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 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. are

DNase positive and Plesiomonas organisms are DNase negative.

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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”

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

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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.

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.

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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

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

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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.