I. Vibrio (Comma/Curved Bacillus) General Biochemical Tests for Vibrio spp.

Description ● Oxidase (+), Nitrate to Nitrite Reducers

● Not part of the human flora; facultatively (Except V. metschnikovii)
anaerobic; monotrichous organism
● Glucose Fermenters, NLF except V.
● Found in brackish or estuarine water vulnificus
(freshwater river or stream meets the ocean),
marine, or salt water ● Motility Test:
○ Broth: Polar sheathed Flagella
● Temperature sensitive (>20 C), isolated
from algae, plankton, fish, and shellfish ○ Solid Media: Peritrichous,
Unsheathed Flagella
● Mode of Acquisition: Eating raw or
undercooked seafoods

● Disease/Infection: Cholera, Wound infection,

Septicemia, Necrotizing Fasciitis

● Halophilic organisms except V. cholerae,

V. mimicus

● Microscopy: G-, short, curved rods

(comma bacillus), asporogenous a. Vibrio cholerae
Characteristics
● Culture:
○ CAP: Smooth, Opaque, Iridescent, ● Causes Cholera/Asiatic Cholera/Epidemic
Greenish Hue Cholera (V. cholerae O1)

○ MAC: NLF except V. vulnificus
➔ Non-Lactose Fermenter (Clear
Colony) is the first distinguishing
feature that differentiates Vibrio from
Enterobacteriaceae
○ BAP: Alpha or Beta Hemolysis
● Caused cholera epidemics (O1 and O139
strains) and seven pandemics (O1 strain)
● V. cholerae subgroups: V. cholerae O1, V.
cholerae O139, V. cholerae Non-01
● Epidemic V. cholerae O1 Biogroups:
Classical and EI Tor

● Susceptibility Test: 150 ug Vibriostatic ○ Classical: VP (-); ○ EI Tor: VP (+),

disk on MHA or TSA (0/129) Do not agglutinate Agglutinate chicken
chicken RBC, RBC; Polymixin B
○ Differentiates Vibrio from Polymixin B (50 ug) (50 ug) Resistant
Aeromonas spp. Susceptible
➔ Susceptible: Vibrio
➔ Resistant: Aeromonas (only infects
amphibians)
● Motility Pattern: Rapid Darting/Shooting
Star Motility (Flagellum covered with LPS
sheath)

● Culture: Smooth, Medium to Large colonies

with Greenish Hue (BAP)

G.A.S.S
● String Test: (+) “Stringing” reaction ○ B Subunit - Intestinal Receptor
➔ Means that it is encapsulated Binding

● TSI: K/A, (-) Has, (-) H2S ● Stimulates hypersecretion of water and
Clwhile inhibiting Sodium absorption - Massive
● LIA: K/K fluid loss (10-15 L) and Electrolyte loss

● Other Biochemical Tests: (+) Oxidase,

Indole, LD/OD

● Virulence Factor: Choleragen (Cholera

Toxin)
b. Vibrio parahaemolyticus
➔ Attaches to the epithelium of the
➔ Isolated in Japan
gastrointestinal tract causing Cholera
Disease or Cholera Infection
● Second most common Vibrio spp. in
Gastroenteritis
➔ Hallmarks of Cholera Infection: Rice-
watery stool sample ● Causes “Summer Diarrhea” in Japan
since during summer, fish is in demand
● Enterotoxins: Cholera Toxin, Zot Toxin, Ace
Toxin ● V. parahaemolyticus serotype O3:K6 -
Pandemic Strain
● Antigenic Structures: Somatic O, Flagellar H
● Mode of Acquisition - Eating contaminated
seafoods

● Virulence Factor - Heat-stable Hemolysin

● Pathogenesis - Kanagawa Phenomenon

Clinical Manifestation of Cholera ○ Kanagawa toxin Positive - Strings
capable of lysing RBCs
● Acute diarrheal disease, spread through
contaminated water ● Selective Medium - Wagatsuma Agar (High-
salt Mannitol medium)
● Ingestion of improperly preserved food
(seafoods, milk, ice cream)

● Hallmark of Cholera: Rice-Watery stool

(10-30x defecation/day)

Choleragen
c. Other Vibrio species
● Protein composed of 2 subunits:
Vibrio vulnificus
○ A Subunit - Enzymatic, enters
epithelial cells and activate adenylate
● Referred as “Lactose-positive” Vibrio
cyclase by addition of ADP-Ribosyl
group
● 2nd to V. cholerae in producing serious type
of Vibrio-associated infection

