6/28/2016

BACTERIAL INFECTION
IN GASTROINTESTINAL
TRACT

Lisa T. Muslich
Microbiology Department
Medical Faculty of Hasanuddin University

Learning Objective
• Bacterial causing infection in GI tract
• Diagnosis of bacterial infection in GI tract
• Microbiology examination of GI tract due to bacterial infection
• Identification and interpretation of the main bacterial cause of GI
tract infection

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Disease of The Gastrointestinal Tract

Foundations in Microbiology, 2012

Disease in GEH system which could be caused by

bacteria that listed in SKDI 2014
• Gastritis  4A
• Gastroenteritis  4A
• Ulcus (gaster, duodenum)  3A
• Appendix abcess  3B
• Typhoid fever  4A
• Food poisoning  4A
• Botulism  3B
• Kolitis  3A
• Bacillary dysentry  4A

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The Family Enterobacteriaceae

• Coliforms in Normal Microbiota : • True Pathogenic Enterics :
- Escherichia coli - Salmonella typhi
- Enterobacter - S. cholerae-suis
- Serratia - S. enteritidis
- Klebsiella - Shigella dysentriae
- Hafnia - S. flexneri
- Citrobacter - S. boydii
• Noncoliforms in Normal - S. sonnei
Microbiota : - Yersinia enterocolitica
- Proteus - Y. pseudotuberculosis
- Providencia • True Pathogenic Nonenteric :
- Morganella - Yersinia pestis
- Edwardsiella

Diarrhea
• Passage of three or more loose or liquid stools per day ( or more
frequent passage than is normal for the individual)
• Three clinical types :
- Acute watery diarrhea – lasts several hours or days (include
cholera);
- Acute bloody diarrhea – also called dysentery; and
- Persistent diarrhea – lasts 14 days or longer
• Causes :
- Infection : bacterial, viral, & parasitic organisms
- Malnutrition
- Source
- Transmission

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Diarrhea in Indonesia

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Escherichia coli
• Gram-negative rod-shaped, motile, non-spore forming bacteria
• Size : 2 x 0.25 - 1µm
• Antigens : O-, K-, and H-antigens (designated by numbers of
antigen presented), eg. O111:K76:H7
• Pili/ fimbriae:
- Type 1 pili  bind to D-mannose (epithelial cell surfaces)
- P Pili (Pap or Gal-Gal)  bind to digalactoside (uroepithelial
cells and erythrocytes)  UTI
- Colonization Factor Antigens (CFAs) or Bundle-forming Pili
(BFP)  bind to human enterocytes

Antigenic structure of E. coli

Sherris Medical Microbiology 6 th ed. 2014

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Escherichia coli
• Toxins :
- α-hemolysin  pore-forming cytotoxin  cytoplasmic contents
leakage  cell death
- Shiga toxin  AB type  B-unit binds to Gb3 receptor; A-unit
modifies 28S-ribosomal RNA of 60S-ribosomal subunit  protein
synthesis blocked  cell death
- Labile toxin (heat labile) 
oB-subunit binds to cell membrane;
oA-subunit catalyzes ADP-ribosylation  G-protein inactivation
 permanent activation of adenylate cyclase system 
chloride secretion and NaCl absorption blockage  water and
electrolytes accumulation  diarrhea
- Stabile toxin (heat stabile)  binds to glycoprotein receptor 
guanylate cyclase activation  cyclic GMP concentration increased

Stx (Shiga) toxin

Sherris Medical Microbiology
6th ed. 2014

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The action of E. coli LT-

toxin
Microbiology-Lippincott’s Illustrated
Review’s 3rd ed. 2012.

Escherichia coli
• Diarrhea-causing E. coli classified due to virulence properties:
1. Enterotoxigenic E. coli (ETEC)
2. Enteropathogenic E. coli (EPEC)
3. Shiga Toxin-producing E. coli (STEC)  includes a subset of
Enterohemorrhagic E. coli (EHEC) strains
4. Enteroinvasive E. coli (EIEC)
5. Enteroaggregative E. coli (EAEC)

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Diarrheagenic E. coli serotypes

Manual of Clinical Microbiology. 2015

Enterotoxigenic E. coli (ETEC)

