Enterobacteriaceae

Chapter 20


Enterobacteriaceae



Objectives



1. Describe the general characteristics of the Enterobacteriaceae, including oxygenation, microscopic Gram staining characteristics, and macroscopic appearance on blood and MacConkey agar.


2. Describe the chemical principle of the media used for the isolation and differentiation of Enterobacteriaceae, including xylose-lysine-deoxycholate agar (XLD), Salmonella-Shigella agar (SS), Hektoen enteric agar (HE), MacConkey agar (MAC), eosin methylene blue agar (EMB), cefsulodin-irgasan-novobiocin agar (CIN), Simmons citrate agar (CIT), gram-negative broth (GN), MacConkey agar with sorbitol (MAC-SOR), lysine iron agar (LIA), and triple sugar iron agar (TSI).


3. Describe the antigens used for serotyping in Enterobacteriaceae, including bacterial location, chemical structure, heat stability, and nomenclature.


4. List the members of the Enterobacteriaceae that are considered intestinal pathogens (rather than extraintestinal pathogens).


5. Compare and contrast infections with the various pathotypes of Escherichia coli (i.e., uropathogenic E. coli [UPEC], meningitis/sepsis–associated E. coli [MNEC], enterotoxigenic E. coli [ETEC], enteroinvasive E. coli [EIEC], enteroaggregative E. coli [EAEC], enteropathogenic E. coli [EPEC], and enterohemorrhagic E. coli [EHEC]), including the route of transmission, types of infection, and pathogenesis.


6. Explain the clinical significance of E. coli O157:H7 and the recommended diagnostic testing for confirmation of infection.


7. Outline the basic biochemical testing procedure to differentiate Enterobacteriaceae from other gram-negative rods.


8. Define ESBL and interpret an antibiotic profile as either positive, negative for ESBL, including corrections required before reporting results.


9. Define MDRTF and the antibiotic susceptibility recommendations associated with identification of an MDRTF isolate.


10. Define an extended spectrum cephalosporin resistance and explain the clinical significance and identification in the clinical laboratory.


11. Describe the modified Hodge test (MHT) procedure, including the chemical principle and clinical significance of the test with regard to carbapenemase resistance.


12. Differentiate Salmonella spp. and Shigella spp. based on biochemical testing.


13. Differentiate Yersinia spp. from the major pathogens among the Enterobacteriaceae.


14. Correlate signs and symptoms of infection with the results of laboratory diagnostic procedures for the identification of a clinical isolate in the Enterobacteriaceae family.



Genera and Species to Be Considered





Because of the large number and diversity of genera included in the Enterobacteriaceae, it is helpful to consider the bacteria of this family as belonging to one of two major groups. The first group comprises species that either commonly colonize the human gastrointestinal tract or are most notably associated with human infections. Although many Enterobacteriaceae that cause human infections are part of our normal gastrointestinal flora, there are exceptions, such as Yersinia pestis. The second group consists of genera capable of colonizing humans but rarely associated with human infection and commonly recognized as environmental inhabitants or colonizers of other animals. For this reason, the discovery of these species in clinical specimens should alert laboratorians to possible identification errors; careful confirmation of both the laboratory results and the clinical significance of such isolates is warranted.




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.



TABLE 20-1


Epidemiology of Clinically Relevant Enterobacteriaceae












































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.


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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.




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.





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.





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.






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.




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.




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.)




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:

 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

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Aug 25, 2016 | Posted by in MICROBIOLOGY | Comments Off on Enterobacteriaceae

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