Comparison of miniaturized multitest systems with conventional methodology for identification of Enterobacteriaceae from foods.

Four miniaturized multiple test systems were compared with tube methodology used to identify Enterobacteriaceae encountered in foods. Identification aids supplied with each system were used to assign names to isolates at the species level. For the 129 strains tested, the Minitek system demonstrated a 96.9 percent agreement with reactions in tubed media. The Inolex, Analytab, and PathoTec test systems exhibited 94.3, 93.8, and 92.7 percent agreement, respectively. Analytab identified 96.1 percent of the isolates to the species level, whereas the Minitek, PathoTec, and Inolex systems were able to identify 78.3, 32.6, and 27.1 percent, respectively. The results indicate that the Analytab and Minitek systems are acceptable substitutes for the tube methodology routinely employed in identifying enterics from foods. Although the PathoTec system might be used to screen isolates for their identity, neither the presently available PathoTec nor the Inolex systems should be substituted for current methodology when definitive identification of foodborne organisms is required.     

Comparison of miniaturized multitest systems with conventional methodology for identification of Enterobacteriaceae from foods.

Four miniaturized multiple test systems were compared with tube methodology used to identify Enterobacteriaceae encountered in foods. Identification aids supplied with each system were used to assign names to isolates at the species level. For the 129 strains tested, the Minitek system demonstrated a 96.9 percent agreement with reactions in tubed media. The Inolex, Analytab, and PathoTec test systems exhibited 94.3, 93.8, and 92.7 percent agreement, respectively. Analytab identified 96.1 percent of the isolates to the species level, whereas the Minitek, PathoTec, and Inolex systems were able to identify 78.3, 32.6, and 27.1 percent, respectively. The results indicate that the Analytab and Minitek systems are acceptable substitutes for the tube methodology routinely employed in identifying enterics from foods. Although the PathoTec system might be used to screen isolates for their identity, neither the presently available PathoTec nor the Inolex systems should be substituted for current methodology when definitive identification of foodborne organisms is required.     

Evaluation of the Minitek System for Identification of Enterobacteriaceae

Clinical isolates (869) and stock cultures (35) of Enterobacteriaceae were tested in parallel with the Minitek and conventional systems. The Minitek correctly identified 822 of 904 cultures. When a deoxyribonuclease plate was inoculated along with the Minitek, it was possible to speciate Enterobacteriaceae within 24 h. False-positive hydrogen sulfide reactions were the major fault with this system. Reactions were clear-cut and easy for technologists to read.     

Identification of Enterobacteriaceae with the Minitek system

A total of 417 strains (361 Enterobacteriaceae, 56 Vibrionaceae) was examined in all the available Minitek system tests. The results were processed through four successive identification schemes devised by the manufacturer and the proportion of strains correctly identified, not identified or incorrectly identified determined for each scheme. From the results, a probability matrix was constructed incorporating all 35 Minitek tests. Test results for each strain were then processed through this matrix to determine its success in identification. From the matrix the order of separating value of the tests was determined. Forty-three of the strains were each tested three times to assess the level of test reproducibility; the corrected error rate was 0.85%.     

Evaluation of the Minitek system for characterization of Bacillus species

Minitek (BBL Microbiology Systems, Cockeysville, Md.) substrate disks were evaluated as alternatives to conventional tests for the characterization of Bacillus species. Results were compared for 10 reference isolates and 87 isolates from food sources. The overall agreement of results between the Minitek and conventional tests was 92% for reference strains and 86% for food isolates.     

Silver-resistant Enterobacteriaceae from hospital patients

The inclusion of agar medium containing 0.5 mM AgNO3 in the hospital laboratory replicating system for routine antibiotic-susceptibility determinations resulted in identification of species of Enterobacteriaceae (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Proteus mirabilis, and Citrobacter freundii) with silver resistance. Since the study began in October, 1975, 11 in-hospital patients receiving silver sulfadiazine for burn wound prophylaxis have yielded silver-resistant bacteria from their infected burns. During this treatment routine burn-site cultures from these patients yielded 230 isolates of Enterobacteriaceae, including 211 which were sulfonamide-resistant, 97 of which were also silver-resistant, and 38 of which were untested for silver resistance. Seven silver-resistant but sulfonamide-sensitive isolates were incidentally recovered from respiratory specimens from four nonburn patients with silver tracheostomy tubes, one silver-resistant sulfonamide-sensitive isolate was recovered from a small infected burn on the foot of an Emergency Room patient. Previous treatment of this burn was unknown. Representative AgNO3-resistant E. coli isolates from four patients were serologically untypable. Serotyping of representative isolates of K. pneumoniae showed a diversity of types except from two patients who had been in the same ward at the same time.     

