20

Vibrio, Aeromonas, Campylobacter, and

Campylobacter-like species
Deborah A. Josko

CHAPTER OUTLINE 7. Compare the epidemiology and clinical infections of each group of
organisms.
Vibrio, 456 8. Compare the conrmatory tests commonly used to identify these
General characteristics, 456 isolates.
Vibrio cholerae, 458 9. Discuss the criteria used to differentiate the bacterial species that
Vibrio parahaemolyticus, 459 cause gastrointestinal illnesses.
Vibrio vulnicus, 460 10. Explain the risk factors associated with acquiring infections by these
Vibrio alginolyticus, 460 organisms.
Laboratory diagnosis, 460 11. Describe the disease states associated with each group of organisms.
Antimicrobial susceptibility, 463
Aeromonas, 463
KEY TERMS
General characteristics, 463
Clinical manifestations, 464 Asiatic cholera Kanagawa phenomenon
Laboratory diagnosis, 464 Cholera toxin Microaerophilic
Antimicrobial susceptibility, 466 Choleragen Thiosulfate citrate bile salt sucrose
Campylobacter and Campylobacter-like species, 467 “Darting” motility (TCBS) agar
Epidemiology, 467 El Tor Type B gastritis
Clinical manifestations, 468 Halophilic Urea breath test
Laboratory diagnosis, 468
Antimicrobial susceptibility, 471 Case in point
Bibliography, 472
A 65-year-old Chinese male patient, in obvious shock, was
examined in the emergency department for a painful swelling
of the left hand. His medical history revealed bilateral knee
OBJECTIVES joint pain and posthepatic cirrhosis. The day before admission,
he had pricked his left index nger while selecting shrimp at
After reading and studying this chapter, you should be able to: a sh market. Initially, he noticed only a local reaction in his
1. Discuss the habitat in which the organisms discussed in this chapter nger, but after 12 hours the left hand began to swell. He later
are found. experienced nausea, vomiting, and diarrhea. The patient was
2. Describe the colony morphology and microscopic characteristics of now clammy with a weak pulse, marked swelling, and bullous
each organism. formation with gangrenous changes apparent on the left hand.
3. Apply the appropriate specimen, transport, and processing for maxi- A diagnosis of septic shock was made, and antimicrobial
mum recovery of each organism. therapy was initiated. Blood and wound cultures subsequently
4. Justify the media of choice for isolation of each organism. grew an oxidase-positive, gram-negative bacillus that produced
5. Discuss key biochemical reactions that will help differentiate among pink colonies on MacConkey (MAC) agar and green colonies on
the various genera and identify different species. thiosulfate citrate bile salt sucrose (TCBS) agar and required
6. Compare each group of organisms with respect to macroscopic and 3% to 6% NaCl for growth. No acceptable identication could
microscopic morphology, and biochemical reactions for identication. be made with a rapid identication system for gram-negative

455
456 PART 2 20 Vibrio, Aeromonas, Campylobacter, and Campylobacter-like species
bacilli, and nal identication was made with conventional
biochemicals supplemented with 1% NaCl. The patient suffered
a cardiac arrest and died 11 hours after admission.
Issues to consider
After reading the patient’s case history, consider:
• Various pathogenic organisms associated with aquatic life
that cause human infections
• Disease spectrum and states associated with these organisms
• Presentation of clinical signs and symptoms
• Media of choice to aid in a rapid diagnosis
• Key biochemical reactions to presumptively and denitively
identify the organism responsible for this infection
• Treatment of choice for this infectious agent
This chapter discusses agents of diarrheal diseases and
other infections caused by species of Vibrio, Aeromonas,
Campylobacter, and Helicobacter. The microscopic character-
istics, epidemiology, and clinical infections caused by each
organism are discussed. Specimen collection, transport,
growth requirements, and key biochemical reactions used
for diagnosis are also included in this chapter. This group of
organisms is important because some of them, the Vibrio spp.
in particular, have 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.
Vibrio
The genus Vibrio resides in the family Vibrionaceae, which
includes 15 genera. To date, only about 15 species within the
family have been found in human clinical specimens: Vibrio
(13 species), Photobacterium (1 species), and Grimontia (1 spe-
cies). Vibrio hollisae is now classied in the genus Grimontia
as G. hollisae, whereas Vibrio damsela has been reclassied as
Photobacterium damselae
At least 150 species of Vibrio are recognized. Vibrio spp.
are commonly found in a wide variety of aquatic environ-
ments, including fresh water, brackish or estuarine water,
and marine or salt water. They are temperature sensitive
in that in temperate climates in which water temperatures
exceed 20° C, as in the summer months, vibrios can easily
be isolated from water, suspended particulate matter, algae,
plankton, sh, and shellsh. However, their numbers decline
markedly in the winter months, and they are generally found
only in the sediments. Therefore the risk of infection from all
Vibrio spp. can be reduced by the avoidance of eating raw
or undercooked shellsh, particularly in warmer summer
months. This is especially pertinent for those who are immu-
nocompromised or have serious underlying liver disease.
Additionally, those who are severely immunosuppressed
should avoid exposure of wounds to fresh, estuarine, and
marine water sources.
Several gastrointestinal and extraintestinal infections,
caused by Vibrio spp. have been reported. Some reasons for
the clinical isolation of this organism include:
• Increased ocean water temperature because of climate
change
• Increased travel to coastal or cholera-endemic areas
• Increased consumption of seafood (particularly raw or
undercooked)
• Increased use of recreational water facilities, which
encourages aquatic exposure
• Larger populations of immunocompromised individuals
• Increased awareness of the existence and signicance of
these organisms in the clinical microbiology laboratory
Pandemics (worldwide epidemics) of cholera, a devastat-
ing diarrheal disease caused by Vibrio cholerae, have been doc-
umented since 1817.
Researchers estimate that approximately 1.3 to 4.0 mil-
lion cases of cholera occur each year, with deaths tolls rang-
ing from 21,000 to 143,000 worldwide. Although in 2018,
the World Health Organization (WHO) reported a 60%
decrease in cholera cases worldwide, the number of cases
reported to the WHO remains high over the past few years.
In 2020, 323,369 cholera cases resulting in 857 deaths were
reported from 24 countries. The discrepancy in these num-
bers was attributed to limited surveillance systems where
cases are not being recorded. Nevertheless, in 2017, the
Global Task Force on Cholera Control partners launched
a strategy for cholera control that aims to reduce cholera
deaths by 90% and to eliminate cholera in as many as 20
countries by 2030. In addition, oral vaccine campaigns have
been implemented in endemic areas and in areas experi-
encing an outbreak. The WHO has prequalied three oral
cholera vaccines for use; however, they are not available in
the United States.
General characteristics
Clinical manifestations
Vibrio spp. can be isolated from a number of clinical sources,
and most species have been implicated in more than one dis-
ease process, ranging from mild gastroenteritis to cholera and
from simple wound infections to necrotizing fasciitis and fatal
septicemia. Table 20.1 lists the various clinical infections asso-
ciated with Vibrio spp. The four Vibrio spp. most often encoun-
tered in the clinical laboratory are V. cholerae, V. parahaemolyticus,
V. vulnicus, and V. alginolyticus.
Because most laboratories, except those in coastal areas,
have a fairly low frequency of isolation of Vibrio spp., a good
medical history is extremely important. Often the best indica-
tion of a possible Vibrio infection is the presence of recognized
risk factors, such as:
• Recent consumption of raw seafood (especially oysters)
• Recent immigration or foreign travel
• Accidental trauma incurred during contact with fresh,
estuarine, or marine water or associated products (e.g.,
shellsh, oyster or clam shells, shhooks)
Microscopic morphology
Vibrio spp. are asporogenous, gram-negative rods that mea-
sure approximately 0.5 to 0.8 µm in diameter by 1.4 to 2.6 µm
in length. Most organisms possess polar, sheathed, mono-
trichous or multitrichous agella when grown in broth; how-
ever, V. parahaemolyticus and V. alginolyticus have the ability
to swarm on solid media because of the production of lateral
 Vibrio 457

