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Bacterial Diarrhea

Microbiology and Parasitology


Ludivina b. Tesoro-solis, md, fpsp
DIARRHEA

Acute syndrome of GIT


Increase in volume fluid (25%) and
frequency of bowel movement
Symptom (gastroenteritis)
Caused by several pathologic states:
Infection
Intestinal disorders
Food poisoning
WHO(April 2013): 2nd leading cause of death
among children < 5 y/o due to dehydration
in the world
WHO (2008): In Philippines, 10,000 Filipino kids
die of diarrhea every year
PhilHealth (2011): 4th cause of hospitalization
WHO (2008): Philippines is the 2nd largest
contributor to diarrhea morbidity next to
China

INFECTIOUS DIARRHEA
Two Basic Mechanisms
1.

2.

Toxigenic type
Bacteria release enterotoxins
Binds to Rc of the small intestine
Toxins disrupts physiology of epithelial
cells
Leads to increase electrolyte and water
secretion (secretory diarrhea)
Increase in volume with little or no blood
Invasive diarrhea
Invade wall of small or large intestine
Damage
of
mucosal
lining
and
underlying tissue layers
Pain in rectum, bloody stools, ulceration
of inner lining

Antigenic Structures/Virulence Factor

Escherichia spp.

Enterobacteriaceae
Vibrio spp.
Campylobacter spp.

ENTEROBACTERIACEAE FAMILY

Escherichia spp.
Salmonella spp.
Shigella spp.
Characteristics

Found worldwide, soil, water, vegetation


Normal intestinal flora of man and animals
Gram negative bacilli
0.3-1.0 x 1.0-6.0 m; non-spore formers
Non-motile or motile
Facultative anaerobes
Grow on non-selective/selective media
Ferment glucose
Reduce nitrate
Catalase
positive
(except
Shigella
dysenteriae)
Oxidase negative (except Plesiomonas
shigelloides)

Spidey Transcriptions

Kingdom: Eubacteria
Domain: Bacteria
Phylum: Proteobacteria
Class: Gamma Proteobacteria
Order: Enterobacteriales
Family: Enterobacteriacea
Genus: Escherichia
Species: E. coli
Binomial name: Escherichia coli
Epidemiology

BACTERIAL DIARRHEA

Lipopolysaccharides (LPS)
Heat-stable
Composed of:
Somatic O polysaccharide
Core polysaccharide
Lipid a
Surface antigens:
O antigen polysaccharide part of LPS
H antigen flagellar antigen
K antigen polysaccharide capsular
antigen

Common Gram negative bacilli in GIT


Transmitted more commonly from person to
person
Incidence
related
to
hygiene,
food
processing, general sanitation, opportunity
for contact
Most infections are endogenous
Strain causing gastroenteritis are acquired
exogenously
Prominent as a normal intestinal bacterium,
thus is an indicator to monitor fecal
contamination in water, food and dairy
products
If E. coli is present in water sample then
fecal pathogens like Salmonella, viruses
and protozoans may also be present
If small amount of E. coli is present then
water is unsafe to drink
Diseases: sepsis, UTI, meningitis, intraabdominal infections, pneumonia, prostatitis
Virulence Factors

Adhesins:
P pili
Colonization factor antigens (ETEC)
Aggregative adherence fimbriae
Bundle forming pili
Intimin
Ipa protein
Toxins:
Heat stable toxins
Heal labile toxins
Verotoxins (Shiga-like toxins)
Hemolysin cause tissue destruction
Endotoxin
Invasive capacity

1 jcc

E. coli serotypes

Enterotoxigenic E. coli (ETEC)


Enteropathogenic E. coli (EPEC)
Enteroinvasive E. coli (EIEC)
Enterhemorrhagic E. coli (EHEC)/ Shiga toxin
Producing E. coli (STEC)
Enteroaggregative E. coli (EAEC)
Diffusely adherent E. coli (DAEC)
ENTEROTOXIGENIC E. coli

Disease: travellers diarrhea, infant diarrhea


Site of action: small intestine
Produces:
Heat-labile toxins (LT-I, LT-II)
Heat-stable toxins (STa, STb)
Secretory diarrhea
1-2 days incubation (lasts 3-4 days)
Heat-labile toxins: A and B subunit
B subunit binds to Rc GM1 ganglioside

