Fluorogenic substrates for the detection of microbial nitroreductases.

AIMS: To synthesize and evaluate fluorogenic substrates for the detection of microbial nitroreductases. These substrates, all based on 7-nitrocoumarin, may be reduced to form fluorescent aminocoumarins. METHODS AND RESULTS: Thirty pathogenic microbial strains, including both bacteria and yeasts, were examined for nitroreductase activity in a whole-cell assay. All strains readily reduced each of the seven substrates to generate fluorescence, suggesting the widespread presence of nitroreductase activity in pathogenic bacteria. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: These novel substrates facilitate the direct detection of nitroreductase activity and have potential as sensitive indicators of microbial growth.

Differentiation of Corynebacterium amycolatum, C. minutissimum, and C. striatum by carbon substrate assimilation tests.

We tested the carbon substrate assimilation patterns of 40 Corynebacterium amycolatum strains, 19 C. minutissimum strains, 50 C. striatum strains, and 1 C. xerosis strain with the Biotype 100 system (bioMérieux, Marcy-l'Etoile, France). Twelve carbon substrates of 99 allowed discrimination among the species tested. Additionally, assimilation of 3 of these 12 carbon substrates (maltose, N-acetyl-D-glucosamine, and phenylacetate) was tested with the API 20 NE identification system (bioMérieux). Since concordant results were observed with the two systems for these three carbon substrates, either identification system can be used as a supplementary tool to achieve phenotypic differential identification of C. amycolatum, C. minutissimum, and C. striatum in the clinical microbiology laboratory.

Comparison of the new API Candida system to the ID 32C system for identification of clinically important yeast species.

API Candida was evaluated in comparison with the ID 32C system for the identification of 619 yeast isolates. The sensitivity of API Candida for the identification of the 15 species it claims to identify with and without additional tests was 97.4% (593 of 609) and 75.2% (458 of 609), respectively. The API Candida system is easy to use and rapid (result in 18 to 24 h).

Biochemical differences among human and animal streptococci of Lancefield group C or group G.

Pyogenic streptococci of Lancefield group C or group G from human or animal sources were examined with a view to increasing the number of diagnostic tests useful for their differentiation. Human strains of group G produced L-prolyl-L-arginine aminopeptidase but isolates of Streptococcus equisimilis (group C) did not. Tests for alpha-L-glutamate aminopeptidase together with fermentation of glycogen or sorbitol distinguished S. dysgalactiae from strains of S. equisimilis isolated from animals. It was confirmed that fermentation tests were helpful in the study of S. equi and S. zooepidemicus and that enzyme reactions helped distinguish between S. canis and the human strains of group G.

Evaluation of API NH, a new 2-hour system for identification of Neisseria and Haemophilus species and Moraxella catarrhalis in a routine clinical laboratory.

API NH is a new 2-h system (bioMérieux, La Balme-les-Grottes, France) for the identification of most Neisseria and Haemophilus spp. of clinical significance and of Moraxella catarrhalis and for the detection of penicillinase production. Furthermore, this system allows the biotyping of Haemophilus influenzae and Haemophilus parainfluenzae. Three hundred eighteen strains belonging to these species, previously identified by conventional methods, were tested. Among the 305 strains belonging to species included in the data base, 225 (73.8%) were identified without additional tests, 79 (25.9%) were correctly identified after extra tests, and 1 strain (0.3%) was misidentified. For 131 (90.3%) of the 145 H. influenzae and H. parainfluenzae strains, results of biotyping were in agreement with results of standard methods. API NH is an accurate and reliable method for the routine identification of these bacteria in a clinical laboratory, for biotyping of Haemophilus spp., and for the detection of penicillinase-producing strains. The system is ready to use and time saving; inoculation of the system and reading of results are easy.

Phenotypic and genomic analyses of human strains belonging or related to Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve.