agella. They have been described typically as curved or Physiology

comma-shaped gram-negative rods, but this morphology is The vibrios are facultatively anaerobic, and all 13 clinically
often seen only in the initial Gram stain of the clinical spec- signicant species are catalase and oxidase positive and able
imen (Fig. 20.1). Vibrios usually appear as small, straight, to reduce nitrate to nitrite, except for V. metschnikovii, which is
gram-negative rods, but they can be highly pleomorphic, negative for all three tests. Most species are generally suscep-
especially under suboptimal growth conditions. tible to the vibriostatic compound O/129 (2,4-diamino-6,7-di-
isopropylpteridine), exhibiting a zone of inhibition to a 150-µg
vibriostatic disk on Mueller-Hinton or trypticase soy agar
(Fig. 20.2). However, this test is not as valuable with isolates
Table 20.1 Clinical infections associated with Vibrio, Grimontia,
from India and Bangladesh, in which resistance to both 10-
and Photobacterium species
and 150-µg disks to O/129 is common in V. cholerae isolates.
Species Clinical infection Frequency Most vibrios, including V. cholerae, also exhibit a positive
V. cholerae O1 Cholera, gastroenteri- Common string test observed as a mucoid “stringing” reaction after
tis, wound infections, emulsication of colonies in 0.5% sodium deoxycholate. This
bacteremia occurs as a result of the release of deoxyribonucleic acid (DNA)
V. cholerae O139 Cholera Common from lysed cells. All species, except for V. cholerae and V. mim-
V. cholerae non-O1 Gastroenteritis, septice- Relatively icus, are halophilic, or salt-loving, and require the addition of
mia, brain abscesses, ear common Na+ for growth. Vibrios can be differentiated from the similar
infections genera Aeromonas and Plesiomonas (now classied in the order
Enterobacterales, see Chapter 19) by key biochemical and
Vibrio Gastroenteritis, wound Common
parahaemolyticus infections growth requirement characteristics, as shown in Table 20.2
Vibrio vulnicus Septicemia, wound Common
infections Antigenic structure
Vibrio alginolyticus Wound infections, ear Common Little is known about the antigenic structure of Vibrio except
infections, conjunctivitis, for V. cholerae, V. parahaemolyticus, and, to a lesser degree, V.
respiratory infections, uvialis and V. vulnicus. Over 200 serogroups of V. cholerae
bacteremia
Vibrio mimicus Gastroenteritis, ear Uncommon
infections
Vibrio uvialis Gastroenteritis Uncommon
Vibrio furnissii Gastroenteritis Uncommon
Vibrio Meningitis, bacteremia Rare
cincinnatiensis
Vibrio Septicemia, peritonitis Rare
metschnikovii
Vibrio harveyi Wound infections Rare
Grimontia hollisae Gastroenteritis Uncommon
Photobacterium Wound infections (cellu- Uncommon
damselae litis; necrotizing fasciitis),
bacteremia
From Tarr C. L., et al. (2015). Vibrio and related organisms. In J. H. Jorgensen,
et al. (Eds.), Manual of clinical microbiology (11th ed., p. 762). Washington, DC: Fig. 20.2 O/129 susceptibility test for Vibrio sp. showing a resistant result (left) and
ASM Press. a susceptible result (right). (Courtesy J. Michael Janda.)

A B
Fig. 20.1 A Microscopic morphology of Vibrio sp. on Gram-stained smear (×1000). B Acridine orange stain of Vibrio cholerae (×1000). (A, Courtesy J. Michael Janda; B,
courtesy Rita R. Colwell.)
458 PART 2 20 Vibrio, Aeromonas, Campylobacter, and Campylobacter-like species

Table 20.2 Key features for the identication of Vibrio, in recent history. The WHO and the Pan American Health
Aeromonas, and Plesiomonas Organization (PAHO) reported in January 2020 that the coun-
Feature Vibrio Aeromonas Plesiomonas try has achieved 1 year free from conrmed cases of cholera,
with the last case reported in January 2019. Cases of cholera
Gram stain reaction − − − are not commonly reported in the United States, and most
Oxidase activity + + + cases are considered imported cases.
O/129a susceptibility
150 µg S R S Clinical manifestations
Growth on TCBS agar + − − Cholera is an acute diarrheal disease that spreads mainly
0% NaCl − /+ + + through contaminated water. However, improperly preserved
6.5% NaCl + − NA and handled foods, including sh and seafood, milk, ice
cream, and unpreserved meat, have been responsible for out-
Acid from:
breaks. The disease manifests in acute cases as a severe gastro-
Glucose + + + enteritis accompanied by vomiting and followed by diarrhea.
Inositol − − + The stools produced by patients with cholera are described as
Mannitol + + −
rice water stools, and the number of stools, which are watery
and contain numerous ecks of mucus, may be as many as
Sucrose +/− +/− − 10 to 30 per day. If left untreated, cholera can result in a rapid
Gelatin liquefaction + + − uid and electrolyte loss that leads to dehydration, hypovole-
a
Vibriostatic agent (2,4-diamino-6,7-diisopropylpteridine). mic shock, metabolic acidosis, and death in a matter of hours.
+, most strains positive; −, most strains negative; + /− or − /+, predominant The devastating clinical scenario is the result of a power-
reaction rst; NA, not available; R, resistant; S, susceptible; V, variable. ful enterotoxin known as cholera toxin, or choleragen. Once
From Tarr, C. L., et al. (2019). Vibrio and related organisms. In K. C. Carroll, et al. ingested, the bacteria colonize the small intestine, in which
(Eds.), Manual of clinical microbiology (12th ed., pp. 775−786). Washington, DC:
ASM Press. they multiply and produce choleragen. The toxin consists of
two toxic A subunits and ve binding B subunits. The toxin
initially binds to the GM 1 ganglioside receptor on the cell
membrane via the B subunits. The A2 subunit then facilitates
have been reported. The three major groups of V. cholerae are V. the entrance of the A1 subunit. Once inside the cell, the active
cholerae O1, V. cholerae O139, and V. cholerae non-O1, non-O139, A1 subunit stimulates the production of adenylate cyclase
all of which share a common agellar (H) antigen and somatic through the inactivation of G protein. This leads to an accumu-
(O) antigen. Only serogroups O1 and 0139 have been respon- lation of cyclic adenosine monophosphate (cAMP) along the
sible for pandemics and large epidemics. Based on the compo- cell membrane, which stimulates hypersecretion of electrolytes
sition of the O antigen, V. cholerae O1 organisms are divided (Na+, K+, HCO3 ) and water out of the cell and into the lumen
into the following serotypes: Ogawa (A, B), Inaba (A, C), and of the intestine, as shown in Fig. 20.3. The net effect is that
Hikojima (A, B, C; very rarely isolated). Strains that phenotyp- the gastrointestinal tract’s absorptive ability is overwhelmed,
ically resemble V. cholerae but fail to agglutinate in O1 antisera resulting in the massive outpouring of watery stools.
are referred to as V. cholerae non-O1. V. parahaemolyticus can Treatment and management of cholera are best accom-
also be serotyped by means of its O and K (capsule) antigens, plished by the administration of copious amounts of intra-
but this is generally done only during epidemiologic studies venous or oral uids to replace uids lost from the severe
conducted by reference laboratories. diarrhea. The administration of antimicrobial agents such as
doxycycline can shorten the duration of diarrhea and thereby
reduce uid loss; however, resistance to doxycycline has been
Vibrio cholerae reported. Therefore administration of additional antimicrobi-
als such as azithromycin and ciprooxacin may be necessary.
Epidemiology Epidemic V. cholerae O1 strains occur in two biogroups,
Vibrio cholerae O1 is the causative agent of cholera, also known classic and El Tor. El Tor has been the predominant biogroup
as Asiatic cholera or epidemic cholera. It has been a disease of in the past two pandemics. Studies in Bangladesh, how-
major public health signicance for centuries. Most epidem- ever, indicate a rapidly occurring reemergence of the clas-
ics occur in developing countries, in which it is endemic. In sic biogroup. In addition, new variants have emerged in
particular, cholera is prevalent in the Bengal region of India Africa and Asia. The El Tor biogroup differs from the classic
and Bangladesh. The seventh and current pandemic of chol- biogroup in that El Tor is Voges-Proskauer positive, hemo-
era originated in Indonesia in 1961. The pandemic strain, V. lyzes erythrocytes, is inhibited by polymyxin B (50 µg), and is
cholerae O1, spread quickly throughout Asia and reached the able to agglutinate chicken red blood cells. The two biogroups
African continent in 1970. Cholera returned to South America also have different bacteriophage susceptibility patterns.
in 1991 for the rst time in almost a century and advanced Numerous cases of Vibrio infections have been reported
northward into Central America and even Mexico. In addi- involving other serogroups, V. cholerae non-O1. Most of
tion, a derivative of the biotype El Tor was responsible for these strains are phenotypically similar to toxigenic V. chol-
causing a major epidemic in Bangladesh in 1992. In October erae O1, but most lack the cholera toxin gene and appear to
2010, a cholera outbreak occurred in Haiti, 10 months after a cause a milder form of gastroenteritis or cholera-like disease.
devastating earthquake. It affected over 820,000 people and However, V. cholerae serogroups O75 and O141 harbor the
killed almost 10,000, and it is considered the worst outbreak cholera toxin gene and have been associated with sporadic,
 Vibrio 459

A2

A1
B B
Cholera toxin
B

INTESTINAL
Na+ K+ LUMEN
- A2 B
HCO3 B
H2O GM1-ganglioside
receptor
B B B
K+ Cell membrane

H2O
Na+ A1
- Gs Gs
HCO3
ADPR
K+ Stimulatory
Na+ H2O AC AC
H2O cAMP Inhibitory
Active Inactive
cAMP
cAMP Gi
cAMP
AC
AC AC
ENTEROCYTE
AC
ATP
ATP

