Clinical Bacteriology Lecture

18

Gram Negative Bacilli || Lesson Reviewer || 3rd Year, 1st Semester [Semi-Finals]

VIBRIO OTHER VIBRIO

GENERAL CHARACTERISTICS
v. parahaemolyticus
Þ Gram-negative, facultative anaerobic, curved or comma-shaped rods
Þ Second most common Vibrio species implicated in gastroenteritis
Þ Require sodium for growth and glucose fermentation
Þ Summer diarrhea in Japan
Þ All species are motile and catalase and oxidase positive except Vibrio
metschnikovii Þ serotype O3:K6 – pandemic strain
Þ Consumption of raw, improperly cooked, or recontaminated seafood,
particularly oysters
EPIDEMIOLOGY
Þ Kanagawa phenomenon
Þ Primary habitat is water
It has been observed that most clinical V. parahaemolyticus strains produce a heat-stable hemolysin that
– Brackish or marine water for Vibrio spp. is able to lyse human erythrocytes in a special, high-salt mannitol medium (Wagatsuma agar). These
strains are considered Kanagawa toxin-positive, whereas most environmental isolates are Kanagawa
– Freshwater for Aeromonas spp. toxin-negative.
– Soil or water for C. violaceum
Þ Transmission to humans is by ingestion of contaminated water, fresh v. vulnificus
produce, meat, dairy products, or seafood by exposure of disrupted skin
and mucosal surfaces to contaminated water. Þ Second most serious types of Vibrio-associated infections
None of these organisms are considered part of the normal human microbiota.
Þ Two categories: primary septicemia and wound infections
Þ Consumption of shellfish, especially raw oysters
Infections caused by V. vulnificus generally fall into two categories, primary septicemia and wound
V. CHOLERAE infections. The former is thought to occur through the gastrointestinal route after the consumption of
shellfish, especially raw oysters.
Þ Causes epidemics and pandemics
Patients with liver dysfunction and syndromes that result in increased serum levels of iron (e.g.,
Þ Diarrheal disease cholera hemochromatosis, cirrhosis, thalassemia major, hepatitis) are particularly predisposed to this scenario.
Patients with V. vulnificus wound infections invariably have a history of some type of traumatic aquatic
Þ Reservoir: humans wound that often presents as a cellulitis.
Þ Has dormant stages
Þ Virulence factors: v. alginolyticus
§ Cholera toxin (CT) / Choleragen
Þ Least pathogenic for humans
§ Zonula occludens (Zot) toxin (enterotoxin)
Þ Originate from extraintestinal sources
§ Accessory cholera enterotoxin (Ace) toxin
Þ Occupational hazard for people in constant contact with seawater such as
§ O1 and O139 somatic antigens
fishermen or sailors
§ Hemolysin / cytotoxins
§ Motility, chemotaxis, mucinase, and toxin coregulated pili (TCP)
Þ 3 major subgroups: SPECIMEN COLLECTION & TRANSPORT
§ V. cholerae O1 Þ ✔CARY-BLAIR
§ V. cholerae O139 Þ ✘Buffered glycerol saline
§ V. cholerae non-O1/non-O139 Þ Strips of blotting paper soaked in liquid stool and placed in airtight
Since 1817, the world has witnessed seven cholera pandemics. In 2010, after a devastating earthquake in Haiti, plastic bags with a few drops of saline to maintain moisture are
more than 604,634 infections and 7436 deaths occurred as a result of V. cholerae O1 infections within a 24- considered viable specimens for up to 5 weeks.
month period. During these outbreaks the organism was spread among people by the fecal-oral route due to
poor sanitation. Evidence suggests that the bacillus has dormant stages that allow its long-term survival in Whenever possible, body fluids, pus, or tissues should be submitted, but swabs are acceptable if they
brackish water or saltwater environments during interepidemic periods. These dormant stages are considered are transported in an appropriate holding medium, such as Cary-Blair, to prevent desiccation. Buffered
viable but nonculturable. glycerol saline is not recommended as transport or holding medium because glycerol is toxic for vibrios.
To effectively release toxin, the organism first must infiltrate and distribute itself along the cells lining the
mucosal surface of the gastrointestinal tract. Motility and chemotaxis mediate the distribution of organisms,
and mucinase production allows penetration of the mucous layer. Toxin coregulated pili (TCP) provide the
means by which bacilli attach to mucosal cells for release of CT. The enterotoxin zona occludens toxin (Zot) has DIRECT DETECTION METHODS
been shown to disrupt the tight junctions of the intestinal cells, effectively decreasing tissue resistance.
The somatic antigens O1 and O139 associated with the V. cholerae cell envelope are positive markers for • ELISA for V. cholerae toxin
strains capable of epidemic and pandemic spread of the disease. Strains carrying these markers almost always
produce CT, whereas non-O1/non-O139 strains do not produce the toxin and hence do not produce cholera. • Commercially available latex agglutination test
The non-O1/non-O139 strains are associated with nonepidemic diarrhea and extraintestinal infections.
• Rapid antigen tests and Immunochromatographic assays
1. Hypersecretion of water and electrolytes by mucosal cells
2. Profuse, watery diarrhea (“rice water stool”)

