NON-ENTERIC GASTROINTESTINAL PATHOGENS

Non-enteric gastrointestinal pathogens are important group of microorganisms because some of them, the Vibrio spp. in
particular, have been associated with large epidemics and pandemics. On top of that, Campylobacter spp. Infection may
be associated with Guillain-Barré syndrome (GBS), and Helicobacter pylori may cause ulcers and is associated with
gastric carcinoma. This unit addresses diarrheal disease agents and other diseases caused by Vibrio, Aeromonas,
Plesiomonas, Campylobacter, and Helicobacter. This will also describe the microscopic features, epidemiology, and
clinical infections caused by each organism. Specimen collection, transport, growth requirements, and key biochemical
reactions used for diagnosis are also included in this unit.

Topic 1: Vibrio
- short, curved, gram (-) rods (0.5 to 0.8 μm diameter x 1.4 to 2.6 μm length)
- facultatively anaerobe, catalase and oxidase (+), nitrate reduction (+) except for V. metschnikovii
- polar, sheathed flagella (broth)MONOTRICHOUS
- exhibit (+) string test (0.5% sodium desoxycholate)
- Halophilic except V. cholerae and V. mimicus
- found in fresh water, brackish or estuarine water, and marine or salt water
- temperature-sensitive (>20° C) isolated from water, algae, plankton, fish, and shellfish
MOT: consumption of raw or undercooked seafoods
Common isolate: V. cholerae (serogroups O1 and non-O1), V. parahaemolyticus, V. vulnificus, and V. alginolyticus

Vibrio cholerae
- causative agent of cholera (Asiatic cholera or Epidemic cholera)
- rapid DARTING OF “SHOOTING-STAR” MOTILITYsingle thick polar flagellum
- small, facultative, gram (-) rodsSLIGHTLY CURVED OR COMMA-SHAPED
Antigenic Structure: Flagellar (H) Ag and Somatic (O) Ag

Major Subgroups
1. V. cholerae O1 – classic or epidemic cholera; biochemically divided into Classic and El Tor
2. V. cholerae O139 – biochemically similar to O1 and type with O1antisera but does not produce the classic cholera
toxin
3. V. cholerae non-O1 – phenotypically resemble V. cholera but fail to agglutinate in O1 antisera

V. Cholerae O1 Serotypes (Based on the composition of O antigen)

a. Ogawa (A, B) or Variant F – common in India
b. Inaba (A, C) or Original J – common in Philippines
c. Hikojima (A, B, C) or Middle/Intermediate  common in Japan

V. cholerae O1 strains biogroups

Test Classic El Tor
Soluble Hemolysin
VP Test
Chicken RBC Agglutination
Polymyxin B (50 μg) Sensitivity
Bacteriophage IV

VIRULENCE FACTORS
a. CHOLERA TOXIN or CHOLERAGEN
- enterotoxin, consists of two toxic A subunits and five binding B subunits
- stimulates hypersecretion of electrolytes (Na+, K+, HCO3 −) and water (fluid loss of 10-15 L and
electrolytes)
- Poorly immunogenic leading to recurring infection
b. Zonula occludens (Zot) toxin – enterotoxin
c. Accessory cholera enterotoxin (Ace) toxin
d. Motility and chemotaxis
e. Mucinase –penetration of the mucous layer
 f. Toxin coregulated pili (TCP) – attachment

RELATED DISEASES AND INFECTIONS

Cholera
- acute diarrheal disease spread mainly through contaminated water
- ingestion of improperly preserved foods, including fish and seafood, milk, ice cream, and unpreserved meat
- severe gastroenteritis accompanied by vomiting and followed by diarrhea
- dehydration, eyes and cheeks appear sunken with diminished skin turgor and a washwoman’s skin appearance
Hallmark: RICE WATER STOOLS (10 to 30/day)
 Vibrio cholera O1 – common cause of epidemic cholera; gastroenteritis, wound infections, bacteremia
 V. cholerae O139 – Cholera
 Vibrio cholera non-O1 – Gastroenteritis or cholera-like disease; septicemia, ear infections
 V. cholerae serogroups O75 and O141 – sporadic, cholera-like diarrhea