A numerical analysis based on phenotypic characteristics (89 enzymatic tests and 49 carbohydrate acidification tests), in which experimental strips from Biomerieux-API, La Balme les Grottes, France, were used, was performed to characterize 82 new isolates belonging or related to Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve. A total of 72 strains were isolated from child or adult feces, and the other strains were obtained from human vaginas and bronchi. In this study we also included 38 type and reference strains that were representative of all species of the genus Bifidobacterium and 6 strains belonging to the genus Lactobacillus. DNA-DNA relationships between B. longum and B. infantis were determined by using 19 strains related to these species, as determined by the numerical analysis. The degree of DNA binding was determined by the S1 nuclease method. The phenotypic study revealed that there were six main clusters, which were subdivided into nine subclusters. Subcluster Va contained the type strains of B. longum and B. infantis. The DNA-DNA relatedness values of some of the new isolates were very similar to the DNA-DNA relatedness values of the type strain of B. longum. On the basis of these data, it was difficult to isolate B. infantis strains and then to define B. infantis as a single species separated from B. longum. Subclusters IVb to IVf comprised reference strains of B. breve. Cluster III and subcluster Ia were not identified.

API Listeria, a new and promising one-day system to identify Listeria isolates.

API Listeria is a new 10-test strip for 24-h biochemical identification of Listeria isolates. With this commercial system, 85% of 646 Listeria strains, including atypical isolates selected for this study, were recognized at the species and subspecies level without a complementary test. A new test differentiates Listeria monocytogenes from L. innocua on the basis of the absence of arylamidase from the former. With this system, 97.7% (252 of 258) of the L. monocytogenes strains tested were correctly identified and differentiated from 99.4% (175 of 176) of the L. innocua strains also tested. Gram-positive bacteria other than Listeria spp. gave quite different biochemical patterns. This system considerably reduced the time needed for conventional identification, since results were available within 18 to 24 h.

[Rapid identification of Candida albicans: evaluation of "Rapidec albicans". Study of 444 yeast strains]. / Identification rapide de Candida albicans: évaluation de "Rapidec albicans". Etude de 444 souches de levures.

Rapid identification of Candida albicans is of great importance as it is the most frequently isolated yeast pathogen. Rapidec albicans, a new 2-h micromethod, performs two fluorescent enzymatic activities: hexosaminidase and proline arylamidase. A total of 444 yeast strains (334 from type culture collections and 110 from recent clinical isolates) were tested. The sensitivity was 98.5% and the specificity 95.8%. When only considering the clinical strains, 47/47 Candida albicans were identified by Rapidec albicans (sensitivity 100%) but only 43/47 by the germ tube test (sensitivity 91.5%). The specificities of the two tests were respectively 98.2% and 100%. This new system is therefore very efficient for the routine diagnosis of Candida albicans in the clinical field. It is easier and quicker than the germ tube test.

Phenotypic differentiation of bifidobacteria of human and animal origins.

The phenotypes of 153 strains belonging or related to the genus Bifidobacterium were studied. These organisms included 38 collection strains and 115 wild strains (41 strains of human origin, 56 strains of animal origin, and 18 strains obtained from rivers or sewage). Our phenotypic analysis revealed seven main groups that were subdivided into 20 subgroups. Seven subgroups contained no type or collection strain. Among the human strains, the type strains of Bifidobacterium pseudocatenulatum and B. catenulatum fell into group I, which contained the type strains of B. adolescentis (subgroup Ib), B. dentium (subgroup Ic), and B. angulatum (ungrouped). The type strain of B. breve belonged to subgroup IIIa1, and the type strains of B. infantis and B. longum fell into subgroup IIIb1. Group VII comprised only wild strains that were isolated from human infant feces. Among the animal strains, group II consisted mainly of bifidobacteria that were isolated from pig feces and contained the type strains of B. suis (subgroup IIb), B. thermophilum (subgroup IIf), B. choerinum, and B. boum (ungrouped). Wild strains belonging to group V were isolated from pig, calf, cow, and chicken feces; this included the type strains of B. animalis (subgroup Va), B. magnum (subgroup Vb), B. pseudolongum, and B. globosum (subgroup Vc). The strains of human origin (groups I, III, and VII) were well separated from the animal strains (groups II, IV, and V). It was not surprising that the wild strains isolated from surface water or sewage were distributed in the animal groups as well as the human groups. Thus, bifidobacteria can be considered to be successful indicators of human or animal fecal pollution when they are correctly classified. The acidification patterns were not adequate to differentiate Bifidobacterium species, as determined previously by Mitsuoka (Bifidobacteria Microflora 3:11-28, 1984) and Scardovi (p. 1418-1434, in P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, vol. 2, 1986). However, enzymatic tests furnished new taxonomic criteria for the genus.

