MLS 223 Clinical Bacteriology Module 6 Unit 2

MODULE 6
GRAM-NEGATIVE BACILLI
Unit 2: Curved, Gram-negative Bacilli
Unit Learning Outcome:
Describe the medically significant curved, gram-negative bacilli as to their general

characteristics, epidemiology, pathogenesis and clinical manifestations, diagnosis,
prevention and control.
Engage
Cholera is one of the oldest diseases with

pandemic potential. The world has
experienced seven cholera pandemics in
the last 194 years. The first of which
occurred between 1817 and 1923 and
largely originating in Jessore, India. The
epidemic continued and eventually
reached other continents including
Europe and the Americas. The seventh
pandemic began in 1961 in the Celebes
Islands, Indonesia, with spread to Asia, the Source: International Centre for Diarrhoeal Disease Research,
Bangladesh (ICDDR,B)
Middle East, and Africa and continues
today.
In the Philippines, the last case of cholera of the pandemic which commenced March
20, 1902, was reported to have occurred March 8, 1904. During that period 166,252
cases, with 109,461 deaths, were reported. Just recently, in 2019, the Department of
Health (DOH) reported a 211% increase of reported cholera cases from 918 in 2018 to 2,
856 cases in 2019. Find the official report as follows:
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Explore
This module unit discusses agents of diarrheal diseases and other infections by species
of Vibrio, Campylobacter, Helicobacter, and Aeromonas. This group of gram-negative,
curved rods are important causes of gastrointestinal diseases. The Vibrio spp. in
particular, has been associated with large epidemics and pandemics; in addition,
Campylobacter spp. infection may play a role in Guillain- Barré syndrome (GBS), and
Helicobacter pylori can cause ulcers and has been linked to gastric carcinoma.
Explain
Vibrio species
GENERAL CHARACTERISTICS
• Gram-negative curved or comma-shaped bacilli.
• Motile (polar, monotrichous flagellum), EXCEPT Vibrio metschnikovii; they exhibit
"darting: or "shooting star" motility
• Non-spore-forming
• Facultative anaerobic
• Aquatic organisms that are found in fresh water, brackish or estuarine water,
and marine or salt water
• Grow in alkaline media
• Most are halotolerant, some are halophilic
• Glucose fermentation
• Catalase(+), EXCEPT Vibrio metschnikovii
• Oxidase(+), EXCEPT Vibrio metschnikovii
Vibrio cholerae
COMMON NAME:
• V. cholerae is commonly called “Kommabacillus” because of the
characteristic curved or comma-shaped appearance of the individual
bacterial cells.
HABITAT AND TRANSMISSION

• Habitat is the human colon.
Man is the only known reservoir of V. cholerae. Human carriage may
persist after untreated infection for months after infection; however,
V. cholerae are sensitive to gastric acid, thus
permanent carrier state is rare. They require high infectious dose if the stomach
can survive and grow in fresh and acid is normal. An estimated 1010 organisms
brackish water. per milliliter are required to survive gastric
• Transmission is by fecal-oral route. passage in healthy persons; only about 100
organisms per milliliter are required in
The most common source of infection
hypochlorhydric persons, either because of
i s c o n t a m i n a t e d w a t e r. F o o d , previous gastrectomy or from ingestion of
especially shellfish (contaminated antacids in treatment of gastric ulcer disease.
from contaminated waters) eaten
raw, have also been a source of
infection.
PATHOGENESIS AND CLINICAL MANIFESTATIONS
• Disease: Cholera, which is also known as Asiatic cholera or epidemic cholera.

- The incubation period is 12 hours up to 3 days depending on the size of
the inoculum ingested.
- Symptoms include sudden onset of nausea, vomiting, abdominal
cramps and profuse "rice water" diarrhea (stool resembles water in
which the rice has been boiled) that may
be as many as 10 to 30 per day, containing
mucus, epithelial cells, and large numbers
of vibrios.
- In severe cholera, infected individual can
lose several liters of fluid, as much as 20-30
liters per day. If left untreated, it can result in
a rapid fluid and electrolyte loss that leads Note the sunken eyes of the child
to dehydration, hypovolemic shock, suffering from severe dehydration.
metabolic acidosis, and death in a matter
of hours.
• Virulence factors:
- Motility and mucinase
To cause disease, cells of V. cholerae must adhere to the gastric
and intestinal mucosal epithelial cells of the host. These bacteria
are motile and secrete mucinase, two properties that aid in the
penetration of the protective mucin layer that coats the surface of
the gastroenteric mucosa.
- Choleragen (cholera toxin)

The cholera toxin is a heat-labile enterotoxin, the genes for which
are encoded in a lysogenic phage.
It consists of 2 subunits: A (active) subunits and binding (B) subunits.
‣ The A subunit is composed of two peptides:

i. A1 with toxin activity; it activates adenylate
cyclase, causing increased levels of cyclic
adenosine monophosphate (cAMP) and
hypersecretion of electrolytes (Na+, K+, HCO3−)
and water out of the cell and into the lumen of the
intestine. The net effect is that the gastrointestinal
tract’s absorptive ability is overwhelmed, resulting in
the massive outpouring of watery stools.
ii. A2, which facilitates penetration of the A1 subunit
into the enterocyte.
‣ B subunit binds the A2 subunit to the GM1 ganglioside

receptor on the cell membrane of the enterocytes. There
are five B subunits per toxin molecule, arranged in a ring
around a central core that contains the enzyme A1.
The action of cholera toxin.
ANTIGENIC STRUCTURE
• H antigen (or flagellar antigen)

- Heat-labile substances that are found in all V. cholerae strains
• O antigen (or somatic antigen)