LABORATORY DIAGNOSIS
Specimen: Stool and Rectal Swabs
Culture – requires alkaline media for growth
 TCBS agar: medium-sized, smooth, opaque, thin-edge yellow colonies which on prolong incubation turns green
(El Tor)
Transport media: Amies and Cary-Blair
Selective Media:
 Alkaline Peptone Water(APW)
 enrichment broth (pH 8.5)
 Tellurite Taurocholate Gelatin Agar (TTGA)
 TCBS
 Gohar, Dieudonne’s Monsur and Aronson Media

a. Cholera Red Test (Nitroso-Indole Reaction)

- 24hr culture in APW with Trytophan and NO3 + conc H2SO4 = RED COLOR (NITROSO-INDOLE)
- Not-specific: (+) result to indole (+) and nitrate (+) organism (Enterobacteriaceae)
b. Bacteriolysis (Pfeiffer’s Phenomenon)
- lysis of V. cholera when inoculated into an immuned guinea pig
c. Grieg’s Test
- demonstrate soluble hemolysin of El Tor
- SRBC + Organism (37°C for 30 mins) = (+) LYSIS OF RBC
d. Direct Fluorescent Ab Technique
e. Darkfield or Phase-Contrast Microscopy – characteristic motility
f. Immobilization Test

BIOCHEMICAL TESTS
 Oxidase (+)
 ODC and LDC (+)
 Glucose and Sucrose Fermenter
 ONPG (+)
 (+) string test
 Vibriostatic compound O/129 (2,4-diamino-6,7-diisopropylpteridine) (150ug) in MHA  susceptible

Vibrio parahaemolyticus
- 2nd most common Vibrio spp. implicated in gastroenteritis
- primary cause of SUMMER DIARRHEA (Japan)
MOT: consumption of raw, improperly cooked, or recontaminated seafood (oysters, clams, crabs, lobsters, scallops,
sardines, and shrimp)
V. parahaemolyticus serotype O3:K6 foodborne outbreaks

Virulence Factor
KANAGAWA PHENOMENON
 - heat-stable hemolysin lyse human RBC in a special, high-salt mannitol medium (WAGATSUMA AGAR) 
Kanagawa toxin-(+)

Related Diseases and Infections

 Seafood Gastroenteritis (sef-limiting)
- explosive watery diarhea without blood or mucus
- headache, abdominal cramps, nausea, vomiting and fever
 Extraintestinal Infections
- localized infection seen in boat workers, seafood cooks and swimmers:
- wounds, ear and eye infections, pneumonia

LABORATORY DIAGNOSIS
Specimens: Stool and Rectal Swabs
Morphology: resembles other Vibrio spp.
Culture – requires alkaline environment same selective media as in V. cholera
 Halophilic and requires 3% NaCl for growth
 TCBS: large, green and smooth; non-sucrose fermenter
Biochemical Tests
 Oxidase (+)
 ODC and LDC (+)
 ONPG (-)
 0/129 (S)
 Urease (+)

Vibrio vulnificus
- known as “LACTOSE(+)” Vibrio spp.
- second most serious types of Vibrio-associated infections

Related Diseases and Infections

 Primary septicemia – gastrointestinal route (consumption of shellfish, especially raw oysters)
 Wound infections – cellulitis, necrotizing fasciitis and/or multiple organ system failure

Vibrio alginolyticus
- least pathogenic for humans and one most infrequently isolated
- inhabitant of marine environments
- strict halophile = at least 1% NaCl
- able to tolerate up to 10% NaCl
- occupational hazard for people in constant contact with seawater

Related Diseases and Infections

 Eye and ear infections
 Wound and burn infections
 Respiratory infections and bacteremia

Extraintestinal Pathogen

OTHER VIBRIO SPECIES

a. Vibrio mimicus – cause diarrhea after ingestion of raw oyster; ear infections
b. Vibrio metschnikovii – Septicemia, peritonitis
c. Vibrio cincinnatiensis – Meningitis
d. Grimontia hollisae (formerly Vibrio hollisae), Vibrio fluvialis, Vibrio furnissii – cause diarrhea
e. Photobacterium damselae (formerly Vibrio damselae) – wound infection

