General Characteristics

Molecular analysis has not proven effective for definitively characterizing all the organisms and genera included within the Enterobacteriaceae family. Therefore, species names and reclassification of organisms continually evolve. In general, the Enterobacteriaceae consist of a diverse group of gram-negative bacilli or coccobacilli; they are non–spore forming, facultative anaerobes capable of fermenting glucose; they are oxidase negative (except for Plesiomonas sp.); and, with rare exception (Photorhabdus and Xenorhabdus spp.), they reduce nitrates to nitrites. Furthermore, except for Shigella dysenteriae type 1, all commonly isolated Enterobacteriaceae are catalase positive.

Epidemiology

Enterobacteriaceae inhabit a wide variety of niches, including the human gastrointestinal tract, the gastrointestinal tract of other animals, and various environmental sites. Some are agents of zoonoses, causing infections in animal populations (Table 20-1). Just as the reservoirs for these organisms vary, so do their modes of transmission to humans.

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).

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.

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.

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.

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, glucose-fermenting, gram-negative, oxidase-negative rods capable of growth on MacConkey agar. The genus contains motile (peritrichous flagella) and nonmotile bacteria. Most E. coli strains are lactose fermenting, but this function may be delayed or absent in other Escherichia spp.

Isolates of extraintestinal E. coli strains have been grouped into two categories: uropathogenic E. coli (UPEC) and meningitis/sepsis–associated E. coli (MNEC). UPEC strains are the major cause of E. coli–associated urinary tract infections. These strains contain a variety of pathogenicity islands that code for specific adhesions and toxins capable of causing disease, including cystitis and acute pyelonephritis. MNEC causes neonatal meningitis that results in high morbidity and mortality. Eighty percent of MNEC strains test positive for the K1 antigen. 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 heat-labile 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 a global aggregative regulator gene, AggR, responsible for cellular adherence. EAEC-associated stool specimens typically are not bloody and do not contain white blood cells. Inflammation is accompanied by fever and abdominal pain.

Ewingella americana

Ewingella americana has been identified from blood and wound isolates. The organism is biochemically inactive, and currently no recommended identification scheme has been identified.

Hafnia alvei

Hafnia alvei (formerly Enterobacter hafniae) has been associated with gastrointestinal infections. The organism, resides in the gastrointestinal tract of humans and many animals It is a motile non–lactose fermenter and is often isolated with other pathogens. Most infections with H. alvei are indentified in patients with severe underlying disease (e.g., malignancies) or after surgery or trauma. However, a distinct correlation with clinical signs and symptoms has not been clearly developed, probably because of the lack of identified clinical cases. Treatment is based on antimicrobial susceptibility testing.

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

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 heat-labile cytotoxin, which has been isolated from patients who have developed a self-limiting antibiotic-associated hemorrhagic colitis. K1 capsular–containing K. pneumoniae organisms are increasingly isolated from 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 described in Chapter 26. The DNase test may also be used to differentiate these organisms. Aeromonas spp. are DNase positive and Plesiomonas organisms are DNase negative.

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

The genera Proteus and Providencia are normal inhabitants of the gastrointestinal tract. They are motile, non–lactose fermenters capable of deaminating phenylalanine. Proteus spp. are easily identified by their classic “swarming” appearance on culture media. However, some strains lack the swarming phenotype. Proteus has a distinct odor that is often referred to as a “chocolate cake” 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

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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