Identification of Enterobacteriaceae with the Minitek system

A total of 417 strains (361 Enterobacteriaceae, 56 Vibrionaceae) was examined in all the available Minitek system tests. The results were processed through four successive identification schemes devised by the manufacturer and the proportion of strains correctly identified, not identified or incorrectly identified determined for each scheme. From the results, a probability matrix was constructed incorporating all 35 Minitek tests. Test results for each strain were then processed through this matrix to determine its success in identification. From the matrix the order of separating value of the tests was determined. Forty-three of the strains were each tested three times to assess the level of test reproducibility; the corrected error rate was 0.85%.     

Evaluation of the Minitek system for characterization of Bacillus species.

Minitek (BBL Microbiology Systems, Cockeysville, Md.) substrate disks were evaluated as alternatives to conventional tests for the characterization of Bacillus species. Results were compared for 10 reference isolates and 87 isolates from food sources. The overall agreement of results between the Minitek and conventional tests was 92% for reference strains and 86% for food isolates.     

Characterization of Ornithine Decarboxylase-Positive, Nonmotile Strains of the Klebsiella-Enterobacter Group

Thirty-seven strains of ornithine decarboxylase-positive, nonmotile Klebsiella-Enterobacter organisms isolated from 36 patients were studied by biochemical and serological testing. Five strains gave biochemical reactions which conformed closely to those of Escherichia coli; three strains gave positive Quellung reactions to specific Klebsiella antisera. (Two of these were thought to be Enterobacter in spite of this typing reaction.) The remaining 29 strains were classified as Enterobacter. These results demonstrate the necessity of doing both an ornithine decarboxylase test and a motility test to differentiate Klebsiella from Enterobacter. Had only a motility test been done, they all would have been called Klebsiella.     

Enterobacteriaceae associated with meats and meat handling.

The source of Enterobacteriaceae on meats was shown to be associated with the meat-handling work surfaces in two packing plants studies. A total of 2,343 Enterobacteriaceae were isolated and identified from meat samples and work surfaces at the packing plants and at the retail facilities. Escherichia coli biotype I and Serratia liquefaciens were detected at all stages of meat handling, indicating that they may be present in meats throughout the meat-handling system. Enterobacter agglomerans and S. liquefaciens were the predominant Enterobacteriaceae at the retail level, but they had limited indicator potential for sanitation and hygiene, Klebsiella pneumoniae was a frequent isolate among Enterobacteriaceae from meats and meat-handling surfaces in the packing plants but not at the retail level, indicating that this organism might signal unhygienic handling of meats at the retail level.     

Evaluation of Modified R-B System for Identification of Members of the Family Enterobacteriaceae

In a paired, double-blind study, the modified ("Beckford tube") R-B system was compared with conventional bacteriological procedures for the identification of members of the family Enterobacteriaceae from clinical isolates and stock cultures. The tests in the R-B system yielding positive reactions comparable to those predicted by Ewing's taxonomic classification of Enterobacteriaceae were production of hydrogen sulfide and presence of lysine and ornithine decarboxylasè activities. The test reactions in the R-B system found to be comparable to those in the conventional method were fermentation of glucose, hydrogen sulfide production, and lysine and ornithine decarboxylase activities. The production of gas from glucose was positive in the R-B system more often than in the conventional method; however, the motility test and the production of indole were positive less often in the R-B system. Adequate preliminary identification of the Enterobacteriaceae with the R-B system is enhanced if Simmons' citrate and Christensen's urea tests are used concomitantly. These findings emphasize the manufacturer's instructions that, in interpretation of results, colonial morphology and biochemical reactions must be used concurrently to make an accurate identification.     

Klebsiella, Enterobacter, and Serratia: Biochemical Differentiation and Susceptibility to Ampicillin and Three Cephalosporin Derivatives

Three hundred twenty-nine strains of the tribe Klebsielleae were compared by several biochemical tests and by susceptibility to selected antibiotics. Biochemical tests included urease, amino acid decarboxylase, and hydrogen sulfide production; fermentation of lactose and dextrose; motility; and tests in the IMViC (indole, methyl red, Voges-Proskauer, citrate) series. The isolates were: Klebsiella species, 67.5%; Enterobacter species, 28%, and Serratia species, 4.5%. Minimal inhibitory concentrations of cephaloridine, cephalothin, and a new cephalosporin, cephalexin, and of ampicillin were determined by the agar dilution procedure. Cephalosporins at 20 mug/ml or less inhibited 90% of the Klebsiella strains but only 15% of the Enterobacter strains. Ampicillin inhibited 27% of Enterobacter strains and 17% of Klebsiella strains. Serratia isolates were insensitive to the cephalosporins and ampicillin. The results suggest that precise identification of this group to the generic level can be accomplished readily in the clinical laboratory and that such information is helpful in the preliminary selection of an antibiotic for treatment of clinical infections.     