LABORATORY DIAGNOSIS
Specimen: Stool, Rectal Swabs, Pus and Tissues
 Stool – collected as early as possible before administration of antimicrobial agents
Culture Media
Transport Media: Amies, Cary-Blair
Buffered glycerol saline – not recommended
 SBA or CHOC agar: medium to large smooth, opaque, and iridescent with a greenish hue colonies
 SBA: α- or β-hemolytic
 MAC: non-lactose fermenters (pathogenic) except V. vulnificus
SELECTIVE MEDIUM
 Thiosulfate Citrate Bile Salt Sucrose (TCBS) agar
- differentiates sucrose-fermenter (YELLOW COLONIES) from nonsucrose-fermenter (GREEN)
- inhibits gram (+) bacteria
- pH indicator: Bromothymol blue and Thymol Blue; 1% Nacl (pH :8.6)
SUCROSE-FERMENTER NONSUCROSE-FERMENTING

 CHROMagar Vibrio
- V. cholera, V. parahemolyticus, and V. vulnificus
- white to pale blue and violet colonies

String Test – differentiate Vibrio spp.(+) from Aeromonas (-)

Reagent: 0.5% Na Desoxycholate
(+) Result: Lysis of cell releases DNA pulled up into a viscous string using a inoculating loop

Vibriostatic compound O/129 (2,4-diamino-6,7-diisopropylpteridine) (150ug) – separate vibrios (S) from other
oxidase(+), glucose fermenters like Aeromonas (R)

BIOCHEMICAL TESTS
 TSIA: A/A, (-) g, (-) H2S
 LIA: K/K
 Inositol non-fermenter (except V. cincinnatiensis and V. metschnikovii(some))
 V. Cholerae: (+) citrate and indole
 V. Vulnificus (+) indole and cellobiose
 V. mimicus: (+) indole

Serology
- screen using POLYVALENT O1 ANTISERUM
- V. parahaemolyticus serotyped by O and K (capsule) Ag

AST
 Disk diffusion (Kirby-Bauer) or Dilutionrecommended
 V. cholera
- susceptible to doxycycline or ciprofloxacin
- Ampicillin, tetracyclines, trimethoprim, and chloramphenicol
 V. vulnificus
- fluoroquinolones alone or combination of ciprofloxacin and cefotaxime
 - Most vibrios are susceptible to gentamicin, tetracyclines, chloramphenicol (except P. damsela),
monobactams, carbapenems, and fluoroquinolones

Topic 2: Aeromonas
- oxidase (+), facultative anaerobe, glucose-fermenting, gram (-) straight rods (1.0 to 3.5 μm long x 0.3 to 1.0 μm
wide)
- distributed in freshwater, estuarine, chlorinated water and marine environments
- grow from 4° C to 42° C and motile spp. posses single flagellum
- causative agent of “RED-LEG” DISEASE in amphibians

Virulence Factors
 Heat labile enterotoxin similar to LT of E. coli and V. cholera enterotoxin
 Heat stable cytotoxic enterotoxin similar to S. dysenteriae
 Extracellular enzymes: protease, amylase, lipase, nuclease
 Hemolysins
 Adherance

Groups
1. MESOPHILIC GROUP (optimal growth around 37°C)
 motile – single polar flagellum
a. A. hydrophila complex
 A. hydrophila
 A. bestiarum,
 motile strains of A. salmonicida
b. A. veronii complex
 A. veronii biovar sobria (formerly A. sobria)
 A. veronii biovar veronii
 A. jandaei
 A. trota
 A. schubertii
c. caviae complex
 A. caviae,
 A. media
 A. eucrenophila
2. PSYCHROPHILIC GROUP (optimal growth around 22°C)
 nonmotile and grows best at 22° C to 25° C
 A. salmonicida – fish pathogen

