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Algorithms for Identification of Aerobic

Gram-Negative Bacteria*
PAUL C. SCHRECKENBERGER AND DAVID LINDQUIST

24
These algorithms are meant to assist in the identification of observed by preparing a wet preparation from a young
organisms that are not readily identified by methods in place colony on a BAP. Decarboxylase reactions are determined
in most clinical laboratories. Microbiologists planning to by using an extremely turbid inoculum in Moeller’s media
identify an unknown gram-negative rod begin with colonies (heavier than usual inoculum). Polymyxin B sensitivity is
on an agar plate. Our definition of “good growth on blood indicated by any zone of inhibition surrounding a 300-U disk
agar plate (BAP)” is the presence of distinct colonies on a BAP. For glucose-nonfermenting rods and other fastid-
(approximately 1 mm) on Trypticase soy agar with 5% sheep ious organisms, the indole test is performed using the Ehrlich’s
blood after 24 h of incubation at 35°C in room atmosphere. extraction method (see chapter 50). “Esculin” refers to hydrol-
Poor growth indicates that more than 24 h of incubation is ysis of esculin in media without bile.
necessary for the development of distinct colonies. If an These algorithms are dichotomous, since many organisms
organism fails to grow on BAP after 72 h, it is considered to may fall into more than one group due to phenotypic vari-
show “no growth.” Morphological and phenotypic criteria ability of a given trait. The presence of two or more atypical
were chosen not only for their discriminatory value but also traits or a major variation from the ideal phenotype depicted
because the methods are available in most laboratories. in an algorithm, due to antibiotic use, auxotrophy, or other
Cellular morphology is determined by using a Gram stain reasons, may limit the algorithm’s utility. These algorithms
from a young colony on a BAP. The description of “tiny coc- are intended as a guide to presumptive identification of
cobacilli” used for Brucella and Francisella in Table 2 implies an unknown isolate. The reference chapter describing the
almost indiscernible cells resembling grains of sand. For organism should be consulted to determine the definitive
many organisms with pleomorphic morphologies, we chose identification. To use the algorithms, start with Table 1 for
to represent the dominant shape. gram-negative bacteria that grow well on blood agar in 24 h
The urea test refers to conventional Christensen’s urea at room atmosphere and Table 2 for fastidious gram-negative
reaction after 24 h of incubation, whereas the rapid urea bacteria. Note that Table 1 consists of three parts, which we
result is read after 4 h. Glucose fermentation refers to an have designated Table 1a, Table 1b, and Table 1c. In each
acid reaction in the butt of a Kligler iron agar (KIA) or triple case begin in the upper left-hand column of the table; if the
sugar iron agar (TSI) tube. “Glucose oxidized” refers to acid test organism matches the given characteristic, then con-
production in the upper portion of oxidative-fermentative tinue horizontally to the right to the next reaction. If the
(OF) media. “BHIserum” refers to brain heart infusion reaction in the box matches your test organism, continue
agar with 10% (vol/vol) serum added. The oxidase test refers moving horizontally until you reach the organisms listed in
to results obtained with the N,N,N,N-tetramethyl-p- the right-hand column. When the reaction in the box does
phenylenediamine dihydrochloride reagent. Motility is best not match your test organism, then move down the column
vertically to find the reaction that matches. Repeat the
process until you reach the right-hand column. Be sure to
* This chapter contains information presented in chapter 23 by Paul C. check all your reactions with the organism characteristics
Schreckenberger and Jane D. Wong in the eighth edition of this Manual. given in the referenced chapter.

