Organism	Habitat (Reservoir)	Mode of Transmission
Escherichia coli	Normal bowel flora of humans and other animals; may also inhabit female genital tract	Varies with the type of infection. For nongastrointestinal infections, organisms may be endogenous or spread person to person, especially in the hospital setting. For gastrointestinal infections, the transmission mode varies with the strain of E. coli (see Table 20-2); it may involve fecal-oral spread between humans in contaminated food or water or consumption of undercooked beef or unpasteurized milk from colonized cattle
Shigella spp.	Only found in humans at times of infection; not part of normal bowel flora	Person-to-person spread by fecal-oral route, especially in overcrowded areas, group settings (e.g., daycare) and areas with poor sanitary conditions
Salmonella serotype Typhi Salmonella serotypes Paratyphi A, B, C	Only found in humans but not part of normal bowel flora	Person-to-person spread by fecal-oral route by ingestion of food or water contaminated with human excreta
Other Salmonella spp.	Widely disseminated in nature and associated with various animals	Ingestion of contaminated food products processed from animals, frequently of poultry or dairy origin. Direct person-to-person transmission by fecal-oral route can occur in health care settings when hand-washing guidelines are not followed
Edwardsiella tarda	Gastrointestinal tract of cold-blooded animals, such as reptiles	Uncertain; probably by ingestion of contaminated water or close contact with carrier animal
Yersinia pestis	Carried by urban and domestic rats and wild rodents, such as the ground squirrel, rock squirrel, and prairie dog	From rodents to humans by the bite of flea vectors or by ingestion of contaminated animal tissues; during human epidemics of pneumonic (i.e., respiratory) disease, the organism can be spread directly from human to human by inhalation of contaminated airborne droplets; rarely transmitted by handling or inhalation of infected animal tissues or fluids
Yersinia enterocolitica	Dogs, cats, rodents, rabbits, pigs, sheep, and cattle; not part of normal human microbiota	Consumption of incompletely cooked food products (especially pork), dairy products such as milk, and, less commonly, by ingestion of contaminated water or by contact with infected animals
Yersinia pseudotuberculosis	Rodents, rabbits, deer, and birds; not part of normal human microbiota	Ingestion of organism during contact with infected animal or from contaminated food or water
Citrobacter spp., Enterobacter spp., Klebsiella spp., Morganella spp., Proteus spp., Providencia spp., and Serratia spp.	Normal human gastrointestinal microbiota	Endogenous or person-to-person spread, especially in hospitalized patients

For species capable of colonizing humans, infection may result when a patient’s own bacterial strains (i.e., endogenous strains) establish infection in a normally sterile body site. These organisms can also be passed from one patient to another. Such infections often depend on the debilitated state of a hospitalized patient and are acquired during the patient’s hospitalization (nosocomial). However, this is not always the case. For example, although E. coli is the most common cause of nosocomial infections, it is also the leading cause of community-acquired urinary tract infections.

Other species, such as Salmonella spp., Shigella spp., and Yersinia enterocolitica, inhabit the bowel during infection and are acquired by ingestion of contaminated food or water. This is also the mode of transmission for the various types of E. coli known to cause gastrointestinal infections. In contrast, Yersinia pestis is unique among the Enterobacteriaceae that infect humans. This is the only species transmitted from animals by an insect vector (i.e., flea bite).

Pathogenesis and Spectrum of Diseases

The clinically relevant members of the Enterobacteriaceae can be considered as two groups: the opportunistic pathogens and the intestinal pathogens. Typhi and Shigella spp. are among the latter group and are causative agents of typhoid fever and dysentery, respectively. Yersinia pestis is not an intestinal pathogen, but it is the causative agent of plague. The identification of these organisms in clinical material is serious and always significant. These organisms, in addition to others, produce various potent virulence factors and can cause life-threatening infections (Table 20-2).

TABLE 20-2

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 changes in cell surface (i.e., loss of microvilli)	Diarrhea in infants in developing, low-income nations; can cause a chronic diarrhea.
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 hemolytic-uremic 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 understood.
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 tissues.	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. • 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. enterocolitica Various factors encoded on a virulence plasmid allow the organism to attach to and invade the intestinal mucosa and spread to lymphatic tissue. Enterocolitis characterized by fever, diarrhea, and abdominal pain; also can cause acute mesenteric lymphadenitis, which may present clinically as appendicitis (i.e., pseudoappendicular syndrome). Bacteremia can occur with this organism but is uncommon. Yersinia pseudotuberculosis 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.

The opportunistic pathogens most commonly include Citrobacter spp., Enterobacter spp., Klebsiella spp., Proteus spp., Serratia spp., and a variety of other organisms. Although considered opportunistic pathogens, these organisms produce significant virulence factors, such as endotoxins capable of mediating fatal infections. 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.