RELATED DISEASES AND INFECTIONS

Gastroenteritis
5 Diarrheal Presentations
1. Acute, secretory diarrhea with vomiting
2. Acute, dysenteric form of diarrhea (like shigellosis) with blood and mucus
3. Chronic diarrhea (>10 days)
4. Cholera-like disease (rice water stools) A. veronii
5. NEBULOUS SYNDROME = TRAVELER’S DIARRHEA (like enterotoxigenic E. coli)

A. caviae – most commonly associated with GIT infections; inflammatory bowel disease
A. hydrophila and A. veronii biovar sobria – HUS or kidney disease

Extraintestinal Infections
 Septicemia, meningitis, and wound infections (cellulitis) – most common
 Osteomyelitis, pelvic abscesses, otitis, cystitis, endocarditis, peritonitis, cholecystitis, keratitis (contact lens wear)
and endophthalmitis
o Wound isolates: A. hydrophila, A. veronii biovar sobria, or A. schubertii
o Sepsis: A. veronii biovar sobria, A. jandaei, and A. hydrophila
LABORATORY DIAGNOSIS
Specimens: Feces, Sterile Body fluids, Tissues and Exudates from wounds
Culture Media
- grow on common laboratory media: BAP, MAC
- large, round, raised, opaque colonies with entire edge and smooth, often mucoid, surface
- strong odor; translucent and white to buff-colored pigment
- pathogenic spp: β-hemolysis
- MAC: LF (most)A. caviae
- TSA with 5% SRBC and 10 or 30 ug/ml Ampicillin
 Ampicillin SBA inhibit A. trota and some A. caviae
 Modified CIN II Plate (4 μg of cefsulodin instead of 15 μg)
- pink-centered colonies = fermentation of mannitol : “BULL’S EYE” apperance

BIOCHEMICAL TESTS
 Oxidase Test
 Aeromonads (+) vs Enterobacteriaceae (-)
 Catalase Test (+)
 Indole Test (+) – A. caviae, A. hydrophila, A. veronii
 String Test (-)
 O/129 (R)
 Inositol Fermentation (-)
 TSI
A/A, (-) g, (-) H2S: A. caviae
A/A, (+) g, (+) H2S: A. hydrophilia, A. verinii
Treatment: Generally self-limiting
 S: Trimethoprim-sulfamethoxazole, aminoglycosides, and quinolones
 R: Penicillin, ampicillin, and carbenicillin

Topic 3: Campylobacter
- small, slender, helically curved, microaerophilic, faintly staining gram(-) rods
- C. rectus and C. curvus are strictly anaerobes
- morphological forms: S shapes, commos, SEAGULL-WINGED and coccoid shapes
- most have single unipolar flgellum CORKSCREW-DARTING MOTILITY on phase contrast or dakfield
microscopy
- require low(5% O2) and increased CO2(10%)
- C. jejuni, C. coli and C. lari are thermophilic(42°C)
- most of pathogenic spp. are oxidase and catalase (+)
- do not ferment or oxidize sugars
- most are S to cephalosporin
- abortion in domestic animals (cattle, sheep, and swine); febrile systemic disease, periodontal disease and
gastroenteritis
MOT: direct contact with animals and handling infected pets and consumption of contaminated water, dairy products
and improperly cooked poultry, sexuallytransmitted
Enteric Campylobacters: C. jejuni, C. coli and C. lari

Campylobacter jejuni
- slow-growing, fastidious and assacharolytic
- DARTING MOTILITY and unable to grow in media with high salt concentration
- most common cause of bacterial gastroenteritis (self-limiting)
- septic arthritis among AIDS patient and Guillain-Barré syndrome
MOT: ingestion of contaminated milk, water and food (does not multiply in food)
Optimum temp: 42°C
Infective dose: >10, 000
Virulence Factors: Invasiveness, cytotoxin, enterotoxin
LABORATORY DIAGNOSIS
Specimen: Stool, Blood, Sterile Body Fluids in Extraintestinal Infections
Culture Media
Incubated at 42°C = 5%O2, 10%CO2 and 85%N2 for 72hrs
Campy-BAP Skirrow’s Butzler’ Charcoal Medium V Campy Campy-CVA
Medium Medium cefoperazone (modification Thio (cefoperazonev
deoxycholate of ancomycinamp
Agar Butzler hotericin
(CCDA) medium) B)

