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PubMed1969

Polynucleotide sequence relationships among members of Enterobacteriaceae.

Polynucleotide relationships were examined among many representatives of the Enterobacteriaceae by means of agar, membrane filter, and hydroxyapatite procedures. The amount of deoxyribonucleic acid (DNA) that reassociated was dependent, especially in interspecific reactions, on the annealing temperature. In only three cases: Escherichia coli-Shigella flexneri, Salmonella typhimurium-S. typhi, and Proteus mirabilis-P. vulgaris, was relative interspecific duplex formation 80% or higher. In most cases interspecies DNA duplex formation was 40% or less of that obtained from intraspecies DNA reassociation reactions. The stability of E. coli-S. flexneri DNA duplexes formed at either 60 or 75 C was virtually identical to that of homologous E. coli DNA duplexes, and the degree of interspecies duplex formation was minimally affected by the temperature increase (86% at 60 C; 77% at 75 C). The thermal stability of DNA duplexes formed at 60 C between DNA from E. coli and DNA from strains of Aerobacter aerogenes, S. typhimurium, S. typhi, and P. mirabilis was about 12 to 14 C below that of reassociated E. coli DNA. At 75 C, the formation of the interspecific DNA duplexes was markedly decreased, but the stability of the DNA able to reassociate at this temperature approximated that of reassociated E. coli DNA. The degree of reassociation and the thermal stability of E. coli-S. flexneri DNA duplexes suggests relatively little evolutionary divergence in these organisms. The other enterobacteria tested, however, have diverged to a point where less than one-half of their DNA can reanneal with E. coli DNA at 60 C and less than 10% reacts at 75 C. The degree of divergence between various enterobacteria does not appear to be uniform along the DNA molecule. Ribosomal ribonucleic acid (RNA)-specific sequences are conserved among most enterobacteria. An examination of messenger RNA relatively specific for the lactose operon suggests that specific chromosomal genes may diverge more or less than the genome as a whole.

Carbon Isotopes↗
PubMed1975

Evaluation of the pathotec Rapid I-D system for identification of Enterobacteriaceae.

The PathoTec Rapid I-D System for identifying Enterobacteriaceae was evaluated with 471 cultures. In 4,910 individual test comparisons, 95.5% of the results agreed, with results of only two test strips, those for esculin hydrolysis and urease production, agreeing with conventional tests in less than 94% of the trials. The PathoTec system exhibited 94.3% accuracy in identifying these cultures in a double-blind study with conventional media and procedures as the alternate system. Two newly developed test strips, for 0-nitrophenyl-beta-D-galactopyranoside and ornithine decarboxylase, were found to be highly reliable.

Classification↗
PubMed1975

Evaluation of tyrosine medium for the identification of Enterobacteriaceae.

Use of the tyrosine medium in the identification of members of the family Enterobacteriaceae form the basis of this paper. Six hundred and twenty-three strains were tested for their property of tyrosine degradation. Three hundred and seventy-two strains were positive in 24 h and an additional eight strains were positive in 48 to 72 h. These positive strains consisted of all 344 strains of the Proteus-Providencia group, all 25 strains of Citrobacter diversus, and three strains of atypical C. freundii. Our findings suggest that tyrosine degradation is a useful property for separating out the Proteus-Providencia group and in specific identification of C. diversus.

Citrobacter↗
PubMed1975

Evaluation of the R/B and Minitek systems for identification of Enterobacteriaceae.

The R/B and Minitek systems for the identification of Enterobacteriaceae were evaluated, and the results were compared with those obtained by conventional methods. Both systems were rapid and allowed correct identification of about 85% of the 294 isolates (23 species) examined. The individual biochemical reactions showed an overall agreement of 92.6% and 93.1% for the R/B and Minitek systems, respectively.

Classification↗
PubMed1980

Evaluation of cefamandole susceptibility testing of Enterobacteriacea by the autobac 1 system.

A total of 509 clinical isolates of Enterobacteriaceae were tested for susceptibility to cefamandole by Autobac 1 and Bauer-Kirby disk diffusion methods, using commercially available 30-micrograms cefamandole disks. Minimal inhibitory concentrations were determined for all organisms showing major or very major discrepancies. Overall agreement between Autobac 1 and disk diffusion was 89.8%, with 5.1% major or very major and 5.1% minor discrepancies. When considering only the genera for which 20 or more isolates were tested, overall agreement was 90.8%. Discrepancies for Escherichia coli showed a trend toward resistance by Autobac 1, with minimal inhibitory concentrations generally in agreement with disk diffusion results. No trends were detected for other genera. The rate of agreement was lower for Enterobacter species (75.4%), but minimal inhibitory concentrations, determined for all discrepancies in this genus, agreed with Autobafc 1 as often a with disk diffusion results.

Cefamandole↗
PubMed1981

Rapid identification of Enterobacteriaceae with microbial enzyme activity profiles.

