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 data bases 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 . For presumptive identification of an organism as E. coli, the characteristic colonial appearance on MacConkey agar, as described in Table 1, 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.

Table1. Colonial Appearance and Characteristics of the Most Commonly Isolated Enterobacteriaceae*

Table 2 provides an overview of common reactions for identifying biochemically unusual enteric pathogens. Figure 1 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 2 provides a systematic algorithm for grouping pathogens into a working identification scheme.

Table2. Biochemical Differentiation of Unusual LDC-, ODC- and ADH-negative Enterobacteriaceae

Fig1. 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. 

Fig2. 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 3.

Table3.  Biochemical Differentiation of Citrobacter Species

Table 4 illustrates the use of biochemical profiles obtained with triple sugar iron (TSI) agar and lysine iron agar (LIA) to presumptively identify enteric pathogens. Organ isms that exhibit the profiles shown in Table 4 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.

Table4. TSI and LIA Reactions Used to Screen for  Enteropathogenic Enterobacteriaceae and Aeromonas/Vibrio spp.

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 anti-sera 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 , 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 deter mine 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.

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اخبار العتبة العباسية المقدسة

مستشفى الكفيل يعلن انضمامه لمشروع الضمان الصحي الحكومي

المجمَع العلمي يقيم مسابقة الكوثر الخاصة بترتيل القرآن الكريم في كربلاء

قسم الشؤون الفكرية والثقافية يُصدر العدد الثالث والعشرين من مجلّة (أوراق معرفية)

قسم التربية والتعليم يُطلق الموسم التدريبيّ الصيفيّ لملاكات مجموعة العميد التربوية

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الآخبار الصحية

اختراق علمي.. دواء يؤخر ظهور السكري من النوع الأول لسنوات

هذا ما يحدث لجسمك إن أهملت نظافة فراشك

لماذا تعتبر النساء أكثر عرضة للإصابة بألزهايمر من الرجال؟

لماذا يحذر الخبراء من الإفراط في تناول الفراولة؟

المزيد

الآخبار التكنلوجية

السيارات الكهربائية أنظف بنسبة 73% من سيارات الوقود

سيارة لوسِد الكهربائية تدخل "غينيس" بأطول مدى في العالم

نظرية جديدة تنسف كل ما نعرفه عن تشكل أول قارة في الأرض

منذ 37 ألف عام.. دراسة تكشف "أقدم أمراض" أصابت البشر

المزيد

مواضيع ذات صلة

Expanded-Spectrum Cephalosporin Resistance and Carbapenemase Resistance

Extended Spectrum β-Lactamase (ESBL) Producing Enterobacteriaceae

Bordetella

Hemophilus

Yersinia

Cultivation of Enterobacteriaceae

Primary Intestinal Pathogens of Enterobacteriaceae

Opportunistic Human Pathogens of Enterobacteriaceae

Vibrio

Salmonella

Escherichia coli

Laboratory Diagnosis for Nocardia, Streptomyces, Rhodococcus, and Similar Organisms