Delay: Cary-Blair, Campy Thio; Buffered Glycerol Saline is toxic

CULTURE: Gray to pinkish or yellowish gray, slightly mucoid and spreading or round and convex; some exihibit
“tailing effect” along the streak line or “runny spreading” growth colonies
Carbolfuchsin – recommended counterstain in GS
Safranin – 2 to 3 minutes
BIOCHEMICAL TEST: Oxidase (+), Hippurate Hydrolysis (+)
SEROTYPING:
a. Penner Method – indirect hemagglutination technique for suluble heat-stable Ag
b. Lior Method – slide agglutination technique for heat-labile Ag
TREATMENT:
 S Erythomycin, Aminoglycosides, Tetracycline and Chloramphenicol
 Cephalothin R and Nalidixic acid S

Campylobacter fetus
- opportunistic pathogen that causes systemic infections n immunocompromised patients; occasionally causes
diarrhea
- isolated from sheep or cattle
- grow at 25°C (37°C); some do not grow at 42°C
- cephalothin sensitive, nalidixic acid resistantresistant to complement lysis
Species
a. C. fetus subsp. Fetus
- Bacteremia and rare cause of GI illness
- recovered in routine blood culture media
- 42° C: inhibited
- smooth, convex, translucent colonies
b. C. fetus subsp. Venerealis
c. C. mucosalis and C. hyointestinalis – dirty yellow pigment
 Campylobacter Growth at Nitrate Sensitivity to
spp. Reduction
25°C 42°C Cepahlotin Nalidixic Indoxyl
Acid Acetate
Hydrolysis
C. jejuni subsp.
jejuni
Campylobacter
jejuni
subsp. doylei
C. coli

C. fetus subsp.
fetus
C.
hydrointestinalis
C. lari

C. fenneliae (now
Helicobacter
fennelliae

Topic 4: Helicobacter
- curved, microaerophilic, gram(-) rods, most are urease(+)
- spiral-shaped, helical (S-shaped) resembling Campylobacter spp.
- motile by monopolar or multi-bipolar flagella
- Oxidase and catalase(+)
- gray and translucent colonies and non-pigmented
Humans isolates:
 H. pylori, H. cinaedi, H. fennelliae, H. heilmannii (formerly Gastrospirillum hominis), H. westmeadii, H. canis,
H. Canadensis sp. nov., H. pullorum, and “H. rappini” (formerly “Flexispira rappini”)

Helicobacter pylori (formerly Campylobacter pylori)

- curved, spiral shaped or bizarre U-shaped mcroaerophilic gram (-)rod, with sheathed tuft of polar flagella exhibits
corkscrew motility
- primary habitat: HUMAN GASTRIC MUCOSA
- colonize mucous layer of antrum and fundus of stomach but not the epithelium
- major cause of TYPE B GASTRITIS
o chronic condition formerly associated primarily with stress and chemical irritants
- produces urease
- antral gastritis, gastric or duodenal ulcer (peptic ulcer disease), and gastric cancer(carcinogen)
- binds to Lewis Ag and Sialic acid

VIRULENCE FACTORS
 VacA – Exotoxin; creates vacuoles in epithelial cells, decreases apoptosis, and loosens cell junctions
 CagA – Pathogenicity Island; Encodes a type IV secretion system for transferring CagA proteins into host cells
 BabA – Encodes outer membrane protein that mediates adherence to blood group antigens on surface of gastric
epithelial cells
 IceA – associated with peptic ulcer disease

H. cinaedi and H. fennelliae

- habitat: human gastrointestinal tract
- human gastroenteritis in immunocompromised patients
- sexual transmission among homosexual men
- proctitis, enteritis, and sepsis in homosexual men
LABORATORY DIAGNOSIS
Specimen: Antral Biopsy (H. pylori=seen in the surface of gastric antral epithelium), urine, feces and dental plaque
 Gastric tissue =best
 Urine= ammonia testing
Transport Media:
 Stuart medium; Isotonic Saline with 4% Glucose
 Cysteine-Brucella broth with 20% glycerol(−70° C)
Stains:
 WARTHIN-STARRY or Silver stains and Giemsa stains
 0.1% basic fuchsin= counterstain in GS
Noninvasive Indirect Test: UREA BREATH TEST
 radioactively labelled (13° C) ureaammonia and labeled HCO3 exhaled as CO2
Immunoassays – monoclonal Ab
 H. pylori stool antigen tests
 one-step immunochromatographic assay
Culture
 incubated at 35-37°C for 1 week, humidified, 5% to 10% O2