- Cell wall lipopolysaccharides that confer serologic specificity to the
bacterial cells
- This forms the basis of a serotyping scheme that classifies the 139 strains
of V. cholerae, designated from O1 to O139.
CLASSIFICATION
Within the species of V. cholerae, there is much dissimilarity among the strains in
both their pathogenic and epidemic potential.
• Serogroups of V. cholerae
V. cholerae strains are designated into 3 major serogroups based on
agglutination with V. cholerae O1 polyvalent antiserum. (see: Serological
Tests on p. 13)
1. V. cholerae O1
- Is agglutinated by V. cholerae O1 antiserum
- Includes virulent strains producing cholera toxin
- Associated with epidemic cholera
2. V. cholerae non-O1
- Is NOT agglutinated by V. cholerae O1 antiserum
- Includes V. cholerae O2 up to V. cholerae O138
- Does NOT produce cholera toxin; but, they appear to produce
an enterotoxin different from cholera toxin.
- Has been associated with isolated cases of diarrheal disease
(NOT epidemic-associated)
3. V. cholerae O139
- Is NOT agglutinated by V. cholerae O1 antiserum
- Represented a new serogroup, as it is a strain that could not be
identified as any of the 138 known types of V. cholerae
- Is also known as “Bengal strain” to indicate its first isolation from
the coastal areas of the Bay of Bengal; it caused large epidemics
of cholera in Bangladesh, India, and neighboring countries in
1992.
- Produces cholera toxin in quantities similar to that produced by V.
cholerae O1
- Makes polysaccharide capsule like other non-O1 strains (V.
cholerae O1 does not make a capsule.)
- Has spread in epidemic proportions across the Indian
subcontinent.
• Biotypes of V. cholerae O1
There are two biotypes of V. cholerae serogroup O1 based on their
differences in biochemical characteristics, which is associated with how
they differ with respect to the severity of the disease they can cause.
1. Classical strain of V. cholerae caused the epidemic cholera from early

1800s to early 1900 – during this time, there were 6 waves of cholera
pandemic that spread across the world.
2. El Tor strain of V. cholerae was isolated in the early 1900s from Mecca-
bound pilgrims at the El Tor Quarantine Station in Sinai Peninsula. These
vibrios resembled classical V. cholerae in many way but caused lysis of
goat or sheep erythrocytes in a test known as the Greig test. The El Tor
strain has been found to be hardier and better capable of surviving in
the environment. It was associated with the epidemic in the Phil in
1961 that has started the 7th pandemic. Currently, the El Tor biotype
of V, cholerae O1 is the predominant cholera pathogen. A number of
tests differentiate them from classical V. cholerae (see: Tests for
Determining Biotypes of V. cholerae on p. 14)
• Serotypes of V. cholerae O1
V. cholerae serogroup, in both the classical and the El Tor biotypes, are
separated into 3 serotypes based on differences in antigen determinants
on the O side chain of the lipopolysaccharide (LPS) antigen. These
serotypes are important for epidemiological studies. These can be
detected by agglutination with specific antiserum (see: Serological Tests
on p. 13)
1. Ogawa has determinants A, and B (Ogawa);
2. Inaba has determinants A, and C (Inaba);
3. Hikojima produces all 3 antigens (A, B, C).
The Ogawa and Inaba strains are considered the predominant epidemic
strains. Hikojima strain has been isolated in outbreaks , but its occurrence
has been rare.
PREVENTION AND CONTROL
• Control rests on education and on improvement of sanitation, particularly of
food and water.
• Patients should be isolated, their excreta disinfected, and contacts followed
up.
• Repeated injection of a vaccine containing either lipopolysaccharides
extracted from vibrios or dense Vibrio suspensions can confer limited protection
to heavily exposed persons (eg, family contacts) but is not effective as an
epidemic control measure.
LABORATORY DIAGNOSIS
Specimens
Feces
- Is the preferred specimen, collected and transported in Cary-Blair
medium.
- Buffered glycerol saline is not acceptable, because glycerol is toxic to
vibrios.
Rectal swabs
- Are also acceptable.
A. Microscopy
- Gram-negative curved or comma-shaped bacilli, 2–4

µm long.
On prolonged cultivation, vibrios may become
straight rods that resemble the Gram-negative
enteric bacteria.
V. cholerae, Gram stain.
B. Cultural method
• Culture media and cultural characterization
‣ ENRICHMENT MEDIUM
Alkaline Peptone Water (APW)
APW
- The 1% (w/v) sodium chloride incorporated in this medium

promotes the growth of Vibrio species, while the alkalinity (pH 8.6)
of this medium inhibits most of the commensal intestinal bacteria.
- Incubate at 35°C for 12-18 hours prior to inoculation of plated
media.
Enrichment in APW is done when few organisms are present, as in specimens from convalescent patients
and asymptomatic carriers.
When the patient is in very early stages of illness and is passing liquid stool, it may not be necessary to
enrich stool specimens.
‣ NONSELECTIVE MEDIA
1. 5% Sheep BAM
Colonies are typically medium to large,

smooth, convex, creamy in consistency,
gray-white, and have entire margins,
smooth, opaque; may be β-hemolytic. Non-hemolytic V. cholerae.
β-hemolytic El Tor biotype

of V. cholerae.
Rough colonies are

occasionally encountered
that adhere to the agar.
2. Gelatin Agar (GA)
Colonies appear transparent and usually

have a characteristic cloudy or opaque
zone (due to gelatinase activity) around
colony, which becomes more definite
after few minutes of refrigeration. When
these colonies are viewed in oblique light
they appear iridescent green to bronze
colored and finely granular
Suspicious colonies can be used directly for further testing. V. cholerae on GA.
‣ SELECTIVE MEDIA
1. Thiosulfate Citrate Bile Sucrose (TCBS) Agar
TCBS. Uninoculated.
- Selective medium of choice for the isolation of V. cholerae and
V. parahaemolyticus as well as other vibrios.
- 1% Sodium chloride stimulates growth of Vibrio species.