Subunit A translocate across membrane, activates


adenylyl cyclase on epithelial cells of small
intestines

Increase cAMP levels

ENTEROINVASIVE E. coli

Disease: Shigella like diarrhea (Shigellosis)


Site of action: colon
Inflammatory
diarrhea:
invasion
and
ulceration

A series of bacterial genes on a plasmid mediate


invasion of colonic epithelium by bacteria
Bacteria lyse phagocytic vacuole and replicate
within cytoplasm
Epithelial cell destruction
Fever, cramping watery diarrhea, dysentery with
scant bloody stools
ENTEROHEMORRHAGIC E. coli (EHEC)/SHIGA TOXIN
PRODUCING E. coli (STEC)

Disease: Hemorrhagic colitis, Hemolytic


Uremic Syndrome (acute renal failure,
thrombocytopenia, hemolytic anemia)
Site of action: large intestine
Produce: Shiga-like toxin (verotoxin) 1 and 2
Common serotype to produce shiga-like
toxin is 0157:H7
Associated with hemorrhagic colitis
Undercooked ground beef

Hypersecretion of water and chloride reabsorption


of sodium

Watery diarrhea, vomitting (rare), cramps, nausea,


low grade fever
Heat-stable toxin (STa)
STa + guanylate cyclase

Bacteria produce Shiga-like toxins


Disrupts protein synthesis, destroy intestinal colinic
villi
Decreased absorption (3-4 days incubation)
Watery diarrhea, vomitting, abdominal pain,
nausea, low grade fever

Increase guanosine monophosphate


Bloody diarrhea, severe abdominal pain (resolution
in untreated patient: 4-10 days)
Hypersecretion of fluids
ENTEROAGGREGATIVE E. coli
ENTEROPATHOGENIC E. coli

Disease: infant diarrhea


Site of action: small intestine

Disease: acute and chronic diarrhea (14


days)
Site: small intestine
Produce: ST-like toxins and hemolysins

Bacteria attach to epithelial cells of small intestine

EAEC

Destruction of microvilli

Mucus production

Malabsorption and diarrhea, fever, nausea,


vomitting, non-bloddy stools

Traps bacteria on epithelium of small intestine


Persistent watery diarrhea, vomitting, dehydration,
low grade fever

Spidey Transcriptions

2 jcc

DIFFUSELY ADHERENT E. coli

Shigella spp.

Disease: infant diarrhea


Site: small intestine
DAEC

Elongation of microvilli with bacteria embeded in


cell membrane

Kingdom: Bacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Enterobacteriales
Family: Enterobacteriacea
Genus: Shigella
Species: S. boydii, S. dysenteriae, S. flexneri, S.
sonnei
Binomial name: S. dysenteriae
Epidemiology

Watery diarrhea (1-5 years old)

Laboratory Diagnosis

BAP, EMB, MAC


Lactose fermenter
MAC: pink colonies
EMB: green metallic sheen
(+) indole
(+/-) lysine decarboxylase
(+/-) motility
Circular, convex, smooth
distinct edges

colonies

with

Biochemical Test

TSI: A/A, gas


Methyl red: positive
Voges-Proskauer: negative
Citrate: negative
Indole: positive
H2S: negative
Urease: negative
Lactose fermentation: positive
Motility positive or negative
LIA: positive
SIM: negative, positive, positive
Other Tests

70% occur in children


Humans are the only reservoir
Usually affects children less than 10 years old
Highly communicable
Spread from person to person, fecal-oral
route
Transmitted via food, fingers, feces, flies
200 bacilli can establish an infection
Diseases:
gastroenteritis,
bacillary
dysentery/shigellosis

Tissue culture cytotoxic assays


Detects/isolate specific toxins
DNA probes
Genes coding for verotoxins

Morphology

Slender, Gram negative rods


Bacilli, coccobacilli
Non-spore former
Facultative anaerobes
Antigenic Structures/Virulence Factors