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 associated with Citrobacter spp. Transmission is typically person to person. Table 20-3 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 20-3

Biochemical Differentiation of Citrobacter Species

Species	Indole	ODC	Malonate	ACID FERMENTATION
Species	Indole	ODC	Malonate	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

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

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

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 second-generation cephalosporins as a result of an inducible AmpC chromosomal β-lactamase. Mutations to the AmpC gene may result in overproduction of β-lactamase, 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.

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. No clear clinical association exists when these organisms are isolated.

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. 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 discussed in this chapter. (See Table 5-1 for general information on specimen collection and transport.)

Specimen Processing

No special considerations are required for processing of the great majority of organisms discussed in this chapter. The one exception is Yersinia pestis. This organism is a select agent. Manipulation of specimens suspected of containing this organism would generate aerosols and should be handled using Biosafety Level 3 (BSL-3) conditions. Refer to Table 5-1 for general information on specimen processing.

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, xylose-lysine-deoxycholate (XLD) agar, and Salmonella–Shigella (SS) agar, are commonly used to cultivate enteric pathogens from gastrointestinal specimens (see Chapter 59 for more information about laboratory procedures for the diagnosis of bacterial gastrointestinal infections). 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.

Table 20-4 presents a complete description of the laboratory media used to isolate Enterobacteriaceae.

TABLE 20-4

Biochemical Media used in the Differentiation and Isolation of Enterobacteriaceae