ATP ATP

Fig. 20.3 The action of cholera toxin. The complete toxin is shown binding to the GM1 ganglioside receptor on the cell membrane via the binding (B) subunits. The active
portion (A1) of the A subunit catalyzes the adenosine diphosphate ribosylation (ADPR) of the Gs (stimulatory) regulatory protein, “locking” it in the active state. Because the
G protein acts to return adenylate cyclase from its active to inactive form, the net effect is persistent activation of adenylate cyclase. The increased adenylate cyclase activity
results in accumulation of cyclic adenosine 3′, 5′-monophosphate (cAMP) along the cell membrane. The cAMP causes the active secretion of sodium (Na+), chloride (Cl−),
potassium (K+), bicarbonate (HCO3−), and water out of the cell into the intestinal lumen. ATP, Adenosine triphosphate. (From Ryan, K. J. [2004]. Vibrio and Campylobacter. In
J. Sherris (Ed.), Medical microbiology: an introduction to infectious diseases [4th ed., p. 375]. New York: McGraw-Hill.)

cholera-like diarrhea and bloodstream infections in the
United States. Other non-O1 serogroup strains have been
implicated in a variety of extraintestinal infections, includ-
ing epidural brain abscesses and septicemia. The emergence
of V. cholerae serogroup O139 in 1992 led to a widespread
occurrence of cholera cases throughout India and Bangladesh.
This was the rst V. cholerae non-O1 strain producing epidemic
disease. The O139 strain contains the cholera toxin gene, ctx,
and produces a disease that closely resembles cholera caused
by V. cholera O1. Of interest is that some of these O139 strains
share cross-reacting antigens with Aeromonas trota, a some-
what uncommon cause of diarrheal disease (discussed later).
Vibrio parahaemolyticus
Epidemiology
Vibrio parahaemolyticus is the second most common Vibrio
species implicated in gastroenteritis after V. cholerae. In the
United States, however, it is the most frequently encountered
species in clinical samples. This species was rst recognized
as a pathogen in Japan in 1950, when it caused a large food
poisoning outbreak. Even today it is the primary cause of
so-called summer diarrhea in Japan. V. parahaemolyticus has
also been isolated in Europe, the Baltic area, Australia, Africa,
Canada, and almost every coastal state in the United States.
Most cases of V. parahaemolyticus reported before 1996 were
associated with various serotypes; however, after 1996, a pan-
demic strain of V. parahaemolyticus serotype O3:K6 emerged
and has since been implicated in numerous foodborne out-
breaks in various parts of the world.
Like other vibrios, V. parahaemolyticus is found in aquatic
environments but appears to be limited to coastal or estuarine
areas, despite a halophilic requirement of 1% to 8% NaCl. V.
parahaemolyticus has an association with at least 30 different
marine animal species, including oysters, clams, crabs, lob-
sters, scallops, sardines, and shrimp. Hence, most cases of
gastroenteritis can be traced to recent consumption of raw,
improperly cooked, or recontaminated seafood, particularly
oysters.
460 PART 2 20 Vibrio, Aeromonas, Campylobacter, and Campylobacter-like species
In 2019, the Centers for Disease Control and Prevention
(CDC) reported a multistate outbreak of gastrointestinal ill-
ness linked to oysters imported from Mexico. A total of 16
individuals from 5 states became ill after ingesting the con-
taminated food. Two people were hospitalized; however, no
deaths were reported.
Clinical manifestations
The gastrointestinal disease caused by V. parahaemolyticus is
generally self-limiting. Patients have watery diarrhea, mod-
erate cramps or vomiting, and low, if any, fever. Symptoms
begin about 24 to 48 hours after ingestion of contaminated
seafood. V. parahaemolyticus has occasionally been isolated
from extraintestinal sources such as wounds, ear and eye
infections, and even cases of pneumonia. Invariably the
patient has a history of recent aquatic exposure or a water-
associated traumatic injury to the infected site.
The pathogenesis of V. parahaemolyticus is not as well
understood as V. cholerae. However, there is a possible asso-
ciation between hemolysin production and virulence, known
as the Kanagawa phenomenon. It has been observed that
most clinical V. parahaemolyticus strains produce a heat-stable
hemolysin that is able to lyse human erythrocytes in a special,
high-salt mannitol medium (Wagatsuma agar). These strains
are considered Kanagawa toxin positive, whereas most envi-
ronmental isolates are Kanagawa toxin negative. There are
exceptions to both observations, however, and the exact role
of this hemolysin in pathogenesis is still not understood. It is
also important to note the recent emergence of atypical ure-
ase-positive V. parahaemolyticus strains from clinical sources
along the Pacic coast of North America.
Vibrio vulnicus
Vibrio vulnicus can be found in marine environments on the
Atlantic, Gulf, and Pacic coasts of North America. After
cholera, the second most serious types of Vibrio-associated
infections are those caused by V. vulnicus. Infections caused
by V. vulnicus generally fall into two categories, primary sep-
ticemia and wound infections. The former is thought to occur
through the gastrointestinal route after the consumption of
shellsh, especially raw oysters. Patients with liver dysfunc-
tion and syndromes that result in increased serum levels of
iron (e.g., hemochromatosis, cirrhosis, thalassemia major,
hepatitis) are particularly predisposed to this scenario. Within
hours, septicemia can develop, with a mortality rate above
50%. Starting in 2007, the CDC required that cases of V. vulni-
cus be reported to local and state public health departments.
Patients with V. vulnicus wound infections invariably
have a history of some type of traumatic aquatic wound
that often presents as a cellulitis. Such cases have also been
documented as progressing to necrotizing fasciitis and/or
Case check 20.1
The patient in the Case in Point had a typical case history and
clinical signs and symptoms of V. vulnificus infection. The patient
suffered from posthepatic cirrhosis, a predisposing factor. In addi-
tion, he had suffered a minor wound after handling shellfish that
rapidly developed into septic shock.
multiple organ system failure. Although such infections can
occur in healthy hosts, the most serious cases are in immuno-
compromised individuals and those who have experienced a
mild to severe injury to the infected site. Infections acquired
from wound infections have a 20% to 30% fatality rate. One
such case involved a fatal episode of multiple organ dysfunc-
tion, only 7 days after a 58-year-old Canadian patient devel-
oped septicemia with V. vulnicus after merely handling raw
tilapia sh he had purchased for dinner.
Vibrio alginolyticus
Of the four major Vibrio spp. likely to be encountered in the
clinical laboratory, V. alginolyticus is the least pathogenic for
humans but is frequently isolated in clinical samples in the
United States. It is a common inhabitant of marine environ-
ments and is a strict halophile, requiring at least 1% NaCl; it is
able to tolerate up to 10% NaCl. Almost all isolates originate
from extraintestinal sources, such as eye and ear infections or
wound and burn infections. The organism can be an occupa-
tional hazard for people in constant contact with seawater,
such as shermen or sailors.
Laboratory diagnosis
Specimen collection and transport
Patients’ history, including travel, underlying health condi-
tions, recent consumption of seafood or contact with marine
water, and gastroenteritis with cholera-like or rice-water
stools, is important in diagnosing vibriosis. Vibrios are not
fastidious, and only a few special collection and processing
procedures are necessary to ensure the recovery from clini-
cal material. Stool specimens should be collected as early as
possible in the course of the illness and preferably before the
administration of antimicrobial agents. Whenever possible,
body uids, pus, or tissues should be submitted, but swabs
are acceptable if they are transported in an appropriate
holding medium, such as Cary-Blair, to prevent desiccation.
Buffered glycerol saline is not recommended as a transport
or holding medium because glycerol is toxic for vibrios.
Strips of blotting paper soaked in liquid stool and placed in
airtight plastic bags with a few drops of saline to maintain
moisture are considered viable specimens for up to 5 weeks.
Culture media
The salt concentration (0.5%) in most commonly used labora-
tory media, such as nutrient agar or sheep blood agar (SBA),
is sufcient to support the growth of many vibrios. On SBA
or chocolate (CHOC) agar, vibrios produce medium to large
colonies that appear smooth, opaque, and iridescent with a
greenish hue. The SBA plate should also be examined for the
presence of α- or β-hemolysis. On MAC agar, the pathogenic
vibrios usually grow as nonlactose fermenters. However,
lactose-fermenting species such as V. vulnicus may be over-
looked and incorrectly considered to be members of the
order Enterobacterales, such as Escherichia coli. It is therefore
imperative to determine the oxidase activity of any suspi-
cious Vibrio-like colony. This can be accomplished by directly
testing colonies from SBA or CHOC agar plates with oxidase