• Traveler’s diarrhea
• Important cause of diarrhea in developing countries (young
children)
• Transmission  contaminated food and water (uncooked
food, marinated meats or vegetables)
• Caused by LT and/or ST enterotoxin producing strain
• Adherence mediated by CFA class of pili.
• No invasion or inflammation of the intestinal mucose
• Natural immunity mediated by IgA spesific for LT and CFAs.
• Prominent symptoms : diarrhea (usually mild watery),
abdominal cramps, sometimes nausea and headache, little
vomiting or fever

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Enteropathogenic E. coli (EPEC)

• 1950s  diarrhea outbreaks in USA and Great Britain
• 20% cases of diarrhea in bottle-fed infants (<1 y.o.)
• Transmission  fecal-oral route
• Non-enterotoxin producing, non invasive
• Two genetic elements of pathogenecity :
- EPEC adherence factor (EAF) plasmid  bundle-forming pilus
- Chromosomal LEE  mediates the A/E phenotype
• Adherence  BFP type of pili  clustered microcolonies 
attachment and effacing (A/E) lesion
• Primarily infect small intestine
• Lack of simple diagnostic

EPEC attachment to epithelial cells (A/E lesion)

Sherris Medical Microbiology 6 th ed. 2014.

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Enterohemorrhagic E. coli (EHEC)

• Important cause of bloody diarrhea
• Regional and national outbreaks  unpasteurized juices and
hamburgers
• Emergence of EHEC : virulence, low infecting dose (100-200
organisms), common reservoir, modern food processing industry
• Primarily attack the colon
• Pathogenicity : Shiga toxin production and A/E lesions
• Extraintestinal feature  Hemolytic Uremic Syndrome (HUS)

Enteroinvasive E. coli (EIEC)

• Identical to Shigella
• Restricted to children < 5 y.o. in developing nation
• Transmission : contaminated food or water
• Infecting dose higher than Shigella
• Less common than ETEC or EPEC in developing country

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Enteroaggregative E. coli (EAEC)

• Protracted (> 14 days) mucoid, watery diarrhea
• Infants and children  developing countries
• Persistent diarrhea in HIV patients
• Symptoms : abdominal pain and fever and stools usually do not
contain blood or fecal leukocytes
• Pathogenesis  not clear

Diarrhea caused by E. coli

• Begin with mild watery diarrhea
• 2 – 4 days incubation
• Duration : few days (except EAEC  protracted diarrhea)
• ETEC & EPEC  watery diarrhea
• EIEC & EHEC  follows by dysenteric illness
• EHEC  often vomits, 1 – 2 days of intense abdominal pain and
bloody diarrhea, fever not prominent

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Microbiology Diagnosis of E. coli

• Gram staining (fresh stool, culture)
• Culture (stool)  MacConkey agar
• Biochemical test (lactose-fermenting,
catalase pos., oxidase neg., indole pos.,
MR pos., VP neg., reduce nitrate, citrate
neg., urease neg.)
• Toxins and genes associated virulence 
immunoassay & nucleic acid methods
(ELISA & PCR) Escherichia coli, Enterobacter
aerogenes, Shigella sonnei, P.
mirabilis (clockwise from top)
in MacConkey agar.
A photographic atlas for microbiology
laboratory. 2011

Biochemical test kit

API®20E
Biomerieux-USA

Enterotube
(Roche diagnostic)
Foundations in microbiology.
2012

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Vibrio spp.
• More than 30 species
• Gram-negative, facultative anaerob,
straight, curved, or comma-shaped rods
• Size : 0.5 – 0.8 x 1.4 – 2.6 µm
• Medically important Vibrio :
- V. cholerae serogroups O1 & O139
- V. cholerae serogroups non-O1/
non-O139
- V. parahaemolyticus
- V. vulnificus

Vibrio cholerae
• 130 serogroups (LPS O-antigen difference)
• Two pathogenic mechanism:
- Cholera toxin
- Toxin-co-regulated pili
• Virulence factors :
a. ctx gene  cholera enterotoxin
b. tcpA gene  toxin-coregulated pilus major protein subunit
• V. cholerae O1 subtypes : Inaba & Ogawa, Hikojima
• V. cholerae O1 biotypes : Classic & El Tor

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Pathogenesis of V. cholerae infection

1. Toxin secretion
2. Toxin-receptors binding
3. Toxin’s active portion
transported
4. System signaling to activate
adenyl cyclase
5. ATP  cAMP
6. Cl- and HCO3- pump 
intestinal lumen
7. Na+, K+ & H2O  intestinal
lumen

Foundations in Microbiology. 2012

Clinical features of Vibrio cholerae infection

• Vomiting
• Copious watery feces (secretory diarrhea contains flecks of mucus
 rice-water stool)  fluid losses up to 50% BW
• Muscle cramps, severe thirst, flaccid skin, sunken eyes
• In children  come and convulsions
• Hypotension, tachycardia, cyanosis and collapse

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Microbiology Diagnosis for Vibrio spp.