Evaluation of a commercial beta-glucuronidase test for the rapid and economical identification of Escherichia coli

A commercial beta-glucuronidase (beta-GUR) test for the rapid and economical identification of Escherichia coli was evaluated. A total of 762 clinical strains and 228 environmental isolates were studied. More than 95% of the E. coli strains were found to be beta-GUR positive. Thirty-one clinical isolates of Shigella sonnei, 10 of Enterobacter cloacae, eight of Enterobacter aerogenes, nine of Citrobacter freundii and one of Salmonella enteritidis also gave positive results. The enzyme beta-GUR was also detected in two environmental strains of E. cloacae and one C. freundii. A comparative study between the beta-GUR test and the conventional identification system was carried out in 233 consecutive isolates of lactose positive enterobacteria. Agreement was observed in 223 cases and 190 E. coli strains were correctly identified using this test. Discrepancies were found in 10 cases: nine E. coli were beta-GUR negative and one C. freundii was beta-GUR positive. Escherichia coli was the only species positive for both beta-GUR and indole tests. This procedure permits a rapid, easy, precise and inexpensive identification of E. coli. beta-GUR positive Enterobacter strains have not previously been described.     

Molecular characteristics of extended-spectrum beta-lactamases and qnr determinants in Enterobacter species from Japan

The incidence of extended-spectrum β-lactamases (ESBLs) has been increasing worldwide, but screening criteria for detection of ESBLs are not standardized for AmpC-producing Enterobacteriaceae such as Enterobacter species. In this study, we investigated the prevalence of ESBLs and/or AmpC β-lactamases in Japanese clinical isolates of Enterobacter spp. and the association of plasmid-mediated quinolone resistance (PMQR) determinants with ESBL producers. A total of 364 clinical isolates of Enterobacter spp. collected throughout Japan between November 2009 and January 2010 were studied. ESBL-producing strains were assessed by the CLSI confirmatory test and the boronic acid disk test. PCR and sequencing were performed to detect CTX-M, TEM, and SHV type ESBLs and PMQR determinants. For ESBL-producing Enterobacter spp., pulsed-field gel electrophoresis (PFGE) was performed using XbaI restriction enzyme. Of the 364 isolates, 22 (6.0%) were ESBL producers. Seven isolates of Enterobacter cloacae produced CTX-M-3, followed by two isolates producing SHV-12. Two isolates of Enterobacter aerogenes produced CTX-M-2. Of the 22 ESBL producers, 21 had the AmpC enzyme, and six met the criteria for ESBL production in the boronic acid test. We found a significant association of qnrS with CTX-M-3-producing E. cloacae. The 11 ESBL-producing Enterobacter spp. possessing bla(CTX-M), bla(SHV), or bla(TEM) were divided into six unique PFGE types. This is the first report about the prevalence of qnr determinants among ESBL-producing Enterobacter spp. from Japan. Our results suggest that ESBL-producing Enterobacter spp. with qnr determinants are spreading in Japan.     

Evaluation of a commercial β-glucuronidase test for the rapid and economical identification of Escherichia coli

A commercial β-glucuronidase (β-GUR) test for the rapid and economical identification of Escherichia coli was evaluated. A total of 762 clinical strains and 228 environmental isolates were studied. More than 95% of the E. coli strains were found to be β-GUR positive. Thirty-one clinical isolates of Shigella sonnei , 10 of Enterobacter cloacae , eight of Enterobacter aerogenes, nine of Citrobacter freundii and one of Salmonella enteritidis also gave positive results. The enzyme β-GUR was also detected in two environmental strains of E. cloacae and one C. freundii. A comparative study between the β-GUR test and the conventional identification system was carried out in 233 consecutive isolates of lactose positive enterobacteria. Agreement was observed in 223 cases and 190 E. coli strains were correctly identified using this test. Discrepancies were found in 10 cases: nine E. coli were β-GUR negative and one C. freundii was β-GUR positive. Escherichia coli was the only species positive for both β-GUR and indole tests. This procedure permits a rapid, easy, precise and inexpensive identification of E. coli. β-GUR positive Enterobacter strains have not previously been described.     