CHOLERA 3. Dramatic fluid loss GRAM STAIN

TOXIN 4. Severe dehydration and hypotension Þ Gram-negative, straight or slightly curved rods
CT will trigger the hypersecretion of water… leading to the production of profuse,
watery diarrhea (hallmark of cholera toxin activity), which would produce a dramatic Þ Rapid darting or shooting-star motility (dark-field microscopy)
fluid loss to a patient, and then leading to severe dehydration and hypotension. If the
fluid is not replaced, it can lead to death.
Transcribed from Ma’am Abigail Paulan’s lecture video _________________________________________________________________________________ O_Ongkay Caylo
 Clinical Bacteriology Lecture
19

CULTURE MEDIA A E R O M O N A S S P P.
Þ Thiosulfate citrate bile salts sucrose (TCBS) agar – for stool cultures AEROMONAS
– Sucrose fermenters – yellow colonies Þ Oxidase-positive, glucose-fermenting, gram-negative rods
– Non-sucrose fermenters – green
Þ Widely distributed in freshwater, estuarine, and marine environments
Þ Alkaline peptone water (pH 8.4) – enrichment broth worldwide
Þ MAC / SS: non-lactose fermenters except V. vulnificus Þ Isolated from retail produce sources and animal meat products
– Perform oxidase test from SBA plate Þ Typically grow from 10o–24oC
TCBS contains 1% sodium chloride, bile salts that inhibit the growth of gram-positive organisms, and sucrose Þ Classified into one of two groups
for the differentiation of the various Vibrio spp. Bromothymol blue and thymol blue pH indicators are added
to the medium. The high pH of the medium (8.6) inhibits the growth of other intestinal microbiota. § Mesophilic group – A. hydrophilia complex, A. veronii complex,
Alkaline peptone water (pH 8.4) may be used as an enrichment broth for obtaining growth of vibrios from stool. A. caviae complex
After inoculation, the broth is incubated for 5 to 8 hours at 35°C and then subcultured to TCBS. Oxidase testing
is unreliable when performed on colonies grown on TCBS media. § Psychrophilic group – A. salmonicida
SO
To evaluate an SBA plate, always accompany with oxidase test because lactose-positive colonies from selective
K192-07 LWBK192-Hubbard
differential media, such as MAC agar, can give false- positive oxidase reactions. These
November organisms
21, 2008 14:10are
considered mesophiles because they grow well at 37oC. They are all motile by
means of a single polar flagellum.
The psychrophilic group (optimal growth around 22oC) consists of only one species, A. salmonicida,
268 A Concise Review of Clinical Laboratory Science
STRING TEST which is a fish pathogen with several subspecies. This organism is nonmotile and grows best at 22o to
25oC. Psychrophilic nonmotile strains are not considered human pathogens.
Þ 0.5% SODIUM DEOXYCHOLATE Table 7–17 Isolation of Vibrio Species on TCBS Agar
Species Colony
Þ Lyses Vibrio cellsO1(+), but notYellow
those of Aeromonas spp. (—)
V. cholerae
V. cholerae non-O1 Yellow CLINICAL MANIFESTATIONS
The string test can beV.used to differentiate Vibrio spp.
alginolyticus from Aeromonas spp. In this test, organisms are emulsified in
Yellow

intestinal infections
0.5% sodium deoxycholate, which lyses Vibrio cells, Dark
V. parahaemolyticus but not those of Aeromonas spp. Cell lysis releases deoxyribonucleic
blue-green
acid (DNA), which canV.bevulnificus
pulled up into a string withDark
an inoculating
blue-greenloop.
TCBS = thiosulfate-citrate-bile salts-sucrose.
§ A. caviae – neonate and pediatric populations
of the clinical specimen. Isolated colonies are straight to pleomorphic gram-negative
VIBROSTATIC TEST (0129)
rods. The organisms are usually isolated from stool specimens. V. cholerae can be
enriched by using alkaline peptone water (pH 8.4). This suppresses the growth of
§ A. hydrophila – HUS or kidney disease
other organisms. All Vibrio species grow well on routine media. The Vibrio species are
Þ Vibrios – Susceptible “halophilic” or “salt-loving,” and with the exception of V. cholerae and V. mimicus,
§ A. veronii biovar sobria – also HUS and cholera-like disease
all species require salt for growth. The differential medium of choice is Thiosulfate
Þ Oxidase-positive glucose fermenters – Resistant
citrate bile salts surcose (TCBS) agar (see Web Color Images 7–65 and 7–66). All
isolates are indole and oxidase-positive (see Web Color Image 7–38), and have a
Þ V. cholerae O1 – Susceptible
fermentative metabolism. The reactions of the various Vibrio species on TCBS are extraintestinal infections
summarized in Table 7–17.
404 PART
Þ V. cholerae non-O1 - Resistant
III Bacteriology
7. Aeromonas and Plesiomonas are found in fresh and salt waters. They may cause Þ Septicemia and wound infections are the most common – A.
diarrheal disease as well as other miscellaneous infections. Normally, only patients
TABLE
who have underlying disease are treated. This type of diarrhea diarrheal infection is hydrophila subsp. Hydrophilia
25-4 usually
Key Biochemical and self-limiting.
Physiologic The role of
Characteristics Aeromonas
ofVibrio and
spp. and Plesiomonas
Grimontia in diarrheal disease is
hollisae
not well established. These organisms grow well on blood and MacConkey agar,
Þ Surgical wound infections involving the use of leeches for medicinal
Ornithine Decarboxylasea

are oxidase positive, and ferment glucose. These bacteria are gram-negative rods.
Lysine Decarboxylasea