• Culture : Thiosulfate Citrate Bile salts
Sucrose (TCBS) agar
• Biochemical test : Oxidase pos. (except V.
metschnikovii & V. gazogenes), Catalase
pos., VP neg.  classical biotypes, VP pos.
 El Tor
• Serology  agglutination with spesific
antisera
• MALDI-TOF Mass Spectrometry V. Cholerae in TCBS agar.
A photographic atlas for
• NAATs microbiology laboratory. 2011

Shigella
• Gram-negative rods, non-motile,
non-spore-forming
• Acid-resistant, facultative anaerob
• Size : 0.3 – 1 x 1 – 6µm
• Four species based on biochemical
reactions and spesific O antigens:
1. Shigella dysentriae (serogroup A)
 15 serotypes
2. Shigella flexneri (serogroup B) 
6 serotypes
Foundations in Microbiology, 2012.
3. Shigella boydii (serogroup C) 
20 serotypes
4. Shigella sonnei (serogroup D) 
1 serotypes

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Clinical features of Shigella infection

• Frequent watery stools, often occult to obvious blood  dysentery
• Mucus in stools
• Fever
• Intense abdominal pain
• Nausea
• vomiting

Microbiology Diagnosis for Shigella spp.

• Culture : Xylose Lysine Desoxycholate
(XLD) agar, Salmonella-Shigella (SS)
agar
• Biochemicals test  considerable
variation occur between species and
types within species
- Ferments sugar w/o gas (except S.
flexneri & S. boydii)
- Lack of mannitol fermentation for S. Escherichia coli, Shigella flexneri,
Salmonella typhimurium,
dysentriae, Enterococcus faecalis (clockwise
- ODC- & ONPG-pos. for. S. sonnei, from top) in SS agar.
A photographic atlas for microbiology
- gas production from D-gucose for laboratory. 2011
some strains of S. flexneri type 6 and
S. boydii type 14

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Shigella (left) and Salmonella (right) in XLD agar.

Faculty of Health and Medical Science, University of
Copenhagen, Denmark.

Epidemiology
• Transmission : person to person under poor sanitary condition
(fecal-oral route), contaminated food or water
• No animal reservoir
• Low infecting dose
• Invasion and destruction of colonic mucosa  acute inflammatory
response  mucosal ulceration + abscess formation
• Watery diarrhea  intense colitis + fever + frequent small-volume
stools (blood and pus)

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Mechanism of Shigella infection causing diarrhea

Microbiology-Lippincott’s Illustrated Reviews 3 rd ed. 2012

Microbiology Diagnosis of Shigella

• Gram staining (fresh stool, culture)
• Culture  MacConkey, XLD, SS agar
• Biochemical test (Urease neg., oxidase neg., H2S neg., ferment
mannitol except Shigella dysentriae & some Shigella flexneri
serotype)
• Molecular method :
- Polymerase Chain Reaction (PCR)
- Multilocus Sequence Typing (MLST)
- Multiple-locus Variable Number Tandem Repeat Analysis
(MVLA)
- Whole Genome Sequencing (WGS)

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Salmonella
• Serotyping classification :
1. Salmonella Enteritidis
2. Salmonella Typhi
3. Salmonella Paratyphi
4. Salmonella Typhimurium
• Species :
1. Salmonella bongori
2. Salmonella enterica :
- Salmonella enterica subspecies enterica (subspecies I)
- Salmonella enterica subspecies salamae (subspecies II)
- Salmonella enterica subspecies arizonae (subspecies IIIa)
- Salmonella enterica subspecies diarizonae (subspecies IIIb)
- Salmonella enterica subspecies houtenae (subspecies IV)
- Salmonella enterica subspecies indica (subspecies VI)

Salmonella
• Rod-shaped,
• Non-spore-forming,
• Gram negative rods
• Motile
• Size : 0.7 – 1.5 x 2 – 5 µm
• Glucose fermenting
• Facultative anaerob

Foundations in
microbiology. 2012

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Sherris Medical
Microbiology, 2014.