Media	Selective	Differential	Nutritional	Purpose
Blood agar (sheep) (SBA, BAP)		Hemolysis of RBCs: Beta: Complete lysis Alpha: Partial, greening Gamma: Nonhemolytic	Routinely used to cultivate moderately fastidious organisms; TSA with 5% to 10% defibrinated blood.	Screening colonies for the oxidase enzyme
Cefsulodin-irgasan-novobiocin agar (CIN)	Selective inhibition of gram-negative and gram-positive organisms	Fermentation of mannitol in the presence of neutral red. Macroscopic colonial appearance: colorless or pink colonies with red center.		Isolation of Yersinia enterocolitica
Citrate agar, Simmons (CIT)		Citrate as the sole carbon source, ammonium salt as nitrate. Ammonium salt alteration changes pH to alkaline, bromthymol blue shifts from green to blue.		Detect organisms capable of citrate utilization
Decarboxylases (ornithine, arginine, lysine)		Incorporate amino acid as differential media (e.g., lysine, arginine, or ornithine). Decarboxylation yields alkaline, pH-sensitive bromcresol purple dye. Basal medium serves as a control. Incubate for up to 4 days. Fermentative organisms turn media yellow, using glucose. [H+] increases, making optimal conditions for decarboxylation. Conversion of the aa to amines raises the pH, reversing the yellow to purple. Nonfermenters turn the purple a deeper color.		Differentiate fermentative and nonfermentative gram-negative bacteria.
Eosin/methylene blue agar (EMB)	Eosin Y and methylene blue dyes inhibit the growth of gram-positive bacteria.	Lactose and sucrose for differentiation based on fermentation. Sucrose is an alternate energy source for slow lactose fermenters, allowing quick differentiation from pathogens.		Identification of gram-negative bacteria. E coli: Lactose fermenter, forms blue-black with a metallic green sheen. Other coliform fermenters form pink colonies. Nonfermenters: Translucent, either amber or colorless.
Gram-negative broth (GN)	Deoxycholate and citrate salts inhibit gram-positive bacteria.		Increasing mannitol, which temporarily favors the growth of mannitol-fermenting, gram-negative rods (e.g., Salmonella and Shigella spp.)	Enhances the recovery of enteric pathogens from fecal specimens
Hektoen enteric agar (HEK)	Bile salts inhibit gram-positive and many gram-negative normal intestinal flora.	Differential lactose, salicin, and sucrose with a pH indicator bromthymol blue and ferric salts to detect hydrogen sulfide (H₂S). Most pathogens ferment one or both sugars and appear bright orange to salmon pink because of the pH interaction with the dye. Nonfermenters appear green to blue green. H₂S production produces a black precipitate in the colonies.		Detection of enteric pathogens from feces or from selective enrichment broth
Lysine iron agar (LIA)		Contains lysine, glucose, and protein, bromocresol purple (pH indicator) and sodium thiosulfate/ferric ammonium citrate. Purple denotes alkaline (K), red color (R), acid (A). K/K: Organism decarboxylates but cannot deaminate, ferments glucose, first butt is yellow. Decarboxylates lysine producing alkaline; changes back to purple. K/A: Organism fermented glucose but was unable to deaminate or decarboxylate lysine. Bordeaux red and yellow butt. R/A: Organism deaminated lysine but could not decarboxylate it. The lysine deamination combines with the ferric ammonium citrate, forming a burgundy color. Blackening of the butt indicates production of H₂S.		Measures three parameters that are useful for identifying Enterobacteriaceae (lysine decarboxylation, lysine deamination, and H₂S production)
MacConkey agar (MAC)	Bile salts and crystal violet inhibit most gram-positive organisms and permit growth of gram-negative rods.	Lactose serves as the sole carbohydrate. Lactose fermenters produce pink or red colonies, may be precipitated bile salts may surround colonies. Non–lactose fermenters appear colorless or transparent.		Selection for gram-negative organisms and differentiating Enterobacteriaceae
MacConkey-sorbitol (MAC-SOR)		Same as regular MacConkey except D-sorbitol is substituted for lactose. Sorbitol-negative organisms are clear and may indicate E. coli O157:H7.		Used to isolate Escherichia coli O157:H7
Motility test medium		Nonmotile organisms grow clearly only on stab line, and the surrounding medium remains clear. Motile organisms move out of the stab line and make the medium appear diffusely cloudy.		Determine motility for an organism. Identification and differentiation of Enterobacteriaceae. Shigella and Klebsiella spp. are nonmotile; Yersinia sp. are motile at room temperature. Listeria monocytogenes (not an Enterobacteriaceae) has umbrella-shaped motility.
Salmonella–Shigella agar (SS)	Bile salts, sodium citrate, and brilliant green, which inhibit gram-positive organisms and some lactose-fermenting, gram-negative rods normally found in the stool.	Lactose is the sole carbohydrate, and neutral red is the pH indicator. Fermenters produce acid and change the indicator to pink-red. Sodium thiosulfate is added as a source of sulfur for the production of hydrogen sulfide. Also includes ferric ammonium citrate to react with H₂S and produce a black precipitate in the center of the colony. Shigella spp. appear colorless. Salmonella spp. are colorless with a black center.		Select for Salmonella spp. and some strains of Shigella from stool specimens.
Triple sugar iron agar (TSI)		Contains glucose, sucrose, and lactose. Sucrose and lactose are present in 10 times the quantity of the glucose; phenol red is the pH indicator. Turns to yellow when sugars are fermented because of drop in pH. Sodium thiosulfate plus ferric ammonium sulfate as H₂S indicator. Acid/acid (A/A): Glucose and lactose and/or sucrose (or both) fermentation. Gas bubbles: Production of gas. Visible air breaks or pockets in agar. Black precipitate: H₂S. Alkaline/acid (K/A): Glucose fermentation but not lactose or sucrose. Alkaline/alkaline (K/K): No fermentation of dextrose, lactose, or sucrose.		Differentiates glucose fermenters from non– glucose fermenters; also contains tests for sucrose and/or lactose fermentation, as well as gas production during glucose fermentation and H₂S production.
Urea agar		Urea is hydrolyzed to form carbon dioxide, water, and ammonia. Ammonia reacts with components of the medium to form ammonium carbonate, raising the pH, which changes the pH indicator, phenol red, to pink. Limited protein in the medium prevents protein metabolism from causing a false-positive reaction.		Identification of Enterobacteriaceae species capable of producing urease. (Citrobacter, Klebsiella, Proteus, Providencia, and Yersinia spp.)
Xylose-lysine-deoxycholate agar (XLD)	Sodium deoxycholate inhibits gram-positive cocci and some gram-negative rods. Contains less bile salts than other formulations of enteric media (e.g., SS, HEK) and therefore permits better recovery.	Sucrose and lactose in excess concentrations and xylose in lower amounts. Phenol red is the pH indicator. Lysine is included to detect decarboxylation. Sodium thiosulfate/ferric ammonium citrate allows the production of H₂S. The following types of colonies may be seen: Yellow: Fermentation of the excess carbohydrates to produce acid; because of the carbohydrate use, the organisms do not decarboxylate lysine, even though they may have the enzyme. Colorless or red: Produced by organisms that do not ferment any of the sugars. Yellow to red: Fermentation of xylose (yellow), but because it is in small amounts, it is used up quickly, and the organisms switch to decarboxylation of lysine, turning the medium back to red. Black precipitate is formed from the production of H₂S.		Selective media used to isolate Salmonella and Shigella spp. from stool and other specimens containing mixed flora

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, MacConkey 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 20-1).

Figure 20-1 Bull’s-eye colony of *Yersinia enterocolitica (arrow)* on cefsulodin-irgasan-novobiocin (CIN) agar.

Colonial Appearance

Table 20-5 presents the colonial appearance and other distinguishing characteristics (pigment and odor) of the most commonly isolated Enterobacteriaceae on MacConkey, HE, and XLD agars (see Figures 7-4, 7-6, and 7-8 for examples). 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 20-3) as the motile organisms spread from the original site of inoculation.