Fig. 20.4 Vibrio cholerae on thiosulfate citrate bile salt sucrose agar.
(Courtesy S.W. Joseph.)
reagent or by subculturing any suspicious, lactose-ferment-
ing colonies on MAC to an SBA plate for next-day testing.
This is necessary because lactose-positive colonies from selec-
tive differential media, such as MAC agar, can give false-pos-
itive oxidase reactions.
If a selective medium is warranted because of the clinical
history (exposure to seafood or seawater) or for geographic
reasons (coastal area resident or recent foreign travel), TCBS
agar is recommended. It differentiates sucrose-fermenting (yel-
low colonies) species (Fig. 20.4), such as V. cholerae, V. alginolyt-
icus, V. uvialis, V. furnissii, V. cincinnatiensis, V. metschnikovii,
and some V. vulnicus strains, from the nonsucrose-ferment-
ing (green) species, such as V. mimicus, V. parahaemolyticus, P.
damsela, and most V. vulnicus strains. About 50% of V. harveyi
isolates are sucrose-positive. Although TCBS generally inhib-
its all other organisms, it should be monitored with stringent
quality control measures. There is great variation among dif-
ferent lots in regard to performance, and not all Vibrio spp.
grow on TCBS, especially Grimontia (formerly Vibrio) hollisae
If an enrichment procedure is desired to enhance isolation
of vibrios, alkaline peptone water with 1% NaCl (pH 8.5) can
be inoculated (at least 20 mL) and incubated for 5 to 8 hours
at 35° C then subcultured at 6 hours and 18 hours onto TCBS
medium. CHROMagar Vibrio (CHROMagar Microbiology,
Paris) for the isolation and identication of V. cholera, V. par-
ahemolyticus, and V. vulnicus is available and can be used in
Case check 20.2
It is important to use selective media and key biochemical
reactions to differentiate Vibrio from Vibrio-like organisms. Key
biochemical tests to differentiate various Vibrio spp. include reac-
tion on TCBS, agent O/129 susceptibility or resistance, the string
test, and growth in different concentrations of NaCl. The Case in
Point uses the results obtained from these reactions to identify the
responsible organism.
Vibrio 461
detecting these species in environmental samples and sea-
food. Vibrio colonies appear from white to pale blue to violet
in color.
Presumptive identication
Several key tests can aid in the initial identication of a Vibrio
isolate. Vibrios can be easily confused with other genera,
including Aeromonas, Plesiomonas, Pseudomonas, and members
of the order Enterobacterales. Their general susceptibility
to the vibriostatic agent O/129 (150 µg) and positive string
test distinguishes them from Aeromonas. Inability to ferment
inositol (except for V. cincinnatiensis and some strains of V.
metschnikovii) separates them from Plesiomonas. Their positive
oxidase reaction (except for V. metschnikovii) separates them
from the Enterobacterales (excluding Plesiomonas shigelloides),
and carbohydrate fermentation metabolism separates them
from the oxidative Pseudomonas
Denitive identication
Numerous useful biochemical tests aid in the denitive iden-
tication of most isolates to the species level. Table 20.3 out-
lines 8 key differential tests to divide the 10 most clinically
signicant Vibrio spp. into 6 groups as an initial identication
step. Some additional tests necessary to identify eight clinical
Vibrio spp. from groups 1, 5, and 6 are summarized in Table
20.4. It is important to note that with the halophilic vibrios, it
often is necessary to add at least 1% NaCl to most biochemical
media to obtain reliable reaction results.
Rapid and semiautomated identication systems
Although rapid and semiautomated identication systems
contain databases of the more commonly encountered clini-
cal vibrios, they generally are inadequate for accurate iden-
tication of these species, particularly those that are less
commonly encountered. The inoculating suspensions should
contain at least 0.85% NaCl, such as that found with the API-
20E identication strips (bioMérieux Vitek, Hazelwood, MO).
Even then, the halophilic species might grow poorly, if at
all, and they often are confused with other genera, such as
Aeromonas. Identication using commercial systems remains
challenging. One study evaluated six commercially available
systems that resulted in identication of only 63% to 81%
of organisms at the species level. Most authorities advocate
identication with conventional biochemicals, supplemented
with additional NaCl, if warranted; alternatively, the rapid
system identication should be conrmed at a reference labo-
ratory or state public health department laboratory.
The use of 16 S ribosomal ribonucleic acid (rRNA) sequenc-
ing alone is less than ideal because of small differences in
sequences and polymorphisms among the various species. As
databases are developed, matrix-assisted laser desorption/
ionization–time-of-ight (MALDI-TOF) mass spectrometry
might become a rapid, cost-effective means of identication.
This method has demonstrated accurate identication of V.
parahaemolyticus
Serology
Agglutination, vibriocidal, or antitoxin tests performed on
acute and convalescent sera are considered reliable meth-
ods for testing vibrios serologically. In any case, all isolates
 462
PART 2
Table 20.3 Key differential tests for the 6 groups of 12 Vibrio species that occur in clinical specimensa

20
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6
V. cholerae V. mimicus V. metschnikovii V. cincinnatiensis G. hollisae P. damsela V. uvialisb V.furnissii V. alginolyticus V. parahaemo V. vulnicus V. harveyi

Vibrio, Aeromonas, Campylobacter, and Campylobacter-like species

lyticus
Growth in nutrient
broth with:
No NaCl addedc + + − − − − − - − − − −
6 % NaCl added + + + + + + + + + + + +
Oxidase + + − + + + + + + + + +
production
Nitrate reduced + + − + + + + + + + + +
to nitrite
Myo-Inositol − − V + − − − − − − − −
fermentation
Arginine − − V − − + + + − − − −
dihydrolase
Lysine + + V V − V − − + + + +
decarboxylase
Ornithine + + − − − − − − V + V −
decarboxylase
a
Showing reactions of these species in different groups. All data except those for oxidase production and nitrate reduction are for reactions that occur within 2 days at 35° to 37° C; oxidase production and nitrate reduction reactions
are done only at day 1.
b
Includes V. furnissii, which differs from V. uvialis primarily by production of gas in d-glucose.
c
Species that require salt should have salt added to each biochemical tested.
+, 90% to 100% positive; −, negative, 0% to 10% positive; NG, no growth, possibly because the NaCl concentration is too low, even when 1% NaCl is added; V, variable, 11% to 89% positive. See Table 20.4 for the exact percentages.
From Tarr, C. L., et al. (2019). Vibrio and related organisms. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 777). Washington, DC: ASM Press.
 Aeromonas 463

Table 20.4 Key differential biochemicals to separate Vibrio species in groups 1, 5, and 6a
Group 1 Group 5 Group 6
V. cholerae V. mimicus P damsela V. uvialis V.furnissii V. alginolyticus V. parahae- V. vulnicus V. harveyi
molyticus
Voges-Proskauer 75 9 95 0 0 95 0 0 50
(1% NaCl)
Motility 99 98 25 70 89 99 99 99 0
Acid production
from:
Sucrose 100 0 5 100 100 99 1 15 50
Cellobiose 8 0 0 30 11 3 5 99 50
Salicin 1 0 0 0 0 4 1 95 0
a
Numbers indicate the percentages of strains that are positive after 48 hours of incubation at 36° C (unless other conditions are indicated). Most of the positive reactions
occur during the rst 24 hours.
From Tarr, C. L., et al. (2019). Vibrio and related organisms. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 777). Washington, DC: ASM Press.

with a presumptive biochemical identication of V. cholerae

should be promptly reported to the appropriate public health
authorities, and the isolate should be forwarded to the health
department or a reference laboratory for serotyping.