A total of 539 clinical isolates belonging to 10 species of the Enterobacteriaceae family were identified by enzyme activity profiles within 30 min of test inoculation. Each isolate was grown at 37 degrees C for 18 h on Mueller-Hinton agar and suspended to an optical density of 200 Klett units on 0.85% saline. Enzyme activity profiles were obtained by inoculating 18 fluorogenic substrates with the standardized bacterial suspension and monitoring initial rates of hydrolysis over the first 30 min of analysis. Individual enzyme activity profiles were entered into a coded data bank, and identifications were based on the Bayesian theory of probabilities. At a confidence level of 95%, five species were identified with a greater than 90% efficiency, three species were identified between 83 and 88% efficiency, and two species demonstrated a 72 and 75% efficiency of identification. The enzyme activity profile method of bacterial identification is rapid, easily automated, and reproducible.

Aminopeptidases↗
PubMed1982

Evaluation of the Enteric-Tek system for identifying Enterobacteriaceae.

The Enteric-Tek wheel (Flow Laboratories), consisting of 14 different biochemical parameters for rapidly identifying Enterobacteriaceae, was evaluated and compared with the conventional method for completely identifying 301 enteric cultures, representing 36 species. The Enteric-Tek system correctly identified 264 (97.8%) of the 270 common or typical strains and 26 (83.9%) of the 31 unusual or atypical strains tested, demonstrating an overall identification accuracy rate of 96.3%. There were 80 (26.6%) correctly identified strains requiring additional tests. Of the 11 (3.6%) misidentifications, 5 (3 Klebsiella and 2 Salmonella strains) were correctly identified at the genus level. When 4,228 individual tests in the Enteric-Tek wheel were compared with the conventional tubed media, 96.4% of the tests agreed; urease, citrate, adonitol, and lactose agreed less than 97%. The Enteric-Tek system was found to be reliable and accurate in producing identifications at the genus and species level within 18 to 24 h.

Bacteriological Techniques↗
PubMed1982

Use of the automicrobic and enteric-tek systems for identification of Enterobacteriaceae.

Studies were performed to evaluate the use of the Enteric-Tek (ET) and the AutoMicrobic system (AMS) for the identification of 201 Enterobacteriaceae freshly isolated from clinical specimens. All test systems were inoculated simultaneously from the same MacConkey agar plate. Organisms were also identified with conventional media. Identifications with the ET and AMS agreed with those made with conventional biochemicals 97% of the time. At a 95% confidence level, the ET was able to identify 75% of the isolates within 18 h without the aid of additional biochemical tests; the AMS identified 92% in 8 h. Technologist time needed for identifications made with the AMS was reduced approximately 57% compared with the ET. In all instances in which the AMS identification disagreed with the conventional, the ET identified the organism correctly. Similarly, organisms misidentified by the ET were correctly identified by the AMS. The data suggest that the AMS and ET identify clinical isolates with comparable accuracy; however, the AMS offers a significant savings in time.

Bacteriological Techniques↗
PubMed1982

Comparison of micro-ID and API 20E in rapid identification of Enterobacteriaceae.

The effectiveness of Micro-ID and API 20E as same-day identification systems for Enterobacteriaceae was evaluated in comparison with conventional identification by using 315 clinical isolates and 90 stock strains. The API 20E system was heavily inoculated according to manufacturer's recommendations for same-day identification. We found that 83 and 81% of isolates provided adequate inocula for Micro-ID and API 20E, respectively, and purity of the heavy inocula was not a problem with either system. Overall agreement with conventional identification at genus and species levels was 93.5% with Micro-ID and 90.2% with API 20E. However, when Klebsiella pneumoniae and K. oxytoca were considered as a single species and Proteus morganii was equated with Morganella morganii, agreement was 95.8 and 90.5%, respectively. Only 83.% of isolates were identified on the day of inoculation by API 20E, in contrast to 94.3% with Micro-ID. The remaining isolates required supplementary overnight testing. Provisional (low selectivity) determinations were constant with conventional identification with 49.3% of isolates with API 20E and 82.6% with Micro-ID. Telephone consultations with the manufacturers to resolve unprinted octal codes required a maximum of 15 min with Micro-ID and from 2 to greater than 48 h with API 20E.

Bacteriological Techniques↗
PubMed1983

ompA gene in the detection of Escherichia coli and other Enterobacteriaceae by nucleic acid sandwich hybridization.