G.A.S.S

● Infections: Septicemia, Wound Infections
● Mode of Acquisition: Eating raw oysters
and fish (Tilapia)
Vibrio alginolyticus
■ Bile salts is a selective ingredient to
prevent the growth of Gram Positive
bacteria
○ Non-Sucrose Fermenters (Green,
TCBS): V. mimicus, V. vulnificus, V.
parahaemolyticus, V. damsela

● Least pathogenic Vibrio for humans (not as Other Tests

clinically significant)
● Samples: Stool, Rectal Swab, Pus, Tissue
● Strict Halophile (1-10% NaCl) (Cary-Blair Medium)

● May be an occupational hazard (Fishermen, ● Gram Stain: G-, Straight or Slightly

Sailors)
● Infections: Eye, Ear, Wound Infections
Laboratory Diagnosis for Vibrio spp.
Culture
● Media: TCBS (Thiosulfate Citrate Bile
Salts Sucrose), Alkaline Peptone Water, Cary-
Blair, MacConkey, BAP
● Growth of Vibrio requires media containing
0.5% NaCl except V. cholerae, V. mimicus
● V. alginolyticus: Halophile (Up to 10% NaCl)
● Pathogenic Vibrio grows as NLF on
MacConkey Agar
Curved Rods - “Comma-shaped bacilli or
curved-shaped”
● String Test (0.5% Na Desoxycholate)
○ Differentiates Vibrio from
Aeromonas
○ (+) Lysis of cells (Vibrio)
● Vibriostatic Test (Susceptibility Test) -
O/129 Disk Test
○ Differentiate Oxidase (+) GI
Pathogens
○ Separates Vibrio (S) from other Ox
(+), Glucose Fermenters like
Aeromonas (R)

● Alkaline Peptone Water with 1% NaCl (pH ○ Differentiates V. cholerae O1 and

8.5): Inoculated (at least 20 mL), incubated 5-8 non-O1 (S) from others (R)
hours at 35 C then subculture at TCBS

Note: Thiosulfate-Citrate-Bile Salts-Sucrose

Agar is selective, differential medium for Vibrio
species (Vibrio spp. cannot ferment Lactose,
BIOCHEMICAL TESTS:
but they can ferment sucrose)

○ Sucrose Fermenters (Yellow, TCBS): Citrate (+); Yellow V. cholerae

Colonies on TCBS
V. cholerae, V. alginolyticus, V.
Indole (+) V. cholerae,
metschnikovii
V. mimicus,
V. vulnificus
■ Yellow because the sucrose is
fermented Cellobiose (+) V. vulnificus

G.A.S.S
 API 20E Best method for ●Motile with single polar flagellum (some non-
Vibrio spp. motile)

Serological V. parahaemolyticus
(O and K)

II. Aeromonas spp. a. Other Aeromonas species

Description 2 Groups

● Found in freshwater, estuarine, and ● Mesophilic group:

chlorinated water isolated from meat products A. hydrophila complex
A. veronii complex
● Not part of the normal flora; Facultatively A. caviae complex
Anaerobic
● Psychrophilic group
● Causes Red Leg Disease: Disease among - A. salmonicida (Fish Pathogen), Non-
warm- and cold-blooded animals (fish, reptile, motile group; grows best 22-25 C
amphibian)
● Most common isolate, common isolate in GI
● Causes Traveler’s Diarrhea: Nebulous infections: A. caviae
syndrome in humans
● Common isolates in HUS: A. hydrophila, A.
● Typically grow from 4- 42 C veronii

● Halophilic requirements: Grow in 0% NaCl, ● A. hydrophila (“water loving”) - cause

not in 6% NaCl Gastroenteritis and Cellulitis

● Microscopy: Gram-Negative straight rods Other Details

● Vibriostatic O/129 test: RESISTANT
● Culture: (Aeromonas, Plesiomonas)
○ CIN: “Bull’s Eye colonies”,
“Apron-like” pattern (with 4 ug ● Inositol Fermentation: Aeromonas (-);
Cefsulodin) Plesiomonas (+)

➔ 2 bull’s eye colonies: ● (+) Indole: A. caviae, A. hydrophila, A. veronii

Aeromonas spp. and Yersinia
enterocolitica subsp.
enterocolitica
○ BAP - Large, Round, Raised, White,
Opaque colonies
● TSI:
○ A/A, (-) Gas, (-) H2S: A. caviae
○ A/A, (+) Gas, (+) H2S: A.
hydrophila, A. veronii

○ MAC - Lactose Fermenters (A.

caviae - most common isolated
Aeromonas spp.)