TABLE 20-5

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
	HE	Colorless
	XLD	Red, yellow, or colorless colonies, with or without black centers (H₂S)
Edwardsiella spp.	MAC	NLF
	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
Plesiomonas shigelloides	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
Salmonella spp.	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
	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-lysine-deoxycholate agar.

^*Most Enterobacteriaceae are indistinguishable on blood agar; see text for colonial description.

^†Pink colonies on MacConkey agar with sorbitol are sorbitol fermenters; colorless colonies are non–sorbitol fermenters.

Figure 20-2 Red-pigmented *Serratia marcescens* on MacConkey agar.

Figure 20-3 *Proteus mirabilis* swarming on blood agar (arrow points to swarming edge).

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 20-1) 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 (Table 20-4). Pigments present in the CIN agar will interfere with correct interpretation of the oxidase test results.

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 (see Procedure 13-41). For presumptive identification of an organism as E. coli, the characteristic colonial appearance on MacConkey agar, as described in Table 20-5, is documented along with positive spot indole test 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.

Table 20-6 provides an overview of common reactions for identifying biochemically unusual enteric pathogens. Figure 20-4 depicts the biochemical reactions typically used to differentiate some of the representative enteric pathogens. To aid the development of an understanding of the separation of common enteric pathogens based on groupings, Figure 20-5 provides a systematic algorithm for grouping pathogens into a working identification scheme.

TABLE 20-6

Biochemical Differentiation of Unusual LDC-, ODC- and ADH-negative Enterobacteriaceae

Genus	Gas from Glucose	Motility	KCN	VP	ACID FERMENTATION
Genus	Gas from Glucose	Motility	KCN	VP	L-Arabitol	Sucrose	Trehalose
Budvicia			neg
Ewingella	neg	V	neg	pos	neg	neg	pos
Leclercia	pos	pos	pos	neg	pos	pos	pos
Moellerella	pos	pos	V	neg	pos	neg	pos
Rahnella	pos	neg	neg	neg	neg	pos	pos
Tatumella	neg	neg	neg	neg	neg	pos	pos
Photorhabdus	neg	pos	neg	neg	neg	neg	neg

ADH, Arginine dihydrolase; KCN, potassium cyanide; LDC, lysine decarboxylase; ODC, ornithine decarboxylase; neg, negative 10%. pos, positive 90%; V, variable 11% to 89%; VP, Voges-Proskauer test.

Figure 20-4 Biochemical differentiation of representative Enterobacteriaceae. V, Variability can be equally either positive or negative; +(v), greater probability for positive reaction >50%; −(v), greater probability for negative reaction >50%; (+), positive > 80%; (−), negative > 80%. The pink squares indicate a pattern useful for preliminary recognition. The green squares indicate a key characteristic for biochemical identification.

Figure 20-5 Algorithm for the identification of Enterobacteriaceae. * Denotes variability in lactose fermentation reactions. LF, Late fermenter; C, indicates growth on Simmons citrate agar; U, indicates urease reaction; I, indicates indole reaction; *MR,* methyl red; +, positive > 90%; − indicates ≤ 10% negative; +/−, > 50%; +, −/+, indicates less than 5% positive]; *KI,* Kligler iron agar; *OD,* ornithine decarboxylase positive; *AD,* arginine decarboxylase positive; *LD,* lysine decarboxylase positive; *PPA,* phenylalanine deamination to phenylpyruvic acid; M, mannitol fermentation; *ONPG,* ortho-nitrophenyl-beta-galactoside test. (Algorithm modified from Gould LH et al: Recommendations for diagnosis of Shiga toxin–producing *Escherichia coli* in clinical laboratories, *MMR* 58(RR12):1, 2009.)

Specific Considerations for Identifying Enteric Pathogens

The common biochemical tests used to differentiate the species in the genus Citrobacter are illustrated in Table 20-3.

Table 20-7 illustrates the use of biochemical profiles obtained with triple sugar iron (TSI) agar and lysine iron agar (LIA) to presumptively identify enteric pathogens (see Chapter 13 for information on the principles, performance, and interpretation of these tests). Organisms that exhibit the profiles shown in Table 20-7 require further biochemical profiling and, in the case of Salmonella spp. and Shigella spp., serotyping to establish a definitive identification. Bacterial species not considered capable of causing gastrointestinal infections give profiles other than those shown, but further testing may be required.