Antimicrobial susceptibility
Both Mueller-Hinton agar and broth contain sufcient salt to
support the growth of Vibrio spp. most often isolated from
clinical specimens. The recommended antimicrobial sus-
ceptibility testing methods are standardized disk diffusion
(Kirby-Bauer) or dilution susceptibility testing methods. The
Clinical and Laboratory Standards Institute (CLSI) has inter-
pretive guidelines only for V. cholerae limited to ampicillin,
tetracyclines, folate pathway inhibitors, and chlorampheni-
col. In general, most strains of V. cholerae are susceptible to
doxycycline or ciprooxacin. Increased resistance from the
acquisition of plasmids is relatively uncommon in the United Fig. 20.5 Microscopic morphology of Aeromonas on Gram-stained smear
States, but there are reports of multiple resistant strains from (×1000). (Courtesy J. Michael Janda.)
Asia and Africa.
Because most vibrios grow rather rapidly and are simi-
lar to enteric bacteria in many ways, it is often useful for a Previously, the genus Aeromonas resided in the family
rst approximation to use the interpretive guidelines for the Vibrionaceae. However, phylogenetic evidence from molec-
Enterobacterales when testing Vibrio isolates other than V. ular studies resulted in the proposal of a separate family,
cholerae for agents not currently covered by the CLSI docu- Aeromonadaceae. Currently, there are 36 species of aero-
ments M100 (V. cholerae) and M45 (other species). In general, monads characterized in the latest edition of Bergey’s manual
uoroquinolones alone or the synergistic combination of cip- of systematic bacteriology. Of those species, at least 19 have been
rooxacin and cefotaxime display excellent in vitro activity identied as emerging pathogens in humans. A recent study,
against V. vulnicus strains. Most vibrios are also susceptible however, reported most human pathogens (95.4%) were
to gentamicin, tetracyclines, chloramphenicol (except P. dam- identied from only four species: Aeromonas caviae (37.26%),
sela), monobactams, carbapenems, and uoroquinolones. Aeromonas dhakensis (23.49%), Aeromonas veronii (21.54%), and
Aeromonas hydrophila (13.07%). In the United States, aero-
monads have a seasonal pattern of increased isolation from
Aeromonas May to October, reecting their increased numbers in aquatic
environments in the warmer months.
The genus Aeromonas consists of ubiquitous, oxidase-positive,
glucose-fermenting, gram-negative rods (Fig. 20.5) that are General characteristics
widely distributed in freshwater, estuarine, and marine envi-
ronments worldwide. They are frequently isolated from retail Aeromonads are straight rods (1.0 to 3.5 µm long by 0.3 to
produce sources and animal meat products. Aeromonads are 1.0 µm wide). In the mid 1970s, they were classied into one of
responsible for a diverse spectrum of disease syndromes in two groups, the mesophilic group that causes several human
warm- and cold-blooded animals, including sh, reptiles, infections (motile organisms with optimal growth around 37°
amphibians, mammals, and humans. C) or the psychrophilic group that is linked to sh diseases
464 PART 2 20 Vibrio, Aeromonas, Campylobacter, and Campylobacter-like species
(nonmotile strains with optimal growth around 22° C). The
mesophilic group consists of three different complexes or
groups of species. One is the A. hydrophila complex, which
includes A. hydrophila subsp. hydrophila, A. hydrophila subsp.
ranae, A. bestiarum, and A. dhakensis. Another is the A. veronii
complex, which includes A. veronii biovar sobria (formerly
misidentied as A. sobria), A. veronii biovar veronii, A. jan-
daei, A. trota, A. schubertii, A. diversa, and A. encheleia. The last
mesophilic complex is the A. caviae complex, which includes
the species A. caviae, A. media, A. rivipollensis, and A. eucren-
ophila. These organisms are considered mesophiles because
they grow well at 37° C. They are all motile by means of a
single polar agellum.
The psychrophilic group consists of only one species, A.
salmonicida, which is a sh pathogen with several subspecies.
This organism is nonmotile and grows best at 22° to 25° C.
Psychrophilic nonmotile strains are not considered human
pathogens. All aeromonads, in general, can typically grow
from 10° to 42° C.
Clinical manifestations
Intestinal infections
The aeromonads are recognized as enteric pathogens, albeit
not in the same manner as the more common enteric patho-
gens Salmonella, Shigella, and V. cholerae. The level and pattern
of virulence is more like the multifactorial patterns of the var-
ious E. coli subgroups associated with enteric disease (e.g.,
enterotoxigenic E. coli, enterohemorrhagic E. coli). Therefore
screening of stool specimens for the presence of these organ-
isms, followed by further identication to the species level, is
appropriate. This is especially true in cases of pediatric diar-
rhea or in any immunocompromised individual.
The medical history of patients displaying diarrhea and
harboring aeromonads often, but not always, involves aquatic
exposure, such as an association with untreated groundwa-
ter or consumption of seafood, particularly raw oysters or
clams. Infections have also been linked to fresh produce and
dairy products. Five diarrheal presentations are observed
in patients in whom Aeromonas has been isolated from their
stools:
1. Acute, secretory diarrhea often accompanied by vomiting
2. An acute, dysenteric form of diarrhea (similar to
shigellosis) with blood and mucus
3. Chronic diarrhea usually lasting more than 10 days
4. A cholera-like disease, including rice-water stools
5. The nebulous syndrome commonly referred to as
traveler’s diarrhea (similar to enterotoxigenic E. coli). Most
cases are self-limiting, but in pediatric, geriatric, and
immunocompromised populations, supportive therapy
and antimicrobials are often indicated.
A. caviae is the species most frequently associated with
gastrointestinal infections, especially in neonate and pedi-
atric populations; it has been associated with inammatory
bowel disease. Other species associated with diarrhea include
A. hydrophila and A. veronii (biovars sobria and veronii).
More serious complications, usually from infections with
A. hydrophila and A. veronii biovar sobria, include hemolytic
uremic syndrome or kidney disease that might require kid-
ney transplantation. A. veronii biovar sobria has also been
linked to cholera-like disease characterized by abdominal
pain, fever, and nausea.
Extraintestinal infections
Aeromonads are also responsible for extraintestinal infections;
septicemia and wound infections are the most common. They
have also been implicated in cases of meningitis, osteomyeli-
tis, pelvic abscesses, otitis, cystitis, endocarditis, peritonitis,
cholecystitis, keratitis associated with contact lens wear, and
endophthalmitis in healthy and immunocompromised indi-
viduals. Wound infection invariably involves a recent trau-
matic aquatic exposure, such as a boating or shing accident,
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. hydro-
philia, A. veronii, and A. dhakensis. Among the survivors of
the 2004 tsunami in southern Thailand, Aeromonas spp. were
the most common cause of skin and soft tissue infections.
Elevated numbers of Aeromonas spp. were recorded in ood
water samples in New Orleans after Hurricane Katrina in
2005.
An association between A. veronii biovar sobria and surgi-
cal wound infections involving the use of leeches for medici-
nal therapy after plastic surgery to relieve venous congestion
has been noted. These patients can develop serious aero-
monad wound infections. It appears that the leech Hirudo
medicinalis has a symbiotic relationship with this aeromonad
species within its gut, wherein the organisms aid in the enzy-
matic digestion of the blood ingested by the leech.
Aeromonad sepsis is one of the most invasive type of
Aeromonas infection and similarly has a strong association
with the species A. veronii biovar sobria, A. caviae, A. dhak-
ensis, and A. hydrophila. Such patients are most likely to be
immunocompromised and have a history of liver disease or
dysfunction, hematologic malignancies, hepatobiliary disor-
ders, or traumatic injuries. Also at high risk are individuals
with leukemia, lymphoma, or myeloma. Although the origi-
nal source of infection is often unknown, it is surmised that it
is the gastrointestinal, biliary, or even more rarely, respiratory
tract.
Laboratory diagnosis
Culture media
Aeromonads grow readily on most media used for routine
and stool cultures. After 24-hour incubation at 35° C, aero-
monads appear as large, round, raised, opaque colonies
with an entire edge and a smooth, often mucoid, surface.
Frequently, an extremely strong odor is present, and pigmen-
tation ranges from translucent and white to buff-colored.
Hemolysis is variable on SBA, but most major clinical spe-
cies, such as A. hydrophila, A. veronii biovar sobria, and A. jan-
daei, display strong β-hemolysis (Fig. 20.6). In addition, the
most commonly isolated species, A. caviae, has been showing
increasing incidence of β-hemolysis on SBA. Although aero-
monads grow on almost all enteric media, they often are over-
looked on MAC agar, especially in stool cultures, because a
number of aeromonads ferment lactose. This is of particular

Fig. 20.6 Aeromonas hydrophila exhibiting β-hemolysis on sheep blood agar.
(Courtesy A. J. Horneman.)
Fig. 20.7 Aeromonas caviae exhibiting lactose fermentation on MacConkey agar.
(Courtesy A. J. Horneman.)
concern because the most commonly isolated species, espe-
cially in pediatric cases of diarrhea, is the lactose-fermenting
A. caviae. This particular species is generally overlooked as
normal biota E. coli (Fig. 20.7).
The combined use of SBA with 20 µg of ampicillin per mil-
liliter and a modied cefsulodin-irgasin-novobiocin (CIN)
plate, with only 4 µg of cefsulodin instead of 15 µg, might
yield the highest recovery of aeromonads. However, the
incorporation of ampicillin in the blood agar may inhibit
some A. caviae as well as all A. trota strains because the hall-
mark feature of A. trota is its unusual universal susceptibility
to ampicillin. Therefore SBA without ampicillin is preferred.
On the standard CIN formulation for enteric Yersinia or the
Aeromonas 465
modied CIN media, Aeromonas will form pink-centered col-
onies from the fermentation of mannitol, with an uneven,
clear apron resembling Yersinia enterocolitica. However, an
oxidase test performed on SBA colonies will easily separate
the oxidase-positive aeromonads from the oxidase-negative
yersinias. The use of an enrichment broth is generally not
considered necessary. However, if such a medium is war-
ranted for detecting chronic cases or asymptomatic carriers,
alkaline peptone water is recommended. This can be inocu-
lated, incubated overnight at 35° C, and subsequently subcul-
tured to appropriate plate media.
Presumptive identication
An important screening procedure for aeromonads is to
perform an oxidase test and a spot indole test on suspi-
cious colonies on SBA, especially β-hemolytic colonies. A
positive oxidase test distinguishes aeromonads from the
order Enterobacterales (except for Plesiomonas shigelloi-
des), and most clinically relevant aeromonads are indole-
positive. The presence and type of hemolysis among
multiple aeromonad colony types in a single culture often
are the only clues to an infection involving more than one
species of Aeromonas
Members of the genera Vibrio, Aeromonas, and Plesiomonas
have many similar characteristics. See Table 20.2 for key tests
to help separate these three genera. The best tests to distin-
guish the aeromonads from Vibrio spp. are the string test
(usually negative for aeromonads and positive for vibrios)
and testing for sensitivity to O/129 (aeromonads are usually
resistant, and most vibrios are susceptible; see Fig. 20.2).
A test to separate aeromonads and plesiomonads from
most vibrios is determining the ability to grow in the
presence of NaCl. Aeromonads and plesiomonads grow
well in nutrient broth with 0% NaCl, but not in 6% NaCl.
Conversely, most vibrios, specically the halophilic species,
cannot grow in 0% NaCl but thrive in 6% NaCl and even
higher concentrations of NaCl (Fig. 20.8). 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 dis-
tinguish aeromonads from this major pandemic cholera
species and the less common, sucrose-negative V. mimicus.
Fermentation of inositol can be used for separating aero-
monads from plesiomonads, in which aeromonads are neg-
ative and plesiomonads are positive. Finally, the ability to
ferment glucose, with or without the production of gas, dis-
tinguishes Aeromonas from oxidase-positive, nonfermenting
Pseudomonas isolates.
Denitive identication
Nucleic acid amplication tests (NAATs) are the most accu-
rate in the identication of aeromonads. Denitive iden-
tication is also accomplished with a small number of
conventional and readily available biochemical tests, includ-
ing the API-20E test strip (bioMérieux); however, limitations
in identifying aeromonads to the species level exist with
most commercially available systems. MALDI-TOF mass
spectrometry accurately identies most aeromonads to the
genus level, but identication to the species level is limited.
Two FDA-cleared systems are available: the MALDI Biotyper
466 PART 2 20 Vibrio, Aeromonas, Campylobacter, and Campylobacter-like species

