u i d ,
a n d abdominal pain. A l l patients had p e r m a n e n t i n d w e l l i n g
peritoneal catheters, w h i c h the nurse w i p e d w i t h gauze thar
had been soaked w i t h an iodophor s o l u t i o n e a c h t i m e t h e
catheter was connected to or disconnected f r o m the m a -
chine tubing. Aliquots ot the iodophor w e r e t r a n s f e r r e d
from stock bottles to small in-use bottles. C^ultures f r o m t h e
dialysate concéntrate and the i n t e m a l áreas of the dialysis
machines were negative; iodophor from a s m a l l i n - u s e plás-
tic container yielded a puré culture of P. aeruginosa.
W h a t improper technique led to this infection?
3. Eleven patients received injections of methylprednisolone
and lidocaine to relieve the pain and inflammation of
arthritis at the same orthopedic surgery office. A l l of t h e m
developed septic arthritis caused by Serrana marcescens.
Unopened bottles of methylprednisolone f r o m the same lot
numbers tested sterile; the methylprednisolone was pre-
served w i t h a quat. C o t t o n balls were used t o wipe m u 1 tiple-
use injection vials before the medication was drawn i n t o a
disposable syringe. The site of injection o n each patient was
also wiped w i t h a c o t t o n ball. T h e c o t t o n balls were soaked
i n benzaikonium chloride, and fresh c o t t o n balls were added
as the jar was emptied. Opened methylprednisolone con-
tainers and the jar of c o t t o n b a l k contained S. marcescens.
H o w was the infection transmitted? W h a t part of the
routine procedure caused the contamination?.
LEARNING W I T H TECHNOLOGY
The Microbiology Place CD-ROM/website includes many
resources to help you study and succeed i n this course. I n addi-
tion to self-quizzes, web links, and flashcards, you'll find the fol-
lowing activities for this chapter i n the " A c t i v i t i e s " section ot
The Microbiology Place:
Case Study: Microbial C o n t a m i n a t i o n of Cosmetics
234 PART O N E Fundamentáis of M i c r o b i o l o g y
i n o c u l a t e d w i t h b a c t e r i a . I n a d d i t i o n , m i x t u r e s s u c h as
w i n e , b l o o d , smoke coiidensates, and extracts o f foods c a n
also be tested t o see i f t h e y c o n t a i n m u t a g e n i c substances.
For a n e x a m p l e o f t h e use o f t h e A m e s test i n expíoring t h e
role o f bacteria i n cáncer, see t h e box o n page 235.
A b o u t 9 0 % o f t h e substances f o u n d by the A m e s test t o
be m u t a g e n i c h a v e also been s h o v m t o be c a r c i n o g e n i c in
animáis. By t h e same t o k e n , t h e m o r e m u t a g e n i c sub-
stances h a v e g e n e r a l l y been f o u n d t o be more c a r c i n o -
genic.
Genetic Transfer and
Recombination
Learning Objectives
• Compare the mechanisms of genetic recombination in
bacteria.
• Differentiate between horizontal and vertical gene transfer.
C c i i e t i c r e c o m b i n a t i o n refers t o t h e exchange o f genes
between t w o D N A molecules t o form new combinations o f
genes o n a c h r o m o s o m e . Figure 8.23 shows o n e type o f ge-
netic r e c o m b i n a t i o n occurring between t w o pieces o f
D N A , w h i c h w e w i l l c a l i A a n d B a n d regard as c h r o m o -
somes f o r t h e sake o f s i m p l i c i t y . I f these t w o chromosomes
b r e a k a n d r e j o i n as s h o w n — a process c a l l e d c r o s s i n g
o v e r — s o m e o f t h e genes c a r r i e d b y these chromosomes
are shuíiSed. T h e o r i g i n a l chromosomes h a v e r e c o m b i n e d ,
so t h a t each n o w carries a p o r t i o n o f t h e other's genes.
I f A a n d B represent D N A f r o m d i f f e r e n t individuáis,
h o w are t h e y b r o u g h t cióse e n o u g h together t o r e c o m b i n e ?
I n eukar>otes, g e n e t i c r e c o m b i n a t i o n is a n ordered process
t h a t usually occurs as p a r t o f t h e sexual cycle o f t h e o r g a n -
i s m . R e c o m b i n a t i o n g e n e r a l l y takes place d u r i n g t h e for-
m a t i o n c : r e p r o d u c t i v e cells, such t h a t these cells c o n t a i n
recombir.ant D N A . I n bacteria, genetic r e c o m b i n a t i o n
J3n h a p r e n i n a n u m b e r o f ways, w h i c h we w i l l discuss i n
:';^.e r'ollev"ir\ sections.
Like ~ utation, genetic recombination contributes t o a
i c p u l a r i m ' s g e n e t i c d i v e r s i t y , vvhich is t h e source o f v a r i -
--.:;on ir. t v o l u t i o n . I n h i g h l y e v o l v e d organisms s u c h as
r r e s e n M - v m i c r o b e s , r e c o m b i n a t i o n is more l i k e l y t h a n
:v.ULacicr. :o be b e n e f i c i a l because r e c o m b i n a t i o n w i l l less
. . k c l v ¿fr":roy a gene's f u n c t i o n a n d may b r i n g t o g e t h e r
j . r r . r i r . i : _ :¡ns o f genes r h a t enable t h e organism t o carry
o u t 2 \"¿..:dble n e w f u n c t i o n .
T.c r-.ajor p r o t e i n t h a t c o n s t i t u t e s t h e flagella o f Sal-
-.on¿l^ also o n e . o f che p r i m a r y p r o t e i n s t h a t causes o u t
i r . i m u n r r/stems co r e s p o n d . H o w e v e r , chese bacteria h a v e
: h e c a p s : d i t y o f p r o d u c i n g t w o different flagellar p r o t e i n s .
-•^.-í o u r r - m u n e system m o u n t s a response againsc those
Chromosome A
Chromosome B C
Crossing over
Recombinant ^
chromosomes
FIGURE 8 . 2 3 G e n e t i c r e c o m b i n c i t i o n b y c r o s s i n g
o v e r b e t w e e n t w o r e l a t e d c h r o m o s o m e s . Chromo-
somes A a n d B each carry one versión of genes E t h r o u g h
I. The chromosomes cross over b y b r e a k i n g a n d r e j o i n i n g ,
sometimes in more than o n e locotion. The result is t w o
recombinant chromosomes, each o f w h i c h carries genes
originating from both chromosomes.
• Genetic recombination is the exchange o f genes between
tvyo D N A molecules to form new combinations o f genes.
cells c o n t a i n i n g o n e f o r m o f t h e flagellar p r o t e i n , those
organisms p r o d u c i n g t h e second are n o t affected. W h i c h
flagellar p r o t e i n is p r o d u c e d is d e t e r m i n e d b y a r e c o m b i n a -
t i o n e v e n t t h a t a p p a r e n t l y occurs s o m e w h a t randomly
w i t h i n t h e c h r o m o s o m a l D N A . T h u s , b y a l t e r i n g t h e fla-
gellar p r o t e i n p r o d u c e d , Salmonella c a n better avoid the
defenses o f t h e host.
V e r t i c a l g e n e t r a n s f e r occurs w h e n genes are passed
f r o m a n o r g a n i s m t o its offspring. Plants a n d animáis t r a n s -
m i t t h e i r genes b y v e r t i c a l t r a n s m i s s i o n . B a c t e r i a c a n pass
t h e i r genes n o t o n l y t o t h e i r offspring, b u t also laterally, t o
o t h e r microbes o f the same g e n e r a t i o n . T h i s is k n o w n as
h o r i z o n t a l g e n e t r a n s f e r . H o r i z o n t a l gene transfer be-
tween^bacteria occurs i n several ways. I r ^ a l l o f t h e m e c h a -
nisms, t h e ttansfer i n v o l v e s a d o n o r c e l l t h a t gives a
p o f t i o n o f its t o t a l D N A t o a r e c i p i e n t c e l l . O n c e t r a n s -
ferred, part of t h e donor's D N A is usually i n c o r p o r a t e d
i n t o t h e recipient's D N A ; che t e m a i n d e r is degraded by
c e i l u l a r enzymes. T h e recipient cell that incotporates
d o n o r D N A i n t o its o w n D N A is called a recombinant. The
transfer o f g e n e t i c m a t e r i a l b e t w e e n b a c t e r i a is b y n o
means a frequent event; i t may o c c u r i n o n l y l % o r less oí
an e n t i r e p o p u l a t i o n . Let's e x a m i n e i n d e t a i l t h e specific
types o f genetic transfer.
 CHAPTER 8 Microbial Genetics 235

V M I C RÓB-I O L O G V I N T H E Ñ E W S :>":í

T h e R o l e of .Bacteria in Cáncer y.

' n 1996, the Internationa! Agency for
Research on Cañcerclassified the
bacterium Helicobacter pylori as a car-
cinogen. No specific mechanism was pro-
posed to explain the relationship
between H . pylori and gastric cáncer. Re-
searchers have known since the early
1970s that there is a relationship be-
tween diet and certain types of cáncer.
However, it may not be the diet itself
that causes cáncer but the interaction
between diet and normal microbiota, the
hundreds óf species of microorganisms
that flourish i n the average person.
Tne importance of microorganisms in
cáncer inductions was first noticed when
rats were fed cycasin, a substance that
occurs naturally i n the nut of the cycad
plant.The bacterial enzyme /3-glucosidase
converts cycasin to methylazoxymethanol,
which caused cáncer in the rats. Germ-
free rats were unaffected by the cycasin,
but rats with the usual intestinal micro-
biota developed colon cáncer.
To determine the role of intestinal
bacteria in converting chemicals to
carcinogens, Elena McCoy and her co-
workers at New York Medical CoUege used
die Ames test to compare the muragenic
abiliries of 2-aminofluorene when acti-
\'ated by liver cell enzymes, intestinal cell
enzymes alone, Bacteroides fraff.hs eruymes
alone, and intestinal cell enzymes plus B.
froffiis enzymes. They found that liver en-
rames produced the highest conversión of
this chemical to a mutagenic form. Intesti-
nal enzymes alone and bacterial enzymes
alone produced some mutagenic conver-
són. A n d a combination of intestinal and
bacterial enzymes had an effect almost as
great as that produced by liver enzymes.
To determine the role of bacteria in
cladder cáncer, reíearchers at the Kyushu
Medical Center in japan used the Ames
test to evaluííEe'the mutagenic abilities of
human uriñe activated by bacteria. The
bacteria were isolated from patients with
urinary tract infections. The bacterial
isolates were tested for their ability to re-
duce nitrate ion ( N 0 3 ~ ) to nitrite ion
{ N 0 2 ~ ) . The mutagenic activity seen
w i t h the addition of nitrite ion indicates
that nitrite is mutagenic (see Table A ) .
Their results suggest that a chemical
reaching the bladder can be activated to
a carcinogen by bacterial enzymes.
In 2000, researchers used the Ames
test to determine whether H . pylori causes
cáncer by inducing mutations. H . pylori
TABLEA
Relative A m o u n t s of Bacterial
Test S u b s t a n c e
Normal human uriñe
Uriñe + N O a " -
reducing bacterio
Uriñe + non-NOs"-
reducing bacterio
Uriñe + NO2"
TABLE B
H. pylori cells
H. pylori supematant
Sterile culture médium
Distilled water
Methyinitcosourea (o carcinogen)
cultures were isolated from patients with
gastric cáncer and patients with gastritis or
peptic ulcers and were grown in a culture
médium. After incubation, the culture was
centrifuged to separare d-ie cells from their
supematant (culture médium). The Ames
test was performed on the bacteria and on
the supematant; distilled water provided a
negative control, and a carcinogen the
positive control. The bacteria isolated
from patients were not mutagenic (Table
B). There was slightly more ScdmoneUa
growth after treatment w i t h the super-
natant, compared to the sterile culture.
This suggests that some substance secreted
by H . pylañ has mutagenic activit>'.
G r o w t h i n A m e s Test
N o grov/th
+ + +
+
++
N u m b e r of
Colonies in
A m e s Test
211
330
240
246
507

Transformation in Bacteria transferred f r o m one b a c t e r i a l c e l l t o a n o t h e r , b u t study o f

this p h e n o m e n o n e v e n t u a l l y led t o t h e conclusión t h a t
C v r i n g the ptocess o f t r a n s f o r m a t i o n , genes are trans-
DNA is the genetic m a t e r i a l . T h e i n i t i a l e x p e r i m e n t o n
ferred f r o m (me b a c t e r i u m t o a n o t h e t as " n a k e d " D N A in
t r a n s f o r m a t i o n was performed by Frederick G r i f f i t h i n Eng-
5c. j t i o n . T h i s process was first d e m o n s t t a t e d over 70 years
land i n 1928 w h i l e he was w o r k i n g w i t h t w o strains o f Strep-
3 2 : , a l t h o u g h it was n o t u n d e r s t o o d at t h e t i m e . N o t o n l y
incoccus pneumoniae. One, a virulent (pathogenic) stram,
d : i t r a n s f o r m a t i o n show t h a t genetic m a t e r i a l c o u l d be
has a {x-)lysaccharide capsule t h a t prevents phagocytosis.
236 PART O N E Fundamentáis of Microbiology

Recom'bination

Living encapsulated ^ Living nonencapsulated ^ Heat-killed encapsulated ^ Living nonencapsulated and

bacteria injected into bacteria injected into bacteria injected into heat-killed encapsulated
mouse mouse mouse bacteria injected into mouse

^ I-

^ Mouse remained healthy

Coícnies of e n c a p s u l a t e d ^ A few cotonies of n o n e n c a p - ^ No colonies were isolated ^ Colonies of e n c a p s u l a t e d

bacteria w e r e isolated from sulated bacteria w e r e isolated from mouse bacteria w e r e Isolated from
dead mouse f r o m mouse; ptiagocytes dead mouse
destroyed nonencapsulated
bacteria

(a) (b) (c) (d)

FIGURE 8 . 2 4 Griffith's experíment demonstrating genetic transformation.

