Use of enzyme tests in characterization and identification of aerobic and facultatively anaerobic gram-positive cocci.

The contribution of enzyme tests to the accurate and rapid routine identification of gram-positive cocci is introduced. The current taxonomy of the genera of aerobic and facultatively anaerobic cocci based on genotypic and phenotypic characterization is reviewed. The clinical and economic importance of members of these taxa is briefly summarized. Tables summarizing test schemes and kits available for the identification of staphylococci, enterococci, and streptococci on the basis of general requirements, number of tests, number of taxa, test classes, and completion times are discussed. Enzyme tests included in each scheme are compared on the basis of their synthetic moiety. The current understanding of the activity of enzymes important for classification and identification of the major groups, methods of testing, and relevance to the ease and speed of identification are reviewed. Publications describing the use of different identification kits are listed, and overall identification successes and problems are discussed. The relationships between the results of conventional biochemical and rapid enzyme tests are described and considered. The use of synthetic substrates for the detection of glycosidases and peptidases is reviewed, and the advantages of fluorogenic synthetic moieties are discussed. The relevance of enzyme tests to accurate and meaningful rapid routine identification is discussed.

Multicenter evaluation of the MicroScan Rapid Gram-Negative Identification Type 3 Panel.

The accuracy and performance of the revised MicroScan Rapid Gram-Negative Identification Type 3 Panel (Dade MicroScan Inc., West Sacramento, Calif.) were examined in a multicenter evaluation. The revised panel database includes data for 119 taxa covering a total of 150 species, with data for 12 new species added. Testing was performed in three phases: the efficacy, challenge, and reproducibility testing phases. A total of 405 fresh and stock gram-negative isolates comprising 54 species were tested in the efficacy phase; 96.8% of these species were identified correctly in comparison to the identification obtained either with the API 20E system (bioMérieux Vitek, Hazelwood, Mo.) or by the conventional tube method. The number of correctly identified isolates in the challenge phase, including new species added to the database, was 221 of 247, or 89.5%, in comparison to the number correctly identified by the conventional tube method. A total of 465 isolates were examined for intra- and interlaboratory identification reproducibility and gave an agreement of 464 of 465, or 99.8%. The overall reproducibility of each individual identification test or substrate was 14,373 of 14,384, or 99.9%. The new Rapid Gram-Negative Identification Type 3 Panel gave accurate and highly reproducible results in this multiple-laboratory evaluation.

Fluorogenic and chromogenic substrates used in bacterial diagnostics.

Methods based on the application of chromogenic and fluorogenic substrates enable specific and rapid detection of a variety of bacterial enzymatic activities. By using these techniques, enzymatic reactions can be examined simultaneously or individually, either directly on the isolation plate or in cell suspensions. For this purpose, various testing principles and test kits for clinical and food microbiology have been introduced successfully during the last few years. In this paper we present a survey of different enzymes of microbial origin that are utilized for microbiological identification and differentiation and the corresponding methods. Particular emphasis is given to the examination of Escherichia coli and the description of the different techniques as used in routine analysis.

Rapid antimicrobial susceptibility testing of gram-negative bacilli using Baxter MicroScan rapid fluorogenic panels and autoSCAN-W/A.

The MicroScan Rapid Neg MIC/Combo panels and autoSCAN-W/A (Walk Away) system utilize automated fluorescence technology for rapid antimicrobial susceptibility testing of Gram-negative bacilli. In a three site clinical study eleven antimicrobial agents were evaluated by comparing results obtained with 741 clinical isolates, using rapid fluorogenic expanded dilution MIC panels and corresponding frozen microdilution reference panels determined visually. Results for 31%, 40%, 12% and 9% of the isolates were available within 3.5, 4.5, 5.5 and 7.0 hours respectively. Results for 7.3% were not available within that time period. For the seven drugs analyzed using a Minimum Inhibitory Concentration range of dilutions, overall agreement (+/- 1 dilution) was 94%, with 1.5% very major, 0.9% major and 2.5% minor errors. For the four drugs analyzed using a Breakpoint range of dilutions, overall agreement (+/- 1 dilution) was 97%, with two percent very major, and one percent major errors. The MicroScan Rapid Neg MIC system is an accurate and rapid method for same day determination of susceptibility of Gram-negative bacilli.

Rapid antimicrobial susceptibility testing of gram-positive cocci using Baxter MicroScan rapid fluorogenic panels and autoSCAN-W/A.

The Microscan Rapid Pos MIC/Combo panels and autoSCAN-W/A (Walk Away) system utilize automated fluorescence technology for rapid antimicrobial susceptibility testing of staphylococci, streptococci, and Listeria. In a three site clinical study, panels containing 26 antimicrobial agents were evaluated by comparing results obtained with 605 clinically significant isolates, using rapid fluorogenic expanded dilution MIC panels and corresponding frozen microdilution reference panels. Results for 16%, 40%, 13%, 9%, 8%, 11% and 1% of the isolates were available within 3.5, 4.5, 5.5, 7.0, 8, 11 and 15 h respectively. Results for 2% were not available within that time period. Overall agreement (+/- 1 dilution) for the 14,609 efficacy comparisons was 96%, with 1% each for very major, major and minor errors. Interlaboratory reproducibility testing of 25 isolates in triplicate in each site, showed an overall essential agreement of 97%. The MicroScan Rapid Pos MIC System is an accurate, reproducible and rapid method for same-day determination of susceptibility of Gram-positive cocci.