system (Bruker, Billerica, MA) and the IVD 3.0 Vitek MS
(bioMérieux), but again, limitations occur in both systems
because of limited database content. Typing systems such
as PFGE and PCR-based techniques are also available for
identication. Serologic assays (i.e., immunoblotting and
enzyme-linked immunosorbent assay [ELISA]) yield low sen-
sitivity and are not readily dependable.
Conventional biochemical tests as shown in Table 20.5 are
useful in the identication of aeromonads to the species level;
however, they have the longest turnaround time of all meth-
ods described with the lowest performance.

Fig. 20.8 Both Aeromonas and Plesiomonas spp. will grow in nutrient broth with
0% NaCl (left), whereas neither will grow in nutrient broth with 6% NaCl (right)
(Courtesy A. J. Horneman.)
Antimicrobial susceptibility
Although most cases of Aeromonas-associated gastroenteri-
tis are self-limiting, antimicrobial therapy is often indicated.
It is always warranted in wound infections and septicemia.
As a genus, aeromonads are almost uniformly resistant to
penicillin, ampicillin, and cephalothin or cefazolin, except
for the susceptibility of A. trota to ampicillin. Aeromonads
are variable with regard to ticarcillin or piperacillin and
cefoxitin.
Thus far, A. veronii biovar veronii isolates still appear to be
susceptible to cephalothin, but only A. veronii biovar veronii
is 100% susceptible to cefoxitin. Otherwise, aeromonads are
generally susceptible to trimethoprim-sulfamethoxazole,
aminoglycosides, and quinolones, although A. veronii biovar
veronii is moderately resistant to tobramycin. It should be
noted that there have been reports of a case of sepsis with
an extended-spectrum β-lactamase (ESBL)-producing A.
hydrophila and a case of necrotizing fasciitis, with the probable
in vivo transfer of a TEM-24 plasmid-borne gene coding for
an ESBL from Enterobacter aerogenes
CLSI antimicrobial susceptibility studies should always
be determined by the standard Kirby-Bauer disk diffusion
method. This is because of possible serious discrepancies in
β-lactamase detection by rapid minimal inhibitory concentra-
tion methods that would directly affect the proper treatment
of aeromonad infections.

Table 20.5 Differential characteristics for clinical Aeromonas species

Characteristic Aeromonas species
A. hydrophila A. veronii bio- A. veronii biovar A. caviae A. schubertii A. jandaei A. trota
var sobria veronii
Esculin hydrolysis + − + + − − −
Voges-Proskauer + + + − − + −
Pyrazinamidase activity V ND ND + ND ND ND
Fermentation
Arabinose + − − + − − −
Cellobiose − V V + − V +
Mannitol + + + + − + V
Sucrose + + + + − − V
Susceptibility
Ampicillin R R R R R R S
Ticarcillin or piperacillin V V V V V V S
Cephalothin −R =S S −R S −R R
Glucose (gas) + + + − − + V
+, Positive; −, negative; ND, not determined; R, resistant; S, susceptible; V, variable.
From Lamy, B., & Horneman, A. J. (2019). Aeromonas. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 771). Washington, DC: ASM Press.
 Campylobacter and Campylobacter-like species 467

Case check 20.3
Although Aeromonas and Plesiomonas are similar to Vibrio spp.,
the key biochemical results used in the Case in Point were able
to rule out Aeromonas and Plesiomonas as the infecting organism
and aided in definitively identifying Vibrio as the pathogen causing
the disease.
Campylobacter and Campylobacter-like
C. jejuni, C. coli and C. lari also cause gastrointestinal disease
(the enteric campylobacters).
Campylobacter fetus subsp. fetus has been isolated most
frequently from blood cultures and is rarely associated with
gastrointestinal illness. Most infections occur in immuno-
compromised individuals, during pregnancy, and in older
adults. Arcobacter spp. have been isolated from a variety
of animals: cattle, pigs, sheep, birds, etc. In humans, they
have been linked to bacteremia, endocarditis, peritonitis,
and diarrhea. Table 20.6 summarizes the clinical signicance
of Campylobacter, Arcobacter, and Helicobacter organisms.