( a ) Living e n c a p s u l a t e d b a c t e r i a caused disease a n d d e a t h w h e n injected into a mouse.
(b) Living n o n e n c a p s u l a t e d b a c t e r i o a r e r e a d i l y d e s t r o y e d b y the p h a g o c y t i c defenses of
the host, so the mouse r e m a i n e d healthy after i n j e c t i o n . (c) After b e i n g killed b y heat,
e n c a p s u l a t e d b a c t e r i a lost the a b i l i t y to cause disease. (d) However, the c o m b i n a t i o n of
l i v i n g r o n e n c o p s u l o t e d bacterio a n d heat-killed e n c a p s u l a t e d b a c t e r i a (neither of w h i c h
a l o n e cause disease) d i d cause disease. Somehow, the live n o n e n c a p s u l a t e d b a c t e r i a
v/ere •'cnsformed b y the d e a d encapsulated bacterio so that they a c q u i r e d the a b i l i t y to
form c capsule a n d therefore cause disease. Subsequent experiments p r o v e d the trons-
r o r m i - factor to b e D N A .

V / r . did encapsulated bacterio kill the mouse while nonencapsulated bacterio d i d not? W h a t
<^ier •he mouse in (d)?

: : t e r i a grow a n d cause p i t e i m i o n i a . T h e other, a n capsulated b a c t e r i a (Figure 8 . 2 4 b ) o r dead e n c a p s u l a t e d

' • s t r a i n , lacks t h e capsule a n d does n o t cause dis- b a c t e r i a d i d n o t k i l l t h e mouse ( F i g u r e 8 . 2 4 c ) . H o w e v e r ,
w h e n t h e dead e n c a p s u l a t e d b a c t e r i a w e r e m i x e d w i t h
.•nth was i n t e r e s t e d i n d e t e r m i n i n g w h e t h e r i n j e c - live nonencapsulated bacteria and injected into the
tic: • h e a t - k i l l e d b a c t e r i a ot t h e encapsulated s t r a i n m i c e , m a n y o f t h e m i c e d i e d . I n t h e b l o o d o f t h e dead
COL 'e used t o v a c c i n a t e m i c e against p n e u m o n i a . A s mice, Griffith fouiid living, encapsulated bacteria.
•ícced, i n j e c t i o n s o f l i v i n g encapsulated b a c t e r i a H e r e d i t a r y m a t e r i a l (genes) f r o m t h e dead b a c t e r i a h a d
:.-ie mouse ( F i s u r e 8.24a); it-ijcctions o f l i v e n o n e n - e n t e r e d t h e liVe cells a n d c h a n g e d t h e m g e n e t i c a l l y so
 CHAPTER 8 Microbial Genetics 237

t h a t their progeny were encapsulated and therefore v i r u - Recipient cell

l e n t {Figure 8.24d).
S u b s e q u e n t i n v e s t i g a t i o n s based o n G r i f f i t h ' s research /b
r e v e a l e d t h a t b a c t e r i a l t r a n s f o r m a t i o n c o u l d be c a r r i e d
out w i t h o u t mice. A b r o t h was inoculated w i t h live
nonencapsulated bacteria. Dead encapsulated bacteria ^
DNA fragments Chromosomal DNA
were t h e n added t o t h e b r o t h . A f t e r i n c u b a t i o n , t h e c u l -
from donor cells
t u r e was f o u n d t o c o n t a i n l i v i n g b a c t e r i a t h a t w e r e e n c a p -
sulated a n d v i r u l e n t . T h e n o n e n c a p s u l a t e d b a c t e r i a h a d ^ Recipient cell takes
up donor DNA
been transformed; they had acquired a new hereditary
r r a i t by i n c o r p o r a t i n g genes f r o m t h e k i l l e d e n c a p s u l a t e d
bacteria.
T h e n e x t step was t o e x t r a c t v a r i o u s c h e m i c a l c o m p o -
n e n t s f r o m t h e k i l l e d cells t o d e t e r m i n e w h i c h c o m p o n e n t
caused t h e t r a n s f o n n a t i o n . T h e s e c r u c i a l e x p e r i m e n t s were
p e r f o r m e d i n t h e U n i t e d States by O s w a l d T . A v e r y a n d h i s
associates C o l i n M . M a c L e o d a n d M a c l y n M c C a r r y . A f t e r ^ Recombination occurs
years o f research, t h e y a i i n o u n c e d i n 1944 t h a t t h e c o m p o - Degraded between donor DNA
unrecombined DNA and recipient DNA
n e n t responsible for t r a n s f o r m i n g harmless S. prxeumoniae
i n t o v i r u l e n t strains was D N A . T h e i r results p r o v i d e d o n e

o f the conclusiva indications t h a t D N A was i n d e e d t h e
carrier o f g e n e t i c i n f o r m a t i o n .
Since the t i m e o f Griffith's experiment, considerable
i n f o r m a t i o n has b e e n g a t h e r e d a b o u t t r a n s f o r m a t i o n . I n
n a t u r e , some b a c t e r i a , perhaps after d e a t h a n d c e l l lysis, re-
léase t h e i r D N A i n t o t h e e n v i r o n m e n t . O t h e r b a c t e r i a c a n
t h e n encounter the D N A and, depending o n the particu-
l a r species a n d g r o w t h c o n d i t i o n s , t a k e u p fragments of
D N A a n d intégrate t h e m i n t o t h e i r o v m chrorriosomes by
recombination. A recipient cell w i t h this new combina-
t i o n o f genes is a k i n d o f h y b r i d , o r r e c o m b i n a n t c e l l ( F i g -
ure 8.25). A l l t h e descendants o f s u c h a r e c o m b i n a n t c e l l
w i l l be i d e n t i c a l t o i t . T r a n s f o r m a t i o n occurs n a t u r a l l y
a m o n g very few g e n e r a o f b a c t e r i a , i n c l u d i n g Bacillus,
Haemophilus (hé-má'fi-lus), Neisseria, Acinetobacter (a-si-
ne'to-bak-tér), a n d c e r t a i n strains o f t h e genera Streptococ-
cus zná Staphylococcus.
T r a n i f o r m a t i o n w o r k s best w h e n t h e d o n o r a n d r e c i p i -
e n t c e l b are very closely related. E v e n t h o u g h o n l y a s m a l l
p o r t i o n j f a cell's D N A is transferred t o t h e r e c i p i e n t , t h e
m o l e c u k t h a t must pass t h r o u g h t h e r e c i p i e n t c e l l w a l l
2ind m e - . b t a n e is s t i l l very large. W h e n a r e c i p i e n t c e l l is
i n a p h . b i o l o g i c a l state i n w h i c h i t c a n take up t h e d o n o r
DNA, is said t o be c o m p e t e n t . C o m p e t e n c e results
f r o m al-erations i n t h e c e l l w a l l t h a t m a k e i t permeable to
large D N ' A molecules.
"Hí-r A .11-uiiderstood and w i d e l y used b a c t e r i u m E. coli
is n o t n i c u r a l l y c o m p e t e n t for t r a n s f o r m a t i o n . ' H o w e v e r , a
simple h b o r a t o r y t r e a t m e n t enables E . coli to readily take
up DK.-.. T h e discover^' o f t h i s t t e a t m e n t has e n a b l e d te-
searchen t o use E. coli for genetic e n g i n e e r i n g , discussed i n
C h a p t t : 9.
i)
Genetically transformed celi
The mechanism of genetic transfor-
FIGURE 8 . 2 5
mation in bacteria.
• A recipient cell with o new combination of genes is a hy-
b r i d or recombinant cell.
Conjugation in Bacteria
A n o t h e r m e c h a n i s m by w h i c h genetic m a t e r i a l is t r a n s -
ferred f r o m one b a c t e r i u m t o a n o t h e r is k n o w n as c o n j u -
g a t i o n . C o n j u g a t i o n is m e d i a t e d by one k i n d o f plasmid, a
c i r c u l a r piece o f D N A t h a t replicares i n d e p e n d e n t l y f r o m
t h e cell's c h r o m o s o m e (discussed o n page 2 4 0 ) . H o w e v e r ,
plasmids differ f r o m b a c t e r i a l c h r o m o s o m e s i n t h a t the
genes t h e y carry are usually n o t essential for t h e g t o w t h of
the c e l l u n d e r n o r m a l c o n d i t i o n s . T h e plasmids responsi-
ble f o r c o n j u g a t i o n are transmissible b e l w e e n cells d u r i n g
conjugation.
C o n j u g a t i o n differs f r o m t r a n s f o r m a t i o n i n t w o major
vv'ays. First, c o n j u g a t i o n requires d i r e c t c e l l - t o - c e l l c o n t a c t .
Second, the c o n j u g a t i n g cells must generally be o f o p p o -
site m a t i n g type; d o n o r cells must carry t h e p l a s m i d , and
r e c i p i e n t cells usually do n o t . I n g r a m - n e g a t i v e bacteria,
the p l a s m i d carries genes t h a t code for t h e synthesis o f sex
pili, projections f r o m the donor's cell surface t h a t c o n t a c t
the r e c i p i e n t and h e l p b r i n g the t w o cells i n t o d i r e c t c o n -
tact. G r a m - p o s i t i v e ' b a c t e r i a l cells produce s t i c k y surface
238 PART O N E F u n d a m e n t á i s of Microbiology
8SÍ ^ T ; ¡ r
F I G U R E 8.26 B a c t e r i a l conjugation. Tfie sex pilus con-
necting Hiese celU undergoing conjugation allows the trans-
fer of genetic information, At the actual time of genetic
exchange, the cells' connecting bridge contracts and the
cells are much closer together. Notice that one cell has
numerouS fimbriae.
• In grom-negative bacteria, the plasmid carries genes that
code for the synthesis of sex pili.
molecules that-cause cells to come into direct contact w i t h
each ocher. í n the process of conjugation, the plasmid is
replicated during the transfer of a single-stranded copy of
the plasmid D N A to the recipient, where the complemen-
tary strand is synthesized.
Because most experimental work o i i conjugation has
been done w i t h E. coli, we will describe the process i n this
organifm. I n E. coli, the F factor (fertility factor) was the
nrst pla¿mid obser\'ed to be transferred bet\veen cells during
conjugadon (Figure 8.26). Donors carrying F factors (F"^
ceíls) irmsfer che plasmid to recipients (F~ cells), which
recome F"^ cells as a result (Figure 8.27a). I n some cells car-
rying F ractors, the factor integrares into the chromosome,
con\g the F"^ cell to an H f r cell (high frequency of re-
Cí)mb¡r..ar.ion) (Figure 8.27b). W h e n conjugation occurs
hecuetr. an Hfr cell and an F~ cell, the Hfr cell's chromo-
s(;me • .v.ch its integrated F factor) replicates, and a parental
-rranJ A the chromosome is transferred to the recipient
cell 1 F'.^-ire 8.27c). Replication of the Hfr chromosome be-
'4in5 ir. -he midc"e ot the integrated F factor, and a small
piecc rhe F fíictor leads the chromosomal genes into the
F Ce... Usually, the chromosome bréales before it is com-
pletelv -ransferred. Once withia the recipient cell, donor
DN.". :.in recombine with the recipients D N A . (Donor
I)N.". 'nac i.s not integrated is degraded.) Therefore, by
conjugation w i t h an Hfr cell, an F~ cell may acquire nev
versions of chromosomal genes (just as i n transformation;
However, it remains an F~ cell because it did not receive
complete F factor during conjugation.
Conjugation is used to niap the location of genes on a
bacterial chromosome (see Figure 8.1b). N o t i c e that gene^
for the synthesis of threonine (thr) and leucine (leu) are
ftrst, reading clockwise from 0. Their locations were deter-
mined by conjugation experiments. Assume that conjuga-
tion is allowed for only 1 minute between an Hfr stran.
that is his"*", pro"*", thr"*" and l-^u"^, and an F~ strain that i -
his~, p r o " , thr~, and l e u " . If the F~ acquired the ability to
synthesize threonine, then the thr gene is located early in
the chromosome, between O and 1 minute. I f after 2 min-
utes the F~ cell now becomes thr"^ and leu"^, the order of
these two genes o n the chromosome must be thr, leu.
Transduction in Bacteria
A third mechanism of genetic transfer between bacteria is
transduction. I n this process, bacterial D N A is trans-
ferred from a donor cell to a recipient cell inside a virus
that infects bacteria, called a bacteriophage, or phage.
(Phages w i l l be discussed íiirther i n Chapter 13.)
To understand how transduction works, we w i l l consider
the life cycle of one type of transducing phage of E. coU; this
phage carries out generalized transduction (Figure 8.2S
on page 240).
O I n the process of infection, the phage attaches to the
donor bacterial cell wall and injects its D N A into the
bacterium.
0 The phage D N A acts as a témplate for the synthesis of
new phage D N A and also directs the synthesis o f
phage protein coats. During phage development inside
the infected cell, the bacterial chromosome is broken
apart by phage enzymes, and
Q at least some pieces of bacterial D N A are mistakenly
packaged inside phage ptotein coats. (Even plasmid
D N A or D N A of another virus that is inside the cell
can be given phage protein coats.) T h e resulting
ptjLajge particles then carry bacterial D N A instead of
phage D N A .
O W h e n the released phage particles later infect a new
population of bacteria, bacterial genes will be transferred
to the newly infected recipient cells at low frequency.
0 Transduction of cellular D N A by a virus can lead to
recombination between the D N A of the donor host
cell and the D N A of the recipient host cell. T h e
process of generalized transduction is typical of bacte-
riophages such as phage Pl of £ . coíi and phage P22 oi
Salmonella. '
 CHAPTER 8 Microbial G e n e í i c s 239

Bacterial Sex pilus

chromosome

Replication
and transfer
of F factor
O
F+ cell F - cell F-^ cell F+ cell

(a) When an F factor (a plasmid) is transferred from a donor (F"*") to a recipient (F~). the F~ cei! is converted into an F"*" cei!.

o
Recomljinatlon between
F factor and chromosome
occurs at a specific site insertion of F factor
on each into chromosome results

Integrated F factor

F+ celi Hfr cell

(b) When an F factor becomes integrated into the chromosome of an F"^ ceit, it malees the cell a high frequency of recombination (Htr) cei

In the recipient.
recombination
Replication occurs between the
and transfer Hfr chromosome
of part of the fragment and the
chromosome F~ chromosome
^

:ell F-cell Hfr cell Recombinant

F-cell

(c) V/r^- =n Hfr donor passes a portion of its chromosome into an F" recipient, «-recombinant F~ ceil results.