O antigens
Endotoxin
Shiga toxin (S. dysenteriae, EIEC)
Exotoxin (acts as enterotoxin and
neurotoxin)
Disrupts protein synthesis; endothelial cell
damage
May cause HUS
Invasive property
Invasion plasmid antigens (surface proteins)
promote adherence and phagocytosis by
epithelial cells of intestinal mucosa)
S. dysenteriae

Treatment

Rehydration (ORS) hydrite


Antispasmodics
E CAT
Case-to-case
basis
(trimethoprimsulfamethoxazole/ampicillin/cephalosporins)
Avoid developing resistant strains
Prevention and Control

Proper cooking of beef products


Avoid unpurified water
Restrict use of antibiotics
Proper hygiene

SHIGELLA
Species

S. dysenteriae
S. flexneri (shigellosis in developing countries)
S. boydii
S. sonnei (industrial world)

Spidey Transcriptions

Oral-fecal route
Incubation: 1-2 days
Colonize small intestine
Invade and replicate in
colonic mucosa
Lower abdominal cramps, profuse watery diarrhea,
tenesmus, pus/mucus/bloody stools
Self-limiting

Acidosis
Dehydration
Death in elderly and
children

3 jcc

Laboratory Diagnosis

Specimen
Fresh stool
Mucus flecks
Rectal swab
Cultural/Growth Characteristics
Facultative anaerobes
MacConkey/EMB
Hektoen enteric agar, SS agar/XLD
medium
Convex, circular, transparent colonies
Non-lactose fermenter (except S. sonnei)
Non-motile
(-) lysine decarboxylase
(-) H2S
(+) acid not gas in butt

Salmonalla spp.

TSI: K/A (with or without gas) no H2S


Methyl red: positive
Voges-Proskauer: negative
Citrate: negative
Indole: variable
H2S: negative
Urease: negative
Lactose fermentation: negative
LIA: negative
SIM: negative, positive/negative, negative
Cannot ferment mannitol

Kingdom: Bacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Enterobacteriales
Family: Enterobacteriacea
Genus: Salmonella
Species: S. bongori, S. enterica
Binomial name: S. enterica
Epidemiology

Biochemical Test

S. typhimurium

Pathogenic for humans/animals


Direct fecal-oral spread
Common sources: poultry, eggs, dairy
products, food and water contaminated by
infected food handlers, recreational drugs
Infective dose: 105 -108
Three main types of disease:
Enteric fever (S. typhi)
Bacteremia (S. choleraesuis)
Enterocolitis/Salmonellosis
(S. typhimurium, S. enteritidis)
Morphology

Gram negative bacilli


Motile with peritrichous flagella
Facultative anaerobe

Treatment

Antigenic Structures/Virulence Factors

Fluid replacement
Ciprofloxacin
Shigella cattc
Ampicillin
Tetracycline
TMP-SMX
Chloramphenicol

Prevention and Control

Sanitary control of water, food, milk


Sewage disposal
Isolation of patient and disinfection
excreta
Screening of food handlers
Fly control
Hand washing
Antibiotic treatment of infected persons

SALMONELLA

of

O and H antigens
Capsular (K) antigen
(Vi) antigen protects organism from action
of complement (protects from antibody
attack)
Pili adherence to epithelium
Endotoxin
Tolerant to acids in phagocytic vesicles
Can survive in macrophages
Pathogenesis: Enterocolitis
S. enteritidis (incubation: 8-48 hours)
Inflammatory response cause release of
prostaglandins

Stimulates cAMP and active fluid secretion

Species

S. enterica
Subspecies
Enterica
Salamae
Arizonae
Diarizonae
Houtinae
Indica
S. bongori

Nausea, vomitting, headache, profuse non-bloody


diarrhea, low grade fever, few leukocytes in stools
Resolve after 2-3 days

Serogroup

Subspecies enterica:
S. choleraesuis
S. typhi
S. paratyphi A
S. paratyphi B
S. enteritidis

Spidey Transcriptions

4 jcc

Treatment
S. typhi

Ingestion

Fluid and electrolyte replacement


Antimicrobial therapy
Needed in enteric fever and bacteremia
Enterocolitis: prolong SX and excretioon
Neonates, chronic disease
Invasive
infections:
ampicillin,
trimethoprim
sulfamethoxazole,
3rd
generation cephalosporin
3 cats eat salmon