● Biochemical Tests: (+) Oxidase, Catalase;

Glucose Fermenters;

G.A.S.S
 Differential Characteristics ● Microscopy: Faintly staining G- small,
TEST
Oxidase
Vibrio
+
Aeromonas
+
Plesiomonas
+
curved, S-shaped rod (may appear as
Vibriostatic Susceptible Resistant Resistant coccobacilli in old cultures)
O/129 (150
ug)
○ Long Spirals or Seagull wing
Growth, 0% -/+ + +/- shaped (Enteric)
NaCl
Growth, 6% + - -
NaCl
Fermentation +/-/+/v +/-/v/v +/+/-/-
● Culture: Gray, flat, glistening, irregular with a
Test: G/I/M/S “tailing effect along the streak line” or
Gelatin + + -
Liquefaction “runny spreading” colonial growth

III. Campylobacter spp.

Description

● Most recognized cause of Guillain-Barre

Syndrome (Test + for Antibodies to a. Campylobacter jejuni
Campylobacter) ● Most common cause of Gastroenteritis
worldwide (Global diarrhea)
➔ Syndrome in which there is nerve
● Also causes septic arthritis (AIDS)
conduction, weakness/neuropathy in
the lower extremities, and antibody
● Acquisition: Eating contaminated chicken
production due to Campylobacter
and turkey (does not replicate in food)
antigen
● Invades epithelium of small intestine causing
● Animal Pathogen (Cattle and Swine) -
inflammation
Sterility and Abortion
● Secretes toxin similar to the Cholera toxin
● Mode of Acquisition: Ingestion of
contaminated water, poultry, dairy products;
● Slow growing, fastidious, Asaccharolytic;
handling of pets
darting motility
● Species: C. jejuni, C. coli, C. fetus subsp.
● Unable to grow in 3.5% NaCl
fetuss, C. sputorum, C. concisus, C. curvus, C.
rectus
● Optimum growth: 42 C
○ Enteric: C. jejuni, C. coli, C. lari
● Infective Dose: >10,000 organisms
○ From Blood Cultures: C. fetus subsp.
Laboratory Diagnosis
fetus
Specimen and Microscopy
○ STDs: C. fetus subsp. fetus, C. fetus
● Specimen: Feces, Rectal Swab, Blood
subsp. venerealis
● Microscopy:
● Motile (Single Polar Flagellum), Non-Spore
○ Recommended Counterstain: Carbol
Forming
Fuchsin
■ Safranin - Applied 2-3 minutes
● Microaerophilic (5-10% O2) except C. rectus
and C. curvus (Strict Anaerobes); Oxidase (+)
○ Hanging Drop Motility: (+) Darting
Motility (TSB)