FIGURE 3 . 2 7 C o n j u g a t i o n ¡ n f. co/i.

• How cees conjugation differ from transformation?

A i . ¿-enes contained w i t h i n a bacterium infected by a
genera!.:ed transducing phage are equally likely to be
packac:td in a phage coat and transferred. I n another
t\'pe c: -ransduction, called specialized t r a n s d u c t i o n ,
Only ct.-rnin bacterial genes are transferred. I n one type
of specialized transduction, the phage codes for certain
toxins produced by their bacteria! hosts, such as
Oor-^nehacizx'mm diphtheriae ( k ó r ' - i - n é - b a k - t i - r é - u m dif-
t h i ' - r e - i ) for diphtheria toxin, Streptococcus pyogenes for
erythrogenic toxin, and E. coli O l 5 7 : H 7 for Shiga t o x i n
240 PART O N E F u n d a m e n t á i s o ' Microbiology -1^
-Phage protein coat

CC3-GD Recombination
Bacterial chromosome

Donor cell
Phage DNA

Q A phage infects the donor Q Phage DNA and proteins are made
bacterial cell. the bacterial chromosome is broke--
into pieces.

Bacterial DNA

Donor bacterial DNAv^ Recipient bactena 3NA

Recipient cell Recombinant cell

^ Occasionally during pliage assembly,
pieces of bacterial DNA are packaged in
a ptiage capsid. Tfien ttie donor cell lyses
and releases phage particles containing
bacterial DNA.
^ A phage carrying bacterial Recombination can occur, producing a
DNA infects a new host cell, recombinant cell with a genotype difieren:
the recipient cell. from both the donor and redpient cells.

F I G U R E 8 . 2 8 T r a n s d u c t i o n b y a b a c t e r i o p h a g e . Shown here is general-

ized transduction, in which any bacterial D N A c a n be transferred from one
cell to another.

• W h a t is transduction?

that causes bloody diarrhea. Specialized transduction
wül be discussed i n Chapter 13 o n page 3 9 0 . I n addition
to m u t a t i o n , transformation, and conjugation, transduc-
tion is another way bacteria acquire new genotypes.
Plasmids and Transposons
i íearning Objective
I • Describe the hjnctions of plasmids and transposons.
Fla.>tTiids and transposons are genetic elements that pro-
v í i c addicional mechanisms for genetic change. They oc-
cu: n both prokars-otic and eukaryotic organisms, but this
d i í c j s s i o n focuses on their role i n genetic change i n
prCí'-iryotes.
P'csmids
Rccall from Chapter 4 (page 92) that plasmids are self-
r e r ' x a t i n g , gene-concaining circular pieces of D N A about
1 - : o che sizc of the bacterial chromo.some (Figure 8.29a).
Tr.-.i are found mainly in bacteria but also i n some eu-
karyotic microorganisms, such as Saccharomyces cerevisiae.
The F factor is a conjugative plasmid that carries genes
for sex pili and for the transfer of the plasmid to another
cell. A l t h o u g h plasmids are usually dispensable, under cer- '
tain conditions genes carried by plasmids can be crucial ro
the survival and growth o f the cell. For example, dissimi-
lation plasmids code for enzymes that trigger the catabo-
lism of certain unusual sugars and hydrocarbons. Some
species of Pseudomonas can actually use such exotic sub-
stances as toluene, camphor, and hydrocarbons o f petm-
leiHn as primary carbón and energy sources because thcv
have catabolic enzymes encoded by genes carried o n pla.-»-
mids. Such specialized capabilities permit the survival f t
those microorganisms i n very diverse and challenging on-
vironments. Because of their ability to degrade and detti.x-
ify a variety of unusual compounds, many of them are
being investigated for possible use i n the cleanup of e n \ i -
ronmental wastes. See the box i n Chapter 2 o n page 34.
Other plasmids code for proteins that enhance rhe
pathogenicity of a bacterium. T h e strain of E. coli rh;'i
causes infant diarrhea and traveler's diarrhea carries plasmul-
 CHAPTER 8 Microbio! Genetics 241

origin of replication mer

10 nm
origin of transfer tet

FIGURE 8 . 2 9 R factor, o t y p e of p l a s m i d . (a) Plasmids isolated from tfie

boderia Bacteroides fragilis that encode resistance to the antibiotic clindamycin.
(b) A diagram of an R factor, which has two parts: The RTF contains genes
fcneeded for plasmid replication and transfer of the plasmid by conjugation; the
r-determinant carnes genes for resistance to four different antibiotics.

¿ W h y are R factors important in the treatment of infectious diseases?

that code for toxiaproduction and for bacterial attachment
tpintestinal cells. W i t h o u t these plasmids, E. coli is aharm-
^ í e s s resident of the large intestine; w i t h them., it is patho-
^ f e n i c Other plasmid-encoded toxins include the
^exfoliative toxin of Staphybcoccus aureus, Clostridium tetani
W neurotoxin, and toxins of Bacillm anthracis. Still other plas-
inids contain genes for the synthesis of bacteriocins, toxic
- ^ í proteins that k i l l other bacteria. These plasmids have been
fixtnd in many bacterial genera, and they are useful markers
for the identiftcation of certain bacteria i n clinical laborato-
^ i ¿ ríes.
j . Resistance factors (R factors) are plasmids that have
signincant medical importance. They were first discovered in
Japan m the late 1950s after several dysentery epidemics. In
some of these epidemics, the infectious agent was resistant to
usual antibiotic. Following isolation, the pathogen was
tcund to be resistant to a number of different antibiotics.
•í;.Inadcition, other normal bacteria from the patients (such as
'^'•-) proved to be resistant as well. Researchers soon dis-
r cove:^ that these bacteria acquired resísiance through rhe
.i- spreac of penes from .^ne organism to another. The plasmids
i- "ediated this transfer are R factors.
' J ; ^ ^ ^ • -actors carry genes that confer upon their host cell re-
Sistar.ce to antibiotics, heavy metáis, or cellular toxins.
R factors contain two groups of genes. One group is
.,;-^calle: the resistance transfer factor (RTF) and includes
genes for plasmid replication and conjugation. The other
group, the r-determinant, has the resistance genes; it codes
for the production of enzymes that inactivate certain drugs
or toxic substances (Figure 8.29b). Different R factors, when
present in the same cell, can recombine to produce K jctors
with new combinations of genes i n their r-determinants
In some cases, the accumulation of resistance genes
within a single plasmid is quite remarkable. For example, Fig-
ure 8.29b shows a genetic map of resistance plasmid RlOO.
Carried on this plasmid are resistance genes for sulfonamides,
streptomycin, chloramphenicol, and tetracycline, as well as
genes for resistance to mercury. This particular plasmid can
be transferred between a number of enteric species, includmg
Escherichia, Kkbsieüa, and Salmonella.
R factors present very serious problems for the treatment
of infectious diseases with antibiotics. The widespread use ot
antibiotics in medicine and agriculture (many types ot ani-
mal feed contain antibiotics) has led to the preferentia! sur-
vival (selección) of bacteria that have R factors, sc^-
populations of resistant bacteria grow larger and larger. The
transfer of resistance between bacterial cells ot a populación,
and even becween bacteria of different genera, also con-
tributes to the problem. The ability to reproduce sexually
with members of its own species defines a eukar^'otic species.
However, a bacterial species can conjúgate and transfer plas-
mids to other species. Neisseria may have acquired its
242 PART O N E F u n d a m e n t á i s of Microbiology

IS1
A C T T A C T G A T A T C A G T A A G T
T G A A T G A C T A
Transposase gene T A G T C A T T C A

inverted repeat Inverted repeat

(a) insertion sequence "IS1"

Tn5

^ Kanamycin resistance J
1
IS1 IS1
(b) Complex transposon "Tn5" (c) R plasmid

F I G U R E 8 . 3 0 T r a n s p o s o n s a n d insertion s e q u e n c e s . (a) An insertion sequence (IS),

the simpiest transposon, contains a gene for transposase, the enzyme that catalyzes transpo-
sition. The transposase gene is bounded at each end by inverted repeat (IR) sequences that
f u n c í i o n a s recognition sites for the transposon. ISl is one exampie of an insertion sequence,
shown here with simplified IR sequences. (b) Complex transposons carry .other genetic mater-
ial in addition to transposase genes. The example shown here, Tn5, carries the gene for
kanamycin resistance and has complete copies of the insertion sequence ISl at each end.
(c) Transposition of the transposon Tn5 into RlOO plasmid. Notice that a plasmid can
acquire IS elements.

• Transposons are smali segments of D N A that can move from one r e g i ó n of a DNA molecule
to another

penicillinase-producing plasmid from Streptococcus,
Agrobacterium can transfer plasmids to plant cells (see Figure
9.18 o n page 268). Nonconjugative plasmids may be trans-
ferred from one cell to another by inserting themselves into
a conjugative plasmid or a chromosome or by transforma-
tion when released from a dead cell. Insertion is made possi-
ble by an insertiori sequence, which will be discussed shortly.
Plasmids are an important tool for genetic engineering,
discussed i n Chapter 9 on page 256.
Transposons : enzyme recognizes as recombination sites between the
Transposons are small segments of DN.A that can move
(be "'transposed") from one región of a D N A molecule to an-
oif.tr. These pieces of D N A are 700-40,000 base pairs long.
In the 1950s, American geneticist Barbara McClintock
dis-:overed transposons i n corn, but they occur i n all or-
gar.isms and have been studied most thoroughly in m i -
cri'-jrganisms. They may move from one site to another
si:t un the same chromosome, or to another chromosome
or riasmid. As you might imagine, the frequent movement
oí :ransposons could wreak havoc inside a cell. For exam-
ple. as transposons move about on chromosome^, they may
:n;crt themselves u^itfiin genes, inactivating them. Fortu-
Hí-.tly, transposition occurs relatively rarely. The fre-
and quency o f transposition is comparable to the spontaneous
mutation rate that occurs i n bacteria—that is, from 10"^
to 10"'' per generation.
A l l transposons contain the information for their own
transposition. As shown i n Figure 8.30a, the simpiest
transposons, also called insertion sequences (IS), con-
tain only a gene that codes for an enzyme (transposase,
which catalyzes the cutting and resealing of D N A that oc-
curs in transposition) and recognition sites. Recogiiiiion
sites are short inverted repeat sequences of D N A that the
transposon and the chromosome.
Complex transposons also carry other genes not con-
^D^cted with the transposition process. For example, bacte-
rial transposons may contain genes for enterotoxin or tor
antibiotic resistance (Figure 8.30b). Plasmids such as R
factors are frequenrly made up of a collection of trans-
posons (Figure 8.30c).
Transposons with antibiotic resistance genes are oí
practical interese, but there is no limitation on the kinds oi
genes that transposons can have. Thus, transposons pro-
vide a natural mechanism for the movement of genes troiv.
one chromosome to another. Furthermore, because t h e
may be carried-between cells on plasmids or viruses, the-.