TABLE 20-7

TSI and LIA Reactions Used to Screen for Enteropathogenic Enterobacteriaceae and Aeromonas/Vibrio spp.*^†

TSI Reactions^‡	LIA Reactions^‡	Possible Identification
K/ or K/A H₂S +	K/K or K/NC H₂S+	Salmonella serotypes Edwardsiella spp.
K/A H₂S+	K/K or K/NC H₂S+	Salmonella serotypes (rare)
K/	K/K or K/NC	Salmonella serotypes (rare)
K/A, H₂S	K/K or K/NC H₂S+	Salmonella typhi (rare)
K/	K/A H₂S+	Salmonella paratyphi A (usually H₂S–)
K/	K/A or A/A	Escherichia coli Salmonella paratyphi A Shigella flexneri 6 (uncommon) Aeromonas spp. (oxidase positive)
K/A	K/K or K/NC	Plesiomonas sp. (oxidase positive) Salmonella typhi (rare) Vibrio spp. (oxidase positive)
K/A	K/A or A/A	Escherichia coli Shigella groups A-D Yersinia spp.
A/ H₂S+	K/K or K/NC H₂S+	Salmonella serotypes (rare)
A/A	K/A or A/A	Escherichia coli Yersinia spp. Aeromonas spp. (oxidase positive) Vibrio cholerae (rare, oxidase positive)
A/A	K/K or K/NC	Vibrio spp. (oxidase positive)

A, Acid; , acid and gas production; H₂S, hydrogen sulfide; K, alkaline; LIA, lysine iron agar; NC, no change; TSI, triple sugar iron agar.

^*Vibrio spp. and Aeromonas spp. are included in this table because they grow on the same media as the Enterobacteriaceae and may be enteric pathogens; identification of these organisms is discussed in Chapter 28.

^†TSI and LIA reactions described in this table are only screening tests. The identity of possible enteric pathogens must be confirmed by specific biochemical and serologic testing.

^‡Details regarding the TSI and LIA procedures can be found in Chapter 13.

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 (see Chapter 26), might cross-react 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 (Meridian Diagnostics, Cincinnati, Ohio; Oxoid, Ogdensburg, New York). Some national reference laboratories therefore are simply performing tests for Shiga toxin rather than searching for 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 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 (Table 20-8).

TABLE 20-8

Therapy for Gastrointestinal Infections Caused by Enterobacteriaceae

Organisms	Therapeutic Strategies
Enterotoxigenic Escherichia coli (ETEC) Enteroinvasive E. coli (EIEC) Enteropathogenic E. coli (EPEC) Enterohemorrhagic E. coli (EHEC) Enteroaggregative E. coli (EAEC)	Supportive therapy, such as oral rehydration, is indicated in cases of severe diarrhea; for life-threatening infections, such as hemolytic-uremic syndrome associated with EHEC, transfusion and hemodialysis may be necessary. Antimicrobial therapy may shorten the duration of gastrointestinal illness, but many of these infections resolve without such therapy. Because these organisms may develop resistance (see Table 20-7), antimicrobial drug therapy for non–life-threatening infections may be contraindicated
Shigella spp.	Oral rehydration; antimicrobial drug therapy may be used to shorten the period of fecal excretion and perhaps limit the clinical course of the infection. However, because of the risk of resistance, using antimicrobial drug therapy for less serious infections may be questioned.
Salmonella serotypes	For enteric fevers (e.g., typhoid fever) and extraintestinal infections (e.g., bacteremia), antimicrobial agents play an important role in therapy. Potentially effective agents for typhoid include quinolones, chloramphenicol, trimethoprim/sulfamethoxazole, and advanced-generation cephalosporins, such as ceftriaxone; however, first- and second-generation cephalosporins and aminoglycosides are not effective. For nontyphoidal Salmonella bacteremia, a third-generation cephalosporin (e.g., ceftriaxone) is frequently used. For gastroenteritis, replacement of fluids is most important. Antimicrobial therapy generally is not recommended either for treatment of the clinical infection or for shortening the amount of time a patient excretes the organism.
Yersinia enterocolitica and Yersinia pseudotuberculosis	The need for antimicrobial therapy for enterocolitis and mesenteric lymphadenitis is not clear. In cases of bacteremia, pseudotuberculosis piperacillin, third-generation cephalosporins, aminoglycosides, and trimethoprim/sulfamethoxazole are potentially effective agents. Y. enterocolitica is frequently resistant to ampicillin and first-generation cephalosporins, whereas Y. pseudotuberculosis isolates are generally susceptible

For extraintestinal infections, antimicrobial therapy is a vital component of patient management (Table 20-9). Although a broad spectrum of agents may be used for therapy against Enterobacteriaceae (see Table 12-6 for a detailed list), every clinically relevant species is capable of acquiring and using one or more of the resistance mechanisms discussed in Chapter 14. The unpredictable nature of any clinical isolate’s antimicrobial susceptibility requires that testing be done as a guide to therapy. As discussed in Chapter 12, several standard methods and commercial systems have been developed for this purpose. Table 20-10 presents intrinsic patterns of resistance identified in Enterobacteriaceae.

