Section I– Microbiology By Nada Sajet

Cronobacter sakazakii

Cronobacter sakazakii, formerly Enterobacter sakazakii, is a pathogen associated with bacteremia, meningitis,

and necrotizing colitis in neonates. The organism produces a yellow pigment that is enhanced by incubation at

25°C.

C.sakazakii may be differentiated from Enterobacter spp. As Voges-Proskauer, arginine dihydrolase, ornithine

decarboxylase positive. In addition, the organism displays the following fermentation reactions: D-sorbitol

negative, raffinose positive, L-rhamnose positive, melibiose positive, D-arabitol negative, and sucrose positive.

C. sakazakii is intrinsically resistant to ampicillin and first- and secondgeneration cephalosporins as a result of

an inducible AmpC chromosomal β-lactamase. Mutations to the AmpC gene may result in overproduction of βlactamase, conferring resistance to third-generation cephalosporins.

Edwardsiella tarda

Edwardsiella tarda is infrequently encountered in the clinical laboratory as a cause of gastroenteritis. The

organism is typically associated with water harboring fish or turtles. Immunocompromised individuals are

particularly susceptible and may develop serious wound infections and myonecrosis. Systemic infections occur

in patients with underlying liver disease or conditions resulting in iron overload. Enterobacter spp.

(E. aerogenes, E. cloacae, E. gergoviae, E. amnigenus, E. taylorae)

 Enterobacter spp. are motile lactose fermenters that produce mucoid colonies. Enterobacter spp. are reported

as one of the genera listed in the top 10 most frequently isolated health care–associated infections by the

National Healthcare Safety Network. The infections are typically associated with contaminated medical

devices, such as

respirators and other medical instrumentation. The organism has a capsule that provides resistance to

phagocytosis. Enterobacter spp. may harbor plasmids that encode multiple antibiotic resistance genes, requiring

antibiotic susceptibility testing to identify appropriate therapeutic options.

Escherichia coli (UPEC, MNEC, ETEC, EIEC, EAEC, EPEC and EHEC)

Molecular analysis of E. coli has resulted in the classification of several pathotypes as well as commensal

strains. The genus consists of facultative anaerobic, glucosefermenting, gram-negative, oxidase-negative rods

capable

of growth on MacConkey agar. The genus contains motile (peritrichous flagella) and nonmotile bacteria. Most

E. coli strains are lactose fermenting, but this function may be delayed or absent in other Escherichia spp.

Isolates of extraintestinal E. coli strains have been grouped into two categories: uropathogenic E. coli (UPEC)

and meningitis/sepsis–associated E. coli (MNEC).

UPEC strains are the major cause of E. coli–associated urinary tract infections. These strains contain a variety

of pathogenicity islands that code for specific adhesions and toxins capable of causing disease, including

cystitis and acute pyelonephritis. MNEC causes neonatal meningitis that results in high morbidity and

mortality. Eighty percent of MNEC strains test positive for the K1 antigen.

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The organisms are spread to the meninges from a blood infection and gain access to the central nervous system

via membrane-bound vacuoles in microvascular endothelial cells.

As mentioned, intestinal E. coli may be classified as enterohemorrhagic (or serotoxigenic [STEC], or

verotoxigenic [VTEC]), enterotoxigenic, enteropathogenic, enteroinvasive, or enteroaggregative. EHEC is

recognized

as the cause of hemorrhagic diarrhea, colitis, and hemolytic uremic syndrome (HUS). HUS, which is

characterized by a hemolytic anemia and low platelet

count, often results in kidney failure and death. Unlike in dysentery, no white blood cells are found in the stool.

Although more than 150 non-O157 serotypes have been associated with diarrhea or HUS, the two most

common

are O157:H7 and O157:NM (nonmotile). The O antigen is a component of the lipopolysaccharide of the outer

membrane, and the H antigen is the specific flagellin associated with the organism. ETEC produces a heatlabile

enterotoxin (LT) and a heat-stable enterotoxin (ST) capable of causing mild watery diarrhea. ETEC is

uncommon in the United States but is an important pathogen in young children in developing countries.

EIEC may produce a watery to bloody diarrhea as a result of direct invasion of the epithelial cells of the colon.

Cases are rare in the United States. EPEC typically does not produce exotoxins. The pathogenesis of these

strains is associated with attachment and effacement of the intestinal cell wall through specialized adherence

factors. Symptoms of infection include prolonged, nonbloody diarrhea; vomiting; and fever, typically in infants

or children.

EAEC has been isolated from a variety of clinical cases of diarrhea. The classification as aggregative results

from the control of virulence genes associated with aglobal aggregative regulator gene, AggR, responsible for

cellular adherence. EAEC-associated stool specimens typically are not bloody and do not contain white blood

cells. Inflammation is accompanied by fever and abdominal pain.

Ewingella americana

Ewingella americana has been identified from blood and wound isolates. The organism is biochemically

inactive, and currently no recommended identification scheme has been identified.

Hafnia alvei

Hafnia alvei (formerly Enterobacter hafniae) has been associated with gastrointestinal infections. The

organism, resides in the gastrointestinal tract of humans and many animals It is a motile non–lactose fermenter

and is often

isolated with other pathogens. Most infections with H.alvei are indentified in patients with severe underlying

disease (e.g., malignancies) or after surgery or trauma.

However, a distinct correlation with clinical signs and symptoms has not been clearly developed, probably

because of the lack of identified clinical cases. Treatment is based on antimicrobial susceptibility testing.

Klebsiella spp. (K. pneumoniae, K. oxytoca)

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Klebsiella spp. are inhabitants of the nasopharynx and gastrointestinal tract. Isolates have been identified in

association with a variety of infections, including liver abscesses, pneumonia, septicemia, and urinary tract

infections. Some strains of K. oxytoca carry a heatlabile cytotoxin, which has been isolated from patients who

have developed a self-limiting antibiotic-associated

community-acquired pyogenic liver abscess worldwide.