can also spread from one organism—or even species—to
another. Transposons are thus a potentially powerful medi-
ator of evolution i n organisms.
Genes a n d Evolution
Leorning Objective
• Discuss how genetic mutation and recombination provide
material for natural selection to act on.
We have now seen how gene activity can be controlled by
the cell's i n t e m a l regulatory mechanisms and how geries
STUDY OUTLINE
Structure and Function of the
Genetic Material (pp. 211-222)
1. Genetics is the study of what genes are, how they carry
information, how their information is expressed, and how
they are replicated and passed to subsequent generations or
other organisms.
2. D N A i n cells exists as a double-stranded helix; the two
strands are held together by hydrogen bonds between spe-
ciAc nitrogenous base pairs: A T and CG.
3. A gene is a segment of DNA, a sequence of nucleotides,
that codes for a functional product, usually a protein.
4. When a gene is expressed, D N A is transcribed to produce
RNA; mRNA is then tíanslated into proteins. ,
5. The D N A in a cell is duplicated before the cell divides, so
each daughter cell receives the same genetic information.
Genotype and Phenotype (p. 211)
1. Genotype is the genetic composition of an organism, its
entire complement of DNA.
2. Phenotype is the expression of the genes: the proteins of the
cell and the properties they confer on the organism.
DNA ond Chromosomes (pp. 211-212)
1. ~he D N A in a chromosome exists as one long double
r.clix associated with various proteins that regúlate genetic
iccivity.
2. Bacterial D N A is circular; the chromosome of E. coli, for
rxample, contains about 4 million base pairs and is approxi-
-acely 1000 times longer than the cell.
3 jenor.ni:s is the nnolecular characterization of genomes.
4. ^nformarion contained in the D N A is transcribed into
r .^J A and translated into proteins.
DNA Replication [pp.2]2-2U)
1. luring DNA replication, the two strands of che double helix
-.párate at che replication fork, and each strand is used as a
CHAPTER 8 Microbial Genetics 243
themselves can be altered or rearranged by m u t a t i o n ,
transposition, and recombination. A l l these processes pro-
vide diversity i n the descendants o f cells. Diversity pro-
vides the raw material for evolution, and natural selection
provides its driving forcé. Natural selection w i l l act on d i -
verse populations to ensure the si rvival of those fit for,that
particular environment. The different kinds of microor-
ganisms that exist today are the result of a long histor\ o f
evolution. Microorganisms have continually changed by
alterations i n their genetic properties and acquisition o f
adaptations to many different habitats.
témplate by DNA polymerases to synthesize two new strands
of D N A according to the mies of nitrogenous base pairing.
2. The result of D N A replication is two new strands of D N A ,
each having a base sequence complementary to one of the
original strands.
3. Because each double-stranded D N A molecule contains one
original and one new strand, the replication process is
called semiconservative.
4. D N A is synthesized in one direction designated 5' 3'.
A t the replication fork, the leading strand is synthesized
continuously and the lagging strand discontinuously.
5. D N A polymerase proofreads new molecules of D N A and
removes mismatched bases before continuing D N A synthesis.
6. Each daughter bacterium receives a chromosome that is
virtually identical to the parent's.
RNA and Protein Synthesis (pp. 217-222)
1. During transcription, the enzyme R N A polymerase synthe-
sizes a strand of RNA from one strand of double-stranded
DNA, which serves as a témplate.
2. R N A is synthesized from nucleotides containing the bases
A , C, G, and U , which pair with the bases of the D N A
strand being transcribed.
3. The starting point for transcription, where R N A
polymerase binds to DNA, is the prometer; the región of
D N A that is the end point of transcription is the termina-
- tor; RNA is synthesized in the 5' ¿r> 3' direction.
4. Translación is the process in which the information in the
nucleotide base sequence of mRNA is used to dictare the
amino acid sequence of a protein.
5. The mRNA associates with ribosomes, which consist of
rRNA and protein.
6. Three-base segmencs of mRNA that specify amino acids are
called codons.
7. The genetic code refers to che relationship among che nu-
cleocide base sequence of DNA, che corresponding ccxJons
of mRNA, and che amino acids for which che codons code.
244 PART O N E F u n d a m e n t á i s of Microbiology
8. The genetic code is degenerate; that is, most amino acids
are coded for by more than one codon.
9. Of the 64 codons, 61 are sei se codons (which code for
amino acids), and 3 are non ense codons (which do not
code tor amino acids and ari stop signáis for translation).
l ü . The scart codon, AUG, cod s for methionine.
11. Specific amino acids are attíched to molecules of tRNA.
Another portion of the cRNA has ; base triplet called an
ancicodon.
12. rhe base pairing of codon and anticodon at the ribosome
results in specific amino acids being brought to the site of
protein synthesis.
13. The ribosome moves along the mRNA strand as amino
acids are joined to form a growing polypeptide; mRNA is
read in the 5'—> 3'direction.
14. Translation ends when the ribosome reaches a stop codon
on the mRNA.
15; In prokaryotes, translation can begin before transcription is
complete.
The Regulatíon of Bacterial
Gene Expression (pp. 222-224)
1. Regulating protein synthesis at the gene level is energy-
efficient because proteins are synthesized only as they are
needed.
2. Constitutive enzymes produce products at a fixed rate.
Examples are genes for the enzymes in glycolysis.
3. For these gene regulatory mechanisms, die control is aimed
at mRNA synthesis.
Repression and Induction (p. 223)
1. Repression controls the synthesis of one or several (repress-
1 ble) enzymes.
2. "-Cn^en cells are exposed to a particular end-product, the
ivnthesis of eruymes related to that product decreases.
3. '.n the presence of certain chemicals (inducers), cells syn-
-.nesize more enzymes. This process is called induction.
4. -.n example of induction is the producción of j3-galactosi-
:ase by E. coli in che presence of laceóse; laceóse can then
:e mecabolized.
The Operon Model of
Gene Expression (pp. 223-224)
1. "Trie íormation ot enzymes is decermined by scructural genes.
2. bacteria, a group of coordinacely regulated scruccural
:.-:nes with related n.orabolic tunctions, plus che promocer
;--d operacor sites chac concrol cheir transcription, are called
operon.
3. che operon model for an inducible syscem, a regulacory
^tne codes for che repressor procein.
4. .V hen the inducer is absenc, the repressor binds ro che
cerator. and no niRNA is synchesized.
5. When che inducer is presenc, ic binds co che repressor so
chac ic cannoc bind co che operacor; chus, mRNA is made.
and enzyme synchesis is induced.
6. In repressible syscems, che repressor requires a corepre^^or ¡n
order co bind co che operacor sice; chus. che corepressor
concrols enzyme synchesis.
7. Transcripción of scruccural genes for catabolic enzvmes
(such as /3-galaccosidase) is induced bv che absence ot glu-
cose. Cyclic AMP and CRP muse bind co a promocer m the
presence of an alcernacive carbohydrace.
8. The presence of glucose inhibirs che mecabolism of alcerna-
cive carbón sources by cacabolice repression.
Mutation: Change in the
Genetic Material (pp. 224-234)
1. A mucacion is a change in che nicrogenous base sequence of
DNA; that change causes a changé in the product coded for
by the mutated gene.
2. Many mutations are neutral, some are disadvantageous, and
others are beneficial.
Types of Mutations (pp. 22Ó-228)
1. A base substitution occurs when one base pair i n D N A is
replaced with a different base pair.
2. Alterations in D N A can result in missense mutations
(which cause amino acid substitutions) or nonsense muta-
tions (which créate stop codons). ,
3. In a frameshift mutation, one or a few base pairs are deleted
or added to DNA.
4. Mutagens are agents in the environment that cause perma-
nent changes in DNA.
5. Spontaneous mutatioris occur without the presence of any
mutagen.
Mutagens (pp. 228-230)
1. Chemical mutagens include base-pair mutagens (for example,
nitrous acid), nucleoside analogs (e.g., 2-aminopurine and
5-bromouracil), and frameshift mutagens (e.g., benzpyrene).
2. lonizing radiación causes che forr»acion of ions and free
radicáis chac react wich DNA; base substicucions or breakage
of che sugar-phosphace backbone resulcs.
3._^Ulcraviolec (UV) radiación is nonjonizing; ic causes bond-
ing becween adjacenc chymines.
4. Damage co DNA caused by UV radiación can be repaired b\
enzymes chac cur out and replace che damaged porción of
DNA.
5. Lighc-repair enzymes repair chymine dimers in che presence
of visible light.
The Frequency of Mutation (p 231)
1. Mucacion rate is the probabilicy thac a gene will mucate when
a cell div}des;.the nire is expressed as 10 co a negative power.

2. Mutations usually occur randomly along a chromosome.
3. A low rate of spontaneous mutations is beneficial in provid-
ing the genetic diversity needed for evolution.
Identifying Mutants {p. 23])
1. Mutants can be detected by selecting or testing for an ai--^
rered phenotype.
2. Positive selection involves the selection of mutant cells and
the rejection of nonmutated cells.
3. Replica plating is used for negative selection—to detect, for
example, auxotrophs that have nutritional requirements not
possessed by the parent (nonmutr-ed) cell.
Identifying Chemical Carcinogens (pp. 232-234)
1. The Ames test is a relatively inexpensive and rapid test for
identifying possible chemical carcinogens.
2. The test assumes that a mutant cell can revert to a normal
cell in the presence of a mutagen, and that many mutagens
are carcinogens.
3. Histidine auxotrophs of SalmoneUa are exposed to an enzy-
matically treated potential carcinogen, and reversions to
the nonmutant state are selected.
Genetic Transfer and
Recombination (pp. 234-243)
1. Genetic recombination, the rearrangement of genes from
sepárate groups of genes, usually involves D N A firom differ-
ent organisms; it contributes to genetic diversity.
2. In Crossing over, genes from two chromosomes are recom-
binéd into one chromosome containing some genes from
each original chromosome.
3. Vertical gene transfer occurs during reproduction when
genes are passed from an organism to its offspring.
4. Horizontal gene transfer in bacteria involves a portion of
the cell's D N A being transferred from donor to recipient.
5. When some of the donor's DNA has been integrated into the
recipients DNA, the resultant cell is called a recombinaiit.
Transformation in Bacteria (pp. 235-237)
1. Ouring this process, genes are transferred from one bac-
terium to another as "naked" D N A m solución.
STUDY QUESTIONS
Review
1. E:.-:fly describe che componencs of DNA, and explain its
iv,r.:rional relacionship to RN.^ and procein.
2. Ct^vV a diagram showinjí a portion of a chromosome under-
'¿. ni^' replication.
CHAP'^^R 8 Microbio! Genetics 245
2. This process wasfirscdemonstraced in Sn-¿/)tocüccu5 pneumo-
niae, and occurs nacurally among a few genera of bacceria.
Conjugation in Bacteria {pp 237-238)
1. This process requires contact between living cells.
2. 'One cype of genecic donor cell is an F"; recipient cells are
r~. F cells concain plasmids called F factors; chese are trari>-
ferred to the F~ cells during conjugación.
3. When che plasmid becomes incorporaced inco che chrom->- .
some, che cell is called an Hfr (high frequency of recombi-
nation) cell.
4. During conjugation, an Hfr cell can transfer chromosomal
DNA to an F~cell. Usually, the Hfr chromosome breaks
before it is fully transferred.
Transduction in Bacteria (pp 238-240)
1. In this process, D N A is passed from one bacterium to an-
other in a bacteriophage and is then incorporated into the
recipient's DNA.
2. I n generalized transduction, any bacterial genes can be
transferred.
Plasmids and Transposons (pp. 240-243) í
1. Plasmids are self-replicating circular molecules of D N A carry-
ing genes that are not usually essential for the cell's survival.
2. There are several types of plasmids, including conjugative
plasmids, dissimilation plasmids, plasmids carrying genes for
toxins or bacteriocins, and resistance factors.
3. Transposons are small segments of D N A that can move
from one región to another región of che same chromosome,
or to a different chromosome or a plasmid.
4. Transposons are found in the main chromosomes of organ-
isms, in plasmids, and in the genecic macerial of viruses.
They vary from simple (insertion sequences) to complex.
5. Complex transposons can carry any cype of gene, including
ancibiocic-resiscance genes, and are chus a nacural mecha-
nism for moving genes from one chromosome co anocher.
Genes and Evolution (p. 243)
1. Diversicy is che precondition for evolution.
2. Genecic mutation and recombination provide a diversity ,1
..-organisms, and che process of nacurel selección aüows rhe
growch of chose best adapced to a given environment.
a. Identify the repliCr; ion fork.
b. What is che role o\A pv)lynierase.' O í RN'A polymerase.'
c. hlow does rhis proc -ss represenr semiconservative repli-
:ation.'
246 PART O N E F u n d a m e n t á i s of Microbiology