Bile salts (oxgall) inhibit gram-positive bacteria.
Sodium thiosulfate serves as a sulfur source and, in combination
with ferric citrate, detects hydrogen sulfide production.
Saccharose (sucrose) is included as a fermentable carbohydrate
for the metabolism of vibrios.
The alkaline pH (8.6) of the medium inhibits most of the
commensal intestinal bacteria.
Thymol blue and bromthymol blue are included as indicators of
pH changes.
- During preparation, heat to boiling to dissolve the agar. DO NOT

AUTOCLAVE.
V. cholerae on TCBS. Overnight growth (18 to 24 hours) will

produce large (2 to 4 mm in diameter), slightly flattened,
yellow colonies (due to sucrose fermentation) with opaque
centers and translucent peripheries.
Suspicious colonies for further testing should be subcultured to a noninhibitory medium, such as
gelatin agar, heart infusion agar (HIA), Kligler’s iron agar (KIA), or triple sugar iron agar (TSI).
2. Taurocholate tellurite gelatin agar (TTGA or Monsur’s agar)

Approximate Formula Per Lite
Enzymatic digest of casein...................................................10 g
Sodium Chloride...................................................................10 g
Sodium taurocholate.............................................................5 g
Sodium carbonate.................................................................1 g
Gelatin...................................................................................30 g
Agar.......................................................................................13 g
Potassium Tellurite 0.1% Supplement...................................5 g
- Sodium taurocholate inhibits the contaminating gram-positive

bacteria.
Potassium tellurite is a selective and differential agent. It inhibits
many gram-positive bacteria and due to the reduction reaction
the colonies form a grey to black color.
Sodium carbonate helps in maintaining the elevated pH of the
medium which is also inhibitory to most Enterobacteriaceae and
gram-positive bacteria.
Gelatin acts as an additional carbon and energy source and is
the substrate of bacterial gelatinase causing halo effect around
the colony.
V. cholerae on TTGA. Overnight growth appears as small (1-2 mm),
opaque colonies with slightly dark centers. After 24 hours, the centers
of the colonies become darker, and eventually the entire colony
becomes “gunmetal” grey in color. In addition to the dark coloration,
which is due to the reduction of tellurite surrounded by an opaque
zone which resembles a halo.
Suspicious colonies for further testing should be subcultured
to a noninhibitory medium.
3. MacConkey (MAC) Agar
MAC will also support the growth of some

but not all strains of V. cholerae.
Overnight colonies of V. cholerae on
MAC tend to be small to moderately
sized (1 to 3 mm) and usually appear as
lactose-negative (colorless) or slightly
pink, often resembling colonies of “late”
or “slow” lactose-fermenting organisms. V. cholerae on MAC.
Suspicious colonies can be used directly for further testing.
C. Identification tests
1. Oxidase (+)
- A characteristic that separates them from the
Enterobacteriaceae. Please refer to notes on to Ex. No. 21 -
- Conduct the oxidase test with fresh growth from an Neisseriaceae and Moraxella catarrhalis
for the principle and procedure of
HIA slant or any non-carbohydrate-containing oxidase test.
medium. Do not use growth from TCBS agar.
2. TSI: A/A, no gas, no H2S; KIA:K/A, no gas, no H2S

- Gluc+, Lac-, Suc+ Please refer to notes on to Ex. No. 25 -
Family Enterobacteriaceae and Related
enterobacteria for the principle and
procedure of:
3. Decarboxylase/Dihydrolase Test - Triple Sugar Iron Agar (TSI)
- Lysine Iron Agar (LIA)
- Modified by the addition of 1% NaCl - Decarboxylase test
- Lysine decarboxylation (+) - LIA: K/K
Ornithine decarboxylation (+)
Arginine dihydrolase (-)
4. String Test
- Principle:
Vibrio species suspended in 0.5% sodium desoxycholate will be lysed.
The suspension will lose turbidity, and DNA will be released from the lysed
cells causing the mixture to become viscous.
- Procedure:
18- to 24-hour colonies from HIA or other noninhibitory medium are mixed
with a few drops of 0.5% sodium desoxycholate
on a glass slide.
An inoculating loop is immersed into the mixture
and pulled away from the drop.
- Result:
Vibrio cholerae produces a long string that
becomes more tenacious after 60 seconds or
more (other vibrios may give an initial string
reaction that diminishes or disappears 45 to 60
seconds later.
V. cholerae. Positive string test.
5. Growth in Salt Broths
- Principle:
Vibrios either require NaCl or have their growth stimulated by its
addition.
- Procedure:
The 0% and 1% salt broths are inoculated very
lightly from fresh growth.
The inoculum should be light enough to prevent
visible turbidity before incubation of the broths.
The broths are incubated at 35˚ to 37˚C and
read at 18 to 24 hours. In the absence of
overnight growth, they may be incubated for up
to 7 days
- Result:
V. cholerae in nutrient broth base
V. cholerae grows in nutrient broth base without with 1.0% NaCl (A), and without
NaCl, and stimulated by addition of 1.0% NaCl NaCl (B).
Negative control (C).
6. Vibriostatic Test
- Uses 2,4-diamino-6,7-diisopropyl-pteridine phosphate
(referred to as O/129)-impregnated disks.
- V. cholerae: O129-susceptible
Other Vibrio species - O129-resistant
However, recent strains of V. cholerae O1
and V. cholerae O139 have demonstrated
resistance; therefore the dependability of this
test is questionable.
O129 susceptibility of V. cholerae.
7. Other biochemical tests:
a. Cholera red test (Nitroso-indole test)