TABLE 20-9

Antimicrobial Therapy and Susceptibility Testing of Clinically Relevant Enterobacteriaceae

Organism	Therapeutic Options	Potential Resistance to Therapeutic Options	Testing Methods*	Comments
Escherichia coli, Citrobacter spp., Enterobacter spp., Morganella spp., Proteus spp, Providencia spp., Serratia spp.	Several agents from each major class of antimicrobials, including aminoglycosides, beta-lactams, and quinolones, have activity. See Table 12-7 for a list of specific agents that should be selected for in vitro testing. For urinary tract infections, single agents may be used; for systemic infections, potent beta-lactams are used, frequently in combination with an aminoglycoside.	Yes; every species is capable of expressing resistance to one or more antimicrobials belonging to each drug class.	As documented in Chapter 12; disk diffusion agar dilution and commercial systems	In vitro susceptibility testing results are important for guiding broth dilution and therapy.
Yersinia pestis	Streptomycin is the therapy of choice; tetracycline and chloramphenicol are effective alternatives.	Yes, but rare	See CLSI document M100-515; testing must be performed only in a licensed reference laboratory.	Manipulation of cultures for susceptibility testing is dangerous for laboratory personnel and is not necessary.

^*Validated testing methods include standard methods recommended by the Clinical and Laboratory Standards Institute (CLSI) and commercial methods approved by the U.S. Food and Drug Administration (FDA).

TABLE 20-10

Intrinsic Antibiotic Resistance in Enterobacteriaceae *

	Escherichia hermannii	Hafnia alvei	Serratia marcescens	Yersinia enterocolitica	K. pneumoniae	CITROBACTER		ENTEROBACTER		PROTEUS			PROVIDENCIA
	Escherichia hermannii	Hafnia alvei	Serratia marcescens	Yersinia enterocolitica	K. pneumoniae	C. freundii	C. koseri	E. cloacae	E. aerogenes	P. vulgaris	P. mirabilis	P. penneri	P. rettgeri	P. stuartii
Ampicillin	R	R	R	R	R	R	R	R	R	R		R	R	R
Amoxicillin/ clavulanate		R	R	R		R	R	R	R				R	R
Ampicillin/sulbactam		R	R			R	R	R	R
Piperacillin							R
Ticarcillin	R			R	R		R
Cephalosporins I: cefazolin and cephalothin		R	R	R		R		R	R	R		R	R	R
Cephamycins cefoxitin, and cefotetan		R	R			R		R	R
Cephalosporins II: cefuroxime			R			R		R	R	R		R
Tetracyclines										R	R	R	R	R
Nitrofurantoin			R							R	R	R	R	R
Polymyxin B Colistin			R							R	R	R	R	R

^*Cephalosporins III, cefepime, aztreonam, ticarcillin/clavulanate, piperacillin/tazobactam, and the carbapenems are not listed because the Enterobacteriaceae have no intrinsic resistance in to these antibiotics.

Modified from Clinical and Laboratory Standards Institute (CLSI): Performance standards for antimicrobial susceptibility testing—nineteenth informational supplement, CLS Document M100-S21, Wayne, Pa, 2011.

Extended Spectrum β-Lactamase (ESBL)–Producing Enterobacteriaceae

Enterobacteriaceae are capable of producing β-lactamases that hydrolyze penicillins and cephalosporins, including the extended spectrum cephalosporins (cefoxime, ceftriazone, ceftizoxime, and ceftazidime). These enzymes are referred to as ESBLs. A chromogenic agar has been developed for the detection of ESBLs. The agar chrom ID ESBL (bioMerieux, Marcy l’Etolle, France) uses cefpodoxime as a substrate to increase the recovery and sensitivity of CTX-M type ESBL isolates. Some limitations must be considered in the use of this medium, including hyperproducing AmpC (Enterobacter and Citrobacter spp.) and hyperproducing penicillinase (K. oxytoca) false positives. In addition, both Vitek 2 (bioMerieux, Durham, North Carolina) and Phoenix (Becton Dickinson, Sparks, Maryland) have ESBL panels, with expert interpretation available for clinical diagnostic use. Table 20-11 presents an example of an ESBL pattern from a clinical isolate that may require technical interpretation and correction before the results are reported.

TABLE 20-11

Extended Beta-Lactamase Antibiotic Resistance Pattern Based on Vitek 2 Gram-Negative Susceptibility AST-GN24 of an E. coli Isolate