3. The following is a code for a strand of DNA'. presenc, a protein muse be bound cightlv -
the site. When che inducer is presenc, ir
will bind co che so chat _ car.
DNA 3' ATAT T T T _ _
1 2 3 4 5 6 7 8 910111213141516171819 occur.
mRNA ^ C G U UGA b. If v.nzyme a ib reprensible, end-product C, called a
tRNA U G G .' , causes the to bind co the
Amino acid "Met :i-r"" Whac causes derepression?
ATAT = Promofer sequence c. If enzyme a is constitutive, what effece, if any, will che
presence of A or C ha\-e on ic?
10. Idencify chree ways ot prevencing miscakes in D N A .
a, Using the genetic code provided in Figure 8.9, fill inthe
11. Define che following cerms:
blanks to complete the segment of DNA shown.
a. genocype í
^ b. Fill in the blanks to complete the sequence of amino
b. phenocype :
acids coded for by this strand of DNA.
c. recombinacion
c. Write the code for the complementary strand of DNA
completed in pare a. 12. Which sequence is che bese carget for damage by U V radia-
d. What would be the effece if C was subseieueed for T at ción: A G G C A A , C T T T G A , or G U A A A U ? Why aren'c all
base 10? bacceria killed when chey are exposed co sunlighc?
e. What would be the effect if A was substituted for G at 13. You are provided wich cultures with the following charac-
base 11? ceriseics:
f. What would be the effect if G was substituted for T ae Culture 1: F"", genotype A ^ C^
base 14? Culture 2: F~, genotype A ~ B~ C~
g. What would be the effect if C was inserted between bases a. Indicate the possible genotypes of a recombinant cell re-
9 and 10? sulting firom the conjugation of cultures 1 and 2.'
h. How would U V radiation affect this strand of DNA? b. Indicare the possible genotypes of a recombinant cell re-
i . Identify a noriser\se sequence in this strand of DNA. sulting íix)m conjugation of the two cultures after the F"*"
4. Describe translation, and be sure to include the following has become an Hfr-cell.
eerms: ribosome, rRNA, amino acid activation, tRNA, 14. Why are semiconservative replication and degeneracy of
anticodon, and codon. the genetic code advantageous to the survival of species?
5. E.xplain how you would find an ai\tibiotic-resistant mutane 15. Why are mutation and recombination important in the
by direct selection and how you would find an antibiotic- process of natural selection and the evolution of organisms?
sensitive mutant by indirect selection.
6. Match the following examples of mutagens. .
Múltiple Cholee
A mutagen that is a. Frameshift mutagen
Match the following terms to the definitions in questions 1
incorporaced into DNA b. Nucleoside analog and 2.
in place of a normal base c. Base-pair mutagen a. conjugation * d. transformation
A mutagen that causes d. lonizing radiación b. transcription e. translation
che formación of highly e. Nonionizing c. transduction
reaceive ions radiation
A mucagen chac alcers 1. The transfer of DNA from a donor co a recipient cell by a
adenine so chat it base- bacteriophage.
pairs wich cycosine 2. The cransfer of DNA from a donor eo a recipiene as naked
A m.ucagen chat causes DNA in solución.
insercions 3. Feedback inhibición differs from repression because feed-
A mucagen chat causes che back inhibición
formación ot pyrimidine is less precise. , d . scops che synchesis
dimers b. is slower accing. of new enzymes.
7. I-scribe che principie of che Ames cese for idencitying c. scops che acción of e. all of che above
i.'cmical carcinogens. preexisring enzymes.
S. _i-hne plasmids, and explain che relacionship becween F 4. Bacceria can acquire ancibiocic resiscance by
'icrors and cor\jugacion. a. mucacion. d. all of che above
9. -e the following metabolic pathway to answer che ques- b. inserción of cranspost)ns. e. none ot che alxwe
: .ns chat foUow ic. c. acquiring plasmids.
5. Suppose you inoculare chree tlasks of minimal sales broch
enzyme a en:yme h
wich E. coli. Flask A concains jílucose. Flask B concains
: ;bstrate A ' Intermediare B » End-product C i'lucose and laceóse. Flask C ct)ncains laceóse. Afcer a tew
li enzyme a is inducible and is not being synthesized at

hours of incubation, you test the flasks for the presence ot
/3'galactosidase. Which flask(s) do you predict will have
this enzyme?
a. A d. A and B
b. B ' . . e. B a n d C
c. C
6. Plasmids differ from transposons because plasmids
a. become inserted into chromosomes.
b. are self-replicated outside the chromosome.
c. move from chromosome to chromosome.
d. carry genes for antibiotic resistance. '
e. none of the above
Use the following choicer *.o answer questions 7 and 8.
a. catabolite repression d. repression
b. D N A polymerase e. translation
c. induction
7. The .mechanism by which the presence of glucose inhibits
the \ac operon.
8. The mechanism by which lactose controls the lac operon.
9. Two daughter cells are most likely to inherit which one of
the following firom the parent cell?
a. a change in a nucleotide in mRNA
b. a change in a nucleotide in t R N A
c. a change in a nucleotide in rRNA
d. a change in a nucleotide in D N A
e. a change in a protein
10. Which of the following is not a method of horizontal gene
transfer?
• a. binary fission
b. conjugation
c. integration of a transposon
d. transduction
e. transformation
Crítical Thinking
1. Nucleoside analogs and ionizing radiation are used in the
treatment of cáncer These mutagens can cause cáncer, so
how do you suppose they are used to treat the disease?
2. Replication of che E. coíi chromosome takes 40-45 min-
utes, but the organism has a generation time of 26 minutes.
Kow dcMrs the cell have time to make complete chromo-
somes for each daughter cell? For each granddaughter cell?
3. ?iexiÁomo-\\as has a plasmid containing the mer operon,
which includes the gene for mercurio reductase. This en-
r/me catalyzes the reduction of the mercurio ion H g ' to
che uncharged form of mercury, Hg°. Hg^"^ is quite co.xic to
ctils; Hg""^ is not.
a. What do you suppose is che inducer for this operon?
b. The procein encoded by one of the mer genes binds Hg""^,
rh,' poriplasm and brings ic inco che cell. Why would a
cell bring in a t05¿ir.? •
c. Whac is che valué of che mer operon co ?%eu¿or{\o'i\as^.
CHAPTER 8 Microbial Genetics 247
Clinicai Applications
1. Ciprofloxacin, erychromycin, and acyclovir are used co creat
microbial infeccions. Ciproíloxacin inhibics DN.A gvrase.
Erychromycin binds in fronc of che A sice on che 505 sub-
unic of a ribosome.. Acyclovir is a guanine analog.
a. Whac sceps in procein synchesis are inhibiced by each dru;.\
b. Which drug is more effeccive againsc bacceria? Why?
. c. Which drug is more efíective against viruses? Why?
d. Which drugs will have effeccs on che host's cells? Why?
e. Use the índex lo identify the disease for which acyclovir
is primarily used. Why is it more effeccive than epj-ch-
romycin for creating chis disease?
2. HIV, che vims chac causes AIDS, was isolated from three
individuáis, and the amino acid sequences for the viral coat
were decermined. Of che amino acid sequences shown be-
low, which two of the viruses are most closely related? How
can these amino acid sequences be used co idencifv che
source of a virus?
Patíent Viral Amino Acid Sequence
A Asn Gin Thr Alo Ala Ser Lys Asn He Asp Ala Leu
B Asn Leu His Ser Asp Lys He Asn He lie Leu Leu
C Asn GIn Thr Ala Asp Ser He Vai He Asp A l a Leu
3. Human herpesvims-8 (HHV-8) is common in certain pares
of Africa, the Middle East, and the Mediterranean, but ic is
rare elsewhere except in AIDS patients. Genetic anal^-ses
indicate that the African strain is not changing, whereas
the Western strain isoccumulating changes. Using the
"^portioris of the HHV-8 genomes (shown below) thac encode
oné of the viral proteins, how similar are these two viruses?
What mechanism can account for the changes? What dis-
ease does HHV-8 cause ?
Western 3'ATGGAGTTCTTCTGGACAÁGA
African 3' ATA A A C T T T T T C T T G A C A A C O
LEARNING W I T H TECHNOLOGY
The Microbiology Place CD-ROM/website includes many
resources co help you scudy and succeed in chis course. In addi-
tion co self-quizzes, web links, and flashcards, you'U ñnd the tol-
lowing accivicies for chis chapter in che "A.ccivicies" section ot
The Microbiology Place:
DNA Replicacion
Transcripción: Flcnv of Genetic Information '
• Transcription: How DNA is Transcribed
Mutation ' . '
Recombination
The \ac Operon
Case Study: Mappin^ a Bacterial Chromosome
 C a p í t u l o 12 N u c l e ó í j d o s y á c i d o s nucleicos 331

Figura 12-11 Enlaces de

hidrógeno en los pares
de bases definidos por
Watson y Crick.

C-r Timina
Adenina O \
\
c-r H

\\\

^ H H
\ „--N^ C
U .->', // ü

H-C N- Citosina

(3uanina

H
-1,08'^«^

disolución que contenga la misma concentración de nucleotides libres.

Este fenómeno se denomina efecto h i p o c r ó m i c o .
Los grupos funcionales más importantes de las purinas y pirimidinas son
los grupos carboniio y los átomos de nitrógeno del anillo y los grupos
amino exocíclicos. La formación de enlaces de hidrógeno en los que par-
ticipan los grupos amino y carboniio constituye el segundo tipo principal de
interacción entre bases. Los enlaces de hidrógeno entre bases permiten la
asociación complementaria de dos y en ocasiones de tres cadenas de ácido'
nucleico. Los patrones de formación de enlaces de hidrógeno más impor-
taníes son los definidos por James Watson y Francis Crick en 1953, según
los cuales A se enkiza específicamente con T (o U) y G se enlaza con C
(Fíg. 12-11). Estos dos tipos de pares de bases predominan en el DNA y el
RÍMA de doble cadena, y los tautómeros que .se muestran en la Figura 12-2
son los responsables de estos apareamientos. El apareamiento específico
de las bases permite la duplicación de la información genética por la
síntesis de cadenas de ácido nucleico complementarias a las ya existentes.

Esírmctura de ios ácidos -aiideiccís

El descubrimiento de la estructura del DNA a cargo de Watson y Crick en
1953 fue un acontecimiento trascendental para la ciencia, que dio lugar a la
aparición de disciplinas completamente nuevas y tuvo influencia sobre el
desarrollo de muchas otras. Nuestros conocimientos actuales acerca del
modo en que se almacena y utiliza la información genética de una célula se
basan en investigaciones que han sido posibles gracias a este descubrimien-
to. A pesar de que las rutas de transmisión de ía información no se discuten
hasta la Parte IV, las nociones generales que se dan en los Capítulos 1 y 3
de este libro constituyen actualmente requisitos previos ineludibles para
poder discutir acerca de cualquier área de la bioquímica. En este apartado
nos centraremos en la estructura del DNA, en los acontecimientos que
James Watson
condujeron a su descubrimiento y presentaremos los avances más recientes
en el campo. También introduciremos la estructura del RNA.
Como sucedía en el caso de la estructura de proteínas (Capítulos 6 y 7),
en. ocasiones resulta de utilidad describir ia esíructufa de ácidos nucleicos hrancis uncK
en términos de niveles jerárquicos de complejidad (estructura primaria,
 CHAPTER 8 Microbial Genetics 215

NEW TEMPLATE FIGURE 8.5 A d d i n g a nucleotide

'STRAND STRAND to DNA. W h e n a nucleoside triphos-
phate bonds to the sugar in a grow-
Sugar ing D N A strand, it loses two
phosphates. Hydrolysis of the phos-
Phosphate
phate bonds provides the energy for
the reaction.

• Synthesis of D N A requires dATP,

dTTP, dGTP, dCTP; d stands for
deoxyribose.

Pyrophosphate r~c"
released

Nucleoside
triphosphate

Replication
Q Proteins stabilize the Q The leading strand is
unwound parental DNA. synthesized continuously

Co)(3J -DNA polymerase

by DNA polymerase.
3'

Enzymes unwind the

parental double
helix.

• Replication
fork

• RNA primer
5' RNA
polymerase
'DNA polymerase
3'
.Okazaki fragment
Parental DNA
strand polymerase

Q The lagging strand is DNA polymerase DNA ligase joins

synthesized discontinuously. digests RNA primer the discontinuous
RNA polymerase synthesizes a and replaces it with DNA fragments of the
short RNA primer, which is then lagging strand.
extended by DNA polymerase.

FIGURE 8.6 A s u m m a r y of events ot the DNA replication fork. Enzymes at the

replication fork unwind the parental double helix. D N A polymerase synthesizes a continuous
strand of new D N A , using one of the parental strands as a t é m p l a t e . D N A polymerase also
uses the other parental strand as a t é m p l a t e , but because the o r í e n t a t i o n of the sugars is
opposite, RNA polymerase starts the synthesis by a d d i n g a short stretch of RNA called an
RNA primer. The D N A polymerase digests a w a y the RNA as it makes a small piece of D N A .
The small units are subsequently ¡ o i n e d by D N A ligase.

® W h y is one strand of D N A synthesized discontinuously?

:ión

'on estos métodos en 1966 se habían establecido las secuencias (

s de todas las palabras del código de tripletes para los aminoácidc
le entonces, estas palabras del código se han verificado de much
as distintas. En la Figura 26-7 se da el "diccionario" completo (
)nes de aminoácidos. El descifrado del código genético se considera
or descubrimiento científico de los años sesenta.