Prevention and Control

Absorbed to epithelial cells in terminal portion of


small intestine

Bacteria penetrate cells and migrate to lamina


propia layer of ileo-cecal region

Multiply in lymphoid follicles


Reticuloendothelial hyperplasia and hypertrophy

Good sanitation
Hand washing
Screening of food handlers
Observe strict hygienic precautions
Food thoroughly cooked
Vaccination:
Oral live attenuated vaccine
Vi capsular polysaccharide vaccine
(intramuscularly)

Note: Both vaccines have 50-80% efficacy

(incubation: 10-14 days)

VIBRIO
Species

Fever, malaise, constipation, bradycardia, myalgia

rose spots

Laboratory Diagnosis

Specimen: blood, urine, stool, bone marrow


Growth/cultural characteristics
SS agar, Hektoen enteric agar
Non-lactose fermenter
Enrichment broth-selenite broth for
maximum recovery of organism from
fecal specimen
(+) H2S
Motile
BA: smooth, greenish color, 2-3 mm
Biochemical Test

Widals Test

Detects antibodies against O and H antigen


2 specimens used (7-10 day interval)
Note for rise in antibody titer
Positive: titer against O > 1:320; H > 1:640

Spidey Transcriptions

Other Test
Slide agglutination test
Known sera + unknown culture
clumping
Serotyping: reference lab
Test kits
Tube agglutination test (Widals test)

V. cholerae serogroups non-O1 and non0138


Cholera-like disease, mild diarrhea
V. parahaemolyticus (gastroenteritis; shellfish
ingestion)
V. vulnificus (trauma to skin/shellfish ingestion)
V. mimicus (raw oysters)
V. fluvialis, V. hollisae (diarrhea)
V. damsela, V. anginolyticus, V. metchnicovii
(ear, wound, soft tissue infections)
Vibrio spp.

Note: S. typhi produce little or no H2S

Note: Both seogroups of V. cholerae can be


endemic or pandemic

TSI: K/A, H2S positive


Methyl red: positive
Voges-Proskauer: negative
Citrate: variable
Indole: negative
Urease: negative
Lactose fermentation: negative
LIA: positive
SIM: positive, negative, positive

V. cholerae (fecal-oral)
Serogroups
V. cholerae O1
El Tor
Classical
V. cholerae 0139

Kingdom: Bacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Vibrionales
Family: Vibrionaceae
Genus: Vibrio
Species: V. cholerae
Binomial name: V. cholerae
Epidemiology

Found in marine and water surfaces


worldwide
Diseases: cholera, gastroenteritis, wound
infection, bacteremia
Spread:
cholera

consumption
of
contaminated
food
and
water,
gastroenteritis;
consumption
of
contaminated shellfish, crabs, shrimp
Infectious dose is high (108 -1010)

5 jcc

Morphology
Biochemical Test

2-4 m
Curved or comma shaped
Gram negative bacilli
(+) polar flagella
Aerobic rods
Virulence Factors

Flagella
Cholera toxin enterotoxin; hypersecretion
of electrolytes and water
Coregulated pilus adherence to mucosal
cells
Hemmaglutination protease induces
intestinal inflammation
Siderophores iron sequestration
Neuraminidase increase toxin receptors
Pathogenesis

Treatment

Water and electrolyte replacement


Antibiotic therapy: tetracycline, doxycyline,
azithromycin
Prevention and Control

Ingestion of contaminated water (1-4 days)

Improvement of sanitation
Isolation of patient and
excreta

disinfection

Acute gastroenteritis

Contaminated seafood (raw fish/shellfish) (12-24


hours incubation)

Multiply and liberate toxin

Nausea, vomitting, abdominal cramps, fever,


watery-bloody diarrhea (1-4 days)

Nausea, vomitting, profuse diarrhea (rice water


stools), abdominal cramps, profound dehydration,
circulatory collapse, anuria

Self-limiting

Hypovolemic shock, cardiac arrhythmia, renal


failure

of

V. parahaemolyticus

Intestinal tract (attach to microvilli of the brush


border of epithelial cells)

Oxidase: positive (except V. metchnicovii)