Clinical features of Salmonella infection

• Typhoid fever  typhi variant
• Fever, diarrhea, abdominal pain
• Gastroenteritis or enteric fever  paratyphi, hirschfeldii &
typhimurium
• Vomiting, diarrhea, mucosal irritation

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Microbiology Diagnosis of Salmonella

• Agglutination test (Slide agglutination, Microtiter Agglutination,
Tube Agglutination)
• Biochemical test (Non-lactose-fermenting, Glucose fermented
sometimes with gas, H2S pos., Citrate pos., Urease neg., Oxidase
neg., Indole neg., ONPG gelatinase neg.)

Clostridium difficile
• Gram-positive, anaerobic rods
• Endospore-forming bacteria  under
anaerobic condition
• Small number  normal in human intestinal
tract
• Prolonged use of broad-spectrum antibiotic
• Causing pseudomembranous colitis or
antibiotic-associated colitis
• Produce toxin A (enterotoxin) and toxin B
(cytopathic)  necrosis in intestinal wall
Foundations in
• Symptoms : diarrhea, more severe cases  microbiology. 2012
abdominal cramps, fever, leukocytosis

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Microbiological aspect in C. difficile infection

Two major categories in CDI
diagnostic assay:
• Toxin detection : PCR (for toxin B
gene), latex agglutination and ELISA
(toxin A & B test),
• Organism detection : anaerobic
culture, glutamate dehydrogenase
(GDH) & nucleic acid amplification
tests (NAATs)

C. difficile on CCFA.
• Culture : cycloserine-cefoxitin- Manual of clinical microbiology. 2015

fructose-egg yolk agar

Yersinia enterocolitica
• Gram-negative, non-spore-forming bacilli
• Bipolar staining with Giemsa or Wayson’s dye
• Size : 0.5 – 0.8 x 1 – 3 µm
• Motile  peritrichous or paripolar flagella
• Facultative anaerob
• Growth temp. : 4 – 43 oC
• Possess lipid A-oligosaccharide core –O antigen polysaccharide
• 54 different O antigens and 19 H factors
• Serotypes account for human infection : O:3, O:5,27, O:8 & O:9

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Clinical features of Y. enterocolotica infection

• Primarily infects lymphoid tissue of the small intestine & ileocaecal
junction
• In children : fever, diarrhea, abdominal pain and vomiting
• Most common form of disease is gastroenteritis
• Elderly people with cirrhosis, iron overload or immunosuppressive
 septicemia
• Pneumonia, meningitis  rare
• Post infection complication  erythema nodosum, polyarthritis,
Reiter’s syndrome and thyroiditis
• Pathogenic serotypes are commonly from swine

Microbiological aspect of Y. enterocolica

infection
• Biochemical test : glucose fermenting w/o gas. Catalase pos.,
oxidase neg.
• Culture : MacConkey, blood and chocolate agars
• Serotype : agglutination test, ELISA, PCR

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Campylobacter spp.
• Most common bacterial cause of
diarrhea in USA
• Slender, curved or spiral Gram-negative
rods (Size : 0.2 – 0.9 x 0.5 – 5 µm)
• Non spore-forming
• Polar flagella (one or both poles)  S-
shaped or gull-winged pairs
• Main pathogenic species : C. jejuni & C.
pylori Scanning micrograph of
Campylobacter jejuni
• Reservoirs : poultry, pigs, cattle Microbiology, A system approach. 2012

• Transmission : ingestion of raw milk,

undercooked poultry or meat, and
contaminated water

Clinical features of C. jejuni infection

• Incubation periods : 2-10 days
• Symptoms : frequent watery stools, fever, vomiting, headaches and
severe abdominal pain
• Generally associated with type B gastritis (gastric ulcer or peptic
ulcer)

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Microbiology aspect of C. jejuni

• Microaerophilic  incubation in 5% - 10% CO2
• Thermophilic  incubation at 35oC
• Non fermentative, oxidase pos., hippurate hydrolysis pos.,
• Aerobic and anaerobic blood culture and stool culture
• Culture medium :
a. Blood-free media :
- Charcoal Cefoperazone Deoxycholate Agar (CCDA) 
most sensitive
- Charcoal-based Selective Medium (CSM)
b. Blood-containing media :
- Campy-CVA (cefoperazone, vancomycin, amphotericin)
- Skirrow medium