Catalase Test as an Aid to the Identification of Enterobacteriaceae

Although the catalase test has been used for many years for rapid differentiation of the genera of gram-positive organisms, little has been said about its use in the family Enterobacteriaceae. It was further noted that a wide variety of methods exist for the execution of the catalase test, that there is no universally accepted strength specified for the hydrogen peroxide, and that no gradations for the vigor and speed of the reaction have been mentioned. Under the conditions of the clinical laboratory, we have developed a simple, rapid, and accurate method for the catalase test that has been of great value as an aid in the identification of the Enterobacteriaceae. With 3% H(2)O(2), it was observed that Serratia, Proteus, and Providencia were vigorous catalase reactors. Only Salmonella and rare Escherichia, Enterobacter, and Klebsiella isolates were moderate catalase reactors. Escherichia and Shigella strains were mostly nonreactive, with less than one-third weekly (+) reactive, whereas most Enterobacter strains tended to be weakly reactive. Klebsiella strains were divided equally between nonreactive and weakly reactive. In practice, this test was also of great value in discerning nonpigmented Serratia cultured from the hospital environment and in detecting mixed flora containing nonspreading Proteus.     

Use of reagent-impregnated (“Patho-Tec”) test papers in the identification of Enterobacteriaceae and similar bacteria

A comparative examination of Patho-Tec reagent-impregnated paper strips and the corresponding classical biochemical tests for the identification of Enterobacteriaceae was made. Patho-Tec papers appeared to be reliable for the cytochrome-oxidase and phenylalanine-deaminase tests. The urease test papers gave false negative reactions inCitrobacter andEnterobacter isolates, but never inProteus strains. The lysine-decarboxylase test papers gave too many false positive results. These shortcomings of the strips interfere with fast and reliable identification ofSalmonella-like Enterobacteriaceae.     

Four-Hour Urease Test for Distinguishing Between Klebsiella and Enterobacter

Infections with Klebsiella and Enterobacter have increased among hospitalized patients. To study such infections, relatively simple but precise methods are needed for clinical laboratories to identify the two genera accurately. Moreover, a rapid identification is essential for assisting with the therapy of the patients. For this purpose, a new 4-hr urease test was developed so that colonies could be tested directly from blood-agar plates which have been inoculated with clinical material and allowed to incubate overnight. This 4-hr test was positive with 98.5% of 202 Klebsiella species and negative with 80 Enterobacter species. As a single criterion for distinguishing between the two major genera, the new 4-hr urease test was just as accurate as a motility test (99% of the 282 isolates were accurately identified with either). The 4-hr urease test represents a simple, rapid, and reliable technique which is ideally suited for use in clinical microbiology laboratories.     

宁波地区肠杆菌科细菌碳青霉烯酶基因的检测研究

目的对宁波地区耐碳青霉烯类肠杆菌科细菌的耐药情况和碳青霉烯酶耐药基因进行研究了解。方法收集2010年1月至11月耐亚胺培南(IPM)、美罗培南(MEM)或厄他培南(ETP)的肠杆菌科菌株进行Hodge试验确认,对于阳性试验菌株PCR同时检测blaKPC、blaNDM-1、blaIMI-1、blaGES、blaSME、blaNmcA和blaSHV-387种基因。结果共收集到肠杆菌科细菌256株,其中耐碳青霉烯类肠杆菌科细菌16株,占6.1%;采用改良Hodge试验确认阳性10株,占62.5%株。PCR检测显示10株均携带有blaKPC,其中肺炎克雷伯菌6株,产气肠杆菌2株,阴沟肠杆菌2株。结论宁波地区产blaKPC型碳青霉烯酶是肠杆菌科细菌耐碳青霉烯类药物的关键因素,其编码基因位于可转移质粒进行传播使得目前的耐药情况越来越严峻。     

Oligonucleotide probes specific for the genus Salmonella and for Salm. typhimurium

The Crystal Enteric/Nonfermenter (E/NF) identification kit (Becton Dickinson Microbiology Systems, USA) was evaluated using water-derived bacterial isolates and results compared to those obtained by the API 20E system (BioMérieux, UK). Both the E/NF and 20E systems correctly identified 93% of the Enterobacteriaceae reference cultures. Both systems agreed in the identification of 64·9% of environmental isolates. The E/NF system gave a positive identification to 88·0% of isolates and the 20E to 79·5% of isolates. The principal tests which gave differing reactions between the two systems were arginine dihydrolase, lysine decarboxylase, urease and citrate utilization.