Antibiotic	Vitek 2		Expert*		Final^†
Amikacin	16	S	16	S	16	S
Ampicillin	≥32	R	≥32	R	≥32	R
Ampicillin/sulbactam	≥32	R	≥32	R	≥32	R
Cefazolin	≥64	R	≥64	R	>-64	R
Cefepime	2	S	2	R	2	R
Cefoxitin	≥64	R	≥64	R	≥64	R
Ceftazidime	≥64	R	≥64	R	≥64	R
Ceftriaxone	≥64	R	≥64	R	≥64	R
Ciprofloxacin	≥4	R	≥4	R	≥4	R
Ertapenem	≤0.5	S	≤0.5	S	≤0.5	S
ESBL	Pos	+	Pos	+	Pos	+
Gentamicin	≤1	S	≤1	S	≤1	S
Imipenem	≤1	S	≤1	S	≤1	S
Levofloxacin	≥8	R	≥8	R	≥8	R
Nitrofurantoin	≤16	S	≤16	S	≤16	S
Piperacillin/tazobactam	16	S	16	S	16	S
Tobramycin	≥16	R	≥16	R	≥16	R
Trimethoprim/sulfamethoxazole	≤20	S	≤20	S	≤20	S

Suggested antibiogram correction: Therapeutic interpretations suggest corrections to cefepime; all other cephalosporins were already resistant.

Note: All the cephalosporins except cefepime display a resistance pattern.

^*Expert findings indicate that susceptibility results are fully consistent with the organism identification.

^†Final column indicates that the laboratory technologist corrected the interpretation as indicated before reporting the results to the physician.

ESBLs can occur in bacteria other than Klebsiella spp., E. coli, and Proteus mirabilis. 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 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.

Expanded-Spectrum Cephalosporin Resistance and carbapenemase resistance

The explosion of molecular biology in the past two decades has provided alternatives to phenotypic strategies for the identification of organisms and the genotyping of drug resistance. The bacterial chromosome represents the majority of the genetic make-up or genome within a single organism. However, many genes may be located on extra-chromosomal elements, including transposons and plasmids that are capable of independent replication and movement between organisms. Plasmids exist as double-stranded, closed, circular miniature chromosomes. A single bacterial cell may contain several plasmids. Transposable elements are pieces of DNA that move from one genetic element to another, such as from the plasmid to the chromosome or vice versa (see Chapter 2). Multi-drug resistant organisms are increasing in frequency on a worldwide basis due to the presence of these mobile genetic elements. In addition, these elements may have a complex structure, including the presence of integrans, which are genetic elements specifically designed to take up and incorporate or integrate genes such as those that encode antibiotic resistance.

In the last decade, a very serious emerging mechanism of resistance referred to as carbapenemase resistance has developed in the Enterobacteriaceae family in both hospital and community-acquired infections. Carbapenemase is currently the last treatment option for infections caused by multi-drug resistant bacteria. The various classes of carbapenemases include KPC (Class A) VIM, IMP, NDM (Class B), and OXA-48 (Class D). Class A, C, and D β-lactamases are the enzymes that contain serine at the active site. The metallo-β-lactamases (Class B) require a zinc ion for hydrolysis. Genes encoding the β-lactamase enzymes mutate continuously in response to the heavy pressure exerted by antibiotic use. Amp-C class (Class C) genes that were originally carried on chromosomes are now found on plasmids. The last class of β-lactamases is referred to as oxacillanses (Class D) and contains a higher hydrolysis rate for oxacillin than penicillin.

The resistant mechanism is typically plasmid-borne, and the gene product is capable of hydrolyzing almost all known β-lactam antibiotics. The plasmids that harbor these mobile genetic elements include the various classes of non-typeable plasmids (using current PCR-based replicon typing) and the IncHI family of plasmids. These plasmids demonstrate conjugative transfer (movement between individual bacterial cells) at a higher frequency at 30 °C than at 37 °C. The carbapenemase resistance gene within these plasmids may also be included in a cassette of genes that are flanked by insertion sequences or small transposons that facilitate the movement of the gene between genetic elements. In addition, many of these genes in particular the NDM (Class B) are neither species- nor plasmid-specific, therefore indicating a limitless boundary for spread of this resistance. OXA-48 is carried on a composite transposon known as TN1999 or variants of the transposon known as TN1999.2 and TN1999.3. The metallo-β lactamases are also transferable via a plasmid, and in addition to β-lactamase resistance, the strains are frequently resistant to aminoglycosides and fluorquinolones while remaining susceptible to polymixins.

It appears that these resistant determinants are capable of existing in a very diverse genetic background and able to move from one genetic element to another, one organism to another, and across genus and species lines in an unlimited capacity. It is therefore important for practitioners and laboratorians to not overlook or ignore any new emerging antibiotic patterns of resistance where they least expect them to occur. On February 14, 2013 the Center for Disease Control distributed an official CDC Health Alert through their Health Alert Network indicating that new carbapenem-resistant Enterobacteriaceae warrant additional action by healthcare providers. This alert was based on four key points:

1. While carbapenemase resistance may still be uncommon in some areas, at least 15 unusual biochemical resistance forms have been reported in the United States since July, 2012.

2. This increases the need for healthcare providers to work to aggressively prevent the emergence of CRE.

3. Guidelines are currently available from CDS to prevent CRE (e.g., contact precautions). Guidelines are available at http://www.cdc.gov/hai/organisms/cre/cre-toolkit/index.html.