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BACTERIOLOGY
TABLE 1a Identification algorithm for gram-negative bacteria that grow well on blood agar (part 1)

Yellow-pigmented colonies

Phenylalanine deaminase
Arginine decarboxylase

Growth on MacConkey
Lactose, trehalose, or
Lysine decarboxylase
Fluorescent pigment
Pigmented colonies
Glucose fermented

Sucrose fermented

Glucose oxidized

xylose fermented
Cell morphology

OF mannitol

Nitrate to gas
Polymyxin B

OF maltose
H2S in TSI

H2S in TSI
6% NaCl
Oxidase

Motility

ONPGa

Esculin
Indole

ONPG
Urea
Organism group (chapter)

Rod  Purple Chromobacterium (40)


Other   Vibrio (47)
 Aeromonas (46), Plesiomonas (45)
 Pasteurella (40), Actinobacillus (40)
 Pasteurella bettyae (40)
 EF4a (40)
Pasteurella avium (40), Actinobacillus actinomycetemcomitans (40)
 Vibrio metschnikovii (47)
  Enterobacteriaceae (42–45)
 Providencia, Morganella (42, 45)
 Edwardsiella (42, 45)
Pasteurella bettyae (40)
P. bettyae (40)
Pink Asaia, Azospirillum, Methylobacterium, Roseomonas (50)
Not pink    Shewanella spp. (50)
 Balneatrix alpica (50)
  Pseudomonas aeruginosa, P. fluorescens, P. putida (48)
 Agrobacterium Yellow Grp, O-1, O-2, Sphingomonas spp. (50)
R   Pseudomonas-like group 2 (50)
Burkholderia cepacia complex (49)
 B. stabilis (49)
 B. pseudomallei (49)
 Ralstonia mannolilytica (49)
Pandoraea spp., Ralstonia spp. (49)
S   Ochrobactrum anthropi, Achromobacter groups B and E (50)
  CDC Vb-3 (48), OFBA-1, Ochrobactrum spp. (50)
Ochrobactrum spp. (50), Acidovorax spp. (49), Pseudomonas-like
group 2 (50)
P. mendocina (48), CDC Ic , Ochrobactrum spp. (50)
   Rhizobium radiobacter (50)
Ochrobactrum anthropi, Achromobacter Group F, Halomonas venusta,
Inquilinus limosus (50)

24. Identification of Aerobic Gram-Negative Bacteria ■


 P. stutzeri (48), Ochrobactrum anthropi (50)
Pseudomonas-like group 2, Herbaspirillum, CDC halophilic
nonfermenter group 1 (50)
 CDC group O-3, Inquilinus limosus, Massilia timonae (50),
Brevundimonas vesicularis (49)
 P. stutzeri (48), Achromobacter xylosoxidans (50)
A. xylosoxidans (50), Brevundimonas diminuta (49)
aONPG, O-nitrophenyl-beta-D-galactopyranoside.

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TABLE 1b Identification algorithm for gram-negative bacteria that grow well on blood agar (part 2)

NO2 reduction
decarboxylase

OF mannitol
Polymyxin B

NO3 to NO2
Curved rods
morphology

H2S in TSI

6.5% NaCl
Rapid urea

Acetamide
Pigmented

pigmented

Growth in
fermented

Arginine
oxidized
Oxidase

colonies
colonies

Glucose
Glucose

Motility

Esculin

Gelatin
Yellow-
Indole

Urea
Cell

BACTERIOLOGY
Organism group (chapter)

Rod Not pink   R Pandoraea spp. (49)


S   Laribacter hongkongensis (50)
P. pseudoalcaligenes, P. alcaligenes, Pseudomonas species group 1 (48)
 Bordetella bronchiseptica, Oligella ureolytica, Cupriavidus pauculus (50)
  Pseudomonas species group 1 (48), Achromobacter denitrificans (50)
Alcaligenes faecalis (50)
  Delftia acidovorans (49)
 Achromobacter piechaudii (50)
Bordetella avium (50, 51)
 CDC halophilic group 1 (50)
 P. pseudoalcaligenes, P. alcaligenes (48), Comamonas terrigena, C. testosteroni
(49), Achromobacter piechaudii, Cupriavidus taiwanensis (50)
Brevundimonas diminuta, Brevundimonas vesicularis (49), Bordetella hinzii
(50, 51), P. alcaligenes (48), Cupriavidus gilardii, Cupriavidus respiraculi (50)
  Chryseobacterium indologenes/gleum, Empedobacter brevis, CDC group IIi (50)
 Bergeyella zoohelcum (50)
  Elizabethkingia meningoseptica (50)
CDC group IIc, IIh, IIi (50)
 Weeksella virosa, Empedobacter brevis (50)
CDC group IIe, IIg (50)
   Sphingobacterium spiritivorum, Inquilinus limosus (50)
  Sphingobacterium thalpophilum (50)
Sphingobacterium multivorum (50)
Sphingomonas paucimobilis, Flavobacterium mizutaii (50)
 EF-4b, Paracoccus yeei, Psychrobacter immobilis (50)
EO-3, EO-4 (50)
  Myroides spp. (50)
Oligella ureolytica (50)
 Alishewanella fetalis (50)
Neisseria weaveri, N. elongata, Gilardi rod group 1 (39, 50)
TABLE 1c Identification algorithm for gram-negative bacteria that grow well on blood agar (part 3)

