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Three isolates of an unclassified, oxidase-negative and citrate-positive, gram-negative bacillus belonging to the family Enterobacteriaceae were studied. With the exception of the citrate reactions, these strains most closely resembled Escherichia coli in their biochemical reactivity.
A brief, simplified scheme involving the spot indole test and colonial morphology was evaluated for genus level identification of prompt lactose-fermenting (PLF) members of the Enterobacteriaceae. One hundred and ninety-four consecutive, clinically important PLF gram-negative rods isolated in a clinical microbiology laboratory were identified by this simplified scheme, as well as by standard biochemical tests, and the API 20E (Analytab Products, Inc., Plainview, N.Y.) system. In the simplified scheme a flat, spot indole-positive colony was identified as Escherichia coli. Spot indole-negative organisms forming nucoid colonies were identified as Klebsiella sp. or Enterobacter sp. on the basis of semisolid motility and ornithine decarboxylase tests. Approximately 94% of the study isolates followed reactions typical for E. coli, Klebsiella sp., and Enterobacter sp. as defined by this simplified scheme. When compared with the standard and Analytab Products Inc. identifications, the overall accuracy was 97.4%. The accuracy of identification of E. coli, Klebsiella sp., and Enterobacter sp. was 98.1%, 95.6%, and 87.5%, respectively. This simplified scheme is recommended for identification of selected PLF isolates in the clinical microbiology laboratory.
Eighteen Enterobacteriaceae and Pseudomonas aeruginosa strains, 16 of them with well-defined beta-lactam resistance mechanisms, were sent to 52 Spanish microbiology laboratories. Interpretative categories for 8 extended-spectrum beta-lactams were collected. Participating laboratories used their own routine susceptibility testing procedures (88% automatic systems, 10% disk diffusion, and 2% agar dilution). Control results were established by two independent reference laboratories by applying the NCCLS microdilution method and interpretative criteria. Interpretative discrepancies were observed in 16% of the results (4.4% for cefepime, 3.0% for aztreonam, 2.8% for piperacillin-tazobactam, 1.7% for cefotaxime [CTX] and ceftazidime, 1.1% for ceftriaxone, 0.9% for meropenem, and 0.3% for imipenem). High consistency with reference values (<5% of major plus very major errors) was observed with (i) American Type Culture Collection quality control strains; (ii) strains with low-efficiency mechanisms inactivating extended-spectrum beta-lactams, such as OXA-1-producing Escherichiacoli or SHV-1-hyperproducing Klebsiella pneumoniae; (iii) strains with highly efficient mechanisms, such as SHV-5 porin-deficient K. pneumoniae, CTX-M-10 in Enterobacter cloacae hyperproducing AmpC, and P. aeruginosa with the MexAB OprM efflux phenotype or hyperproducing AmpC. Low consistency (>30% major plus very major errors) was detected in K1-producing Klebsiella oxytoca, CTX-M-9-producing E. coli, and in OprD(-) P. aeruginosa strains. Extended-spectrum beta-lactamase (ESBL)-producing strains accounted for 86% of very major errors. Recognition of the ESBL phenotype was particularly low in Enterobacter cloacae strains (<35%), due to the lack of NCCLS-specific rules in this genus. A K1-producing K. oxytoca was misidentified by 10% of laboratories as an ESBL producer. The use of well-defined resistant strains is useful for improving proficiency in susceptibility testing in clinical laboratories.
We evaluated the Vitek 2 ID-GNB identification card (bioMérieux, Inc., Durham, N.C.) for its ability to identify members of the family Enterobacteriaceae and other gram-negative bacilli that are isolated in clinical microbiology laboratories. Using 482 enteric stock cultures and 103 strains of oxidase-positive, gram-negative glucose-fermenting and nonfermenting bacilli that were maintained at -70 degrees C and passaged three times before use, we inoculated cards according to the manufacturer's directions and processed them in a Vitek 2 instrument using version VT2-R02.03 software. All panel identifications were compared to reference identifications previously confirmed by conventional tube biochemical assays. At the end of the initial 3-h incubation period, the Vitek 2 instrument demonstrated an accuracy of 93.0% for the identification of enteric strains; 414 (85.9%) were correctly identified at probability levels ranging from excellent to good, and an additional 34 (7.1%) strains were correctly identified but at a low level of discrimination. Nineteen (3.9%) strains were unidentified, and 15 (3.1%) were misidentified. The 19 unidentified strains were scattered among 10 genera. Three of the 15 misidentified strains were lactose-positive Salmonella spp. and were identified as Escherichia coli; another was a lactose-positive, malonate-negative Salmonella enterica subsp. arizonae strain that was identified as E. coli. Of the 103 glucose-fermenting and nonfermenting nonenteric strains, 88 (85.4%) were correctly identified at probability levels ranging from excellent to good, and 10 (9.7%) were correctly identified, but at a low level of discrimination, for a total of 95.1% accuracy with this group. Two strains were unidentified and three were misidentified. The errors occurred for strains in three different genera. With the increased hands-off approach of the Vitek 2 instrument and accuracies of 93% for the identification of enteric organisms and 95.1% for the identification of nonenteric organisms with the ID-GNB card, use of this product presents an acceptable method for the identification of most gram-negative organisms commonly isolated in the clinical laboratory. A comparison of these results to those obtained by testing 454 of the same strains with the Vitek GNI+ card revealed no significant difference in the abilities of the two cards to identify these organisms accurately.