All strains of K. pneumoniae are resistant to ampicillin. In addition, they may demonstrate multiple antibiotic

resistance patterns from the acquisition of multidrug-resistant plasmids, with enzymes such as carbapenemase.

Morganella spp. (M. morganii, M. psychrotolerans)

Morganella spp. are found ubiquitously throughout the environment and are often associated with stool

specimens collected from patients with symptoms of diarrhea.

They are normal inhabitants of the gastrointestinal tract. M. morganii is commonly isolated in the clinical

laboratory; however, its clinical significance has not been clearly defined. Morganella spp. are deaminase

positive and urease positive.

Pantoea agglomerans

Pantoea agglomerans appears as a yellow-pigmented colony and is lysine, arginine, and ornithine negative. In

addition, the organism is indole positive and mannitol, raffinose, salicin, sucrose, maltose, and xylose negative.

The organism is difficult to identify using commercial or traditional biochemical methods due to the high

variability of expression in the key reactions. Sporadic infections can occur due to trauma from objects

contaminated with

soil or from contaminated fluids (i.e., IV fluids).

Plesiomonas shigelloides

Plesiomonas shigelloides is a fresh water inhabitant that is transmitted to humans by ingestion of contaminated

water or by exposure of disrupted skin and mucosal surfaces. P. shigelloides can cause gastroenteritis, most

frequently in children, but its role in intestinal infections is still unclear.

P. shigelloides is unusual in that it is among the few species of clinically relevant bacteria that decarboxylate

lysine, ornithine, and arginine. It is important to distinguish Aeromonas spp. from P. shigelloides., since both

are oxidase positive. This is accomplished by using the string test. The DNase test may also be used to

differentiate these organisms. Aeromonas spp. areDNase positive and Plesiomonas organisms are DNase

negative.

Proteus spp. (P. mirabilis, P. vulgaris, P. penneri) and Providencia spp. (P. alcalifaciens, P. heimbachae, P.

rettgeri, P. stuartii, P. rustigianii)

The genera Proteus and Providencia are normal inhabitants of the gastrointestinal tract. They are motile, non–

lactose fermenters capable of deaminating phenylalanine.

Proteus spp. are easily identified by their classic “swarming” appearance on culture media. However, some

strains lack the swarming phenotype. Proteus has a distinct odor that is often referred to as a “chocolate cake”

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or “burnt chocolate” smell. For safety reasons, smelling plates is strongly discouraged in the clinical laboratory.

Because of its motility, the organism is often associated

with urinary tract infections; however, it also has been isolated from wounds and ears. The organism has also

been associated with diarrhea and sepsis.

Providencia spp. are most commonly associated with urinary tract infections and the feces of children with

diarrhea. These organisms may be associated with nosocomial outbreaks.

Serratia spp. (S. marcescens, S. liquefaciens group)

Serratia spp. are known for colonization and the cause of pathagenic infections in health care settings. Serratia

spp.

are motile, slow lactose fermenters, DNAse, and orthonitrophenyl galactoside (ONPG) positive. Serratia spp.

Are ranked the twelfth most commonly isolated organism from pediatric patients in North America, Latin

America, and Europe. Transmission may be person to person but is often associated with medical devices such

as urinary catheters, respirators intravenous fluids, and other

medical solutions. Serratia spp. have also been isolated from the respiratory tract and wounds. The organism is

capable of survival under very harsh environmental conditions and is resistant to many disinfectants. The red

pigment (prodogiosin) produced by S. marcescens typically is the key to identification among laboratorians,

although pigment-producing strains tend to be of lower virulence. Other species have also been isolated from

human infections. Serratia spp. are resistant to ampicillin and first-generation cephalosporins because of the

presence of an inducible, chromosomal AmpC β-lactamase. In addition, many strains have plasmid-encoded

antimicrobial resistance to other cephalosporins, penicillins, carbapenems, and aminoglycosides.

Primary intestinal pathogens

Salmonella (All Serotypes)

Salmonella are facultative anaerobic, motile gram-negative rods commonly isolated from the intestines of

humans and animals. Identification is primarily based on the ability of the organism to use citrate as the sole

carbon source and lysine as a nitrogen source in combination with hydrogen sulfide (H2S) production. The

genus is comprised of two primary species, S. enterica (human pathogen) and S. bongori (animal pathogen). S.

enterica is subdivided into six subspecies: subsp. enterica, subsp. salamae, subsp. arizonae, subsp. diarizonae,

subsp. houtenae,and subsp. indica. S. enterica subsp. enterica can be further divided into serotypes with unique

virulence properties.

Serotypes are differentiated based on the characterization of the heat-stable O antigen, included in the LPS, the

heat-labile H antigen flagellar protein, and the heat-labile Vi antigen, capsular polysaccharide. A DNA

sequence–based method has been developed for molecular identification of DNA motifs in the flagella and O

antigens.

Shigella spp. (S. dysenteriae, S. flexneri, S. boydii, S. sonnei)

 Shigella spp. are nonmotile; lysine decarboxylase–negative;

citrate-, malonate-, and H2S-negative; non–lactose fermenting; gram-negative rods that grow well on

MacConkey agar. The four subgroups of Shigella spp. are: S.dysenteriae (group A), S. flexneri (group B), S.

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boydii(group C), and S. sonnei (group D). Each subgroup has several serotypes. Serotyping is based on the

somatic LPS O antigen. After presumptive identification of a suspected

Shigella species based on traditional biochemical methods, serotyping should be completed, especially in the

case of S. dysenteriae. Suspected strains of Shigella sp. that cannot be typed by serologic methods should be

referred to a reference laboratory for further testing.