Addition of H2SO4 to 24-h culture in APW with tryptophan and nitrate.
(+) result: Red color (due to nitroso indole)
b. Pfeiffer’s phenomenon
Lysis of V. cholerae organisms when inoculated into immuned guinea
pig
8. Serological Tests
- Principle:
Used to identify V. cholerae O1 serogroup, and further subdivide into
serotypes, Ogawa, Inaba, and Hikojima by agglutination with specific
antisera: anti-O1, Ogawa antiserum, and Inaba antiserum.
- Procedure:
i. Agglutination tests may be carried out in a petri dish or on a clean
glass slide.
ii. An inoculating needle or loop, or sterile applicator stick, or tooth pick is
used to remove a portion of the growth from the surface of HIA, KIA,
TSI, or other nonselective agar medium.
iii. Emulsify the growth in a small drop of physiological saline and mix
thoroughly by tilting back and forth for about 30 seconds. Examine the
suspension carefully to ensure that it is even and does not show
clumping due to autoagglutination. If clumping occurs, the culture is
termed “rough” and cannot be serotyped.
iv. Add a small drop of antiserum to the suspension. Usually approximately
equal volumes of antiserum and growth suspension are mixed, but the
volume of suspension may be as much as double the volume of the
antiserum.
v. Mix the suspension and antiserum well and then tilt slide back and
forth to observe for agglutination.
- Result:
(+) reaction: Very strong clumping will appear
within 30 seconds to 1 minute.
Agglutination with Antiserum

V. cholerae O1 serotypes
O1 Ogawa Inaba Antisera to the O1 serogroup of V.
Ogawa + + - cholerae will agglutinate homologous
organisms (left). A normal serum or
Inaba + - + saline control (right) does not show
Hikojima + + + agglutination.
Tests for Determining Biotypes of V. cholerae O1
.. Classical El Tor
β-Hemolytic on sheep blood agar - +
CAMP test - +
Voges-Proskauer test - +
Chicken red blood cell agglutination - +
Susceptibility to 50 U polymyxin B S R
Phage IVsusceptibility S R
+, positive test; −, negative test; S, susceptible; R, resistant.
• CAMP TEST
- For the principle and procedure, please refer to Ex. No. 20 - Streptococcaceae and Enterococcus species.
• Voges-Proskauer (VP) Test

- For the principle and procedure, please refer to Ex. No. 25 - Family
Enterobacteriaceae and Related Enterobacteria.
• Chicken red blood cell agglutination

- Fresh chicken or sheep red blood cells can be used for this assay.
- A 2.5% (vol/vol) suspension of washed (3 times) and packed (by
centrifugation) cells is made in normal saline after the final wash.
- A large loopful of the red cell suspension is placed on a glass slide.
- A small portion of the growth from a nonselective agar slant is added
to the red cells with a needle or loop, and is mixed well.
- In a positive test, agglutination of the red cells occurs within 30 to 60 Classic strains of Vibrio cholerae do not
seconds. agglutinate chicken erythrocytes (top),
in contrast to the El Tor biotype
(bottom), which is capable of
• Susceptibility to 50 U polymyxin B agglutinating the erythrocytes.
- A 50-unit polymyxin B disk is used for this test, and known strains of
classical and El Tor biotypes are used as controls.
• Phage IV susceptibility
- The isolate to be tested is grown overnight in pure culture on a noninhibitory medium.
- From the overnight growth, brain heart infusion (BHI) broth (or Trypticase soy broth) is inoculated and
incubated for 6 hours at 35˚ to 37˚C.
- A lawn of bacteria in log-phase growth (OD=0.1) is then seeded onto the surface of a BHI agar plate by dipping
a cotton swab into the 6-hour broth and lightly inoculating (swabbing) the entire surface of the plate.
- Positive and negative control strains should also be included.
- A drop of the bacteriophage diluted to routine test dilution is applied to the bacterial lawn.
- The plate is incubated overnight and read the next day.
- If the bacteria are susceptible to the bacteriophage they will be lysed, and there will be a zone of lysis in the
bacterial lawn.
Non-cholera VibrIos
A. Vibrio parahaemolyticus
• inhabits seawaters
• first recognized as a pathogen in Japan in 1950, when it caused a large food
poisoning outbreak
• causes gastroenteritis --- it is the second most common Vibrio species
implicated in gastroenteritis after V. cholerae. Infection results from
consumption of raw, improperly cooked, or recontaminated seafood,
particularly oysters. After an incubation period of 12–24 hours, nausea and
vomiting, abdominal cramps, fever, and watery to bloody diarrhea occur and
fecal leukocytes are often observed. The enteritis tends to subside
spontaneously in 1–4 days with no treatment other than restoration of water
and electrolyte balance.
• halophilic organism requiring 2-4% NaCl and grows slowly on nonselective
media
• No enterotoxin has yet been isolated from this organism; however, there is a n
association between hemolysin production and virulence, known as the
Kanagawa phenomenon.
Kanagawa phenomenon
- This refers to the β hemolysis of V. parahaemolyticus from clinical