H. cinaedi and H. fennelliae – selective media used for Campylobacter but without cephalothin
H. pylori
 Nonselective media: CHOC agar and Brucella agar with 5% sheep blood
 Selective: Skirrow’s, MTM

Biochemical Tests
 H. pylori : oxidase, catalase, and rapid urease tests(+); cephalothin(S) and nalidixic acid (R); no sugar
metabolized
AST:
 Agar dilution MHA with 5% aged (>2weeks) SRBC, microaerophilic, read after 3 days
Treatment:
 Metronidazole, a bismuth salt, and either amoxicillin or tetracycline, macrolide (H. pylori)

Topic 5: Miscellaneous Bacteria

Arcobacter
- associated with gastroenteritis
- asaccharolytic
- Media: Campy-CVA = 37° C under microaerobic conditions for 72 hours

A. butzleri
- 4th most common Campylobacter-like organism isolated from stool
- persistent watery diarrhea
- growth at 15°C and 25°C, nitrate reduction(+), Indoxyl Acetate Hydrolysis(+)

Streptobacillus moniliformis
- belongs to Fusobacteriaceae family
- gram(-) rod that requires media containing blood, serum, or ascites fluid and incubation under CO2
- facultative, nonmotile anaerobe that tends to be highly pleomorphic
- develop L forms (bacteria without cell walls)
Natural habitat: URT of wild and laboratory rats (mice, gerbils, squirrels, ferrets, weasels)
MOT: Rat bite, ingestion of contaminated food
Haverhill Fever
- acquired by ingestion
- acute onset of chills, fever, headache, vomiting, and often severe joint pains
- rash on the palms, soles of the feet, and other extremities
 Complications: endocarditis, septic arthritis, pneumonia, pericarditis, brainabscess, amnionitis, prostatitis, and
pancreatitis
LABORATORY DIAGNOSIS
Specimen: Blood or aspirates from infected joints, lymph nodes, or lesions
Culture
 BAP (5% to 10% CO2 48 hours at 37°C) – nonhemolytic
 grows as “fluff balls” or “bread crumbs” in broth
 “fried egg” appearance, dark center and a flattened, lacy edge – L-phase colonies = bipolar-staining coccoid
(Dienes stain)
 BHI with 20% horse serum: small, smooth, glistening, colorless or grayish
 and have irregular edges
Microscopy – straight of variable size or as long tangled chains and filaments with bulbar swellings
Biochemical Tests
 Indole, catalase, oxidase, and nitrate(-)
 Nonmotile and urea, LDC(-)
Treatment: Penicillin – drug of choice for human rat-bite fever

Spirillum minus
- short, thick, gram(-), helical, strictly aerobic with tapering endsand 2-3 spirals resembling Campylobacter
- polytrichous polar flagella
- causes RAT-BITE FEVER in humans “SODOKU”
o arthritis is rarely seen and swollen lymph nodes are prominent
o Manifestation:
 Local lesion – chancre-like indurated ulcer with black crust
 Regional gland swelling
 Skin rashes – purplish maculopapular eruption
 Ralapsing type of fever

LABORATORY DIAGNOSIS
Specimen: Blood, exudate, or lymph node tissues, serum from exanthematous patches, ground-up tissue from lesions
Culture: Non-culturable on synthetic media
Stains:
 Gram Stain
 Giemsa or Wright Blood smears
 Silver Impregnation MethodFontana-Tribondeau (Flagellar stain)
 Dark field or Phase contrast microscopy
Definitive Tes t: injection of lesion material or blood into white mice or guinea pigs and recovery 1 to 3 weeks
Treatment: Penicillindrug of choice