Plesiomonas may show long filamentous forms. Differentiation of Aeromonas and

therapy after plastic surgery to relieve venous congestion – A. veronii
Arginine Dihydrolasea

Growth in 0% NaClb

Growth in 6% NaClb

Plesiomonas is summarized in Table 7–19.

Gas from Glucose

Colony on TCBSc

8. Differentiation of Aeromonas, Plesiomonas, and Vibrio is summarized in Table 7–20. biovar sobria
TCBSc Growth
Oxidase

Sucrose

Wound infection invariably involves a recent traumatic aquatic exposure, such as a boating or fishing accident,
Species

Lactose

XIV. GRAM-NEGATIVE NONFERMENTATIVE BACILLI

Indole

and generally occurs on the extremities. The most common presentation is cellulitis. Aeromonad sepsis is one
of the most invasive type of Aeromonas infection and similarly has a strong association with the species A.
Grimontii hollisaeA. This large
! group
! of"organisms" uses
" biochemical
" " pathways
" " other! than
Veryfermentation.
poor They
Greenmay
be oxidizers asaccharolytic (nonoxidizers). veronii biovar sobria, A. jandaei, and A. hydrophila. Such patients are most likely to be immunocompromised.
Vibrio alginolyticus ! v (see "
Web Color
" Image
! !7–67)
" or they
v may
" be ! Good Yellow
Gram-negative nonfermentative bacilli account for approximately 15% of gram-negative
Vibrio cholerae ! ! " v ! ! " ! ! v Good Yellow
rod-shaped bacteria isolated in the clinical laboratory. They are found in the environment
Vibrio cincinnatiensisd ! v " " ! v " " " ! Very poor Yellow
and cause disease in immunocompromised individuals. Virtually all of these organisms
Photobacterium damsela are opportunists.
! " "An isolate
" v
"that grows!on blood
" agar
" but Reduced
! not at 36°C agarGreen
MacConkey should
e
CULTURE MEDIA
Vibrio fluvialis be suspected
! v of being
" a"nonfermenter.
! " This
! is "especially
" true
! ifGood
the isolate is also Yellow
oxidase
Vibrio furnissii positive.
! Nonfermenters
v ! "that do
! grow" on !MacConkey
" "agar!appear
Goodas lactose negative.
Yellow Þ Display strong β–hemolysis – A. hydrophila, A. veronii biovar sobria,
Vibrio harveyi
B.
! ! " " v !
The most commonly isolated gram-negative, nonfermentative, rod-shaped bacteria are listed
" " " ! Good Yellow
and A. jandaei
Vibrio metschnikovii
in Box"7–6. v " v ! v v " " v May be reduced Yellow
Vibrio mimicus ! ! " v " ! " ! ! v Good Green
Þ Most commonly isolated species – A. caviae
C. The
Vibrio parahaemolyticus most
! frequently
! " isolated
" "gram-negative
! " nonfermenter
! " ! is Pseudomonas
Good aeruginosa.
Greenf
Vibrio vulnificus This organism
! ! is commonly
" (!) seen
" in!patients
" who " serious
! have ! burns and cystic fibrosis.
Good Greeng Þ Ferments lactose in MAC
Þ May resemble Yersinia enterocolitica in CIN
!, #90% of strains are positive; ", #90% of strains are negative; (!), delayed; V, variable.
Table
a 7–18
1% NaCl added toIdentification
enhance growth. of Vibrio Species
b
Nutrient broth with 0% or 6%Growth
NaCl added.in NaCl Acid from: Susceptible to O/129
c
Thiosulfate citrate bile salts sucrose agar.