4. Many of these organisms have been identified in patients within the United States following previous treatment and/or medication outside of the United States. These isolates should be referred to a reference laboratory for confirmatory susceptibility testing that should minimally include an evaluation for KPC and NDM carbapenemases.

Procedure 20-1 Modified Hodge Test (MHT)

Purpose

This test is used to identify carbapenemase production in Enterobacteriaceae.

Principle

Carbapenemase production is detected by examining a test isolate for the production of the enzyme, allowing growth of a carbapenem-susceptible organism to grow toward the carbapenem disk. The resulting growth pattern appears as a cloverleaf-like indention.

Method

1. This is a standard disk diffusion test using either a 10 µg meropenem or 10 µg ertapenem disk placed on a Mueller-Hinton agar plate.

2. The control organism and the test isolate are diluted to a 0.5 McFarland standard. Before inoculation of the agar plate, dilute the control organism McFarland standard to a 1 : 10 with sterile saline.

3. Streak the Mueller-Hinton agar plate with the control organism as a standardized disk diffusion assay.

4. Place the antibiotic disk on the plate.

5. Take a loopful of the test isolate and streak it from the disk to the edge of the plate.

6. Incubate the plate overnight at 37°C.

7. Record the results.

Expected Results

Positive result: Indented growth of the Escherichia coli control strain toward the carbapenem disk.

Negative result: No indention of growth of the E. coli control strain from the zone of inhibition.

Limitations

Interpretation is subjective.

Assay cannot differentiate the presence of different carbapenemases.

False-positive results have been reported for some extended spectrum beta-lactamases (ESBLs) and for AmpC-producing organisms.

Quality Control

E. coli ATCC 25922 carbapenem susceptible

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 trimethoprim-sulfamethoxazole, 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 Ty2la or a parenteral single-dose 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).

Chapter Review

1. All of the following organisms are considered normal intestinal floral except:

a. C. freundii

b. Y. enterocolitica

c. E. coli

d. E. aerogenes

2. Enterobacteriaceae are typically gram negative and:

a. Non–glucose fermenters

b. Capable of reducing nitrates to nitrites

c. Catalase negative

d. Oxidase positive

3. A patient presents with a urinary tract infection. After 24 hours of incubation, the urine culture grows a non–lactose fermenter on MacConkey agar, colorless colonies on HE indole-positive organism. The isolate is most likely:

a. Citrobacter spp.

b. Escherichia spp.

c. Klebsiella spp.

d. Proteus spp.

4. Incubation of which organism at 25°C produces a characteristic yellow pigment?

5. The most common cause of hemolytic uremic syndrome is:

6. Which E. coli produces a heat-labile (LT) enterotoxin and a heat-stable enterotoxin?

7. A patient presents to the physician with pain and frequency of urination. The urine culture reveals a non–lactose fermenting, gram-negative rod with characteristic swarming on blood agar. The biochemical test that would specifically distinguish this organism from other Enterobacteriaceae is:

a. Lactose fermentation

b. Oxidase

c. Phenylalanine deaminase and H₂S

d. Triple sugar iron agar

8. A patient presents with diarrhea and abdominal cramping. The organism isolated from the stool culture is identified as S. dysenteriae (group A). The TSI reaction would have indicated:

9. Which organism is commonly considered an extraintestinal pathogen?

10. Matching: Match each term with the correct description.

a. LPS

b. capsular

c. prodogiosin

d. deoxycholate inhibition

e. MDRTF

f. extended spectrum cephalosporin

k. bile salts and brilliant green

l. pyogenic liver abscess

m. flagella

n. H₂S production

o. mannitol and neutral red

11. Short Answer: Interpret the following susceptibility patterns for two E. coli isolates.

Antibiotic	Results
Antibiotic	Organism 1		Organism 2
Amikacin	≤2	S	16	S
Ampicillin	≤2	S	≥32	R
Ampicillin/Sulbactam	≤2	S	≥32	R
Cefazolin	≤4	S	≥64	R
Cefepime	≤1	S	≤1	S?
Cefoxitin	≤4	S	≤4	S
Ceftazidime	≤1	S	2	S?
Ceftriaxone	≤1	S	8	S?
Ciprofloxacin	≤0.25	S	≥4	R
Ertapenem	≤0.5	S	≤0.5	S
Gentamicin	≤1	S	≤1	S
Imipenem	≤1	S	≤1	S
Levofloxacin	≤0.12	S	≥8	R
Nitrofurantoin	≤16	S	≤16	S
Piperacillin/tazobactam	≤4	S	≥128	R
Tobramycin	≤1	S	≥16	R
Trimethoprim/sulfamethoxazole	≤20	S	≥320	R