Yersinia spp. (Y. pestis, Y. enterocolitica,

Y. frederiksenii, Y. intermedia, Y. pseudotuberculosis)

Yersinia spp. are gram-negative; catalase-, oxidase-, and indole-positive, non–lactose fermenting; facultative

anaerobes capable of growth at temperatures ranging from 4° to 43°C. The gram-negative rods exhibit an

unusual bipolar staining. Based on the composition of the LPS in the outer membrane, colonies may present

with either a rough form lacking the O-specific polysaccharide chain (Y. pestis) or a smooth form containing

the lipid A-oligosaccharide core and the complete O-polysaccharide (Y. pseudotuberculosis and Y.

enterocolitica). Complex typing systems exist to differentiate the various Yersinia spp., including standard

biochemical methods coupled with biotyping, serotyping, bacteriophage typing, and antibiogram analysis. In

addition, epidemiologic studies often include pulsed-field gel electrophoresis (PFGE) studies.

Rare human pathogens

 A variety of additional Enterobacteriaceae may be isolated from human specimens, such as Cedecea spp.,

Kluyvera spp., Leclercia adecarboxylata, Moellerella wisconsensis, Rahnella aquatilis, Tatumella ptyseos, and

Yokenella regensburgei. These organisms are typically opportunistic pathogens found in environmental

sources.

Laboratory diagnosis:

Specimen collection and transport

Enterobacteriaceae are typically isolated from a variety of sources in combination with other more fastidious

organisms. No special considerations are required for specimen collection and transport of the organisms.

Direct detection methods

All Enterobacteriaceae have similar microscopic morphology; therefore, Gram staining is not significant for the

presumptive identification of Enterobacteriaceae.

Generally isolation of gram-negative organisms from a sterile site, including cerebrospinal fluid (CSF), blood,

and other body fluids, is critical and may assist the physician in prescribing appropriate therapy.

Direct detection of Enterobacteriaceae in stool by Gram staining is insignificant because of the presence of a

large number of normal gram-negative microbiota. The presence of increased white blood cells may indicate an

enteric infection; however, the absence is not sufficient to rule out a toxin-mediated enteric disease.

Other than Gram staining of patient specimens, specific procedures are required for direct detection of most

Enterobacteriaceae. Microscopically the cells of these organisms generally appear as coccobacilli, or straight

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rods with rounded ends. Y. pestis resembles a closed safety pin when it is stained with methylene blue or

Wayson stain; this is a key characteristic for rapid diagnosis of

plague.

Klebsiella granulomatis can be visualized in scrapings of lesions stained with Wright’s or Giemsa stain.

Cultivation in vitro is very difficult, so direct examination is important diagnostically. Groups of organisms are

seen in mononuclear endothelial cells; this pathognomonic entity is known as a Donovan body, named after the

physician who first visualized the organism in such a lesion.

The organism stains as a blue rod with prominent polar granules, giving rise to the safety-pin appearance,

surrounded by a large, pink capsule. Subsurface infected cells must be present; surface epithelium is not an

adequate specimen.

P. shigelloides tend to be pleomorphic gram-negative rods that occur singly, in pairs, in short chains, or even as

long, filamentous forms.

Cultivation

Media of Choice

Most Enterobacteriaceae grow well on routine laboratory media, such as 5% sheep blood, chocolate, and

MacConkey agars. In addition to these media, selective agars, such as Hektoen enteric (HE) agar, xyloselysine-deoxycholate (XLD) agar, and Salmonella-Shigella (SS) agar, are commonly used to cultivate enteric

pathogens from gastrointestinal The broths used in blood culture systems, as well as thioglycollate and brain

heart infusion broths, all support the growth of Enterobacteriaceae.

Cefsulodin-irgasan-novobiocin (CIN) agar is a selective medium specifically used for the isolation of Y.

enterocolitica from gastrointestinal specimens. Similarly, MacConkey-sorbitol agar (MAC-SOR) is used to

differentiate sorbitol-negative E. coli O157:H7 from other strains of E. coli that are capable of fermenting this

sugar alcohol. Klebsiella granulomatis will not grow on routine agar media. Recently, the organism was

cultured in human monocytes from biopsy specimens of genital ulcers of patients with donovanosis.

Historically, the organism has also been cultivated on a special medium described by Dienst that contains

growth factors found in egg yolk. In clinical practice, however, the diagnosis of granuloma inguinale is made

solely on the basis of direct examination.

Incubation Conditions and Duration

Under normal circumstances, most Enterobacteriaceae produce detectable growth in commonly used broth and

agar media within 24 hours of inoculation. For isolation, 5% sheep blood and chocolate agars may be incubated

at 35°C in carbon dioxide or ambient air. However, Mac- Conkey agar and other selective agars (e.g., SS,

HE,XLD) should be incubated only in ambient air. Unlike most other Enterobacteriaceae, Y. pestis grows best

at 25° to 30°C. Colonies of Y. pestis are pinpoint at 24 hours but resemble those of other Enterobacteriaceae

after 48 hours. CIN agar, used for the isolation of Y. enterocolitica, should be incubated 48 hours at room

temperature to allow for the development of typical “bull’s-eye” colonies (Figure 1).

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Colonial Appearance

Table 4 presents the colonial appearance and other distinguishing characteristics (pigment and odor) of the most

commonly isolated Enterobacteriaceae on MacConkey, HE, and XLD agars.