species
Campylobacter and Campylobacter-like species, which include Table 20.6 Clinically signicant Campylobacter species and
Helicobacter, Arcobacter, and Wolinella, have undergone changes Campylobacter-like organisms
in taxonomy. Campylobacters were formerly classied with Species Clinical Signicance
the vibrios because of their positive oxidase and characteristic
Arcobacter butzleri Associated with diarrheal disease and
microscopic morphology, but DNA homology studies showed
bacteremia in humans and in children
that Campylobacter spp. do not belong with the vibrios. In addi- with recurring gastrointestinal illness
tion, unlike the vibrios, which are fermentative, most campylo- (abdominal cramps), endocarditis, and
bacters are asaccharolytic. The genus Campylobacter belongs to peritonitis
the family Campylobacteraceae, whereas the genera Helicobacter
Arcobacter Isolated from cases of human bactere-
and Wolinella are members of the family Helicobactereaceae. cryaerophilus mia and diarrhea
The genus Acrobacter is in the family Arcobactereaceae.
Based on rRNA sequence studies, Wolinella recta and Arcobacter skirrowii Gastroenteritis
Wolinella curva were transferred to the genus Campylobacter as Campylobacter jejuni Most common cause of bacterial diar-
C. rectus and C. curvus. Although they may appear to be strict rhea worldwide
anaerobes, they have been grown in a microaerophilic envi- Campylobacter coli Gastroenteritis, bacteremia in immuno-
ronment. Microaerophilic organisms, such as Camplyobacter, compromised patients
require oxygen, but at a concentration less than that of room Campylobacter fetus Bacteremia in immunocompromised
air; 5% is normally optimal. To date, there are 43 species of subsp. fetus patients and extraintestinal infections
Campylobacter. There are 39 species of Helicobacter, most of during pregnancy
which colonize the stomachs of mammals. Campylobacter Involved in periodontal disease; has also
concisus been recovered from individuals with
Epidemiology gastrointestinal illness
Campylobacter Gastroenteritis, periodontal disease
Campylobacter spp. have been known to cause abortion in curvus
domestic animals, such as cattle, sheep, and swine, and are
primarily zoonotic organisms. Although these organisms Campylobacter Bacteremia, soft tissue abscesses
gracilis
were suspected of causing human infections earlier, campylo-
bacters were not established as human pathogens until sensi- Campylobacter Gastroenteritis
tive isolation procedures and media were developed. Today, hyointestinalis
the most common cause of bacterial gastroenteritis world- Campylobacter lari Enteritis very similar to that caused by
wide is Campylobacter jejuni. The transmission of campylobac- Campylobacter jejuni, also septicemia
terioses has been attributed to direct contact with animals and Campylobacter rectus Gastroenteritis; associated with peri-
handling infected pets, such as dogs, cats, and birds, and indi- odontal disease
rect contact by consumption of contaminated water and dairy Campylobacter Lung, axillary, groin abscesses
products and improperly cooked poultry. Person-to-person sputorum biovar
transmission has been reported, and some Campylobacter spp. sputorum
are also sexually transmitted.
Helicobacter pylori Common cause of duodenal ulcers and
In 2019, the Foodborne Diseases Active Surveillance type B gastritis; risk factor in gastric
Network estimated that approximately 20 cases of cam- carcinoma, and MALT lymphoma
pylobacteriosis are diagnosed every year for each 100,000
Helicobacter bilis Bacteremia, sepsis
people in the population. The CDC reported 236 foodborne
outbreaks of Campylobacter from 2010 to 2017, accounting for Helicobacter Gastroenteritis
2381 illnesses. Poultry, raw milk, and untreated water were canadensis
the sources most often identied. Because of unreported or Helicobacter cinaedi Colitis, cellulitis, and sepsis
misdiagnosed cases, it is believed campylobacteriosis affects Helicobacter felis Gastritis and ulcers
over 1.5 million individuals each year. The populations that
Helicobacter Colitis, sepsis
most often manifest the disease include infants and young
fennelliae
adults, although all age groups are at risk. In addition to
468 PART 2 20 Vibrio, Aeromonas, Campylobacter, and Campylobacter-like species
Helicobacter pylori is strongly associated with gastric, peptic,
and duodenal ulcers and with gastrointestinal carcinoma. H.
pylori has been identied in as many as 80% of gastric ulcer
patients. Although the organism was previously found in
human gastric tissue, it was difcult to assess their signi-
cance because the samples were taken at autopsy. It is esti-
mated that 50% of the population worldwide is infected with
H. pylori. In developing countries in Africa, Asia, and South
America, the incidence is reported to be as high as 80% to
90%. This higher incidence is attributed to poor sanitary
conditions, and it appears that infection occurs early in life.
Some data suggest human-to-human transmission and the
possibility of human reservoirs. Although it is not conclu-
sively proven, fresh groundwater is the likely source of many
infections.
Clinical manifestations
Campylobacter
Several Campylobacter spp. have been implicated in human
infection, the most common being C. jejuni, C. coli, C. fetus
subsp. fetus, C. lari, and C. upsaliensis. Patients infected with
C. jejuni present with a diarrheal disease that begins with
mild abdominal pain within 2 to 10 days after ingestion of
the organism. Cramps and diarrhea (with or without blood/
fecal white cells) often follow the initial signs and symptoms.
Patients may experience fever and chills and, rarely, nausea
and vomiting. In most patients, the illness is self-limiting and
usually resolves in 2 to 6 days. Untreated patients can remain
carriers for several months. Other enteric Campylobacter infec-
tions (those caused by C. coli, C. lari, and C. upsaliensis) have
similar clinical manifestations.
The C. fetus species contains three subspecies, C. fetus
subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. testu-
dinum. C. fetus subsp. fetus is primarily linked to bacteremia
in pregnant women and patients who are immunocompro-
mised. It is also associated with gastroenteritis. However,
cases are underreported because this species does not grow at
42° C, the temperature stool cultures are generally incubated
to recover the enteric campylobacters.
Strong evidence suggests that Campylobacter infection plays
a role in GBS, an autoimmune disorder characterized by acute
paralysis caused by damage to the peripheral nervous sys-
tem. Many patients with GBS test positive for antibodies to
Campylobacter, with an estimated 1 in 1000 individuals diag-
nosed following a Campylobacter infection. 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.
Helicobacter pylori
H. pylori has been primarily linked to gastric infections and
peptic ulcer disease. Once acquired, H. pylori colonizes the
stomach for a long time and can cause a low-grade inam-
matory process, producing a chronic supercial gastritis.
Although it does not invade the gastric epithelium, the infec-
tion is recognized by the host immune system, which initiates
an antibody response. The antibodies produced are not pro-
tective, however.
H. pylori is also recognized as a major cause of type B
gastritis, a chronic condition formerly associated primar-
ily with stress and chemical irritants. In addition, there is a
strong association between long-term H. pylori infection and
gastric cancer. H. pylori causes a rare stomach cancer called
mucosa-associated lymphoid tissue (MALT). This rare type of
cancer is the only cancer that may be cured with antimi-
crobial agents. Other species of helicobacters, including H.
cinaedi and H. fennelliae, have been associated with human
gastroenteritis and bacteremia, generally in immunocom-
promised patients. In addition, enterohepatic helicobacters
infect humans and inhabit the intestinal and hepatobiliary
tracts of various mammals and birds. It is believed that
transmission of these enterohepatic species occurs from ani-
mals to humans.
Laboratory diagnosis
Specimen collection and transport
C. fetus subsp. fetus can be recovered in several routine blood
culture media and is often associated with bacteremia and
extraintestinal infections. Campylobacter spp. that cause enteric
illness are isolated from stool samples and rectal swabs, the
less preferred specimen. If a delay in processing the stool spec-
imen is anticipated, it can be placed in a transport medium
such as Cary-Blair to maintain the viability of the organisms,
modied Cary-Blair, or Fecal Enteric Plus. A common stool
transport medium, buffered glycerol saline, is toxic to enteric
campylobacters and should therefore be avoided.
H. pylori can be recovered from gastric biopsy materials.
Samples must be transported quickly to the laboratory. Stuart
medium can be used to maintain the viability of the organisms
if a delay in processing is anticipated. Tissue samples may
also be placed in cysteine-Brucella broth with 20% glycerol
and frozen at −70° C or Portagerm pylori media (bioMérieux).
Culture media
An enriched selective agar, Campylobacter blood agar
(Campy blood agar) is a commonly used medium to isolate
C. jejuni and other enteric campylobacters. This commercially
available medium contains Brucella agar base, 10% sheep
red blood cells, and a combination of antimicrobials: van-
comycin, trimethoprim, polymyxin B, amphotericin B, and
cephalothin. Other selective media that have been success-
ful in recovering Campylobacter spp. are Butzler medium and
Skirrow’s medium. Table 20.7 shows the composition of each
of these selective media.
Campy-CVA (cefoperazone-vancomycin-amphotericin
B) medium has been reported to provide better suppression
of fecal biota, even when this medium is incubated at 37°
C. Incubation at 37° C allows the recovery of Campylobacter
spp. that are inhibited at 42° C. C. fetus subsp. fetus, C. rec-
tus, and C. curvus can be isolated using routine culture media.
Charcoal-based, blood-free media, such as charcoal cefoper-
azone deoxycholate agar (CCDA) and charcoal-based selec-
tive media (CSM), are also available. Studies have shown that
blood-free, charcoal-containing selective medium is superior
in isolating Campylobacter spp. compared to media contain-
ing blood. A combination of media that contains either CCDA
or CSM can achieve the highest yield of Campylobacter spp.
 Campylobacter and Campylobacter-like species 469

Table 20.7 Selective media for the cultivation of Campylobacter

species
Medium Base Antimicrobial agent
Campy blood Brucella agar Vancomycin
agar plate 10% sheep red blood Trimethoprim
cells
Polymyxin B
Amphotericin B
Cephalothin
Skirrow’s Heart infusion Vancomycin
Lysed, debrinated Trimethoprim
horse red blood cells Polymyxin B
Butzler Columbia blood Bacitracin
Novobiocin
Fig. 20.9 Microscopic morphology of Campylobacter on Gram-stained smear (×1000).
Horse blood, Cycloheximide
debrinated
Colistin
EZ Gas Generating Pouch System can hold up to four plates
Cefazolin but requires at least two plates per resealable pouch. One
CCDA Nutrient agar Cefoperazone sachet is added to each pouch and is activated on exposure
Charcoal Amphotericin B to air, similar to the previous procedure. For optimal results,
a paper towel or moistened cotton ball with 5 mL of water
Sodium
should be placed inside the pouch. The pouch is sealed by
desoxycholate
closing the zipper part of the pouch and then incubated at the
CCDA, Charcoal cefoperazone deoxycholate agar (Becton Dickinson, Franklin
Lakes, NJ). appropriate temperature.

in stool samples. To recover H. pylori, a combination of a
nonselective medium, such as CHOC agar or Brucella agar
with 5% horse red blood cells, and a selective medium, such
as Skirrow’s agar or Wilkins-Chalgren agar, may be used.
It is important that the inoculated medium be fresh and
moist and that the culture be incubated in a microaerophilic
environment, with increased humidity.
Incubation
There is a double purpose for incubating stool cultures at
42° C to recover C. jejuni. First, C. jejuni and other enteric
campylobacters grow optimally at 42° C. Second, growth of
colon microbiota is inhibited at this higher temperature. C.
fetus subsp. fetus, on the other hand, is a rare stool isolate, and
growth is suppressed at 42° C; therefore to isolate this organ-
ism, media should be incubated at 37° C.
Enteric Campylobacter and Helicobacter spp. require a
microaerophilic and capnophilic environment. The ideal
atmospheric environment for these organisms is a gas mix-
ture of 5% O2, 10% CO2, and 85% N2 for Campylobacter spp.
and 4% O2, 5% CO2, 5% H2, and 86% N2 for Helicobacter spp.
Except for C. rectus and C. curvus, a strict anaerobic environ-
ment does not support the growth of most Campylobacter
spp. Several methods can be used to obtain the required
environment for campylobacters. With the GasPak EZ Gas
Generating Container System (BD Diagnostic Systems,
Sparks, MD), specimen plates along with a sachet are placed
into a clear plastic incubation container, sealed, and incubated
at the appropriate temperature. The sachet is activated on
exposure to air once the foil pouch is removed. The GasPak
Presumptive identication
Microscopic morphology
Campylobacter spp. are curved, non–spore-forming, gram-neg-
ative rods that measure approximately 0.2 to 0.9 µm × 0.5 to
5.0 µm (Fig. 20.9). Enteric campylobacters may appear as long
spirals or S- or seagull-wing shapes. These organisms may
appear as coccobacilli in smears prepared from older cul-
tures. On Gram-stained smears, these organisms stain poorly.
For better visualization, carbol-fuchsin is recommended as a
counterstain; if safranin is used, counterstaining should be
extended to 2 to 3 minutes.
Campylobacter spp. exhibit a characteristic “darting” motil-
ity on hanging drop preparations or when visualized under
phase-contrast microscopy. It should be noted that the sen-
sitivity using phase-contrast microscopy to observe motile
organisms has not been widely studied; therefore consider-
able skill is needed to identify these organisms. To observe the
typical motility, organisms should be suspended in Brucella or
tryptic soy broth. Distilled water and saline seem to inhibit
motility.
Arcobacter spp. have a microscopic morphology similar to
that of Campylobacter spp. Unlike the single polar agellum
of campylobacters, Helicobacter spp. have either a single polar
agellum or multiple agella at one pole.
Colony morphology
The typical colony morphology of C. jejuni and other enteric
campylobacters is moist, runny looking, and spreading.
Colonies are usually nonhemolytic; some are round and
raised, and others may be at. C. fetus subsp. fetus produces
smooth, convex, translucent colonies. A tan or slightly pink
470 PART 2 20 Vibrio, Aeromonas, Campylobacter, and Campylobacter-like species