specimens when grown on a special medium, Wagatsuma agar which is
a high-salt (7%) blood agar (defibrinated human or rabbit blood) medium
containing D-mannitol as the carbohydrate source.
- It is well recognized to be closely associated with human pathogenicity of
V. parahaemolyticus due to the expression of Thermostable Direct
Hemolysin (TDH). TDH damages the erythrocyte membrane by acting as
a pore-forming toxin
- Most environmental strains of V. parahaemolyticus do NOT manifest
Kanagawa phenomenon.
B. Vibrio vulnificus
• Causes the second most serious types of Vibrio-associated infections after

cholera
• Infections generally fall into two categories:
(1) Bacteremia
- Thought to occur through the gastrointestinal route after the
consumption of shellfish, especially raw oysters and in persons
who have alcoholism or liver disease.
- Often proceeds rapidly, with development of severe disease and
about 50% of the patients die.
(2) Wound infections
- May occur among normal or immunocompromised persons who
are in contact with water where the bacterium is present.
‐
- Infections may be mild but often proceed rapidly (over a few
hours), with development of bullous skin lesions, cellulitis, and
myositis with necrosis.
• Halophilic organism requiring 1% NaCl; shows good growth on blood agar
Key Identification Characteristics of Some Clinically-important Vibrio Species.

Growth in nutrient
Colony on broth with
Vibrio species Oxidase Lactose Sucrose Lys Orn Arg
TCBS
0% NaCl 1% NaCl
V. cholerae Yellow + V + + + - + +
V. parahaemolyticus Green + - - + + - - +
V. vulnificus Green + + - + V - - +
V. parahaemolyticus on TCBS.
Campylobacter species
The campylobacters were formerly classified with the vibrios because of their positive
oxidase and characteristic microscopic morphology but has been reclassified because
of DNA homology studies.
• They are primarily zoonotic organisms causing diseases collectively referred to as

campylobacterioses (s. campylobacteriosis).
•
• They have been known to cause abortion in domestic animals, such as cattle, sheep,
and swine.
Campylobacter jejuni
• Gram-negative, curved, S-shaped or gull-winged rods or long spiral forms
• Motile (single unsheathed polar flagellum)
• Microaerophilic
• Capnophilic
• Grows best at 42oC
• Catalase (+)
• Oxidase (+)
• Non-fermentative, non-oxidative

• The campylobacters are found worldwide as inhabitants of the GI tract and
oral cavity of humans and animals including wild or domesticated cattle,
sheep, swine, goats, dogs, cats, and fowl, especially turkeys and chickens.
• Transmission is by fecal-oral route. Infections
are acquired by ingestion of contaminated
beverages and improperly cooked foods, C. jejuni is susceptible to gastric acid, and
especially poultry; or direct contact with ingestion of about 104 organisms is usually
infected animals or animal products and their necessary to produce infection.
excreta.

• Disease: Gastroenteritis.
C. jejuni is known as the most common cause of bacterial gastroenteritis
worldwide.
- When ingested, the bacteria multiply in the mucosa of the last
segment of the small intestine (ileum); they invade the epithelium,
and produce inflammation.
- Symptoms become apparent after an incubation period of 1 to 7
days which include headache, fever, chills, abdominal pain
followed by cramps and bloody or watery diarrhea; and rarely
accompanied by nausea and vomiting.
- Stools often contain red and white blood cells.
- In most patients, the illness is self-limited and usually resolves in 5 to 8
days.
- Occasionally, the bloodstream is invaded, and a clinical picture of
enteric fever develops.
- Localized tissue invasion
- Endotoxin
- Campylobacter jejuni toxin (CJT)
A heat-labile enterotoxin that stimulates a secretory diarrhea like
that of cholera.
Campylobacter jejuni is a common antecedent to Guillain-Barré Syndrome.
- Guillain-Barré syndrome (GBS) is an autoimmune

disorder characterized by acute ascending paralysis
caused by damage to the peripheral nervous system.
- Strong evidence suggests that Campylobacter infection
plays a role in GBS since many patients with GBS test
positive for antibodies to Campylobacter,
- It is believed that antibodies produced during a
Campylobacter infection bind to gangliosides found on
peripheral nerves.
- Cross-reactivity with these nerve cells in an
autoimmune response may be responsible for this
debilitating nerve disorder.
Specimens
- Feces
- Rectal swabs
A. Microscopy
• Gram staining: Gram-negative, curved, S-shaped or

gull-winged, or long spiral forms.
C. jejuni, Gram stain.
B. Cultural method
• Selective media
- Contain antibiotics that suppress the growth of normal fecal flora.
Antimicrobial
Medium Base Additives
Agents
Blood-based media
Bacitracin
Novobiocin
Butzler’s selective Fluid thioglycollate Agar (3%) Colistin
medium Sheep blood (10%) Cephalothin
Actidione
Vancomycin
Blood agar base
Skirrow’s blood agar Lyzed horse blood (7%) Polymixin B
Trimethoprim
Vancomycin
Polymixin B
Blaser’s medium Brucella agar base Sheep blood (10%) Trimethoprim
(Campy-BAP) Cephalothin
Amphotericin
Cefoperazone
Brucella agar base
Campy CVA Agar Sheep blood (10%) Vancomycin
Amphotericin B
Blood-free media
Activated charcoal Cefoperazone
Campy CSM Agar Columbia Agar Base Hematin Cycloheximide
Sodium pyruvate Vancomycin
Preston Nutrient broth No. 2 Bacteriologic charcoal
Campylobacter blood- 1.2% New Zealand Sodiumdeoxycholate Cefoperazone
Ferrous sulfate
free medium agar Sodiumpyruvate
Casein hydrolysate
• Inoculation
Stool or rectal swab specimen is directly inoculated onto the surface of one
of the recommended selective agar media.
Formed stool specimens may also be processed by emulsifying a small
portion (peanut sized) in phosphate-buffered saline or broth before
inoculating one or two drops to the surface of the agar with a Pasteur
pipette; similarly, one or two drops of liquid stool specimens can be
inoculated directly.
• Incubation
- At 42°C for 24, 48, and 72 hours
- Microaerophilic atmosphere of 5% O2, 10% CO2, and 85% N2, which can
be achieved by:
i. Disposable gas generators (e.g., BBL CampyPak Plus disposable
gas generator envelope in GasPak jar)
ii. Evacuation-replacement procedures
The use of a CO2 incubator is not recommended for cultivating