e
d
Ferments myoinositol.
Species
5% yellow.
0% 8% VP Lactose Sucrose Arabinose 10 mg 150 mg Ferments lactose in MAC so perform an oxidase test, especially when it is taken from SBA to easily separate
V. alginolyticus
f
1% yellow. − + + + − − − V oxidase-positive Aeromonads from the oxidase-negative Yersinia.
g
0% yellow.
V. cholerae + − V + − − V +
V. parahaemolyticus − + − − − + − V
V. vulnificus − − − V + − + +
amount of salt is sufficient to allow
V = variable; VP = Voges-Proskauer; O/129 = 2,4,diamino-6,7-diisopropylpteridine phosphate; + = positive; – = negative.
organism.
growth of the halophilic PRESUMPTIVE IDENTIFICATION
Several key tests can aid in the initial identification of a VibrioMatrix-assisted isolate. Vibrios candesorption
laser be easilyionization
confusedtime-of-flight
with other
genera, including Aeromonas, Plesiomonas, Pseudomonas and massmembers of the(MALDI-TOF
spectrometry family Enterobacteriaceae.
MS) could provide a Þ Oxidase positive
rapid identification method for the identification of vibrios
– Their general susceptibility to the vibriostatic agent O/129isolated (150 μg) fromand positive string test
In adistinguishes them
from Aeromonas.
clinical specimens. recent study, Cheng
et al. (2015) evaluated the identification of V. vulnificus,
Þ Indole – positive
– Inability to ferment inositol (except for V. cincinnatiensis &V.some parahaemolyticus, V. fluvialis, andseparates
strains of V. metschnikovii) 33 speciesthem
of O1from
and
Þ Grow well in nutrient broth with 0% NaCl, but not in 6% NaCl
non-O139 V. cholerae. The system was not able to identify
Plesiomonas. the serogroup types O1 and non-O139 to the species level.
– Their positive oxidase reaction (except for V. metschnikovii) separates
The ability themcommercial
of most from the identification
Enterobacteriaceae
systems Þ Ferments glucose with or without drugs
(excluding Plesiomonas shigelloides), and to accurately identify Aeromonas organisms to the species
A B level is limited and uncertain, and with some kits, difficulty
– Carbohydrate fermentation metabolism separates them from A test to separate aeromonads and plesiomonads from most vibrios is determining the ability to grow in the
arisesthe
in oxidative
separatingPseudomonas.
Aeromonas spp. from Vibrio spp. There-
presence of NaCl. Aeromonads and plesiomonads grow well in nutrient broth with 0% NaCl, but not in 6% NaCl.
All species, except V. cholerae and V. mimicus, are halophilic,fore or salt-loving,
identification and ofrequire the addition
potential pathogensof sodium
should forbe
growth. Interspecies differentiation can be done through growth in Sodium chloride andbiochemical
confirmed using conventional productiontestsof acid from
or serotyp- Conversely, most vibrios, specifically the halophilic species, cannot grow in 0% NaCl but thrive in 6% NaCl and
lactose, sucrose, and arabinose. ing. Tables 25-4 and 25-5 show several characteristics that even higher concentrations of NaC.
can be used to presumptively group Vibrio spp., Aeromonas
spp., and C. violaceum. New technologies such as nucleic
____________________________________________________________________________________________ O_Ongkay Caylo
• Figure - Colonies of Vibrio cholerae (A) and V. parahaemolyticus
(B) on thiosulfate citrate bile salts sucrose (TCBS) agar. acid–based testing and MALDI-TOF MS for additional
 Clinical Bacteriology Lecture
20

However, because V. cholerae and V. mimicus are nonhalophilic and grow well without additional salt, any salt
tolerance test must be used in conjunction with the string test and O/129 disk to distinguish aeromonads from
this major pandemic cholera species and the less common, sucrose-negative V. mimicus. Fermentation of
inositol can be used for separating aeromonads from plesiomonads, in which aeromonads are negative and
plesiomonads are positive. Finally, the ability to ferment glucose, with or without the production of gas,
distinguishes Aeromonas from oxidase-positive, nonfermenting Pseudomonas isolates.
CHAPTER 20 Vibrio, Aeromonas, and Campylobacter Species
TABLE 20.5 Differential Characteristics for Mesophilic Clinical Aeromonas Species
CAMPYLOBACTER & HELICOBACTER
Campylobacter and Campylobacter-like species, which include Helicobacter and Wolinella, have undergone
changes in taxonomy. Campylobacters were formerly classified with the vibrios because of their positive
oxidase and characteristic microscopic morphology, but DNA homology studies showed that Campylobacter
spp. do not belong with the vibrios. In addition, unlike the vibrios, which are fermentative, most
459
campylobacters are asaccharolytic (meaning they do not utilize any sugar).

Aeromonas Species CAMPYLOBACTER

Characteristic A. hydrophila A. veronii biovar sobria A. veronii biovar veronii A. caviae A. schubertii A. jandaei A. trota

Esculin hydrolysis + − + + − − −
Þ Microaerophilic
Voges-Proskauer + + + − + − −
Pyrazinamidase activity
Fermentation
V ND ND + ND ND ND Þ Oxidase positive
Þ Gram-negative curved shaped bacilli
Arabinose + − − + − − −
Cellobiose − V V + − V +
Mannitol + + + + − + V
Sucrose
Susceptibility
+ + + + − − V
Þ Assacharolytic
Ampicillin R R R R R R S
Ticarcillin or Piperacillin V V R V V V S Micro-aerophilic organisms require oxygen, but at a concentration less than that of room air; 5% is
Cephalothin
Glucose (gas)
V
+
V
+
V
+
V
−
V
−
V
+
V
V
normally optimal.
+, Positive; −, negative; R, resistant; S, susceptible; V, variable CHAPTER 20 Vibrio, Aeromonas, and Campylobacter Species 451
Horneman AJ: Aeromonas. In Jorgensen JH, et al, editors: Manual of clinical microbiology, ed 11, Washington, DC, 2015, ASM Press, p. 755.