Segunda letra del codón

U C A G
iiliiililJ-illiliÉiili^
ÜUU
ÜUC
Ph e
Ph e
:lÍllÍÍBlÍ UAU
UAC
Tyr
Tyr
UGU
ÜGC
Cys"
Cys
u IÍIÍÍIISB
UUA Leu•••lili UAA m p i UGA Stop
i K l í B i liiilIBII UAG Sfcop UGG Trp

CUÜ Leu l i l l i l B i l CAÜ His^ C G Ü Arg

CUC Leu l l l l l l l l l l CAC His CGC Arg

CUA Leu COA Pro CAA Gln CGA Arg

Primera GÜG Leu CCG Pro CAG Glja CGG Arg
tra del codón
^extremo 5') ÁUU lie ACÜ Thr A A U Ásn AGU Ser
AUC lie ACC T l i r , Á A C ; ' ; .Asn AGC ^

ACÁ Thr AAA Lys AGA Arg

AGG Thr AAG Lys AGG Arg

GUÜ ^U Ala GAU Asp Gly

G V C 2 Ala GAC Asp GGC Gly
G
~GUA \ \la GAA Glu G Ú A Gly
GUG ^ GAG Glu GGG Gly
i ' •[?! 'l J i ^
 10.2 • B A S E S M O L E C U L A R E S P E LA M U T A C I Ó N a 269

Las mutaciones pueden ser espontáneas o inducidas. de bases que pueden ocurrir en una estrecha región del
Las mutaciones e s p o n t á n e a s pueden surgir como conse- D N A de u n gen que codifica para una pro teína. El error en
cuencia de la acción de la radiación natural (por ejemplo, ra- el D N A se transcribe al mRNA, y este RNA erróneo se usa,
Crecen a su vez como molde en la traducción a proteína. (Puesto
yos cósmicos) que alteran la estructura de las bases del
todas las
colonias DNA. Las mutaciones espontáneas pueden ocurrir duran- que sólo se utiliza una de las bandas del D N A como mol-
jg la replicacion, como resultado de errores en el aparea- de para el mRNA, u n par de bases AT no significa lo mis-
miento de las bases, originando cambios en el D N A mo que un par TA.) El código triplete que dirige la inserción
replicado. de un aminoácido a través de un RNA de transferencia será,
Las mutaciones que implican un par de bases (o unos por tanto, incorrecto. ¿Cuáles son las consecuencias de las
pocos pares), se conocen generalmente como mutaciones sustituciones de pares de bases?
puntuales. Las mutaciones puntuales pueden ser resultado Para interpretar los resultados de la m u t a c i ó n , debe-
¿esustituciones de pares de bases en el D N A o de la inserción mos recordar primero que el código genético es degene-
odeleción de un par de bases {microinserciones o microdele- rado {véase Sección 7.13). Por ello, no todas las mutaciones
ciones). Como en toda mutación, el cambio fenotípico deri- en los genes que codifican p r o t e í n a s causan cambios en
vado de una mutación puntual depende de d ó n d e ocurra las proteínas. Esto se ilustra en la Figura 10.3, que mues-
exactamente la mutación en el gen, qué cambio de nucleó- tra varios resultados posibles cuando el D N A que codifi-
completo tido tuvo lugar, y q u é producto codifica dicho gen. ca exclusivamente un codón de tirosina sufre una mutación.
Como se indica, u n cambio en el R N A de U A C a U A U no
Sustituciones d e p a r e s d e b a s e s tendría efecto porque U A U es t a m b i é n u n c o d ó n de tiro-
Si dentro de la región codificante de un gen que codifica sina. Las mutaciones que originan este tipo de cambio se
para una proteína ocurre una mutación puntual, cualquier denominan mutaciones silenciosas. N ó t e s e que la muta-
cambio en el fenotipo de la célula es probablemente conse- ción silenciosa casi siempre ocurre en la tercera base del
cuencia de un cambio en la secuencia de la proteína produ- c o d ó n (arginina y leucina pueden tener t a m b i é n muta-
É^íM Figura 10.3 muestra diversas sustituciones de pares ciones silenciosas en la primera posición). Como se v i o
en el Capítulo 7 (véase Tabla 7.3), cambios en la primera o
segunda base del triplete conducen con mucha m á s fre-
cuencia a cambios significativos en la pro teína. Por ejem-
plo, u n simple cambio de base de U A C a A A C (Figura
10.3) ocasiona u n cambio en la proteína de tirosina a as-
parragina. Esto se conoce como una m u t a c i ó n con senti-
Replicacion do e r r ó n e o , porque el «sentido» q u í m i c o (secuencia de
normal aminoácidos) del p o l i p é p t i d o resultante ha cambiado. Si
el cambio ocurriera en una posición crítica en ia caderi^á
polipeptídica, la proteína p o d r í a ser inactiva o tener acti-
vidad reducida. Sin embargo, no todas las mutaciones que
ocasionan la sustitución de u n a m i n o á c i d o por otro con-
TAG TAT TAC ducen necesariamente a p r o t e í n a s no funcionales. El re-
ATC ATA ATG
sultado depende del lugar de la cadena polipeptídica en
el que ha ocurrido la sustitución y cómo afecta al plega-
miento y a la actividad catalítica de la proteína. Una m u -
Transcripción tación con sentido erróneo puede conducir a una enzima
que es sensible a la temperatura. Por ejemplo, se conocen
mutantes bacterianos sensibles a la temperatura que
AAG UAG UAU UAC funcionan norpialmente a 30°C pero no pueden crecer a
Codón de C o d ó n de C o d ó n de C o d ó n de 40°C, aunque el tipo silvestre crezca bien a las dos tem-
«sparagina terminación tirosina tirosina
íí-tados por el m*- peraturas. Tales sustituciones se denominan también con-
3l¡cada están mar dicionalmente letales porque la bacteria no puede crecer
lias marcabas cor bajo una condición pero puede hacerlo bajo otra. Los fe
Traducción
notipos sensibles a la temperatura ocurren frecuentemente
V V V porque la proteína m u í a n t e puede mantener su confor-
mación correcta a la temperatura m á s baja pero se des-
teína Proteína Proteína Proteína pliega parcialmente (desnaturaliza) a la temperatura m á s
i mutación *^^8ctuosa incompleta normal normal alta.

urgen en las<^ Mutación Mutación Tipo Otro resultado posible de la sustitución de pares de ba-
? bases del 5* cambio sin silenciosa silvestre ses es la formación de un codón de parada, causaría la ter
asentido sentido minación prematura de la traducción, originando una
:hos casos, 1^'
y en el organi-*-. pro teína incompleta que, casi seguro, no sería funcional (Fi-
mbios son p^^", Posibles efectos de sustituciones de pares de bases gura 10.3). Las mutaciones de este tipo se llaman mutacio-
Tibien cambii^] gen que codifica una proteína: tres proteínas diferentes origina- nes sin sentido porque el cambio es de un codón para un
^ cambios en el DNA de un único codón del DNA. aminoácido (codón con sentido) a un codón de terminación
 MmOBÍAí
WNETICS\^n

Phage DNA
viruses
of pro-
Neck
Protein coat
rium, it
. At the
/sis and Bacterial
chromosome
er other
)me and Phage Bacterial Donor Ceil
;re for a
ive pro- A phage invades the Phage DNA and
e condi- donor bacterial cell proteins are made
ains in a
i phage,
ach, and

/sogenic
with the Assembly of
random transducing
Bacterial chromosome virus
V phages Bacterium 2 is broken into pieces
mtaining is transduced
len these
i A to the
Bacterial DNA
NA from
be trans- Phage DNA
also the
Donor DNA Cell lysis and
ransduc- integrales into phage reléase
*Vhen the host chromosome
jr, it may
percent).
Infection of
;is of new new host cell
ed. When Bacterium 2
rial genes
Generalized transduction involving a bacterial virus
md donor (bacteriophage) and a donor bacterium.
Lce the re-
rare event • Figure 18 •
.0 not eas-

:K REVIEW MICROBIOLOGY
 2. El organismo debe aislarse en cultivos puros, aparte de estar presente en el cuerpo del
animal.
3. Tales cultivos, cuando son inoculados en animales susceptibles, deben desencadenar los
síntomas característicos de la enfermedad.
4_ El organismo debe ser reaislado de estos animales experimentales y cultivado
nuevamente en el laboratorio, después de lo cual dtbQ seguir siendo el mismo.

Los postulados de Koch no sólo proporcionaron un medio para mostrar que organismos
específicos producen enfermedades especificas, sino que también dio un tremendo ímpetu
al desarrollo de la ciehda de la microbiología recalcando la importancia del empleo de los
cuJtivos de laboratorio.

Quimiotet-ápicos.

A la ve2 que se aislaban eñ rápida sucesión los agentes causales de las enfermedades
infecciosas,-se descubrían ¡os mecanismog de la inmunidad y los métodos para combatirlas,
como las vacunas, los sueros antitóíucos y los quimioterápicos.

Aunque e! empleo de sustancias químicas para el tratamiento de las enfermedades

infecciosas se inició hace ya siglos por métodos empíricos, el verdadero fiindador de la
quimioterapia fije Paul Ehrlich, en Alemania, mientras estudiaba medicina en 1870. Ehrlich
introdujo colorantes que todavía se usan en histología para la tinción selectiva de los
componentes basófílos y acidófilos celulares y posteriomiente formuló la teoría de la
"cadena lateral" para explicar las interacciones extraordinariamente específicas de los
anticuerpos y tos antígenos. Ehrlich, apoyándose en esta teoria y en la acción de los
colorantes, consideró que cualquier sustancia tóxica, para poder ejercer su acción, debía
íljaise químicamente sobre la materia viva, ya fijeran células del organismo o agentes
patógenos. Esto íe impulsó a buscar sustancias que presentaran capacidad de fijación
selectiva, es decir, que siendo capaces de fijarse sobre los patógenos no mvieran afinidad
por las células del huésped., Después de un gran número de ensayos llegó el descubrimiento
de un arsenical, el Salvarsan, que ñie utilizado durante mucho tiempo en el tratamiento de
la sífilis. Este descubri.miento fue seguido por el de otros quimioterápicos, arsenicales
orgánicos, anfimoniales y antipalúdicos de síntesis, hasta que, en 1935, Domagk, en
Alemania desarrollo un colorante, el Prontosil, cuyo núcleo activo, la p-
aminobencenosulfonamida, por sustitución de diversos radicales ha dado lugar al
aislamiento de gran número de compuestos, que se conocen con el nombre genérico de
sulfamidas. Por otra parte, en 1929, Fleming realizó la observación de un fenómeno de
antagonism^o bacíenano entre un hongo [Penicillium notatum) y los estafilococos: al
contaminar espontáneamente las esporas del hongo, vehiculadas por ei aire, una placa de un
cultivo de estafilococos, habían inhibido el desanollo de estos organismos. Esta
observación fundamental determinó que, en 1940, Florey y Chain aislaran el xíúmtí
antibiótico conocido, la penicilina, v 'que en años más tarde por el estudio de uk
actinomiceto del suelo {Streptomices griseus) se llegará al descubrimiento de la
estreptomicina por Waksmann. En las décadas siguientes, la investigación exhaustiva de
microorganismos del suelo ha permitido el aislamiento de varios centenares de antibióticos,
de los cuales alrededor de cien se han demostrado de utilidad clínica.''^"'*

IL-ANTEVnCROBÍANOS

Los compuestos químicos con acción antimicrobiana pueden ser clasificados en dos
grandes grupos, antibióticos y quimioterápicos. El término "antibiótico" se utiliza siempre
para conceptuar todas las sustancias orgánicas producidas por microorganismos que fueran
capaces de actuar sobre otros microorganismos inhibiendo su crecimiento o destmyéndolos,
mientras que el término "quimioterápico" se utiliza para aquellas sustancias obtenidas por
síntesis química, aunque la tendencia actual es la de agmparlos bajo el título de "agentes
antimicrobianos". Para que una sustancia pueda ser considerada como "antibiótico" debe
cumplir, de acuerdo con Waksman y cols.(1941), las siguientes condiciones:

i) Especificidad: Se traduce en el espectro de acción del antibiótico, consistente en su

actuación frente a un gmpo detemiinado y limitado de microorganismos. Este
comportamiento se debe a que los antibióticos actúan en un lugar anatómico concreto de la
bacteria, que es específico de cada antibiótico.

Unos se fijan en una molécula y bloquean su acción, y otros alteran una línea metabólica
ñindaniental para la bacteria. Es el sitio de acción, o "blanco molecular" , del antibiótico,
cuyo bloqueo o modificación se traduce en cambios metabólicos trascendentes para la vida
del microorganismo.''^

ii) Elevada potencia biológica: Es decir, que sea activo a pequeñas concentraciones;
actividad que suele expresarse como concentración inliibitoria mínima (CIM), equivalente a
la cantidad mínima necesaria para impedir el crecimiento de un microorganismo en
condiciones estandarizadas. Para la mayoría de los agentes antimicrobianos de eficacia
clínica, las CIM fíente a microorganismos totabiiente sensibles varían entre 100 y 0.01
mg/mL o cifras inferiores, y, para una terapia eficaz, habitualmente parecen requerirse
niveles superiores a la CIM en el lugar donde se produce la infección.''^

iii) Toxicidad selectiva: Un antibiótico ideal presenta toxicidad selectiva. Este término
implica que el medicamento es nocivo para el microorganismo infectante sin serio para el
huésped. En muchos casos, la toxicidad es relativa, lo que significa que un medicamento
puede dañar a un microorganismo a una concentración que el huésped puede tolerar. La
toxicidad selectiva, por lo general depende de la inhibición de fenómenos bioquímicos que
se presentan en o que son esenciales en el microorganismo, pero no para el huésped.*^

-7-

1
1 i
 1. Clíisiñcación de los antlmicrobhmos.

Los criterios de claiificación de los antimicrobianos son diversos,, lo que origina varías
claves que han permitido agiiiparlos seg-úii su origen, estnictura química y actividad.