All Enterobacteriaceae produce similar growth on blood and chocolate agars; colonies are large, gray, and

smooth. Colonies of Klebsiella or Enterobacter may be mucoid because of their polysaccharide capsule. E. coli

is often beta-hemolytic on blood agar, but most other genera are nonhemolytic. As a result of motility, Proteus

mirabilis, P. penneri, and P. vulgaris “swarm” on blood and chocolate agars. Swarming results in the

production of a thin film of growth on the agar surface (Figure 3) as the motile organisms spread from the

original site of inoculation. Colonies of Y. pestis on 5% sheep blood agar are pinpoint at 24 hours but exhibit a

rough, cauliflower appearance at 48 hours. Broth cultures of Y. pestis exhibit a characteristic “stalactite pattern”

in which clumps of cells adhere to one side of the tube.

Y. enterocolitica produces bull’s-eye colonies (dark red or burgundy centers surrounded by a translucent

border; see Figure (1) on CIN agar at 48 hours. However, because most Aeromonas spp. produce similar

colonies on CIN agar, it is important to perform an oxidase test to verify that the organisms are Yersinia spp.

(oxidase negative).

The oxidase test should be performed on suspect colonies that have been subcultured to sheep blood agar.

Pigments present in the CIN agar will interfere with correct interpretation of the oxidase test results.

Table (4) Colonial Appearance and Characteristics of the Most Commonly Isolated Enterobacteriaceae

Organism Medium Appearance

Citrobacter spp. MAC Late LF; therefore, NLF after 24 hr; LF after 48 hr; colonies are light pink after 48

hr

MAC Colorless

Edwardsiella spp. MAC NLF

Figure( 1) Bull’s-eye colony of Yersinia enterocolitica on cefsulodin-irgasan-novobiocin (CIN) agar

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HE Colorless

XLD Red, yellow, or colorless colonies, with or without black centers (H2S)

Enterobacter spp. MAC LF; may be mucoid

HE Yellow

XLD Yellow

Escherichia coli MAC Most LF, some NLF (some isolates may demonstrate slow or late fermentation);

and generally flat, dry, pink colonies with a surrounding darker pink area of

precipitated bile salts†

HE Yellow

XLD Yellow

Hafnia alvei MAC NLF

HE Colorless

XLD Red or yellow

Klebsiella spp. MAC LF; mucoid

HE Yellow

XLD Yellow

Morganella spp. MAC NLF

HE Colorless

XLD Red or colorless

Plesiomonas

shigelloides

BAP Shiny, opaque, smooth, nonhemolytic

MAC Can be NLF or LF

Proteus spp. MAC NLF; may swarm, depending on the amount of agar in the medium; characteristic

foul smell

HE Colorless

XLD Yellow or colorless, with or without black centers

Providencia spp. MAC NLF

HE Colorless

XLD Yellow or colorless

Salmonella spp. MAC NLF

HE Green, black center as a result of H2S production

XLD Red with black center

Serratia spp. MAC Late LF; S. marcescens may be red pigmented, especially if plate is left at 25°C

(Figure 20-2)

HE Colorless

XLD Yellow or colorless

Shigella spp. MAC NLF; S. sonnei produces flat colonies with jagged edges

HE Green

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XLD Colorless

Yersinia spp. MAC NLF; may be colorless to peach

HE Salmon

XLD Yellow or colorless

HE, Hektoen enteric agar; LF, lactose fermenter, pink colony; MAC, MacConkey agar; NLF, non–lactose

fermenter, colorless colony; XLD, xylose-lysinedeoxycholate agar.

*Most Enterobacteriaceae are indistinguishable on blood agar. Pink colonies on MacConkey agar with sorbitol

are sorbitol fermenters; colorless colonies are non–sorbitol fermenters.

Approach to identification

In the early decades of the twentieth century, Enterobacteriaceae were identified using more than 50

biochemical tests in tubes; this method is still used today in reference and public health laboratories. Certain

key tests such as indole, methyl red, Voges-Proskauer, and citrate, known by the acronym IMViC, were

routinely performed to group the most commonly isolated pathogens.

Today, this type of conventional biochemical identification of enterics has become a historical footnote in most

clinical and hospital laboratories in the United States.

In the latter part of the twentieth century, manufacturers began to produce panels of miniaturized tests for

identification, first of enteric gram-negative rods and later of other groups of bacteria and yeast. Original panels

were inoculated manually; these were followed by semiautomated and automated systems, the most

sophisticated of which inoculate, incubate, read, and discard the panels. Practically any commercial

identification system can be used to reliably identify the commonly isolated Enterobacteriaceae. Depending on

the system, results are available within 4 hours or after overnight incubation. The extensive computer databases

used by these systems include information on unusual biotypes.

The number of organisms used to define individual databases is important; in rare cases, isolated organisms or

new microorganisms may be misidentified or not identified at all.

The definitive identification of enterics can be enhanced based on molecular methods, especially 16S ribosomal

RNA (rRNA) sequencing and DNA-DNA

hybridization. Through the use of molecular methods, the genus Plesiomonas, composed of one species of

oxidase-positive, gram-negative rods, now has been

included in the family Enterobacteriaceae. Plesiomonas sp. clusters with the genus Proteus in the

Enterobacteriaceae by 16S rRNA sequencing. However, like all other Enterobacteriaceae, Proteus organisms

are Oxidase negative.

The clustering together of an oxidase-positive genus and an oxidase-negative genus is a revolutionary concept

in microbial taxonomy.

In the interests of cost containment, many clinical laboratories use an abbreviated scheme to identify commonly

isolated enterics. E. coli, for example, the most commonly isolated enteric organism, may be identified by a

positive spot indole test For presumptive identification of an organism as E. coli, the characteristic colonial

appearance on MacConkey agar, as described in( Table 4), is documented along with positive spot indole test

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result. A spot indole test can also be used to quickly separate swarming Proteae, such as P. mirabilis and P.

penneri, which are negative, from the indole-positive P. vulgaris.