returned to South HELICOBACTER merica in 1991PYLORI for the first time in almost a
forCampylobacter and
hydrophilia and A. dhakensis sp. nov., comb. nov, from a uatic
TABLE 20.2 Key Features
ounds, A. veronii biovar sobria, A. hydrophilia, and A. jandaei the Identification
Campylobacter -Like Species of Þ Strongly associated with gastric, peptide, and
century and advanced north ard into duodenalmerica,
Central ulcers asand welleven
as
from septicemia and meningitis, A. caviae from pediatric diarrhea,
Vibrio, Aeromonas,
and A. veronii biovar sobria from medicinal leech therapy cases.
he disease associations, coupled ith differences in antimicrobial
and
Campylobacter and Plesiomonas
Campylobacter-like species, hich include
Helicobacter and Wolinella, have undergone changes in ta onomy.
intowith Megastrointestinal
ico. In addition, carcinoma a derivative of the biotype El or as
Þresponsible for causing a ma or epidemic in Bangladesh in 1992.
susceptibilities among the species, strongly suggest that conven- Campylobacters ere formerly classified ith the vibrios because
tional identification to the species level and antimicrobial suscep- of their positive o idase and characteristic microscopic morphology, High incidence
Feature
tibilities using the Kirby-Bauer method should be done on all Vibrio but DN Aeromonas
homology studies sho ed thatPlesiomonas
Campylobacter spp. do
Cases of cholera are not commonly reported in the nited States,
clinical aeromonad isolates, if possible. not belong ith the vibrios. In addition, unlike the vibrios, hich
are fermentative, most campylobacters are asaccharolytic. ÞandType most B gastritis
cases are considered imported cases.
Antimicrobial
Gram stainSusceptibility reaction − Based on rRN −se uence studies, Wolinella recta−and Wolinella