Glucose oxidized

Brown diffusible

Growth at 42oC
decarboxylase

decarboxylase
or OF lactose

NO2 reduced
NO3 reduced
morphology

OF maltose
Pigmented
fermented

Mannitol
Oxidase
colonies

pigment
Glucose

Motility

Gelatin
Esculin
DNase

Lysine

Lysine
Urea
Cell

Organism group (chapter)

Rod Not pink     Burkholderia cepacia complex (48)


Both negative   Pseudomonas luteola (47)
P. oryzihabitans (47)
Burkholderia gladioli (48)
 Stenotrophomonas maltophilia (48)
Sphingomonas paucimobilis (49)
 Bordetella ansorpii (50, 51)
 Kerstersia gyiorum (50)
Bordetella trematum (50, 51)
 Acinetobacter baumannii, EO-5 (50)
 Bordetella parapertussis (50, 51)

24. Identification of Aerobic Gram-Negative Bacteria ■


 CDC NO-1 (50)
 Bordetella holmesii (50, 51)
Acinetobacter lwoffii (50)
Diplococci  Neisseria (39), Moraxella catarrhalis (50)
Acinetobacter (50)
Coccobacilli   Oligella ureolytica (50)
  EF-4b, Paracoccus yeei, Psychrobacter immobilis (saccharolytic) (50)
EO-3, EO-4 (50)
 Moraxella canis, M. catarrhalis (50)
 Psychrobacter phenylpyruvica (50), Brucella spp. (52)
 Oligella urethralis (50)
 Moraxella lacunata, M. nonliquefaciens, M. osloensis, P. immobilis
(asaccharolytic) (50)
M. atlantae, M. lincolnii, M. osloensis, P. immobilis (asaccharolytic) (50)
 Acinetobacter baumannii, EO-5 (50)
 Bordetella parapertussis (50, 51)
 CDC NO-1 (50)
 Bordetella holmesii (50, 51)

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Acinetobacter lwoffii (50)
376 ■ BACTERIOLOGY

TABLE 2 Identification algorithm for gram-negative bacteria with poor or no growth on blood agara

Growth H2S Cauliflower-


Growth Cellular Pigmented 6% O-shaped Require Organism group
on Urea Oxidase in like
only on: morphology colonies NaCl cells X±V (chapter)
BAP TSI colonies

Poor Tiny coccobacilli  Brucella (52)


  Francisella
philomiragia (52)
Bordetella (50)
Francisella (52)
Rods Pink Asaia, Azospirillum,
Methylobacterium,
Roseomonas (50)
Other  Bartonella (54)
Haemophilus
aphrophilus (41),
Cardiobacterium,
Dysgonomonas,
Eikenella,
Kingella,
Simonsiella,
Suttonella (40)
Diplococci or  Paracoccus yeei (50)
coccobacilli
Neisseria (39),
Moraxella (50)
None Chocolate Diplococci or Neisseria (39)
coccobacilli
Fusiform rod Capnocytophaga (40)
Rod  Bartonella, Afipia (54)
 Haemophilus (41)
Francisella (52)
BCYE Long gram-negative Legionella (53)
rods
Regular rods  Bordetella (51)
Francisella (52)
BHI Pleomorphic, beaded Streptobacillus (40)
serum filamentous rods
Small rods Bartonella, Afipia (54)
a Abbreviations: BCYE, buffered charcoal yeast extract agar; X, hemin; V, nicotinamide adenine dinucleotide.