Table 20.8 Biochemical tests to differentiate Campylobacter, Arcobacter, and Helicobacter species
Species Catalase Nitrate Urease H2S pro- Hippurate Indoxyl Growth at Susceptibility to
reduction duction hydrolysis acetate 15° 25° 42° Nalidixic Cephalothin
(TSI) hydrolysis C C C acid (30 µg) (30 µg)
Campylobacter + + − − + + − − + + −
jejuni subsp.
jejuni
Campylobacter V − − − V + − − − + +
jejuni subsp.
doylei
Campylobacter + + − V − + − − + + −
coli
Campylobacter + + V − − − − − + − −
lari
Campylobacter + + − − − − − + − − +
fetus subsp. fetus

Campylobacter − + − − − + − − + + +
upsaliensis
Campylobacter − + − V − − − − + − −
concisus
Campylobacter − + − V V V − − + + ND
curvus
Campylobacter V + − − − + − − W + ND
rectus
Arcobacter V + − − − + + + − V V
butzleri
Helicobacter + − + − − − − − − R S
pylori
Helicobacter + − − − − + − − − S S
fennelliae
Helicobacter + + − − − − − − − S I
cinaedi
+, Positive result; −, negative result; I, intermediate; ND, not determined; R, resistant; S, susceptible; TSI, triple sugar iron agar slant; V, variable result; W, weak.
From Nachamkin, I. (2019). Campylobacter and Arcobacter. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 1032). Washington, DC: ASM Press;
and Couturier, M. R. (2019). Helicobacter. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 1046). Washington, DC: ASM Press.

coloration is observed in some enteric campylobacter colonies.
Other Campylobacter species produce colonies similar to those
of C. jejuni. Although most do not produce pigment, C. muco-
salis and C. hyointestinalis can produce a dirty yellow pigment.
Denitive identication
Isolates from stool specimens and rectal swabs can be pre-
sumptively identied as Campylobacter spp. by a posi-
tive-oxidase, the characteristic Gram-stained microscopic
morphology, and the characteristic motility. The micro-
scopic morphology is important because it differentiates
Campylobacter from other bacteria, such as Aeromonas and
Pseudomonas, which are oxidase-positive and can grow at
42° C in a microaerophilic environment. A positive hippu-
rate hydrolysis is an important characteristic for the iden-
tication of C. jejuni. Table 20.8 lists the biochemical tests
most useful for denitively identifying the most commonly
encountered Campylobacter, Arcobacter, and Helicobacter spe-
cies. Campylobacter identication can be achieved using
fecal antigen detection methods and NAATs, including real-
time PCR, Prodesse ProGastro SSCS (Hologic GenProbe,
San Diego, CA), Luminex xTAG GPP (Luminex Molecular
Diagnostics), Verigene EP (Luminex Molecular Diagnostics),
BioFire Film Array GP (bioMérieux), and BD Max EB (Becton
Dickinson, Franklin Lakes, NJ) assays. Typing systems such
as PFGE and multilocus sequence typing (MLST) are also
available. MALDI-TOF mass spectrometry can be used to
identify common and uncommon species of Campylobacter;
however, growth conditions must be optimized for success-
ful identication of Campylobacter spp.
Helicobacter infections usually are identied by nonculture
methods. H. pylori can be presumptively identied in a gas-
tric biopsy specimen by a rapid urease test (Fig. 20.10). The
collected tissue sample is placed onto Christensen’s urea
medium and incubated at 37° C for 2 hours. A color change
suggests the presence of H. pylori. Other rapid, commer-
cially available tests include the CLOtest Rapid Urease Test
(Kimberly Clark/Ballard Medical Products, Neenah, WI) and
a paper strip test by PyloriTek (BARD, Murray Hill, NJ).

Fig. 20.10 Urease reactions. Left to right, Negative, weak positive,
positive.
Urease activity can also be detected by the urea breath
test, which is reportedly sensitive and specic and is rec-
ommended for monitoring therapy. In this test, the patient
is given 13C-labeled urea orally. Urea degraded by the urease
activity of H. pylori in the stomach releases 13CO2, which is
absorbed into the bloodstream and detected in the exhaled
breath by a scintillation counter. H. pylori infection can also
be diagnosed by fecal antigen detection using enzyme immu-
noassay methodology, microscopic examination of stained
gastric tissue, and DNA amplication tests (e.g., PCR assay).
Immunologic assays
Latex agglutination tests are available for the rapid identi-
cation of colonies of enteric campylobacters on primary
isolation media. Two commercial kits are available in the
United States for culture identication, Campy JCL (Scimedx,
Denville, NJ) and DrySpot Campylobacter test kit (Thermo
Fisher Scientic, Waltham, MA). These kits can detect the
presence of C. jejuni, C. coli, C. upsaliensis, C. lari, and some-
times C. fetus subsp. fetus. However, neither system differen-
tiates among the Campylobacter spp. Commercial kits are also
available for detecting Campylobacter antigen in fecal samples.
Specic antibodies in serum can be detected by ELISA or
indirect immunouorescent assay methods. These methods
have been reported to be reasonably sensitive and specic
indicators of Campylobacter and H. pylori infections. Serologic
testing is useful for epidemiologic studies for Campylobacter
but is not recommended for routine diagnosis. Serologic test-
ing measuring IgG antibodies using ELISA kits is an import-
ant screening method for the diagnosis of H. pylori infection.
Antimicrobial susceptibility
C. jejuni and C. coli exhibit variable susceptibilities to
antimicrobials such as macrolides, uoroquinolones,
Campylobacter and Campylobacter-like species 471
aminoglycosides, chloramphenicol, and tetracycline. Both
species are usually susceptible to macrolides; however,
it must be noted that erythromycin resistance rates have
increased and are more common in C. coli compared to C.
jejuni. Most patients usually recover without antimicrobial
intervention. Ciprooxacin and other quinolones can also be
used; however, ciprooxin resistance is high among isolates
in the United States. Ampicillin, aminoglycosides, imipenem,
and chloramphenicol can be used to treat systemic C. fetus
infections.
The standard triple therapy for treating H. pylori infections
consists of clarithromycin, either amoxicillin or metronida-
zole, and a proton pump inhibitor. An alternative regimen
consisting of uoroquinolone, levooxacin, and a rifamycin
(rifabutin) can be used.
POINTS TO REMEMBER
• Most species in this chapter are found in fresh, estuarine, or
marine water.
• Thirteen species of Vibrio have been implicated in human
infection, and most are agents of diarrheal disease.
• V. cholerae produces a powerful enterotoxin and is responsi-
ble for large numbers of epidemics and pandemics.
• Other Vibrio spp. are common causes of diarrheal disease
related to the consumption of raw shellsh or related to
aquatic wound infections with serious sequelae, such as
septicemia and death.
• TCBS agar is the medium of choice to isolate and differenti-
ate the Vibrio spp. This medium distinguishes sucrose-
fermenting strains from nonsucrose-fermenting strains.
• Aeromonas spp. can cause several types of diarrhea and a
variety of extraintestinal infections that can lead to septice-
mia, meningitis, and death.
• Human Campylobacter spp. are generally responsible for
gastroenteritis; C. jejuni is one of the most common causes of
bacterial diarrhea worldwide.
• Patients suffering from GBS often test positive for
Campylobacter antibodies. GBS is believed to be an auto-
immune disorder resulting from cross-reactivity of
Campylobacter antibodies with the nerve ganglia.
• H. pylori is strongly associated with gastric and duodenal
ulcers and has been implicated in cases of gastric carcinoma.
• Campylobacter and Helicobacter are microaerophilic, requir-
ing oxygen at concentrations less than that found in ambi-
ent air.
LEARNING ASSESSMENT QUESTIONS
1. A gram-negative bacillus isolated from a stool specimen
produces clear colonies on MacConkey agar and yellow
colonies on thiosulfate citrate bile salt sucrose medium.
The isolate is subcultured to a sheep blood agar plate with
an O/129 disk. The isolate is sensitive to O/129 and is oxi-
dase-positive. Which organism would you suspect?
a. Vibrio parahaemolyticus
b. Vibrio cholerae
c. Plesiomonas
d. Aeromonas
472 PART 2 20 Vibrio, Aeromonas, Campylobacter, and Campylobacter-like species

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