G.A.S.S
 Culture
● Selective Media: CAMPY-BAP, Butzler Agar,
Skirrow’s Media (for non-enteric pathogens),
Charcoal Cefoperazone Deoxycholate Agar
(CCDA)
➔ Helicobacter pylori grows in
Skirrow’s Media which is why it should
be differentiated from Campylobacter
● Transport Medium: Cary-Blair Medium
● Blood culture- 2 weeks of incubation (blind
culture)
● Detected effectively by the CO2 monitoring
system
● EIA - Direct detection of C. jejuni and C. coli
in fecal specimens
● C. jejuni - susceptible to nonspecific
bactericidal activity of human serum
IV. Helicobacter spp.
Description
● Found in the GIT of mammals and birds
● Species: H. pylori, H. cinaedi, H. fennelliae,
H. rappini (Former: Flexispira rappini)
● Transmission: Oral-Oral or Fecal-Oral routes
● Motile (Monopolar or Multi-bipolar);
Microaerophilic (can withstand acidic pH)
● Microscopy: G- spiral-shaped (S-shaped)
rods
● Culture: Gray with translucent colonies
● Biochemical Tests: (+) Oxidase, Catalase;
(+) Urease (Rapid Urease producers)
G.A.S.S
Note: Urease is a strong alkaline
enzyme that alters the pH of the
stomach
Helicobacter pylori
● Major cause of Type B Gastritis, Peptic
Ulcer, Gastric Carcinoma
● Primary Habitat: Human Gastric Mucosa
(Mucous Layers of the Antrum and Fundus of
the stomach)
● Binds to Lewis Antigen and to Sialic Acid
Note: Those with Lewis Antigen and
Sialic Acid are susceptible to
Helicobacter infection
● Biochemical Tests: Strong-Urease
producer; (+) Oxidase, Catalase
• Important factors for survival:
Motility - allows organism to escape the
acidity
Helicobacter cinaedi and Helicobacter
fenneliae
● Isolated from blood of patients with
bacteremia and patients with HIV
Laboratory Tests for Helicobacter spp.
Specimen and Gram Stain
● Specimens:
○ Tissue Biopsy Material (Stuart’s
Medium), Urine (Ammonia testing),
Feces, Dental Plaques
Note:
• One of function of liver: Detoxification
• Test to check this function of liver:
conversion of ammonia to urea.
 • If the body can’t transform ammonia
to urea, there will be accumulation of
ammonia and may lead to hepatic
comatose - meaning that there is a
problem with the detox capability of the
liver.
● Susceptibility Test: Agar dilution using MHA +
5% Aged (≥ 2 weeks) sheep blood, incubated
at microaerophilic conditions
Differential Test for Campylobacter and
Helicobacter

• Accumulated Ammonia will go up the TEST Campylobacter

jejuni
Helicobacter pylori

brain and cross the blood-brain barrier Catalase Test + +

Nitrate Reduction + V
and accumulate there and lead to Test
comatose Urease Test - +
H2S Production - -
(TSI)
Hippurate + -
• H. pylori reconverts urea to ammonia. Hydrolysis
-Feces Indoxyl Acetate + -
Hydrolysis
-Dental Plaques Susceptibility
Testing:
Cephalothin (30 ug) Resistant Susceptible
● Gram Stain: Nalidixic (30ug) Susceptible Resistant
○ 0.1% Basic Fuchsin counterstain Growth at:
15 C - -
25 C - -
42 C + V
○ Stains for Biopsy:
Warthin-Starry
Note: Campylobacter and Helicobacter grow
Silver Stain
best at 42 C
Giemsa Stain
REVIEW QUESTIONS:
Note: since it causes ulceration and
carcinoma, biopsy is also one of the
1. Test that differentiates Aeromonas from
specimens for Helicobacter
Vibrio spp.?
-Vibriostatic disk
Culture
2. In Vibrio spp. all are non-lactose
● Media: CAP, MTM (Modified Thayer-Martin
fermenter except?
Agar), Skirrow Agar, 5% Sheep’s Blood
-V. vulnificus
Brucella Agar

● Transport: Stuart’s Media, Cysteine-Brucella 3. What is the reaction of Vibrio spp. in

Broth with 20% Glycerol (Tissue samples), oxidase test?
Isotonic Saline with 4% Glucose -Oxidase +

● May require >5 days incubation, Capnophilic 4. It is a gram-negative bacilli, comma

environment shaped with rapid/ darting shooting star
motility and is halophilic.
● Hemin - stimulates further growth -Vibrio

Other Tests 5. Virulence factor/toxin of Vibrio

● Urea Breath Test - Excellent sensitivity cholerae?
and specificity -Choleragen (Cholera Toxin)

● PCR - Sensitive method for H. pylori

6. What is the hallmark of cholera?
● H. pylori - Metronidazole susceptible -Rice-Watery stool (10-30x
defecation/day)

G.A.S.S
 7. It causes “summer diarrhea”
-Vibrio parahaemolyticus

8. Vibrio spp. that is endemic in Japan?

-Vibrio parahaemolyticus

9. It is the only lactose-positive Vibrio.

-Vibrio vulnificus

10. Agar used for selective isolation

of Vibrio cholerae and other
enteropathogenic Vibrios.
-Thiosulfate Citrate Bile Salts
Sucrose (TCBS) Agar

11. Causative agent of “red leg” disease in

amphibians
-Aeromonas

12. Motility of Campylobacter?

-Darting motility

13. Selective medium for Vibrio

parahaemolyticus
-Wagatsuma Agar

G.A.S.S