Dogs, cats, rodents,

rabbits, pigs,

sheep, and cattle; not part of

normal human microbiota

Yersinia

enterocolitica

Ingestion of organism during contact with infected

animal or from contaminated food or water

Rodents, rabbits, deer, and birds;

not

part of normal human microbiota

Yersinia

pseudotuberculosis

Endogenous or person-to-person spread, especially in

hospitalized patients

Normal human gastrointestinal

microbiota

Citrobacter spp.,

Enterobacter spp.,

Klebsiella spp.,

Morganella spp.,

Proteus

spp., Providencia

spp., and Serratia spp

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However, because they generally do not initiate disease in healthy, uncompromised human hosts, they are

considered opportunistic.

Although E. coli is a normal bowel inhabitant, its pathogenic classification is somewhere between that of the

overt pathogens and the opportunistic organisms. Diuretic strains of this species, such as enterotoxigenic

E. coli (ETEC), enteroinvasive E. coli (EIEC), and enteroaggregative E. coli (EAEC), express potent toxins and

cause serious gastrointestinal infections. Additionally, in the case of enterohemorrhagic E. coli (EHEC) also

referred to as verocytotoxin producing E. coli (VTEC) or Shiga-like toxin producing E. coli (STEC), the

organism may produce life-threatening systemic illness. Furthermore, as the leading cause of

Enterobacteriaceae nosocomial infection, E. coli is likely to have greater virulence capabilities than the other

species categorized as “opportunistic” Enterobacteriaceae.

Table (1-2 )Pathogenesis and Spectrum of Disease for Clinically Relevant Enterobacteriaceae

Organism Virulence Factors Spectrum of Disease and Infections

Escherichia coli

(as a cause of

extraintestinal

infections)

Several, including endotoxin,

capsule

production pili that mediate

attachment to host cells

Urinary tract infections, bacteremia, neonatal

meningitis, and

nosocomial infections of other various body sites.

Most common

cause of gram-negative nosocomial infections.

Enterotoxigenic

E. coli

(ETEC)

Pili that permit gastrointestinal

colonization. Heat-labile (LT)

and

heat-stable (ST) enterotoxins

that

mediate secretion of water and

electrolytes into the bowel

lumen

Traveler’s and childhood diarrhea, characterized by

profuse, watery

stools. Transmitted by contaminated food and water.

Enteroinvasive

E. coli (EIEC)

Virulence factors uncertain, but

organism invades enterocytes

lining

the large intestine in a manner

nearly

identical to Shigella

Dysentery (i.e., necrosis, ulceration, and

inflammation of the large

bowel); usually seen in young children living in

areas of poor

sanitation.

Enteropathogenic

E. coli (EPEC)

Bundle-forming pilus, intimin,

and other

factors that mediate organism

attachment to mucosal cells of

the

small bowel, resulting in

Diarrhea in infants in developing, low-income

nations; can cause a

chronic diarrhea.

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changes in

cell surface (i.e., loss of

microvilli

Enterohemorrhagic

E. coli (EHEC,

VTEC, or STEC)

Toxin similar to Shiga toxin

produced by

Shigella dysenteriae. Most

frequently

associated with certain

serotypes,

such as E. coli O157:H7

Inflammation and bleeding of the mucosa of the

large intestine (i.e.,

hemorrhagic colitis); can also lead to hemolyticuremic syndrome,

resulting from toxin-mediated damage to kidneys.

Transmitted by

ingestion of undercooked ground beef or raw milk.

Enteroaggregative

E. coli (EAEC)

Probably involves binding by

pili, ST-like,

and hemolysin-like toxins;

actual

pathogenic mechanism is

unknown

Watery diarrhea that in some cases can be prolonged.

Mode of

transmission is not well understoo

Shigella spp. Several factors involved to

mediate

adherence and invasion of

mucosal

cells, escape from phagocytic

vesicles, intercellular spread,

and

inflammation. Shiga toxin role

in

disease is uncertain, but it does

have

various effects on host cells.

Dysentery defined as acute inflammatory colitis and

bloody diarrhea

characterized by cramps, tenesmus, and bloody,

mucoid stools.