/j/'a»-i w or/gen, se pueden dividir en: , : . ,

a) Biológicos. Son producidos por microorganismos, lo que permite subdividirlos a su

vez en: antimicrobianos elaborados por bacterias típicas (p. ej. poíimixinas por Bacilhts
polimixa), actiiiomicetos (p.ej. cloranfenicol por S. venezvelae) y hongos (p.ej. penicilina
•por Penicillium ¡íotatum). ' ,

b) Sintéticos. Son producidos exclusivamente por síntesis química. Son ejemplos de

ellos los nitrofuranos y las sulfamidas.

c) Sem i sintéticos. En ia aciisalidad constituyen el giiipo más numeroso e irnportarite.

Scbre el núcleo básico del antibiótico, producido por el microorganismo, se engarzan en la
posición más adecuada radicales obtenidos por síntesis, confiriendo: mejora del espectro,
de ía fennacocinética, disminución de la toxicidad, etc.''"^'^

ii) Por su espectro de acción. El rango de actividad de un antimicrobiano se denomina

espectro, ténnino utilizado para describir los géneros y especies frente a los cuales ha
demostrado ser activo. De acuerdo a esta característica pueden clasificarse en:

a) De amplio espectro, como el cloranfenicol y las íetiaciclinas, que interfieren en el

crecimiento de numerosas especies bacterianas.

b) De espectro intennedio, que actúan frente a un número más limitado de especies (p.tíj.
penicilinas, macrólidos).

c) De especíTO -'educido, que, como las poíiinixinas, sólo tienen un compoiiamieriío añc^z
freiiíc a un número limitado de especies.^

iii) Per su forma de acción, piiQÚendiviünsQ CYi:

a) ,Bacterío3íáticos: que bloquean el desarrollo y multiplicación de las bacterias, poro no

las lísan, por lo que al retirar el antimicrobiano su efecto es reversible.

b) Bactericida: que provocan la muerte bacteriana, con lo que el ;:receso es

irreversible.'*'^''^

ivj Por el mecanismo de acción. Para la m.ayor parte de los antibióticos, no se

comprenden por completo sus mecanisraos de acción. No obstante, para ñnes de discusión,
es conveniente presentar los mecanismos bajo cuatro diferentes encabezados:

-8- • •

%
 a) Inhibición de la síntesis de la pared celular.

b) Alteración de la permeabilidad de la membrana celular o el transporte activo a través

de la misma.

c) Inhibición de la síntesis de proteínas (es decir, la inhibición de la traducción y

transcripción del material genético).

d) Inhibición de la síntesis de ácido nucleico.

v) Por la estructura química. Es la más utilizada y puede intioducir otros parámetros para
una correcta adecuación clínico-microbiológica. Sobre la base de su estnictura y
composición, los antimicrobianos se agrupan en familias que tienen caracteristicas comunes
no sólo químicas, sino también mecanismo de acción y espectro semejantes. Dentro de cada
familia pueden separarse subgrupos por matices, como espectro de acción, y estabilidad
enzirnática, diferenciándose en el grupo sobre todo con base en la tolerancia,
farmacocinética., etc. Las familias y grupos más importantes son:''*

I. {3-lactámicos
Penicilinas
Cefalosporinas
Cefamdcinas
Oxacefeminas
Clavaminas
Carbapeneminas
Monobactámicos
Etc.
n. AmÍDOcicIítoles
Espectinomicina
Aminoglicósidos
ffl.
IV.
Folipeptidleos
Tetracscünas
V. Cíoraafenicoi y derivados
VT. Macrólidos
VIL Lineosa midas
yin. Rifamicíoas
ÍX. Fosfomicioa
X. Siílfamidas
XI. Nitrofurantoína
XII. Quinoionas
XIÍI. Derivados nitroheterocíclicos
XIV. AníifÜQgicos
 2. Mecanismos de acción de los andmicrobianos.

Para que un antimicrobiano ejerza su acción, es necesario que üegiae al foco infeccioso,
penetre en las bacterias y alcance íntracelulamiente la concentración necesaria. La entrada
en la bacteria se puede lograr por diñisión o transporte activo. En el aspecto molecular, los
antimicrobianos de uso en clínica pueden ejercer su acción en las siguientes estructuras o
funciones:

a) Acción anÉibiótica por inhibición de la síntesis de la pareJ celular (Bscitracina,

cefalosporiíias, cicloseriaa, peaieillnas, vaiicomieina).

En contraste con las células animales, las bacterias poseen una capa extema rígida
denominada pared celular, la cual rodea por completo la membrana celular citoplásmica.
Mantiene la forma del microorganismo y "contiene" a la célula bacteriana, que poses una
presión osmótica interna inusualmente alta En las bacterias gramnegativas, la paite rnás
extema de la pared celular está fomiada por una bicapa de lípidos llamada membr¿ina
extema. La presión iníema es 3 a 5 veces mayor en las bacterias gramposiíivas que en las
bacterias gramnegativas. La pared celular contiene un polímero compiejo entrelazado,
químicamente diferenciable, denominado peptidoglucano, compuesto de polisacáxidos y
péptidos. Por lo general los polisacáridos se componen de los aminoácidos N -
aceíilglucosamiaa y ácido acetiímurámico; este último sólo se localiza en las bacterias. A
los aminoaziicares se les unen pequeñas cadenas peptídicas. La rigidez fínal de ia pared
celular dependerá del entrelazamiento de las cadenas pepíídíc-as como resultado de las
reacciones de transpeptidación llevadas a cabo por vaiias enzimas. La capa de
peptidoglucaiios es mucho más gruesa en la pared celular de las bacterias grampositivas
que en la de las gramnegativas.''''^

i) Inhibición de la transpeptidación (beta-lacíámicos)

Todas las penicilinas y cefalosporinas (antibióticos beta lactámicos) son írjjv.bidoies

selectivos de la sí:ilesi¿ de la pared celular bacteriana. Aunque v.'sta es sólo -^¡la de las
diversas actividades de estos medicamentos, quizá es la mejor coinprcndida. El paso Miic'al
de la acción del fármaco consiste en su fijación a ciertos receptores celulares, llamados
proteínas iljadoras de penicilina (PBP, del inglés pemalh.n-binümg-proteins), los Ci!;?Jes,
llegan a ser de 3 a 6 en muchas bacterias. Distintos receptores (PBP) pueden poseer
diferentes afinidades para un medicamento, y cada uno puede mediar im tipo distinto de
acción.

- 10-
 Las PBP se encuentran bajo control microsómico, y las mutaciones pueden alterar su
número y afinidades por los medicamentos beta lactámicos especifícos. Después de que un
beta lactámico se ha unido a sus receptores, se inhibe la reacción de transpeptidación y se
bloquea la síntesis de peptidoglucanos. El siguiente paso probablemente implica la
eliminación o inactivación de un inhibidor de las enzimas autolííicas (hidrolasas) en la
pared celular. Esto activa a las enzimas líticas en algunos microorganismos y puede causar
la lisis si el ambiente es isotónico. En un ambiente hipertónico (por ejemplo sacarosa al
20%), los microbios pueden cambiar a protoplastos o esferobíastos cubiertos sólo por una
fi-ágil membrana celular.

La inhibición de las enzimas de transpeptidación por las penicilinas y cefalosporinas

puede deberse a una similitud estructural de estos medicamentos a la acil-D-alanil-D-
alanina. La notable falta de toxicidad de las antibióticos beta-Iactámicos para las células de
los mamíferos se atribuye a la ausencia de ima pared celular de peptidiglucano en estás.

ii) Inhibición de la síntesis precursora de peptidoglucanos. „

Otros medicameiitos, inhiben los primeros pasos de la biosíníesis de los peptidoglucanos.

Puesto que los primeros pasos de la síntesis tienen lugar dentro de la membrana
citoplásmica, estos medicamentos deben penetrar la membrana para ser eficaces. Ejemplos
de éstos son:

- Bacitracina: Inhibe ía hidrólisis o desfosforilación del lípido pirófosfato, así éste no

puede reutilizarse de nuevo en el transporte de polímeros lineales de disacárido-
pentapéptido a través de la membrana citoplásmica. .

- Vancomicina: Impide la transferencia del disacárido pentapéptido, i.nido al lípido

portador de la membrana citoplásmica, al aceptor de la pared celular. Esto se consigue
uniéndose a la D-alanil-D-alanina terminal del pentapéptido disacárido, ío que impide la
actuación del peptidoglicanosintetasa. De fonr^a similar actúa la listocetina.

- Fosfomicina; Interfiere la condensación de la UDP-GlcNAc con el fosfoenol-pimvato

para foimar el éter enólico del UDP-GlcNAc-pimvato. Esta reacción es inhibida porque la
fosfomicina se une covalentemente con la transferasa que la cataliza, a causa de su analogía
estructural con el fosfoenol-pimvato. ,

- Cicloserina, un análogo de la D-alanina, bloquea la acción de la alanina racemasa, una

enzima esencial en la incorporación de la D-alanina en el pentapéptido del
peptidoglucano. ''^'^
 b) Acción aüíibiótica por inhibición de ía función de ía membrana celular (poüenos y
poíimixinas).

La membrana cehilar o citoplásmica es vital para todas las células, ya que entre sus
propiedades incluye ei actuar como b.anera de permeabi'iiJad selectiva, controlando de esta
forrna la composición del medio interno celulai'. Las sustancias que alteran este estructura
modifican la permeabilidad, permiten la .iabda de iones K^""^ y müxrromoléculas, como los
ácidos nucleicos y causan un efecto lítico.

Las poíimixinas se comportan corno detergentes catiómcos. Son polip-áptidos con un

extremo liposoíuble y otro hidrosoluble. El primero se une a los fosfolípidos de ía
niembraiia c:í-jplásmica bacíeriaiia y el segundo penetra en la pane bJdtonlica. De esta
fonna se dc¿orgam"za esta esímctura y aumenta su penneabilidad, con la pérdida de
metaboliíos esenciales y miienc bacteriana como resultado ílnal. Las bacterias más
susceptibles son las que tienen en su membrana mayor cantidad de fosfolípidos
(giamnegativas). La insensibilidad o resistencia está en relación con la impermeabilidad de
la pared celular para estos fámiacos, como es el caso de las grampositivas que tienen una
pared celular muy gruesa.

Los antibióticos poliénicos (nistatina y anfotericina B) son activos frente a hongos, poseen
una parte de su molécula con numerosos enlaces (pclieno) hidrofóbica y, además, otra
hidroíilica. La porción hidrofóbica se une con los esteróles de la membrana celular de los
hongos, introduciendo a su vez la parte hidrofílica. De esta forma no sólo se altera la forma
de la rnenibrana, sino que, además, se forman poros hidrófílicos y se rnodiñca la
penneabilidad íiormal de esta esímctura.

Todos estos antibióticos son Uticos, incluso en bacterias en reposo, y tienen cierto
poíencial tóxico, especialmcuíe la anfotericina B, ya que sc*ñ capaces de unirse con los
lípdos de las membranas ciíopiásmicas de las células de los marniisros.'"''''^

c) Accióa afiíibióífCíí por infeibiclóa de la síntesis de proísíftas ( AmísioglucósMos,

íeíi'aciclinas, macrólidos [eritromicmas], cioramfeiikol, ímcoMÍcíJias).

Se ha establecido que los arninoglucósidos, íetraciclinas, cloranrifenicol, macrólidos y

lincomicinas pueden inliibir la síntesis de proteínas medianíe la acción sobre los ribosomias
en las bacterias.

Las bacterias tienen ribosomas 70S, en tanto que las células de mamíferos tienen
ribosomas SOS. Las submiidades de cada tipo de ribosoma, su composición química y LUS
especificidades funcionales son lo suficientemente diferentes para explicar por qué los
antibióticos pueden inhibir la síntesis de proteínas en los ribosom.as bacterianos, sin tener
raayor efecto sobre los ribosomas de ios mamíferos.
 En la síntesis normal de proteínas microbianas el mensaje del mRNA es leído, de manera
simultánea, por varios nbosomas que se extienden a lo largo de la cadena de mRNA. estos
son los llamados polisomas.

i) Aminoglucósidos. -

De hecho, se ha estudiado más el modo de acción de la estreptomicina que de cualquier

otro aminoglucósido (Kanamicina, neomicina, gentamicina, neíilmicina, amikacina, etc),
pero probablemente todos actúan de manera similar. El primer paso es la fijación del
aminoglucósido a una proteína receptora específica ( P 12 en el caso de la estreptomicina)
en la subunidad SOS del ribosoma 70S microbiano. Segundo, los aminoglucósidos bloquean
la actividad normal del complejo de iniciación para la formación de péptidos (mRNA +
formil metionina + tRNA). Tercero, el mensaje del mRNA es mal leído en la región de
reconocimiento del ribosoma y, como resultado, el aminoácido erróneo se inserta en el
péptido, originando una proteína no funcional. Cuarto, la fijación a los aminoglucósidos
causa la degradación de los polisomas y su separación en monosomas incapaces de llevar a
cabo una síntesis de proteínas. Estas acciones se presentan más o menos de manera
simultánea y el efecto global suele ser irreversible, causando la muerte de la célula.

ii) Tetraciclinas. -; ' . .

Las tetraciclinas se fijan a la subunidad SOS de los ribosomas microbianos. Éstas inhiben
la síntesis de proteínas mediante el bloqueo de la fijación del aminoacil-tRNA cargado y
así, evitan la introducción de nuevos aminoácidos en la naciente cadena pepíídíca. La
acción suele ser bacíeriostática y reversible al suspender el medicamento.

iii) Cloramfenicol.