Twelve isolates of Enterobacteriaceae (1 of Klebsiella pneumoniae, 8 of Escherichia coli, 1 of Proteus mirabilis, and 2 of Proteus vulgaris) classified as extended-spectrum beta-lactamase (ESBL) producers according to the ESBL screen flow application of the BD-Phoenix automatic system and for which the cefotaxime MICs were higher than those of ceftazidime were collected between January 2001 and July 2002 at the Laboratory of Clinical Microbiology of the San Matteo University Hospital of Pavia (northern Italy). By PCR and sequencing, a CTX-M-type determinant was detected in six isolates, including three of E. coli (carrying bla(CTX-M-1)), two of P. vulgaris (carrying bla(CTX-M-2)), and one of K. pneumoniae (carrying bla(CTX-M-15)). The three CTX-M-1-producing E. coli isolates were from different wards, and genotyping by pulsed-field gel electrophoresis (PFGE) revealed that they were clonally unrelated to each other. The two CTX-M-2-producing P. vulgaris isolates were from the same ward (although isolated several months apart), and PFGE analysis revealed probable clonal relatedness. The bla(CTX-M-1) and bla(CTX-M-2) determinants were transferable to E. coli by conjugation, while conjugative transfer of the bla(CTX-M-15) determinant from K. pneumoniae was not detectable. Present findings indicate that CTX-M enzymes of various types are present also in Italy and underscore that different CTX-M determinants can be found in a single hospital and can show different dissemination patterns. This is also the first report of CTX-M-2 in P. vulgaris.
Inoculation of an automated system for rapid identification (ID) and antimicrobial susceptibility testing (AST) directly from positive blood culture bottles will reduce the turnaround time of laboratory diagnosis of septicemic patients, which benefits clinical outcome and decreases patient costs. Direct test results, however, must always be confirmed by testing a pure overnight culture, which is the "gold standard." We studied the accuracy of direct testing versus repeat testing in order to investigate the possibility of refraining from repeat testing. We also assessed the clinical risk of reporting results based on direct testing only. We inoculated Vitek 2 (bioMérieux) directly from 410 positive BACTEC 9240 (BD) blood culture bottles containing gram-negative rods and studied the ID and AST results. In a comparison of direct inoculation with the standard method, a total of 344 isolates of Enterobacteriaceae and Pseudomonas aeruginosa were tested, and 93.0% were correctly identified. Of the 39 (10.2%) samples that contained bacilli not identifiable by Vitek 2, only 1 gave a conclusive, correct result. The overall MIC agreement among 312 isolates was 99.2%, with 0.8% very major and 0.02% major error rates. Of only three (polymicrobial) samples, the direct susceptibility pattern would be reported to the clinician as too sensitive. Vitek 2 results obtained from direct inoculation of blood culture bottles containing gram-negative bacilli are safe enough for immediate reporting, provided that ID and AST are consistent. Repeat testing is not necessary, unless Gram stain or overnight subculture results raise doubt about the purity of the culture.
Urinary tract infections continue to be among the most common extraintestinal diseases. Cystitis in women is by far the most common urinary tract infection; pyelonephritis in both sexes and prostatitis in men are more severe but less frequent complaints. Escherichia coli is by far the most common cause of urinary tract infection. It is believed that uropathogenic E. coli is adept at colonizing the urinary tract via the production of specific virulence factors. Recently, a novel virulence determinant, Vat, was described for the prototypical uropathogenic E. coli strain CFT073. Vat is a member of the SPATE (serine protease autotransporters of the Enterobacteriaceae) subfamily of the autotransporters. Previously, SPATEs have been described for all pathovars of E. coli, but until recently their presence had been noticeably absent in nonpathogenic E. coli. In this report we describe the prevalence and phylogenetic distribution of the SPATEs among uropathogenic E. coli and the ECOR collection, demonstrating an association between the presence of the SPATEs, including Vat, and uropathogenic E. coli phylogroups. In addition, we describe the distribution of SPATEs among nonpathogenic E. coli.
The ability of Serratia to hydrolyze Tween 80 can be utilized to distinguish this genus from other Enterobacteriaceae.