Early detection of bacterial growth in blood culture by impedance monitoring with a Bactometer model 32.

A Bactometer model 32 was evaluated for use in early detection of bacterial growth. Experiments with simulated cultures showed that 2 ml of broth introduced into the Bactometer module wells could detect 10(2) and 10(6) CFU/ml in 6 h and 2 h respectively. Both Brain Heart Infusion (BHI) and Fastidious Anaerobic broths supported good growth. Detection of nine of 10 organisms inoculated at approximately 10(6) CFU/ml in BHI were detected within 8.5 h. A culture of Bacteroides fragilis failed to grow under these conditions. Of 189 blood cultures, tested by incubation of 2 ml of BHI, 18 were positive by both conventional and Bactometer methods. False-positive or false-negative specimens were not observed using the Bactometer. Use of the Bactometer enables growth detection at least 12 h earlier than culture methods.

Acquisition of new genes by oral Neisseria.

It is suggested that part of the increased pharyngeal carriage of meningococci reported in patients with gonorrhoea is due to misidentification of gonococci which have been transformed to maltose fermenters by DNA from normal throat flora. The distribution of specific aminopeptidases in strains of gonococci, meningococci isolated from the throat and meningococci from systemic infections is consistent with this view. Gonococci oxidising maltose and gonococci with gamma-L-glutamyl aminopeptidase activity, both factors regarded as typical of Neisseria meningitidis, can be produced in vitro by transformation with DNA from N lactamica and N meningitidis. The clinical and theoretical implications of such changes are discussed.

Evaluation of the Lumac kit for the detection of bacteriuria by bioluminescence.

Four hundred and twenty-two urine samples were screened for significant bacteriuria using bioluminescence and microscopy of uncentrifuged urine. A smaller number of false-negatives were seen with bioluminescence (10%) than with microscopy (40%) while both techniques gave a similar number of false-positives (18%). The kit required a large amount of manual preparation, largely pipetting. With this and the short shelf-life of the reconstituted reagents, it is not suitable for small numbers of urines. At 45p per urine, the cost of bioluminescence is too high.

Automated methods for identification of bacteria from clinical specimens.

Automated methods for measuring enzyme activities of bacterial suspensions in saline are described. The methods were applied to bacteria cultured from urine specimens, and specific enzyme profiles characteristic for Escherichia coli, Klebsiella sp, Proteus sp, and Pseudomonas sp were established. Identification of 294 freshly isolated strains by automated and conventional methods were compared. Results from automated identification based on eight enzyme tests and assay of protein content, all performed on a bacterial suspension made from one colony in 1 ml of saline, agreed 100% with those obtained by conventional methods. Identification was achieved in 6 hours.

Immunological relationships of bacterial L-asparaginases.

Rabbit antisera against L-asparaginase preparations from Escherichia coli, Erwinia carotovora, Citrobacter sp. and Chromobacterium violaceum showed on immunoelectrophoresis that only the enzymes from E. coli and Citrobacter are immunologically related. Purified preparations had to be used to determine the immunological cross-reactions. Immunoelectrophoresis at different pH values yielded the zero mobility points of the enzymes. The activity of the Er. carotovora preparation was enhanced up to fourfold by homologous antiserum but not by normal sera. Heterologous antisera also enhanced, but only at a higher concentration. Less enhancement was observed for the other enzymes with antisera as well as with bovine serum albumin. Inhibition was not observed.

The properties and large-scale production of L-asparaginase from citrobacter.

An intracellular L-asparaginase with antitumour activity was purified from a strain of Citrobacter. The optimum conditions for enzyme production by fermentation on scales up to 2700 l were investigated. Highest enzyme yield was obtained in corn-steep liquor medium (9-2%, W/V) at 37 degrees C. Oxygen limitation was not necessary for high enzyme yield. A total recovery of 4-3% from nucleic-acid-free extract and a 180-fold increase in specific activity were obtained after purificaiton. The specific activity of the purified preparation was 45 i.u./mg protein. The enzyme hydrolysed D-asparagine and L-glutamine at 7 and 5%, respectively, of its activity toward L-asparagine, but L-glutaminase activity could be demonstrated only at substrate concentrations above 5 mM. The Km values for L-asparagine and D-asparagine were 2-6 X 10(-5) and 1-4 X 10(-4) respectively. The anti-lymphoma activity of the enzyme was demonstrated with Gardner lymphosarcoma and was found only slightly less potent that Crasnitin, the most active asparaginase so far tested in this system.

A maximal predictive classification of Klebsielleae and of the yeasts.

The concepts of the numerical method of maximal predictive classification are illustrated with classifications of 13 species of enterobacteria and of 434 species of yeast. The method seeks to classify into a specified number of classes (k) such that more correct statements can be made about the constituent members than with any other classification. The best choice of k relates to the separation of the classes as measured by the average number of correct statements made for an individual assigned to a class to which it does not belong. The maximal predictive classifications are compared with previous classifications of the two groups, which seem to be poor predictively (in terms of the characters considered in this study). The results suggest that taxonomists may be more concerned with maximizing class separation rather than with prediction, but many more groups of organisms would need similar study before this view could be held with confidence.