+ anaerobes, Þ H. cinaedi
lthough most cases of Aeromonas-associated gastroenteritis are curva ere transferred to the genus Campylobacter as C. rectus
Oxidase
self-limited, activity
antimicrobial therapy is often indicated. It is al + ays + they may appear to be strict
and C. curvus. lthough
O/129
arranted in ound a
Susceptibility
infections and septicemia. s a genus, they have been gro n in a microaerophilic environment. Micro- Clinical Manifestations
aeromonads are almost uniformly resistant to penicillin, ampicillin, aerophilic organisms re uire o ygen, but at a concentration less It is estimated that 50% of the population worldwide is infected with H. pylori. In developing countries
150 μg
and carbenicillin, e cept for the susceptibility of all A. trota S
and than that of room R air 5 is normally optimal. S
lso as a result
inCholera
Africa, Asia, andis South
an acute
America, thediarrheal disease
incidence is reported to bespread
as high as mainly
80–90%. Thisthrough
higher
a moderate number of A. caviae isolates to ampicillin. eromonads of rRN studies, the genera Arcobacter and l o i ill m ere
Growth
are variable on TCBS
ith regard agar
to ticarcillin or piperacillin and + the placed in the family− Campylobacteraceae along −ith Campylo- incidence contaminated ater.
is attributed to poor sanitaryoconditions
ever, improperly
and preserved
it appears that infection and
occurs early handled
in life.
cephalosporins.
0% NaCl
hus far, A. veronii biovar veronii isolates still appear to be
− /+ bacter. +
the family elicobactereaceae.
+
The genera Helicobacter and Wolinella are members of
Itfoods, including
is a major cause fish and
of type B gastritis, a chronicseafood, milk,associated
condition formerly ice cream, andstress
primarily with unpre-
and
6.5% NaCl
susceptible to cephalothin, but only A. veronii biovar veronii+ is − NA chemical irritants. In addition, H. cinaedi has been isolated from the blood of patients with bacteremia
100 susceptible to cefo itin. ther ise, aeromonads are generally Epidemiology served meat, have been responsible
and patients with human immunodeficiency virus infection.
for outbreaks. he disease
Acidto trimethoprim-sulfametho
susceptible from: a ole, aminoglycosides, Campylobacter spp. have been kno n to cause abortion in domestic
and uinolones, although A. veronii biovar veronii is moderately
Glucose + animals, such as cattle, sheep, and s ine and are primarily oonotic
+ + manifests in acute cases as a severe gastroenteritis accompanied
resistant to tobramycin. It should be noted that there have been organisms. lthough these organisms ere suspected of causing
reports of Inositol
a case of sepsis ith an e tended-spectrum β-lactamase − human infections − earlier, campylobacters ere not+ established as by vomiting and follo ed by diarrhea. he stools produced by
EPIDEMIOLOGY
(ESBL) producing A. hydrophila and a case of necroti ing fasciitis, human pathogens until sensitive isolation procedures and media
Mannitol
ith the probable in vivo transfer of a EM-24 plasmid-borne + ere developed.+/− oday, the most common cause − of bacterial patients ith cholera are described as rice ater stools, and the
ESBL gene from Enterobacter aerogenes. gastroenteritis orld ide is Campylobacter jejuni. The transmission
+/− of campylobacterioses +/− has been attributed to direct−contact ith number of stools, hich are aterysuchand contain numerous ecks
Sucrose
Gelatin liquefaction + +the consumption of contaminated − ater and
animals and handling infected pets, such as dogs, cats, and birds, Þ Cause abortion in domestic animals, as cattle, sheep, and swine and
Case Check 20.3 and indirectly by of mucus, may be as
are primarily zoonotic organisms. many as 10 to 30 per day. If left untreated,
dairy products and improperly cooked poultry. erson to person
Although Aeromonas and Plesiomonas are similar to Vibrio spp., the key
transmission has been reported, and some Campylobacter spp. cholera can result in a rapid uid and electrolyte loss that leads
NA, Not available; R, resistant; S, sensitive;
biochemical results used in the Case in Point were able to rule out
are also V, variable;
se ually transmitted. +, most strains Þto dehydration,
Most commonhypovolemic cause of bacterial gastroenteritis worldwide –
Aeromonas and Plesiomonas as the infecting organism and aided in
identifying −,
positive; Vibriomost strains the disease. +/− or In−2014
negative; /+,thepredominant
oodborne Diseases reaction first.Net ork
ctive Surveillance shock, metabolic acidosis, and death
in aCampylobacter jejuni
definitively as the pathogen causing
estimated that appro imately 14 cases are diagnosed every year
a
Vibriostatic agent (2,4-diamino-6,7-diisopropylpteridine). matter of hours.
From Tarr, et al: Vibrio and Related Organisms. In Jorgensen JH, et al, Þ C.he fetus – blood cultures
devastating clinical scenario is the result of a po erful
editors: Manual of clinical microbiology, ed 11, Washington, DC, 2015,
ASM Press, p. 764.
enteroto in
Þ Guillain–Barré syndrome
kno n as cholera toxin, or choleragen. nce ingested,
the bacteria coloni e the small intestine, in hich they multiply
The transmission of campylobacterioses has been attributed to direct contact with animals and handling
and produce choleragen. he to in consists of t o to ic subunits
infected pets, such as dogs, cats, and birds, and indirectly by the consumption of contaminated water and
of colonies in 0.5 sodium deo ycholate. his occurs as a result dairy and fiveand
products binding
improperlyBcooked
subunits. he totoperson
poultry. Person in initially
transmission hasbinds
been to the andM1
reported,
of the release of deo yribonucleic acid (DN ) from lysed cells. some Campylobacter spp.
ganglioside are also sexually
receptor on the transmitted.
cell membrane via the B subunits.
ll species, e cept for V. cholerae and V. mimicus, are halophilic, There heis a strong evidence suggests
2 subunit thenthatfacilitates
Campylobacter the infection plays a role in
entrance ofGuillain-Barré
the 1 syndrome
which is an autoimmune disorder characterized by acute paralysis caused by damage to the peripheral
subunit.
+
or salt-loving, and re uire the addition of Na for gro th. ibrios nervous ncesystem.
insideManythe cell,
patients withthe
GBSactive
test positive1
forsubunit stimulates
antibodies to Campylobacter.the production
can be differentiated from the similar genera Aeromonas and of adenylate cyclase through the inactivation of protein. his
Plesiomonas (no classified in the family Enterobacteriaceae, leads to an accumulation of cyclic adenosine monophosphate
see Chapter 19) by key biochemical and gro th re uirement (c M ) along SPECIMEN COLLECTIONhich
the cell membrane, & TRANSPORT
stimulates hypersecretion
3−) and
+ +
characteristics, as sho n in able 20.2. Þof Bloodelectrolytes
culture –(Na C. fetus, Ksubsp.
, C fetus ater out of the cell and
into the
Þ Stool samples lumen of the intestine, as sho n in ig. 20.3. he net
Antigenic Structure effect is that the gastrointestinal tract s absorptive ability is
Little is kno n about the antigenic structure of Vibrio e cept for Þover Cary–blair
helmed, resulting in the massive outpouring of atery stools.
V. cholerae, V. parahaemolyticus and, to a lesser degree, i li Þ Avoid bufferedand
reatment glycerol saline
management of cholera are best accomplished
and l ifi he three ma or subgroups of V. cholerae are Þby H. thepylori
administration
– gastric biopsy materials amounts of intravenous or oral
of copious
V. cholerae 1, V. cholerae 139, and V. cholerae non- 1, all uids to replace uids lost from the severe diarrhea. he administra-
Þ Stuart medium
of hich share a common agellar ( ) antigen and somatic ( ) tion of antimicrobial agents such as do ycycline can shorten the
antigen. Based on the composition of the antigen, V. cholerae Þduration Tissue samplesof diarrhea may also and be thereby
placed in reducecysteine—Brucella
uid lossbroth ho with ever,
1 organisms are divided into the follo ing serotypes: ga a 20% glycerol and frozen at -70 o
C .
resistance to do ycycline has been reported. herefore administra-
( , B), Inaba ( , C), and iko ima ( , B, C). Strains of V. cholerae tion of additional antimicrobials such as a ithromycin and cipro-
1 and V. cholerae 139 are associated ith epidemic cholera. o acin may be necessary.
Strains that phenotypically resemble V. cholerae but fail to Epidemic V. cholerae 1 strains occur in t o biogroups, classic
agglutinate in 1 antisera are referred to as V. cholerae non- 1. and El Tor. El or has been the predominant biogroup in the last
____________________________________________________________________________________________ O_Ongkay Caylo
V. parahaemolyticus can also be serotyped by means of its and t o pandemics. Studies in Bangladesh, ho ever, indicate a rapidly
 C. The Vibrionaceae include Vibrio and Aeromonas. Plesiomonas, previously a genus in
the family Vibrionaceae, has been moved to the family Enterobacteriaceae. (Plesiomonas