Three separate hospital clinical microbiology laboratories using three different identification systems participated in the identification of Enterobactericeae from a central pool of 'unknown" clinical isolates. With conventional tubed media, API-20E (Anlytab Products Inc.) and R/B tube (Corning Diagnostics) systems, there was a 91.1% agreement in the species designation. No significant differences at the 95% confidence level were found among the systems. Evaluation of individual tests within the systems used revealed lysine decarboxylase of the conventional and citrate of the API-20E system to be significantly different from the same test within the other two systems. The lysine decarboxylase of the conventional system had species relatedness, whereas the differences in citrate of the API-20E system were not related to a particular species. These individual test variations did not affect final organism identification. Reproducibility, evaluated as the system's ability to designate the same identification on two separate occasions, was 92 to 94% for each system. Exact duplication of selected sets of reactions was 60% for conventional, 45% for API-20E, and 61% for R/B. The variations in sets of reactions differed with the system and with the organism involved. The findings suggest equivalency among the three systems in ability to identify common clinical isolates of Enterobacteriaceae and point out the limited usefulness of these systems for biochemical biotyping.
A biochemical screening scheme on agar media for differentiating Enterobacteriaceae in a hospital laboratory is evaluated. Careful observation of test reactions within the scheme permitted the recognition of 78 biotypic reaction patterns which could contribute to epidemiological surveillance. The limitations of the technique are described and discussed and methods of importance in ensuring the reproducibility of reaction patterns emphasised.
Since the introduction of the API 20E kit a number of identification schemes have been developed by the manufacturer for use with the kit. We evaluated the success of these various schemes in identifying 206 strains belonging to 34 taxa of the family Enterobacteriaceae. Many of the strains were atypical and only 94% could be identified by our own system of 50 conventional tests and a computer program. The most advanced identification scheme so far developed for the API 20E kit (the Analytical Profile Index and complementary Computer Service) allowed 88% of the 206 strains to be correctly identified, although 2% were incorrectly identified. The tests in the API 20E kit and 52 conventional tests were separately evaluated for their ability to discriminate between the 34 taxa considered in this study. Our results suggest that replacing some of the tests in the present API 20E kit might further improve its diagnostic performance.
In the Replireader system for identifying Enterobacteriaceae, plates of biochemical media are inoculated with a replicator and the results are put into a computer. The machine correctly identified 92x2% of 734 strains of Gram-negative bacilli isolated from urine; it was incorrect in 0x8% and failed to recognise 7%. The Replireader was also used to record the results of sensitivity tests using a plate dilution method in which the drugs were provided by impregnated filter papers (Adapads).
An automated microELISA Reader was evaluated for its ability to read and interpret microtitre plates. A total of 309 microtitre plates were investigated by automated and visual methods. There was disagreement between the methods in one hundred and twelve (0.6%) wells. However agreements between the two methods for susceptibility tests and Enterobacteriaceae identification were respectively 98.8% and 89.3%.
Two commercial methods for the identification of the Enterobacteriaceae, API 20E and Enterotube II, were compared using the results obtained with 235 cultures of fresh clinical isolates. Enterotube II was an improvement on the original Enterotube, but the availability of two differing indices, one using the Voges-Proskauer (VP) test result and one without, is criticised.
One hundred strains of Enterobacteriaceae were examined in parallel with the API Rapid 20E and Micro-ID commercial four hour identification systems. With the API Rapid 20E system 78% of the strains were correctly identified, 15% were not identified, and 7% were misidentified. The respective figures with the Micro-ID system were 74%, 11%, and 15%.
Six hundred and twenty one strains (555 Enterobacteriaceae, 46 Vibrionaceae, and 20 Acinetobacter) were examined in the Mast system. The results were consulted in the code book supplied by the manufacturer and those not listed were processed through the manufacturer's full database held on an Apricot microcomputer in our laboratory. The proportion of strains correctly identified was 88%, with 9% not identified, and 3% incorrectly identified.
Of 251 isolates of the Enterobacteriaceae identified to species level by API 20E, 208 (83%) were similarily identified by the Sensititre Autoidentification System. Both systems shared a common problem in that discrimination between species of the genera Klebsiella, Enterobacter, and Serratia was poor. The eight digit biocode generated by the Sensititre system for individual isolates is not reproducible and therefore not of epidemiological value.
AIMS: To evaluate the accuracy and reproducibility of multipoint identification schemes in a multicentre trial. METHODS: Forty two strains of Enterobacteriaceae were distributed to 22 laboratories for identification by routine multipoint methods. Analysis of results enabled inter- and intralaboratory reproducibility of a variety of tests, and the ability of laboratories to identify individual organisms to be determined. RESULTS: Interlaboratory reproducibility of most of the biochemical tests was acceptable. The least reproducible tests, both within and between laboratories, were citrate utilisation, production of urease and beta galactosidase, detection of motility, and decarboxylation of lysine and ornithine. Inconsistent results for these tests were often associated with misidentified strains. Most laboratories performed identifications satisfactorily. Most isolates (72.1%) were identified correctly to species level; 9.6% were incorrectly identified, and 6.4% could not be identified at all. The most difficult organisms to identify were Citrobacter freundii, Enterobacter cloacae, Hafnia alvei and Aeromonas hydrophila. Strains of Enterobacter, Serratia sp, and Providencia sp were difficult to speciate. Several laboratories could not identify organisms exhibiting at least one atypical biochemical reaction. CONCLUSION: This study emphasises the need for quality control of media and reagents for multipoint identification of Gram negative enteric bacilli.