Infections with S. sonnei may produce only watery

diarrhea

Salmonella serotypes Several factors help protect

organisms

from stomach acids, promote

attachment and phagocytosis

by

intestinal mucosal cells, allow

survival

in and destruction of

phagocytes, and

facilitate dissemination to other

Three general categories of infection are seen:

• Gastroenteritis and diarrhea caused by a wide

variety of serotypes

that produce infections limited to the mucosa and

submucosa of the

gastrointestinal tract. S. serotype Typhimurium and

S. serotype

Enteritidis are the serotypes most commonly

associated with

Salmonella gastroenteritis in the United States.

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tissues. • Bacteremia and extraintestinal infections occur by

spread from

the gastrointestinal tract. These infections usually

involve

S. Choleraesuis or S. dublin, although any serotype

may cause these

infections.

• Enteric fever (typhoid fever, or typhoid) is

characterized by prolonged

fever and multisystem involvement, including blood,

lymph nodes,

liver, and spleen. This life-threatening infection is

most frequently

caused by S. serotype Typhi; more rarely, S.

serotypes Paratyphi A, B

or C.

Yersinia pestis Multiple factors play a role in

the

pathogenesis of this highly

virulent

organism. These include the

ability to

adapt for intracellular survival

and

production of an

antiphagocytic

capsule, exotoxins, endotoxins,

coagulase, and fibrinolysin

Two major forms of infection are bubonic plague

and pneumonic

plague. Bubonic plague is characterized by high

fever and painful

inflammatory swelling of axilla and groin lymph

nodes (i.e., the

characteristic buboes); infection rapidly progresses to

fulminant

bacteremia that is frequently fatal if untreated.

Pneumonic plague

involves the lungs and is characterized by malaise

and pulmonary

signs; the respiratory infection can occur as a

consequence of

bacteremic spread associated with bubonic plague or

can be

acquired by the airborne route during close contact

with other

pneumonic plague victims; this form of plague is

also rapidly fatal.

Yersinia

enterocolitica

subsp.

Various factors encoded on a

virulence

plasmid allow the organism to

Enterocolitis characterized by fever, diarrhea, and

abdominal pain; also

can cause acute mesenteric lymphadenitis, which

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enterocolitica attach

to and invade the intestinal

mucosa

and spread to lymphatic tissue.

may present

clinically as appendicitis (i.e., pseudoappendicular

syndrome).

Bacteremia can occur with this organism but is

uncommon

Yersinia

pseudotuber

culosis

Similar to those of Y.

enterocolitica

Causes infections similar to those described for Y.

enterocolitica but is

much less common

Citrobacter spp.,

Enterobacter spp.,

Klebsiella spp.,

Morganella spp.,

Proteus spp.,

Providencia spp.,

and Serratia spp.

Several factors, including

endotoxins,

capsules, adhesion proteins,

and

resistance to multiple

antimicrobial

agents

Wide variety of nosocomial infections of the

respiratory tract, urinary

tract, blood, and several other normally sterile sites;

most frequently

infect hospitalized and seriously debilitated patients

SPECIFIC ORGANISMS:

OPPORTUNISTIC HUMAN PATHOGENS

Citrobacter spp. (C. freundii, C. koseri, C. braakii) Citrobacter organisms are inhabitants of the intestinal

tract.The most common clinical manifestation in patients as a result of infection occurs in the urinary tract.

However,additional infections, including septicemias, meningitis, brain abscesses, and neurologic

complications, have been person to person. Table 3 provides an outline of the biochemical differentiation of

the most common clinically isolated Citrobacter species. C. freundii may harbor inducible AmpC genes that

encode resistance to ampicillin and first-generation cephalosporins.

Table(3) Biochemical Differentiation of Citrobacter Species

Species Indole ODC Malonate ACID

FERMENTATIO

N

Adonitol

Dulcitol Melibiose Sucrose

C. braakii v pos neg Neg v v neg

C. freundii v neg neg Neg neg pos v

C. koseri pos pos pos Pos v neg v

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From Versalovic J: Manual of clinical microbiology, ed 10, 2011, Washington, DC, ASM Press.neg, Negative

< 15%; ODC, ornithine decarboxylase; pos, positive ≥ 85%; V, variable 15% to 84%.

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

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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.

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.

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

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. 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.

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).

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

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