El cloramfenicol se fija a la subunidad SOS del ribosoma, e interfiere con la fijación de los
nuevos aminoácidos a la naciente cadena peptídica, debido en gran parte, a que el
cloramfenicol inliibe la pepíidiltransferasa. El cloramfenicol es principalmente
bacterosíático, y el desarrollo de los microorganismos se reanuda cuando se suspende el
medicamento.

iv) Macrólidos (eriíromicinas).

Estos medicamentos se fijan a la subunidad 50S de los ribosomas y pueden competir con
las lincomicinas por los sitios de fijación (un rRÍ>lA 2SS). Los macrólidos pueden interferir
con la formación de complejos de inicio para la síntesis de cadenas de péptidos o con las
reacciones de aminoacil translocación.

-13-
 v) Lincomicinas (elindamicma).

Las linGomidnns ^.e \m\i a 1» jbi'/iiátd 50S dei nbosor.hi /riíj'-r'-'-'ioo y se coiiriuide'i cc-n
los macrólidos í;n el siíio de i'j:ición, ¿-cíiv'A'.-sd aníibcctc-.raLa y Í . - . C . ' O de acción/

d) Áccic-n anobióüea por ttíhibiciósj de la s'ííilesis de í ü d c j nucleicos (Qtt;kio.ítHias,

!i-iíampí«Ha, siiiíbnaeildas, ínmetoprísu).

Todas las .juino'onss y las f.'üoroquinolonas '-on pot.íníer, inhibidores de la síntesis de

reídos nucleicos. Hloquc?.! h acción de la IDNA ^ira^a ('.opoí^^cmerasa 11), ia enzima
.-ausaníe dei empacanui-r.n:; y dcscmpacamicnto de la ¿.'ipcr-r'^pira! -^e OKA.

La rifampicioa inhibe el desarollo bacteriano mediante sa fuerte fijación a la RNA

polímerasa dependiente de DNx\e las bacterias, así, inhibe la síntesis del RNA bacteriano.
La resistencia a este fármaco se desarroíia como una mutación cromosómica áe alta
frecuencia que resulta en un cambio en la RNA polímerasa.

Para muchos microorganismos, ei ácido p-aminobenzóico (dei inglés p-aniinobcnzoic

pcid, P.ABA) cortsíimye un rnetabolito esencial. Es utilizado por éstos como un precursor
para la síntesis de ácido fólico en la vía que lleva a la síntesis de ácidos nucleicos. Es
probable que el modo de acción especifico del PÁBA implique una condensación de
píurídina con ei PABA, dependiente de trifosfato de adenosina (ATP), para producir ácido
difiidfopteróico, que p»osíerionríente se convieríe en ácido fólico. Las sulfonamidas son
análogos esiiucLTiaies dei PABA e inhiben la dehidropteroato sintetasa.

Las süifonamidas pueden entrar en la reacción en lugar del PÁBA y compedx por el sitio
activo de la encima. Como resultado, se forman análogos no ñmcionales de ácido fólico,
svitaíído el desaxToiiO posterior de las céhilas bacterianas. La acción inhibidora de las
•íulfonarnidas en eí desarrollo bacteriano puede ser contrarrestíids por exceso de PABA
fin el ambiente (inliibición ccínpetitiva). Las células animales no pueden sintetizar ácido
fólico y deben átpmáüí: de fuentes exógenas; por tanto, son resistentes a la acción de las
ru}fonamida.s.

E! ínraetoprira ijihibe a la ácido díhidrofólico leductasa de las bacterias 50,000 veces más
rápido que la misma enzima de los mamíferos. Esta enzima reduce el ácido dehidrofólico
en tetrahidrofólico, una- eíapa que lleva a la síntesis de purinas y por último de DNA. Las
sulfonamidas y el '-n'rocteprirn producen un bloqueo seci'cncial ¡"'e c.^sta vía, ;c que resulta an
una paícnciGción ;-.üL'íb!>=^ (sinergisrno) de aciividad.''''''''^

. 14-
 <>ir\em<Kn
que las directivas publicadas noes-
i esta bacteria.'-" Estudios posterio-
ístabiccer directivas válidas paralas
, y los resultados de las encuestas
as que tienen sensibilidad predeci- ,
í haciendi^ cada \ez más corta. La
i de Huíiuoplnliis influenzae y d2 ,
'ae productoras de p-iactamasa es '
ta como para que \ no pueda pre-
id a los análogos de la penicilina.'"
ilivamente resistentes a la penicili-
iL a 1,0 Liij/mL) o que tienen resis-
jCl.M > 1.0 ug/mL) son cada vez
« Estados Luidos, y las cepas de
de infecciiMies graves deben ser
de resistencia."'" Prácticamente,
\ogcnes aun es susceptible a la pe-
^ bados los aislamientos que produ-
. is bacterias recuperadas de un lí-
I n condiciones normales es estéril
Si la bacteria potencialmente pa-
ma kicali/.ición que contiene flo-
el tracto respiratorio superior o la
ir evaluado con ma\r detalle an-
leba de sensibilidad, en particular
is especies de microorganismos,
frotis teñido con Gram puede de-
: células epiteliales escamosas, lo
ación con secreciones coloniza-
eutrófilos segmentados, que indi-
lesta intlamatoria. E n situaciones
logo no puede determinar la con-
;una prueba de sensibilidad, es
ion el clínico.
EBAS
casos, para orientar el tratamien-
s una prueba de difusión con dis-
pon en caldo. Las ventajas de las
que proporcionan más informa-
ien ser aplicadas a un rango más
que las pruebas por difusión. Sin
go detlnkio de mala interpreta-
rititativo si los médicos no saben
el laboratorio no proporciona la
ira la interpretaci(')n. Las pruebas
tienen un historial largo y exito-
do a usar ilependera de las nece-
s. Para la evaluación de las prue-
ayoría de los investigadores uti-
nciones:
:: paracteri/ación de un aisla-
) susceptible;
Icteri/ación de un aislamiento
tente:
ización de un aislamiento sus-
Dmo intermedio, o caracteriza-
intcrmetlio como susceptible o
SéLECCIÓN DE LOS A G E N T E S ANTIMICROBIANOS
La selección final de antibióticos para el vademécum
hospitalario debe ser decidida por consulta con los
miembros del equipo médico. Las listas de agentes anti-
microbianos .sugeridas por el .N'ational Committec tor
Clinical Laboratorv Standard-^ ( N C C L S ) se detalla en los*
cuadros 15-9 y 1.5-10.*
Nótese que muchos antibióticos se agrupan en clases
debido a que su espectro de actividad es similar. La-- re-
comendaciones recientes del NCCL.S se ocupan de las
pruebas selecti\as y de los informes s e l e c t Í M ) s . recono-
ciendo que estos temas sori complejos y \arían de i i i s i i -
tución a institución. Washington ha proporcionado LIIKI
guía para la agrupación de antimicrobianos ¡luadro
15-11).™ No es necesario probar cada antibiótico de la
lista. Deben conocerse los patrones de uso de aniibuni-
cos y de resistencia bacteriana de cada comunidad \-
ben ser tomados en cuenta cuando se eligen los aniibui-
ticos a probar. Es preciso hacer diferencias entre los an-
tibióticos que se prueban en forma habitual y aquellos
cuyos resultadtis son comuiiicados con a s i d u i d a d a los
médicos.
ESTANDARIZACIÓN
El mayor adelanto en la orientacii'ín proporcíonad.i por
ellaboratorio acerca de las pruebas de susceptibilidad en
las últimas décadas proviene del desarrollo de p r o c e d i -
mientos estandarizados ampliamente adoptados. l[s en
extremo importante seguir los protocolos recomendados
en forma estricta, si se de.sea obtener resultados r e p r o d u -
cibles. E l N C C L S publica continuamente estándares pa-
ra ésta y otras prueb-as. (Pueden obtenerse copias del Per-
formance Standanls for Aiuimicrohícil Disk Susce¡)¡ihi-
lity Tests^^- y del Methods for Dilntion Aniiniiimhidl
Susceptibility Tests for Bacteria that Grow Aeroh'icu¡l\''''
solicitándolos por escrito'al N C C L S . 940 West \ a l l e y
Road, Suite 14(K). Wayne, PA 19087-1 SóS.i Es importan'-
teque se establezcan con rapidez procedimientt>s re\-
dos y recoinendaciones actualizadas en todos los labora-
torios clínicos. La membrcsía de la insiitucicHi en la
NCCLS asegura la recepción en término de todas l a s re-
comendaciones nuevas y revisadas. Washington ha resu-
mido los recursos y procesos de esta organización nacio-
nal por consenso que interesa a los microbiólocos e l í n i -
COS.2^0
Los siguientes son algunos de los aspectos importan-
tes de las pniebas de sensibilidad a antibióticos qiie han
sido estandarizados.
* E l N C C L S ha otorgado la a u t o r i z a c i ó n para us.ir parte del M
(Performance Slandards for .•\ntiniicrobial Susccptibilii) T C S I Í H L ' Sixih
Informational Supplcment). E l M l l { > - S 6 a c l u a i i / a c l .Ñ12-.-\ d V r f o r -
inance Standards for .AnliiTiicrobijl Susceptibilitx Testing - Filth E d i -
tion; Approved Standard) y c l M7-.-\ (Methods for Dilutior. Aiitimi-
crobial Susceptibility Tests for Baeteiia that G r o u .Verobicali)
Edition; ApproNcd Standard). L o s datos de interpretación s(ihi son vá-
lidos S I se sigue la m e t o d o l o g í a descrita en el M2-.\.*; > el
N C C L S actualiza con frecuencia las tablas .M2 > M 7 iiK-d...:in: nuevas
ediciones de e s t á n d a r e s > suplementos. L o s usuariií> d c K - i . ..insultar
las ediciones mas recientes. L o s ev'.ir.darev .ictu.ilc- ixi^den ^. i i'iMcni-
cos en el NCCLS. 440 West V . I I I C N R«...!. S : -e U O O . N^.-.
19087, E E . I T - .
G r u p e o 26oí
P R I H B A S D E S E N S I B I L I D A D A A G E N T E S A.NTIMICROBIA.NOS 785
MEDIO DE CRECIMIENTO '
Se ha elegido el medio de Mueller-Hinton, caldo y
agar. para las pruebas de aislamientos bacterianos aero-
bios y anaerobios facultativos. Estas fórmulas son las que
más se aproximan a los criterios de un medio reproduci-
ble. Contienen infusión de carne deshidratada, digerido
ácido de caseína y almidón de maíz. L a mayoría de los
patógenos se desarrolla de manera satisfactoria, y el me-
dio posee propiedades inhibitorias mínimas para las sól-
fonamidas. trimetoprima y tetraciclina." En algunos lo-
tes de medio se encuentran grandes cantidades de timidi-
na. .Algunos microorganismos pueden utilizar la timidina
para evitar el mecanismo de a'-ción de la trimetoprima y
desarrollarse, aun cuando sean intrínsicamente sensibles
al antibiótico. Este mecanismo afecta en especial a los
enteriK'ocos; pueden aparecer colonias aisladas dentro
del halo de inhibición alrededor de los discos que contie-
nen irimetoprima.
Es evidente, por el examen de la fórmula, que incluso
el medio de .Mueller-Hinton no está químicamente defi-
nido. " E l agar es un compuesto natural que se prepara a
partir de algas rojas. Hay \ariaciones en la composición
del agar de diferentes fabricantes, incluso entre lotes pro-
ducidos p(>r ia misma empresa, segiín el origen de las al-
gas. En el presente continiian los intentos para minimizar
las variaciones entre lotes de agar de .Mueller-Hinton. Un
estándar de referencia para los fabricantes pcxlría propor-
cionar una inayor reproducibilidad.-'- E l N C C L S reco-
mienda, para uso de rutina, un medio especialmente for-
muladt) para probar Haemophilus influenzae.'^*' Este me-
dio de prueba para Haemophilus también puede usarse
para otras especies de este género, como se descnbe más
adelante.'-"'
pH 9
El pH del medio debe estar entre 7.2 y 7,4 a tempera-
tura ambiente. E l pH de los medios líquidos puede ser
comprobado directamente con electrodos para pH, y los
medios sóiidos pueden probarse macerando una cantidad
suficiente de agar de ft)rma que el extremo del electnxio
pueda sumergirse, para permitir que cierta cantidad de
agar se solidifique alrededor del electrodo, o mediante un
electrodo de superficie calibrado de manera adecuada.
SUERO
lx3s antibióticos difieren en gran medida en la intensi-
dad de su unión a las proteínas del torrente circulatorio,
para equilibrar el antibiótico libre con el que se enoocÍD-
ira unido a las proteínas del suero. E l antibiótico lit«e y
unido puede medirse, pero no está claro cuál es el resul-
tado más útil. En el laboratorio pueden obtenerse dife-
rentes valores para antibióticos de alta afinidad por pro-
teínas, si se agrega suero en el medio. E l método del
N C C L S no incluye el agregado de suero debido a las di-
ficultades de estandarización del producto y la falta de
Third ceríeza acerca de c ó m o interpretar los resultados. Perl y
col. estudiaron el efecto del suero sobre 1 1 antibióticos
A.V E l
de amplio espectro utilizados para tratar infecciones no-
socomiales producidas por bacilos gramnegativos.^ Los
resultados fueron idénticos con 9 de los 1 1 antibióticos.
P.\
Sólo en el caso de la ceftriaxona O 95% de imión apro-