Lactose: negative
Sucrose: positive
Lysine decarboxylase: positive
Arginine dihydrolase: negative
Ornithine decarboxylase: positive
H2S and gas: negative

No enterotoxins produced
BA; TCBS: green colonies
V. vulnificus

Death

Note: Mortality without treatment is 25-50%; El Tor


milder disease

Laboratory Diagnosis

Specimen: mucus flecks from stools


Cultural/growth characteristics:
Peptone agar, blood agar, TCBS agar
(thiosulfate citrate bile salt sucrose)
yellow colonies
pH 8-9
Halophilic: salt loving

CAMPYLOBACTER
Species

Vibrio cholerae agglutinating sera test


whether a culture is positive for the O antigen
of the lipopolysaccharides of Vibrio cholerae
O1 that exist in two forms termed Inaba and
Ogawa
To one suspension, use as a control, add 1
drop (40l) of saline and mix
To the other suspension add 1 drop (40l) of
antiserum and mix
Rock the slide for 1 minute and observe for
agglutination

Spidey Transcriptions

C. jejuni
C. coli
C. upsaliense
C. fetus
Campylobacter spp.

Agglutination test

Severe wound infection: bullous lesion within


hours; 50% with bacteremia die
Bacteremia and gastroenteritis
Found in infected oysters
TCBS: blue-green colonies
Treatment: tetracycline, ciprofloxacin

Kingdom: Bacteria
Phylum: Proteobacteria
Class: Epsilonproteobacteria
Order: Campylobacteriales
Family; Campylobacteriaceae
Genus: Campylobacter
Species: C. coli, C. concisus, C. curvus, C.
fetus, C. jejuni
Binomial name: C. jejuni

6 jcc

Epidemiology
Biochemical Test

Infections are zoonotic; reservior: cattle,


sheep, pigs, poultry
C. jejuni and C. coli: contaminated food,
milk, water, infected poultry
C. upsaliensis: domestic dogs
Most
common
cause
of
bacterial
gastroenteritis
Acquired: ingestion of contaminated food,
unpasteurized milk or contaminated water
Improperly prepared poultry
Diseases:
gastroenteritis,
septicemia,
spontaneous abortion, meningitis
Killed when exposed to gastric acid

Hippurate Test

Morphology

0.3-0.6 m
Gram negative rods with comma, S, gullwing shapes
Motile
Dark-field/phase contrast: darting motility
Single polar flagellum
Non-spore former

Lipopolysaccharides
Flagellar antigens
Enterotoxin
Cytotoxic enzymes
Adhesins
Motility

Hippurate test is to be used in the


presumptive identification of Gardnerella
vaginalis, Campylobacter jejuni, Listeria
monocytogenes and group B streptococci
by detecting the ability of the organism to
hydrolyze hippurate
Positive test is indicated by the appearance
of a deep blue/violet color in 30 minutes. A
negative reaction is indicated by a faint blue
color change or no color change
Treatment

Antigenic Structure/Virulence Factor

Hippurate hydrolysis test: positive


Catalase: positive
H2S (TSI): negative
Nitrate reduction: positive
Oxidase: positive

Fluid and electrolyte replacement


Antibiotics:
erythromycin,
azithromycin,
clarithromycin
Resistant drugs: cephalosporin, penicillin
Prevention and Control

Proper food preparation


Avoid unpasteurized dairy products
END

Note: Roles are poorly defined for enterotoxins and


cytotoxic enzymes
Pathogenesis
Ingestion of contaminated food (usually
undercooked chicken)

The different biological tests are not included in this


transcription. They can be found on past
transcriptions that I have made or you can read on
them on the laboratory manual.
Good luck and God bless 2016!

Multiply in small intestine; invades mucosa and


inflammation
Crampy abdominal pain, profuse diarrhea may be
bloody, malaise, fever, headache
Self-limited (5-8 days)
Laboratory Diagnosis

Specimen: stool
Cultural/growth characteristics:
Microaerophiles
5-10% carbon dioxide
Incubation temperature: 42C
Selective media: skirrows medium
(vancomycin, polymyxin B, trimethoprim)
Slow growing (2-3 days)
Gray, flat, irregular, mucoid, nonhemolytic
Watery and spreading or round and
convex

Spidey Transcriptions

7 jcc