campylobacters because only strains that are very aerotolerant grow
in the atmosphere provided.
Likewise, a candle extinction jar is not recommended because the
oxygen level (12% to 17%) is too high for optimal growth of
campylobacters.
• Colonial Characteristics
C. jejuni produces two types of colonies.
i. Small, raised, grayish-brown, smooth and glistening with an entire
translucent edge.
ii. Flat, mucoid, translucent, grayish and has an irregular edge.
A small percentage of strains may appear tan or slightly pinkish.

Colonies tend to spread, especially when initially isolated from fresh
clinical specimens.
Close-up view of Campylobacter jejuni on blood agar

illustrating raised, gray-white, and somewhat mucoid
colonies.
C. Identification Tests
• Hippurate hydrolysis-positive
Close-up view of Campylobacter jejuni on blood agar

illustrating raised, gray-white, and somewhat mucoid
colonies.
• Susceptibility test with nalidixic acid (30 µg) and cephalothin (30 µg
CF
Brucella agar plate showing the growth of C. jejuni

around cephalothin and nalidixic acid disks. Note
that with C. jejuni, a zone of inhibition forms around
NA
the nalidixic acid (NA) disk , indicating that this
species is susceptible to nalidixic acid. But, it is
resistant to cephalothin (CF)
Campylobacter fetus
• Has two subspecies: fetus and venerealis.

- C. fetus subspecies fetus is an opportunistic pathogen that causes systemic
infections in immunocompromised patients and elderly patients; the
gastrointestinal tract may be the portal of entry; it has been isolated most
frequently from blood cultures and is occasionally associated with
gastrointestinal illness.
- C. fetus subspecies venerealis is a rare cause of human disease.
• Disease is associated with several surface array proteins
- form a capsule-like structure on the surface of the organism
- has been compared with the polysaccharide capsules of pathogens such as
Neisseria meningitidis and Streptococcus pneumoniae.
- presence of which is correlated with the ability of the bacteria to cause
bacteremia after oral challenge and cause death in a high percentage of the
animals.
Key differentiation between C. jejuni and C. fetus
C. jejuni C. fetus
Growth at
25 oC - +
35-37 oC + +
42 oC + -
Hippurate hydrolysis + -
Susceptibility to
Nalidixic acid (30 µg) S R
Cephalothin (30 µg) R S
Helicobacter pylori
• Was initially called Campylobacter pyloridis and then renamed Campylobacter pylori.
• Gram-negative, curved or spiral forms
• Highly motile (multiple 4-6 sheathed flagella at one pole)
• Microaerophilic
• Capnophilic
• Grows well at 35-37oC
• Catalase (+)
• Oxidase (+)
• Urease (+)
• Nonfermentative, nonoxidative

• Colonizes mucous lining mainly of the antrum of the
stomach (also the duodenum) in 25% of healthy
middle-aged adults and in >60% of adults over 60
years of age. The major, if not exclusive, reservoir is
humans.
• Exact modes of transmission are not known. Several
theories have been proposed as its means of
transmission including:
- It is acquired early in life and carried
asymptomatically.
- Person-to-person transmission by the oral-oral H. pylori grows optimally at a pH of
6.0–7.0 and would be killed or not
to oral-fecal route is likely because grow at the pH within the gastric
intrafamilial clustering of infection occurs. lumen (1.0 to 2.0).
- Since other animals are likewise susceptible, it
has been proposed that the disease is a
zoonosis transmitted to humans from an
animal reservoir.
- It can also be spread by house flies acting as mechanical vectors
PATHOGENESIS AND CLINICAL MANIFESTATION
• Disease:
- Gastritis
- Peptic ulcers (especially, duodenal)
- Mucinase, a protease, modifies the gastric mucus and aids in the
penetration of the mucous layer of the gastric epithelium where
physiologic pH (about 7.4) is present, and rapidly shifts down to neutral
as it penetrates further.
- Urease activity yields production of ammonia that neutralizes stomach
acid. The ammonia produced is cytotoxic to gastric epithelial cells, too.
- Motility, even in mucus, enables the organism to find its way to the
epithelial surface.
- Vacuolating cytotoxin is associated with injury to the gastric epithelium.
There is also a strong evidence that H. pylori is linked to gastric adenocarcinoma and to the
development of gastric non-Hodgkin’s lymphoma (i.e., mucosa-associated lymphoid tissue [MALT]
lymphomas).
1. Gastric/duodenal biopsy by endoscopy
Silver-stained (Warthin-Starry)
a. Histologic examination tissue section of superficial gastric
- Staining with Warthin-Starry mucosa demonstrating clusters of
- Gram’s, Giemsa’s, or H&E. blue-black staining bacilli along the
epithelial lining, consistent with the
bacillary forms of H. pylori. When
observed in Gram-stained
preparation, the individual cells are
long, thick, and curved.
b. Biopsy Urease Test (BUT), CLO-Test (Campylobacter-like Organism)