Figure( 2) Red-pigmented Serratia marcescens on MacConkey agar.

Figure( 3) Proteus mirabilis swarming on blood agar (arrow points to swarming edge).

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Specific Considerations for Identifying Enteric Pathogens

 The common biochemical tests used to differentiate the species in the genus Citrobacter are illustrated in

(Table 3.)

 In most clinical laboratories, serotyping of Enterobacteriaceae is limited to the preliminary grouping of

Salmonella spp., Shigella spp., and E. coli O157:H7. Typing should be performed from a non–sugar-containing

medium, such as 5% sheep blood agar or LIA. Use of sugar-containing media, such as MacConkey or TSI

agars, can cause the organisms to autoagglutinate.

Commercially available polyvalent antisera designated A, B, C1, C2, D, E, and Vi are commonly used to

preliminarily group Salmonella spp. because 95% of isolates belong to groups A through E. The antisera A

through E contain antibodies against somatic (“O”) antigens, and the Vi antiserum is prepared against the

capsular (“K”) antigen of S. serotype Typhi. Typing is performed using a slide agglutination test. If an isolate

agglutinates with the Vi antiserum and does not react with any of the “O” groups, then a saline suspension of

the organism should be prepared and heated to 100°C for 10 minutes to inactivate

the Vi antigen. The organism should then be retested. S. typhi is positive with Vi and group D. Complete typing

of Salmonella spp., including the use of antisera against the flagellar (“H”) antigens, is performed at reference

laboratories. Preliminary serologic grouping of Shigella spp. is also performed using commercially available

polyvalent somatic (“O”) antisera designated A, B, C, and D. As with Salmonella spp., Shigella spp. may

produce a capsule and

therefore heating may be required before typing is successful. Subtyping of Shigella spp. beyond the groups A,

B, and C (Shigella group D only has one serotype) is typically performed in reference laboratories.

P. shigelloides, a new member of the Enterobacteriaceae that can cause gastrointestinal infections might crossreact with Shigella grouping antisera, particularly group D, and lead to misidentification. This mistake can be

avoided by performing an oxidase test. Sorbitol-negative E. coli can be serotyped using commercially available

antisera to determine whether the somatic “O” antigen 157 and the flagellar “H” antigen 7 are present. Latex

reagents and antisera are now also available for detecting some non-0157, sorbitol-fermenting, Shiga toxin–

producing strains of E. coli.

Some national reference laboratories are simply performing tests for Shiga toxin rather than searchingfor O157

or non-O157 strains by culture. Unfortunately, isolates are not available then for strain typing for epidemiologic

purposes. Laboratory tests to identify enteropathogenic, enterotoxigenic, enteroinvasive, and enteroaggregative

E. coli that cause gastrointestinal infections usually involve animal, tissue culture, or molecular studies

performed in reference laboratories.

The current recommendation for the diagnosis of Shiga toxin–producing E. coli includes testing all stools

submitted from patients with acute community-acquired diarrhea to detect enteric pathogens (Salmonella,

Shigella, and Campylobacter spp.) should be cultured for O157 STEC on selective and differential agar. In

addition, these stools should be tested using either a Shiga toxin detection assay or a molecular assay to

simultaneously determine whether the sample contains a non-O157 STEC. To save media, some laboratories

may elect to perform the assay first, then attempt to grow organisms from broths with an assay-positive result

on selective media. In any case, any isolate or broth positive for 0157STEC, non- 0157STEC, or shiga toxin

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should be forwarded to the public health laboratory for confirmation and direct immunoassay testing. Any

isolate positive for O157 STEC should be forwarded to the public health laboratory for additional

epidemiologic analysis. Any specimens or enrichment broths that are positive for Shiga toxin or STEC but

negative for O157 STEC should also be forward to the public health laboratory for further testing.

Most commercial systems can identify Y. pestis if a heavy inoculum is used. All isolates biochemically grouped

as a Yersinia sp. should be reported to the public health laboratory. Y. pestis should always be reported and

confirmed.

Serodiagnosis

 Serodiagnostic techniques are used for only two members of the family Enterobacteriaceae; that is, S. typhi

and Y.pestis. Agglutinating antibodies can be measured in the diagnosis of typhoid fever; a serologic test for S.

typhi is part of the “febrile agglutinins” panel and is individually known as the Widal test. Because results

obtained by using the Widal test are somewhat unreliable, this method is no longer widely used.

Serologic diagnosis of plague is possible using either a passive hemagglutination test or enzyme-linked

immunosorbent assay; these tests are usually performed in reference laboratories.

Antimicrobial susceptibility testing and therapy:

 For many of the gastrointestinal infections caused by Enterobacteriaceae, inclusion of antimicrobial agents as

part of the therapeutic strategy is controversial or at least uncertain The unpredictable nature of any clinical

isolate’s antimicrobial susceptibility requires that testing be done as a guide to therapy. several standard

methods and commercial systems have been developed for this purpose.

The Clinical and Laboratory

Standards Institute and (CLSI) has created guidelines (CLISI document M-100 and M100-S23) for the

minimum inhibitory concentration (MIC) and disk diffusion breakpoints for aztreonam, cefotaxime,

cefpodoxime, ceftazidime, and ceftriaxone for E. coli, Proteus, and Klebsiella spp., as well as for cefpodoxime,

ceftazidime, and cefotaxime for P. mirabilis. The sensitivity of the screening increases with the use of more

than a single drug. ESBLs are inhibited by clavulanic acid; therefore, this property can be used as a

confirmatory test in the identification process. In addition, with regard to cases

in which moxalactam, cefonicid, cefamandole, or cefoperazone is being considered to treat infection caused by

E. coli, Klebsiella spp., or Proteus spp., it is important to note that interpretive guidelines have not been

evaluated, and ESBL testing should be performed. If isolates test ESBL positive, the results of the antibiotics

listed should be reported as resistant.