Clinical Bacteriology Lecture

PTER 20 Vibrio, Aeromonas, and Campylobacter Species 461 will be discussed in this section.)
21
1. The most commonly isolated species of Vibrio include V. cholerae (O-1 and non-O-1),
V. parahaemolyticus, V. alginolyticus, and V. vulnificus.
m. 2. All of these species are found in water sources and are transmitted by contaminated
ng TABLE 20.7 Selective Media for the Cultivation of food and water. DEFINITIVE IDENTIFICATION
ma Campylobacter Species 3. Vibrio cholerae is the cause of cholera, a disease in which vast quantities of fluid and
Þ electrolytes
H. pylori can be presumptively
are lost from the intestinal identified
tract. The in a gastric
liquid stoolsbiopsy specimen
are often referred toby
as
her testing for the
“rice-water” presence
stools, becauseofthey a rapid urease and
are colorless reaction
contain mucus flecks. Cholera-
Medium Base Antimicrobial Agent causing isolates have a somatic antigen referred to as O-1. Non-O-1 isolates do not

in Þ cause
Ureacholera,
breath testcause other infections. V. cholerae can be differentiated from
but may
Campy blood Brucella agar Vancomycin other species by a positive “string test” when mixed with sodium deoxycholate (see
has agar plate 10% sheep red blood cells Trimethoprim The Web
collected
Colortissue
Image sample
7–63).is placed onto Christensen’s urea
medium
4. Vibrio incubated at 37oC forand
andparahaemolyticus 2 hours.
VibrioA color change suggests
alginolyticus usually cause gastroenteritis follow-
H. Polymyxin B the presence of H. pylori.
ing ingestion of raw or improperly handled seafood and wound infections following
he Amphotericin B exposure
Urease to sea
activity can alsowater. Bothby
be detected organisms require
the urea breath test,salt for isgrowth (halophilic).
which
man Cephalothin 5. Vibrio
reportedly vulnificus
sensitive is an and
and specific extremely virulentfororganism
is recommended monitoringthat causes rapidly progressive
Skirrow’s Heart infusion Vancomycin wound
therapy. infections
In this after is
test, the patient exposure
given 13C-toorcontaminated
14C-labeled ureawater
orally.and septicemia after eating raw
Lysed, defibrinated horse Trimethoprim Urea oysters.
degraded by the urease activity of H. pylori in the stomach
6. The13laboratory
releases CO2 or 14CO2, isolation and identification
which is absorbed of Vibrio
into the bloodstream and is summarized in Tables 7–17
red blood cells Polymyxin B and in
detected 7–18. Vibriobreath
the exhaled species
by are counter. as “curved” gram-negative rods (see
usually described
a scintillation
Butzler Meat extract and peptone Bacitracin Web Color Image 7–64), but this morphology is often only seen in the initial Gram stain
Novobiocin
od Horse blood, defibrinated Cycloheximide Table 7–16 Characteristics of Campylobacter Species
are Colistin Growth at:
Hippurate Nalidixic
red Cefazolin Species 25◦ C 37◦ C 42◦ C Hydrolysis Acid Cephalothin

ed, CCDA Nutrient agar Cefoperazone C. jejuni − + + + S R

Charcoal Amphotericin B C. coli − + + − S R
to Sodium desoxycholate C. fetus + + − − R S
ort S = susceptible; R = resistant; + = positive; − = negative.

ers CCDA, Charcoal cefoperazone deoxycholate agar (Becton Dickinson, Isolates from stool specimens and rectal swabs can be presumptively identified as Campylobacter spp.
France). by a positive-oxidase, the characteristic gram-stained microscopic morphology, and the characteristic
motility. The microscopic morphology is important because it differentiates Campylobacter from other
als. An enriched selective agar, Campylobacter blood agar or Campy blood agar, is a commonly used medium bacteria, such as Aeromonas and Pseudomonas, which are oxidase-positive and can grow at 42°C in a
art to isolate C. jejuni and other enteric campylobacters. This commercially available medium contains Brucella microaerophilic environment. A positive hippurate hydrolysis is an important characteristic for the
agar base, 10% sheep red blood cells, and a combination of antimicrobials: vancomycin, trimethoprim, identification of C. jejuni.
s if gro B,optimally
polymyxin amphotericin at 42 cephalothin.
B, and C. Second, gro thmedia
Other selective of colon
that havemicrobiota
been successful is
in
recovering Campylobacter Inter species identification uh aside from growing at various temperature changes such as temperature
be inhibited at thisspp.higher
are Butzlertemperature.
medium and Skirrow’s medium. subsp. on the range such as 25°C, 37°C, and 42°C. Each species can also be differentiated whether they are susceptible
en other (cefoperazone-vancomycin-amphotericin
Campy-CVA hand, is a rare stool isolate, and has
B) medium grobeenthreported
is suppressed
to provide betterat or resistant to nalidixic acid and cephalothin.
suppression
42 C oftherefore
fecal biota, even
to when thisthis
isolate medium is incubatedmedia
organism, at 37oC. Incubation
should at 37incubated
be
oC allows the