- A mucosal biopsy sample is placed into urea medium, incubated at
37 oC for 2 hours; observe for a color change.
A positive urease test (pink to purple color) is

considered indicative of H. pylori's presence.
c. Culture
- Media:
Brucella agar with 5% sheep blood
Trypticase soy broth with 5% sheep blood
- Incubation:
35-37 oC
Microaerophilic atmosphere (5% O2, 10% CO2, 85% N2)
Prolonged incubation (3-5 days) required.
- Colonies:
Small (1-2 mm), circular, convex, translucent, gray.
2. Urea Breath Test (UBT)
- Principle:
Based on the urease activity of H.
pylori to break down urea, a
chemical made up of nitrogen and
carbon, into carbon dioxide and
ammonia. Carbon dioxide is
absorbed from the stomach and
eliminated in the breath.
- Procedure:
The patient ingests radioactive 14C-
labeled urea, followed by collection of breath samples that are
analyzed for the presence of labeled 14CO2 at 60 minutes by
scintillation counter.
3. Serodiagnosis Helicobacter pylori

Growth at
a. Antibody Detection 25 oC -
- Detects antibodies (IgG) 37 oC +
to H. pylori in blood samples. 42 oC + (some strains)
Hippurate hydrolysis -
b. Antigen Detection Susceptibility to
- Detects H. pylori antigens in Nalidixic acid (30 µg) R
stool samples Cephalothin (30 µg) S
Elaborate
Aeromonas species
• The genus Aeromonas previously resided in the family Vibrionaceae but phylogenetic
evidence from molecular studies resulted in the proposal of a separate family,
Aeromonadaceae.
• Gram-negative straight rods
• Motile by means of a single polar flagellum (with very few exceptions)
• All typically grow from 10° to 42° C
• Nonhalophiles
• Oxidase (+)
• Glucose fermenters

• The Aeromonas species are ubiquitous — they have been isolated in tap water,
freshwater, estuarine, marine environments worldwide, soil, and food.
• They are frequently isolated from retail produce sources and animal meat
products.

• Four categories of human infections have been described to be associated
with Aeromonas species:
(1) Gastroenteritis
- caused mostly by A. caviae complex
- other Aeromonas species associated with diarrhea are A.
hydrophila and A. veronii
- manifestations ranges from acute watery diarrhea to less
commonly a dysenteric type of illness
- five diarrheal presentations are observed in patients in whom an
Aeromonas has been isolated from their stools:
a. an acute, secretory diarrhea often accompanied by
vomiting
b. an acute, dysenteric form of diarrhea (similar to
shigellosis) with blood and mucus
c. a chronic diarrhea usually lasting more than 10 days
d. a cholera-like disease, including rice-water stools
e. Traveler’s diarrhea (similar to enterotoxigenic E. coli).
Most cases are self-limiting, but in the pediatric, geriatric,
and immunocompromised populations, supportive
therapy and antimicrobials are often indicated.
(2) Cellulitis and wound infections

- Invariably involves a recent traumatic aquatic exposure and
generally occurs on the extremities.
- The most common presentation is cellulitis, although there have
been a few cases of myonecrosis and necrotizing fasciitis and
even a rare case of ecthyma gangrenosum associated with
sepsis.
- Aeromonad wound isolates are predominantly A. hydrophila
subsp. hydrophilia
(3) Septicemia
- Is one of the most invasive type of Aeromonas infection
- Has a strong association with the species A. veronii biovar sobria,
and A. hydrophila.
- Occurs in patients who are immunocompromised and have a
history of liver disease or dysfunction, hematologic malignancies,
hepatobiliary disorders, or traumatic injuries.
(4) Miscellaneous
- Aeromonas species have also been implicated in cases of
meningitis, osteomyelitis, pelvic abscesses, otitis, cystitis,
endocarditis, peritonitis, cholecystitis, keratitis associated with
contact lens wear, and endophthalmitis in healthy and
immunocompromised individuals.
• Pathology has been associated with:

- Production of hemolysins
- Production of enterotoxins by some strains
- The ability to invade cells
Aeromonas species A. caviae A. hydrophila A. veronii biovar A. veronii biovar

sobria veronii
Growth on McConkey Agar After 24-hour incubation at 35°C, aeromonads appear as round, raised, opaque
lactose-fermenting colonies with an entire edge and a smooth, often mucoid,
surface.
Growth in nutrient broth

- - - -
with 6% NaCl
Oxidase Test + + + +
Voges-Proskauer Test - + + +
Esculin hydrolysis + + - +
Fermentation
Glucose (gas) - + + +
Sucrose + + + +
Mannitol + + + +
MLS 223 Evaluate_6.2
Matching type. Match the descriptions of diseases in column A with the causative
agents in column B. (5 points)
A B
1. Infections caused may fall into one of four categories A. V. cholerae
which vary depending on the site affected and B. V. parahaemolyticus
frequency. C. V. vulnificus
2. Causes a disease characterized by profuse watery D. C. jejuni
diarrhea with stools likened to “rice water” that may E. C. fetus
be as many as 10 to 30 per day. F. H. pylori
3. Has been linked with malignancies in the GIT and G. Aeromonas specie
lymphoid tissue.
4. Gastrointestinal infections are considered an
antecedent to the development of an autoimmune
disorder.
5. Infection of the blood originates from the
gastrointestinal route after ingestion of contaminated
raw shellfish.
MLS 223_Evaluate S26
MULTIPLE CHOICE: Select the BEST answer.
1. Gram staining of Vibrio cholerae shows:

A. small, Gram-negative rods resembling "gull wings"
B. long, Gram-negative curved or comma-shaped bacilli
C. Gram-positive pleomorphic bacilli
D. Gram-positive cocci in chains
2. Vibrio cholerae can be isolated best in feces on:

A. MacConkey agar
B. Eosin-Methylene Blue Agar
C. Thiosulfate Citrate Bile Sucrose Agar
D. Skirrow's Blood Agar
3. Kanagawa phenomenon-positive Vibrio parahaemolyticus strains have been

considered as:
A. motile
B. sucrose fermenter
C. virulent
D. microaerophilic
4. The optimal incubator temperature for isolation of Campylobacter jejuni is:

A. 4oC
B. 20oC
C. 35oC
D. 42oC
5. To obtain a presumptive evidence of Helicobacter pylori in a gastric biopsy from a

patient with peptic ulcer, a portion of the specimen should be added to:
A. Butzler’s selective medium
B. Urea medium
C. Thiosulfate Citrate Bile Sucrose Agar
D. CIN agar
References:
Carroll, K. C. (2016). Jawetz, Melnick & Adelberg's medical microbiology (27th

ed.). New York: McGraw-Hill Education.
Madigan, M. T., Martinko, J. M., Bender, K. S., Buckley, D. H., & Stahl, D. A. (2015).
Brock Biology of Microorganisms(14th ed.). Glenview, Illinois: Pearson
Education.
McPherson, Richard, and Matthew Pincus. Henry's Clinical Diagnosis and

Management by Laboratory Methods E-Book. 22nd ed., Saunders, 2011.
Pommerville, J. C. (2018). Alcamo's Fundamentals of Microbiology (11th ed.).

Burlington: Jones & Bartlett Learning.
Procop, G. W., Church, D. L., Hall, G. S., Janda, W. M., Koneman, E. W.,
Schreckenberger, P. C., & Woods, G. L. (2017). Color Atlas and Textbook of
Diagnostic Microbiology (7th ed.). Philadelphia: Wolters Kluwer Health.
Talaro, K. P., & Chess, B. (2018). Foundations in Microbiology (10th ed.). McGraw
Hill.
Tortora, G. J., Funke, B. R., Case, C. L., Weber, D., & Bair, W. (2020). Microbiology:
An introduction (12th ed.). Upper Saddle River: Pearson.

MLS 223 Clinical Bacteriology Module 6 Unit 2

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MLS 223 Clinical Bacteriology Module 6 Unit 2

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MODULE 6

Unit 2: Curved, Gram-negative Bacilli

Unit Learning Outcome:

Describe the medically significant curved, gram-negative bacilli as to their general

Cholera is one of the oldest diseases with

HABITAT AND TRANSMISSION

PATHOGENESIS AND CLINICAL MANIFESTATIONS

• Disease: Cholera, which is also known as Asiatic cholera or epidemic cholera.

- Choleragen (cholera toxin)

It consists of 2 subunits: A (active) subunits and binding (B) subunits.

‣ The A subunit is composed of two peptides:

‣ B subunit binds the A2 subunit to the GM1 ganglioside

• H antigen (or flagellar antigen)

• O antigen (or somatic antigen)

1. Classical strain of V. cholerae caused the epidemic cholera from early

- Gram-negative curved or comma-shaped bacilli, 2–4

• Culture media and cultural characterization

Alkaline Peptone Water (APW)

- The 1% (w/v) sodium chloride incorporated in this medium

Colonies are typically medium to large,

β-hemolytic El Tor biotype

Rough colonies are

2. Gelatin Agar (GA)

Colonies appear transparent and usually

1. Thiosulfate Citrate Bile Sucrose (TCBS) Agar

- 1% Sodium chloride stimulates growth of Vibrio species.

- During preparation, heat to boiling to dissolve the agar. DO NOT

V. cholerae on TCBS. Overnight growth (18 to 24 hours) will

2. Taurocholate tellurite gelatin agar (TTGA or Monsur’s agar)

- Sodium taurocholate inhibits the contaminating gram-positive

3. MacConkey (MAC) Agar

MAC will also support the growth of some

Suspicious colonies can be used directly for further testing.

2. TSI: A/A, no gas, no H2S; KIA:K/A, no gas, no H2S

5. Growth in Salt Broths

O129 susceptibility of V. cholerae.

a. Cholera red test (Nitroso-indole test)

Agglutination with Antiserum

• Voges-Proskauer (VP) Test

• Chicken red blood cell agglutination

- This refers to the β hemolysis of V. parahaemolyticus from clinical

• Causes the second most serious types of Vibrio-associated infections after

Key Identification Characteristics of Some Clinically-important Vibrio Species.

• They are primarily zoonotic organisms causing diseases collectively referred to as

HABITAT AND TRANSMISSION

PATHOGENESIS AND CLINICAL MANIFESTATIONS

Campylobacter jejuni is a common antecedent to Guillain-Barré Syndrome.

- Guillain-Barré syndrome (GBS) is an autoimmune

• Gram staining: Gram-negative, curved, S-shaped or

The use of a CO2 incubator is not recommended for cultivating

A small percentage of strains may appear tan or slightly pinkish.

Close-up view of Campylobacter jejuni on blood agar

Close-up view of Campylobacter jejuni on blood agar

Brucella agar plate showing the growth of C. jejuni

• Has two subspecies: fetus and venerealis.

HABITAT AND TRANSMISSION

1. Gastric/duodenal biopsy by endoscopy

b. Biopsy Urease Test (BUT), CLO-Test (Campylobacter-like Organism)

A positive urease test (pink to purple color) is

2. Urea Breath Test (UBT)

3. Serodiagnosis Helicobacter pylori

HABITAT AND TRANSMISSION

PATHOGENESIS AND CLINICAL MANIFESTATIONS

(2) Cellulitis and wound infections