 CLSI has revised the interpretive criteria for cephalosporins (cefazolin, cefotaxime, ceftazidime, ceftizoxime,

and ceftriaxone) and aztreonam. Using the new interpretive guidelines, routine ESBL testing is no longer

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necessary, and it is no longer necessary to edit results for cephalosporins, aztreonam, or penicillins from

susceptible to resistant. ESBL testing will remain useful for epidemiologic and infection control purposes.

Multidrug-resistant typhoid fever (MDRTF)

Multidrug-resistant typhoid fever is caused by S. serotype Typhi strains resistant to chloramphenicol,

ampicillin, and cotrimoxazole. Isolates classified as MDRTF have been indentified since the early 1990s in

patients of all ages. The risk for the development of MDRTF is associated with the overuse, misuse and

inappropriate use of antibiotic therapy.

Susceptibility tests should be performed using the typical first-line antibiotics, including chloramphenicol,

ampicillin, and trimethoprimsulfamethoxazole, along with a fluoroquinolone and a nalidixic acid (to detect

reduced susceptibility to fluoroquinolones), a third-generation cephalosporin, and any other antibiotic currently

used for treatment.

Prevention

Vaccines are available for typhoid fever and bubonic plague; however, neither is routinely recommended in the

United States. An oral, multiple-dose vaccine prepared against S. serotype Typhi strain or a parenteral singledose vaccine containing Vi antigen is available for people traveling to an endemic area or for household

contacts of a documented S. serotype Typhi carrier.

An inactivated multiple-dose, whole-cell bacterial vaccine is available for bubonic plague for people traveling

to an endemic area. However, this vaccine does not provide protection against pneumonic plague. Individuals

exposed to pneumonic plague should be given chemoprophylaxis with doxycycline (adults) or trimethoprim/

sulfamethoxazole (children younger than 8 years of age)

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Vibrio, Aeromonas, Chromobacterium, and Related Organisms

Genera and species to be considered:

Current Name Previous Name

Aeromonas caviae complex

A. caviae

A. media

Aeromonas hydrophila complex

A. hydrophila subsp. hydrophila

A. hydrophila subsp. dhakensis

A. bestiarum

A. salmonicida

Aeromonas veronii complex

A. veronii biovar sobria

A. veronii biovar veronii

A. jandaei

A. schubertii

Chromobacterium violaceum

Photobacterium damselae Vibrio damsela

Grimontia hollisae CDC group EF-13; Vibrio hollisae

Vibrio alginolyticus Vibrio parahaemolyticus biotype 2

Vibrio cholerae

Vibrio cincinnatiensis

Vibrio fluvialis CDC group EF-6

Vibrio furnissii

Vibrio harveyi Vibrio carchariae

Vibrio metschnikovii CDC enteric group 16

Vibrio mimicus Vibrio cholerae (sucrose negative)

Vibrio parahaemolyticus Pasteurella parahaemolyticus

Vibrio vulnificus CDC group EF-3

General characteristics:

The organisms discussed in this lecture are considered together because they are all oxidase-positive

glucosefermenting, gram negative bacilli capable of growth on MacConkey agar. Their individual morphologic

and physiologic features are presented Other halophilic organisms, such as Halomonas venusta and

Shewanella algae, require salt but do not ferment glucose, as do the halophilic Vibrio spp.

Aeromonas spp. are gram-negative straight rods with rounded ends or coccobacillary facultative anaerobes that

occur singly, in pairs, or in short chains. They are typically oxidase and catalase positive and produce acid from

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oxidative and fermentative metabolism. Chromobacterium violaceum is a facultative anaerobic, motile, gram

negative rod or cocci.

The family Vibrionaceae includes six genera, three of which are discussed in this lecture .The Photobacterium

and Grimontia each include a single species. The genus Vibrio consists of 10 species of gram-negative,

facultativeanaerobic, curved or comma-shaped rods. Most species are motile and are catalase and oxidase

positive except

Vibrio metschnikovii. All Vibrio spp. require sodium for growth and ferment glucose.

Epidemiology

Many aspects of the epidemiology of Vibrio spp., Aeromonas spp., and C. violaceum are similar (Table 1). The

primary habitat for most of these organisms is water; generally, brackish or marine water for Vibrio spp.,

freshwater for Aeromonas spp., and soil or water for C. violaceum. Aeromonas spp. may also be found in

brackish water

or marine water with a low salt content. None of these organisms are considered part of the normal human

flora. Transmission to humans is by ingestion of contaminated water, fresh produce, meat, dairy products, or

seafood or by exposure of disrupted skin and mucosal surfaces to contaminated water.

The epidemiology of the most notable human pathogen in this lecture, Vibrio cholerae, is far from being fully

understood. This organism causes epidemics and pandemics (i.e., worldwide epidemics) of the diarrheal disease

cholera. Since 1817 the world has witnessed seven cholera pandemics. During these outbreaks the organism is

spread among people by the fecal-oral route,

usually in environments with poor sanitation.

The niche that V. cholerae inhabits between epidemics is uncertain. The form of the organism shed from

infected humans is somewhat fragile and cannot survive long in the environment. However, evidence suggests

that the bacillus has survival, or dormant, stages that allow its long-term survival in brackish water or saltwater

environments during interepidemic periods. These dormant stages are considered viable but nonculturable.

Asymptomatic carriers of V. cholerae have been documented, but they are not thought to be a significant

reservoir for maintaining the organism between outbreaks.