recovery of Campylobacter spp. that are inhibited at 42oC. C fetus subsp. Fetus, C. rectus, and C. curvus can
at 37 usingC.routine culture media. Charcoal-based, blood-free media, such as charcoal cefoperazone
be isolated
deoxycholate
IMMUNOLOGIC ASSAYS
Enteric(CCDA)
agar and charcoal-basedand
Campylobacter selective media (CSM), are spp.
Helicobacter also available.
re uire A combination
a micro- of
media that contain either CCDA or CSM can achieve the highest yield of Campylobacter spp. in stool samples.
aerophilic Þ Latex agglutination tests are available but cannot differentiate among
py To recover H. pylori, aand capnophilic
combination environment.
of a nonselective he agar
medium, such as CHOC ideal atmospheric
or Brucella agar with 5%
Campylobacter spp.
environment 2, 10
nd horse red blood cells, andfor thesemedium,
a selective organisms is a gas
such as Skirrow’s agar,mi ture
may be used.of
It is5important that the
inoculated medium be fresh and moist and that the culture be incubated in a microaerophilic environment,
um 2, and
withCincreased 85 N2 for Campylobacter spp. and 5 to 10
humidity. 2 Þ ELISA / IIFA
da and 5 to 12 C 2 for Helicobacter spp. E cept for C. rectus Specific antibodies in serum can be detected by enzyme-linked immunosorbent assay or indirect
m, and C. curvus, a strict anaerobic environment does not support
INCUBATION immunofluorescent assay methods. These methods have been reported to be reasonably sensitive and
specific indicators of Campylobacter and H. pylori infections. Serologic testing is useful for
ive the gro th of most Campylobacter spp. Several methods can
epidemiologic studies for Campylobacter but is not recommended for routine diagnosis. Serologic
ter Þbe Incubate
used to stool
obtaincultures
the reat 42 o
C to recover
uired environmentC. jejunifor campylobacters. testing is an important screening method for the diagnosis of H. pylori infection.
s With– the as for
Inhibitory akC.Efetus as subsp. enerating
fetus – grow Container
instead atSystem
37oC (BD
Diagnostic Systems, Sparks, MD), specimen plates along ith
Þ Enteric Campylobacter and Helicobacter spp. require a microaerophilic
B) a sachet are placed into a clear plastic incubation container,
and capnophilic environment.
cal sealed, and incubated at the appropriate temperature. he sachet
on Enteric Campylobacteron
is activated andeHelicobacter
posurespp. torequire a micro- the
air once aerophilic
foilandpouch
capnophilic
is environment.
removed.
The ideal atmospheric environment for these organisms is a gas mixture of 5% O2, 10% CO2, and 85%
are he as ak E as enerating ouch System can hold
N2 for Campylobacter spp. and 5% to 10% O2 and 5% to 12% CO2 for Helicobacter spp. Except upforto
vus C.four
rectusplates but rea strict
and C. curvus, uires at least
anaerobic t o plates
environment does notper resealable
support the growth pouch.
of most
Campylobacter spp.
ed, ne sachet is added to each pouch and is activated on e posure
ate to air, similar to the previous procedure. or optimal results, a
lso paper to el or moistened cotton ball ith 5 mL of ater should
MICROSCOPIC MORPHOLOGY
or be placed inside the pouch. he pouch is sealed by closing the
in Þipper Curved,partnon-spore
of the forming,
pouch and gramthennegative rods at the appropriate
incubated
ive Þtemperature.
Long spirals or S- or seagull-wing shapes
ed Þ Corkscrew motility
may Presumptive Identification
In gram-stained smears, these organisms stain poorly. For better visualization, carbol-fuchsin is
nd Microscopic Morphology. Campylobacter spp. are curved,
recommended as a counterstain; if safranin is used, counterstaining should be extended to 2–3 minutes.
lic non spore-forming, gram-negative rods that measure appro imately
0.2 to 0.9 μm × 0.5 to 5.0 μm ( ig. 20.9). Enteric campylobacters
may appear as long spirals or S- or seagull- ing shapes. hese
organisms may appear as coccobacilli in smears prepared from
C older cultures. n ram-stained smears, these organisms stain
ers poorly. or better visuali ation, carbol-fuchsin is recommended

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