Clinical features of selected diarrheal pathogens infection

WGO. 2012

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Helicobacter pylori
• Gram-negative, non-spore-forming
bacteria
• Curved, helical or spiral or fusiform
rod-shaped
• Size : 0.3 – 0.6 x 1 – 10 µm
• Motile  multiple (4 – 8 /cell)
monopolar sheathed flagella
• Microaerobic with respiratory type
of metabolism
• Lack carbohydrate utilization H. pylori Gram stain, 100x.
World Journal of Gastroenterology.
pathway 2008

• Pathogenesis : stomach epithelial tissue  specific binding 

entrance
• Transmission unknown (probably oral-oral or fecal-oral route)
• Two-thirds of world’s population are infected
• 25% healthy middle-age adults & 65% > 60 y.o. adults
• Animal also susceptible  proposed as zoonosis transmitted
disease

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Clinical features of H. pylori infection

• Colonize in cardia, corpus and antrum of stomach
• Sharp or burning pain emanating from the abdomen
• Acute gastritis (abdominal pain, nausea, vomiting)  cronic
gastritis
• Antrum-predominant gastritis  hyperacidity, duodenal ulcer
predisposed
• Lesion  peptic ulcers  bloody stools, vomiting, or both (worse
at night, after eating, or psychological stress
• Long-term infection  stomach cancer
• Independent risk factor for the development of atrophic gastritis,
gastric ulcer disease, gastric adenocarcinoma and gastric mucosa-
associated lymphoid tissue (MALT) lymphomas

Microbiology aspect
• Culture of gastric biopsy specimen  BHIA, brucella agar, Wilkins
Chalgren agar, Trypticase soy agar (primary), Columbia agar + 10%
defibrinated horse blood (excellent results)
• Cytochrome oxidase producing, urease hydrolysis, nitrate
reduction, indoxyl acetate hydrolysis, alkaline phosphatase activity
• Microbiology examination : ELISA  HpSA (stool antigen test)

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Gastroenteritis and Diarrhea

• When does the stool samples taken?
- If microbiological diagnosis is required : persistent diarrhea/
malabsorption; blood, mucous or pus in the stool; history of
diarrhea and/or vomiting and systemically unwell patient; recent
hospitalization; antibiotic therapy
- Public health sampling : outbreaks or diarrhea and/or vomiting;
public health hazard
- Immunocompromised patients
- Travel history

How to avoid GI tract infection?

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WHO data on vaccination of GEH disease caused

by bacteria
• Available vaccine :
- Cholera
- Typhoid
• Vaccines in development :
- Campylobacter jejuni
- Enterotoxigenic E. coli
- Paratyphoid fever
- Shigella

Definitions
• Strain : genetic variants within a species
• Serotype (or serovar) : the type of a microorganism determined by
its constituent antigens; a taxonomic subdivision based thereon
• Serogroup : an unofficial designation denoting a group of bacteria
containing a common antigen, possibly including more than one
serotype, species, or genus

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References
1. Brooks G. F., et al. Jawetz, Melnick & Adelberg’s Medical Microbiology (25th
ed.). New York: McGraw-Hill. 2010
2. Harvey R. A., Cornelissen C. N., & Fisher B.D. Lippincott’s Illustrated
Reviews: Microbiology (3rd ed.). Philadelphia: Two Commerce Square. 2012
3. Ryan K.J., Ray C. G. Sherris Medical Microbiology (6th ed.). New York:
McGraw Hill. 2014
4. Cowan M. K. Microbiology A Systems Approach (3 rd ed.). New York:
McGraw Hill. 2012
5. Talaro K.P., Chess B. Foundation in Microbiology (8th ed.). New York:
McGraw-Hill. 2012
6. Jorgensen J.H., Pfaller M. A. Manual of Clinical Microbiology. Canada : ASM
Press. 2015
7. Mishra S. K., Agrawal D. A Concise Manual of Pathogenic Microbiology. New
Jersey : John Wiley & Sons, Inc. 2013
8. Goldman E., Green L. H. Practical handbook of microbiology. Danvers: CRC
Press. 2015
9. Farthing M., et al. Acute diarrhea in adults and children : a global
perspective. World Gastroenterology Organization Global Guidelines. WGO.
2012.

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