Species Habitat (Reservoir) Mode of Transmission

Fecal-oral route, by ingestion of

contaminated

washing, swimming, cooking, or drinking

water; also by ingestion of contaminated

shellfish or other seafood

Niche outside of human gastrointestinal

tract between occurrence of epidemics and

pandemics is uncertain; may survive in a

dormant state in brackish or saltwater;

human carriers also are

known but are uncommon

Vibrio cholerae

V. alginolyticus Brackish or saltwater Exposure to contaminated water

Table (1) Epidemiology

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V. cincinnatiensis Unknown Unknown

Photobacterium damsela Brackish or saltwater Exposure of wound to contaminated water

V. fluvialis Brackish or saltwater Ingestion of contaminated water or seafood

V. furnissii Brackish or saltwater Ingestion of contaminated water or seafood

Grimontia hollisae Brackish or saltwater Ingestion of contaminated water or seafood

V. metschnikovii Brackish, salt and freshwater Unknown

V. mimicus Brackish or saltwater Ingestion of contaminated water or seafood

V. parahaemolyticus Brackish or saltwater Ingestion of contaminated water or seafood

V. vulnificus Brackish or saltwater Ingestion of contaminated water or seafood

Ingestion of contaminated food (e.g., dairy,

meat,

produce) or, water; exposure of disrupted

skin or mucosal surfaces to contaminated

water or soil; traumatic inoculation of fish

fins and or fishing hooks

Aquatic environments around the world,

including

freshwater, polluted or chlorinated water,

brackish water and, occasionally, marine

water; may

transiently colonize gastrointestinal tract;

often infect various warm- and coldblooded animal species

Aeromonas spp.

Exposure of disrupted skin to contaminated

soil or water

Environmental, soil and water of tropical

and subtropical regions. Not part of human

flora

Chromobacterium

violaceum

Pathogenesis and spectrum of disease :

 As a notorious pathogen, V. cholerae elaborates several toxins and factors that play important roles in the

organism’s virulence. Cholera toxin (CT) is primarily responsible for the key features of cholera (Table 2).

Release of this toxin causes mucosal cells to hypersecrete water and electrolytes into the lumen of the

gastrointestinal tract.

 The result is profuse, watery diarrhea, leading to dramatic fluid loss. The fluid loss results in severe

dehydration and hypotension that, without medical intervention, frequently lead to death. This toxin-mediated

disease does not require the organism to penetrate the

mucosal barrier. Therefore, blood and the inflammatory cells typical of dysenteric stools are notably absent in

cholera. Instead, “rice water stools,” composed of fluids and mucous flecks, are the hallmark of cholera toxin

activity.

V. cholerae is divided into three major subgroups; V. cholerae O1, V. cholerae O129, and V. cholerae non-O1.

The somatic antigens O1 and O139 associated with the V.cholerae cell envelope are positive markers for strains

capable of epidemic and pandemic spread of the disease.

Strains carrying these markers almost always produce cholera toxin, whereas non-O1/non-O139 strains do not

produce the toxin and hence do not produce cholera. Therefore, although these somatic antigens are not

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virulence factors per se, they are important virulence and epidemiologic markers that provide important

information about V. cholerae isolates. The non-O1/non-O139 strains are associated with nonepidemic diarrhea

and extraintestinal infections.

V. cholerae produces several other toxins and factors, but the exact role of these in disease is still uncertain

(seeTable 2). 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 cholera toxin.

Depending on the species, other vibrios are variably involved in three types of infection: gastroenteritis, wound

infections, and bacteremia. Although some of these organisms have not been definitively associated with

human infections, others, such as Vibrio vulnificus, are known to cause fatal septicemia, especially in patients

suffering from an underlying liver disease.

 Aeromonas spp. are similar to Vibrio spp. in terms of the types of infections they cause. Although these

organisms can cause gastroenteritis, most frequently in children, their role in intestinal infections is not always

clear.

Therefore, the significance of their isolation in stool specimens should be interpreted with caution. Severe

watery diarrhea has been associated with Aeromonas strains that produce a heat-labile enterotoxin and a heatstable enterotoxin. In addition to diarrhea, complications of infection with Aeromonas spp. include hemolyticuremic syndrome and kidney disease.

Species Virulence Factors Spectrum of Disease and Infections

Cholera: profuse, watery diarrhea leading to

dehydration,

hypotension, and often death; occurs in

epidemics and

pandemics that span the globe. May also

cause

nonepidemic diarrhea and, occasionally,

extra intestinal

infections of wounds, respiratory tract,

urinary tract,

and central nervous system

Cholera toxin; zonula occludens (Zot) toxin

(enterotoxin); accessory cholera enterotoxin

(Ace) toxin; O1 and O139 somatic antigens,

hemolysin/cytotoxins, motility, chemotaxis,

mucinase, and toxin coregulated pili (TCP)

pili.

Vibrio cholerae

Ear infections, wound infections; rare cause

of

septicemia; involvement in gastroenteritis is

uncertain

Specific virulence factors for the non–V.

cholerae

species are uncertain

V. alginolyticus

V. cincinnatiensis Rare cause of septicemia

Table (2) Pathogenesis and Spectrum of Diseases

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Wound infections and rare cause of

septicemia

P.damsela

V. fluvialis Gastroenteritis

V. furnissii Rarely associated with human infections

Grimontia hollisa Gastroenteritis; rare cause of septicemia

Rare cause of septicemia; involvement in

gastroenteritis

is uncertain

V. metschnikovii

V. mimicus Gastroenteritis; rare cause of ear infection

Wound infections and septicemia;

involvement in

gastroenteritis is uncertain

V. vulnificus

Gastroenteritis, wound infections,

bacteremia, and

miscellaneous other infections, including

endocarditis,

meningitis, pneumonia, conjunctivitis, and

osteomyelitis

Aeromonas spp. produce various toxins and

factors, but their specific role in virulence is

uncertain

Aeromonas spp.