The applicability of the DNA sandwich hybridization method to detection of bacterial DNA from crude samples is demonstrated using Escherichia coli as a model. In sandwich hybridization the sample DNA mediates the binding of a labeled probe DNA fragment to a second DNA bound on filter. For this study the DNA reagents were prepared by subclonings from a recombinant plasmid containing the E. coli K-12 ompA gene and an adjacent fragment of E. coli DNA. The 5' half of the ompA gene (738 base pairs) in pBR322 served as the filter reagent. The 3' half of the ompA gene (300 base pairs) and the adjacent 1,500 base pairs of E. coli DNA were cloned into the single-stranded phage vector M13mp7, and the resulting recombinant phage DNA was labeled with 125I and used as probe in the hybridizations. For maximal hybridization the DNA reagents had to be present in excess of the sample nucleic acid, which was preferably fragmented before testing. In the optimized test, 3 X 10(6) molecules of E. coli DNA from lysed cells were detected by an overnight reaction; the sensitivity of the test was not affected by the presence of 10(9) unrelated bacteria. With the ompA reagents, all members of the family Enterobacteriaceae tested were detected even if the sensitivity was decreased as compared with that for the homologous bacteria. With all other bacteria, including aerobic and anaerobic species of clinical importance, the test was negative. These findings suggest that it may be possible to find group-specific reagents to be used in diagnostic bacteriology.

Bacterial Outer Membrane Proteins↗
PubMed1984

Prediction of sulfamethoxazole-trimethoprim synergistic action against members of the family Enterobacteriaceae with a two-plate agar dilution breakpoint MIC system.

Synergy between sulfamethoxazole (SMZ) and trimethoprim (TMP) was predicted by a two-plate agar dilution breakpoint MIC system. Comparison of the results of this new system with those of the disk diffusion system (P.M. Waterworth, Postgrad. Med. J. Suppl. 45:21-27, 1969) after tests with 1,518 Enterobacteriaceae isolates showed an overall correlation of 99.8%, a sensitivity of 99.7%, and a specificity of 100%. The method involves spot inoculation of 10(3) organisms onto each of two plates, one containing 160 micrograms of SMZ per ml and the other 8 micrograms of TMP per ml (in Oxoid IsoSensitest medium with 3% agar supplemented with 7% saponin-lysed horse blood), and then incubation overnight at 37 degrees C in air. All but three organisms for which SMZ-TMP was found to be synergistic by disk testing were inhibited on both plates. Three isolates of Proteus mirabilis, which failed to correlate with disk testing by this new system, all showed SMZ MICs of 1,000 micrograms/ml. The SMZ-TMP combination was falsely predicted to be nonsynergistic against these three organisms. There were no false synergy predictions by the breakpoint MIC system. Laboratories should report susceptibility to the SMZ-TMP combination only when there is synergy between the constituents. This simple, reliable agar dilution technique enables laboratories to accurately report synergy between SMZ and TMP.

Drug Resistance, Microbial↗
PubMed1975

Evaluation of the enteric analyzer for identification of Enterobacteriaceae.

The reliability of the Enteric Analyzer for identification of Enterobacteriaceae was evaluated using biochemical results previously obtained for 291 organisms with the conventional, R/B, and Minitek systems. The instrument correctly identified 77.3% of the organisms using conventional system results, 74.2% using R/B results, and 60.5% using Minitek results. The low rate of identification with the conventional system occurs primarily because the instrument is not programmed to consider delayed biochemical reactions. The arbitrary use of 90% and 99% probabilities for test reactions also contributes to a lower percentage of identification. The Enteric Analyzer does not replace the judgment of experienced personnel in the identification of atypical bacteria, but it may prove helpful in speeding up final computer identification of typical microorganisms.

Classification↗
PubMed1975

Evaluation of the Enteric Analyzer, an instrument to aid in the identification of Enterobacteriaceae.

This study evaluated the Enteric Analyzer, an instrument designed to identify Enterobacteriaceae, with data obtained from the Modified r/b Enteric Differential System, other rapid identification systems, or conventional identification systems. It is programmed for 19 reactions with data obtained from Center for Disease Control publications. The instrument is very simple to use. Typical strains from 25 of 28 species were unequivocally identified. With the other three species, two choices were given. A switch allows the user to identify all species possibilities where a given biochemical pattern occurs in more than 1% of the strains or more than 10% of the strains. The instrument is useful both in the clinical laboratory and for teaching purposes.

Classification↗
PubMed1984

Evaluation of the four-hour rapid 20E system for identification of members of the family Enterobacteriaceae.

A study was conducted to compare the API Rapid 20E 4-h system (API System S.A., France; commercially available in the U.S.A. under the name DMS Rapid E System; DMS Laboratories, Darts Mill, Flemington, N.J.), the API 20E 18- to 24-h system (Analytab Products, Plainview, N.Y.), and a conventional media system to measure the ability of each to identify members of the family Enterobacteriaceae. Comparison tables rather than simple percentage agreement tables were generated to define the particular strengths and weaknesses of each system and to allow the laboratory to best use the data. The Rapid 20E compared quite favorably with conventional media. It yielded correct identifications with 95.9% of the isolates tested (API 20E, 98% identification rate). In 2.5% of the isolates, the Rapid 20E gave only genus identifications, and in 1.4% the organisms did not correspond to any key in the code book and could not be identified by the manufacturer's computer service. The ease of inoculation and the 4-h capability make the Rapid 20E system an extremely attractive development in the field of bacterial identification.

Bacteriological Techniques↗