Rare but dangerous infection. Begins with

cellulitis or

lymphadenitis and can rapidly progress to

systemic

infection with abscess formation in various

organs and

septic shock

Endotoxin, adhesins, invasins and cytolytic

proteins have been described.

Chromobacterium

violaceum

C. violaceum is not associated with gastrointestinal infections, but acquisition of this organism by

contamination of wounds can lead to fulminant, life-threatening systemic infections.

Laboratory diagnosis

Specimen collection and transport

Because no special considerations are required for isolation of these genera. However, stool specimens

suspected of containing Vibrio spp. should be collected and transported only in Cary-Blair medium.

 Buffered glycerol saline is not acceptable, because glycerol is toxic for vibrios. Feces is preferable, but rectal

swabs are acceptable during the acute phase of diarrheal illness.

Specimen processing

No special considerations are required for processing of the organisms .

Direct detection methods

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 V. cholerae toxin can be detected in stool using an enzyme linked immunosorbent assay (ELISA) or a

commercially available latex agglutination test but these tests are not widely used in the United States.

Microscopically, vibrios are gram-negative, straight or slightly curved rods (Figure 1).

When stool specimen from patients with cholera are examined using darkfield microscopy, the bacilli exhibit

characteristic rapid darting or shooting-star motility. However, direct microscopic examination of stools by any

method is not commonly used for laboratory diagnosis of enteric bacterial infections.

Aeromonas spp. are gram-negative, straight rods with rounded ends or coccobacilli. No molecular or serologic

methods are available for direct detection of Aeromonas spp. Cells of C. violaceum are slightly curved, medium

to long, gram-negative rods with rounded ends. A polymerase chain reaction (PCR) amplification assay has

been developed for identification of C. violaceum.

Cultivation:

Media of Choice

Stool cultures for Vibrio spp. are plated on the selective medium thiosulfate citrate bile salts sucrose (TCBS)

agar.

TCBS contains 1% sodium chloride, bile salts that inhibit the growth of gram-positive organisms, and sucrose

forthe 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 flora. Although some Vibrio spp. grow very poorly on this medium, those that

grow well produce either yellow or green colonies, depending on whether they are able to ferment sucrose

(which produces yellow colonies).

Figure 1 Gram stain of Vibrio parahaemolyticus

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 Alkaline peptone water (pH 8.4) may be used as an enrichment broth for obtaining growth of vibrios from

stool. After inoculation,the broth is incubated for 5 to 8 hours at 35°C and then subcultured to TCBS.

Chromogenic Vibrio agar, which was developed for the recovery of Vibrio parahaemolyticus from seafood,

supports the growth of other Vibrio spp. Colonies on this agar range from white to pale blue and violet.

Aeromonas spp. are indistinguishable from Yersinia enterocolitica on modified cefsulodin-irgasan-novobiocin

(CIN) agar (4 μg/mL of cefsulodin); therefore, it is important to perform an oxidase test to differentiate the two

genera. Aeromonas agar is a relatively new alternative medium that uses D-xylose as a differential

characteristic.

These organisms typically grow on a variety of differential and selective agars used for the identification of

enteric pathogens. They are also beta-hemolytic on blood agar.

C. violaceum grows on most routine laboratory media. The colonies may be beta-hemolytic and have an

almond like odor. Most strains produce violacein, an ethanolsoluble violet pigment.

All of the genera considered in this lecture grow well on 5% sheep blood, chocolate, and MacConkey agars.

They also grow well in the broth of blood culture systems and in thioglycollate or brain-heart infusion broths.

Incubation Conditions and Duration

 These organisms produce detectable growth on 5% sheep blood and chocolate agars when incubated at 35°C

in carbon dioxide or ambient air for a minimum of 24 hours. MacConkey and TCBS agars only should be

incubated at 35°C in ambient air. The typical violet pigment of C. violaceum colonies (Figure 2) is optimally

produced when cultures are incubated at room temperature (22°C).

Figure 2 Colonies of Chromo bacterium violaceum on DNase agar. Note violet pigment

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Organism Medium Appearance

Large, round, raised, opaque; most pathogenic strains are beta-hemolytic except A.

caviae, which is usually nonhemolytic Both NLF and LF

BA

Mac

Aeromonas spp.

Round, smooth, convex, some strains are beta-hemolytic; most

colonies appear black or very dark purple; cultures smell of ammonium cyanide

(almond-like) NLF

BA

Mac

Chromobacterium

violaceum

Medium to large, smooth, opaque, iridescent with a greenish hue; V. cholerae, V.

fluvialis, and V. mimicus can be beta-hemolytic NLF except V. vulnificus, which may

be LF

BA

Mac

Vibrio spp. and

Grimontia

hollisae

Medium to large, smooth, opaque, iridescent with a greenish hue; may be betahemolytic NLF

BA

Mac

P. damsela

BA, 5% sheep blood agar; Mac, MacConkey agar; LF, lactose fermenter,

NLF, non–lactose fermenter.

Colonial Appearance

 Table 3 describes the colonial appearance and other distinguishing characteristics (e.g., hemolysis and odor)

of each genus on 5% sheep blood and MacConkey agars. The appearance of Vibrio spp. on TCBS is shown in

Figure ( 3).

Table( 3 ) Colonial Appearance and Characteristic

Figure 3 Colonies of Vibrio cholerae (A) and V. parahaemolyticus (B) on TCBS agar.

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Approach to identification

The colonies of these genera resemble those of the family Enterobacteriaceae but can be distinguished notably

by their positive oxidase test result (except for V. metschnikovii, which is oxidase negative). The oxidase test