Description and evaluation of the semiautomated 4-hour ATB 32E method for identification of members of the family Enterobacteriaceae.

A study was performed to compare the rapid identification system ATB 32E (API-bioMérieux SA, La Balme-les-Grottes, France) with conventional biochemical methods for identifying 414 isolates of the family Enterobacteriaceae and the genus Aeromonas, mainly of clinical origin. Overall, 395 strains (95.4%) were correctly identified, with 48 (11.6%) requiring extra tests for complete identification. Ten strains (2.4%) were not identified, and nine (2.9%) were misidentified. The ATB 32E is a suitable alternative for rapid identification of members of the family Enterobacteriaceae.

Evaluation of API Coryne in comparison with conventional methods for identifying coryneform bacteria.

A study was performed to evaluate a new manual miniaturized system, API Coryne (API-bioMérieux, Inc., La Balme les Grottes, France), in which conventional biochemical methods were used to identify 240 isolates of coryneform and related bacteria. A total of 40% of the isolates were excluded from the study because they could not be identified by conventional methods. Identifications of the 240 isolates obtained with API Coryne showed a 97.6% concordance with conventional methods (79% after 24 h of incubation, 21% after 48 h of incubation): 158 (65.8%) isolates were identified with no further testing, and extra testing was required for 76 (31.8%) isolates. In three (1.2%) cases, the organisms did not correspond to any key in the code book and could not be identified by the computer service of the manufacturer. Only three (1.2%) strains were misidentified. The system was shown to be reliable and rapid when compared with standard identification methods.

International collaborative evaluation of the ATB 32 staph gallery for identification of the Staphylococcus species.

This international collaborative study evaluates a new system (ATB 32 Staph) for the identification of staphylococci taking into account the new novobiocin-sensitive and -resistant species reported. This study involved eight laboratories and 792 strains were tested. The reproducibility obtained for the cumulative results of the inter- and intra-laboratory tests was more than 90%. For 713 strains relevant of a species 95.5% were correctly identified by the system. Eight strains (1.2%) were misidentified and 24 strains (3.3%) were not identified. For 79 strains initially considered as not-classified, 62% were identified at the species level by the new system. The newer ATB 32 Staph gallery is a performant and useful method for routine identification of the currently described staphylococci species from clinical and animal origin.

[Synthesis of new fluorogenic substrates derivated of 7-amino-4-trifluoromethylcoumarin. Detection of gram-negative bacteria, Streptococci group A and Enterococci]. / Synthèse de nouveaux substrats fluorogéniques dérivés de la 7-amino 4-trifluorométhyl coumarine. Détection des bactéries Gram négatif, des Streptocoques groupe A et des Entérocoques.

The synthesis of substrates having 7-amino 4-trifluoromethyl coumarin as fluorogenic reagent is described. The first group is performed from L-alanine derivative in order to investigate L-alanine aminopeptidase activity for the detection of Gram-negative bacteria. The second group is synthesized from the L-pyrrolidon carboxylic acid. It allows us to detect the PYRase activity of group A Streptococci and Enterococci. These substrates are evaluated with the N-protected form and the amino free one.

Carbon substrate assimilation patterns of clinical and environmental strains of Aeromonas hydrophila, Aeromonas sobria and Aeromonas caviae observed with a micromethod.

The assimilation of carbon substrates by 103 strains of Aeromonas of different origin identified by conventional methods was studied by means of a standardized micromethod containing 147 tests (API system). Six distinct groups could be recognized and the discriminating substrates were determined. 3 species of Aeromonas can be identified by means of conventional method: A. hydrophila, A. sobria and A. caviae. The method has a number of drawbacks: Some media are unreliable, others are difficult to read, strict preservation conditions are essential. The proposed micromethod for carbon substrate assimilation allows, in most cases, a simple separation of the 3 motile Aeromonas species.

RAPIDEC UR, a 2-h miniaturized system for pinpointing uropathogens.

A miniaturized 2-h system (RAPIDEC UR; API System, la Balme-les-Grottes, Montalieu-Vercieu, France) that uses nine enzymatic tests for the diagnosis of most uropathogens was challenged with 330 strains. With few exceptions (Citrobacter, Enterobacter, and Providencia spp., Morganella morganii, and staphylococci other than Staphylococcus saprophyticus), there were distinct patterns that allowed at least a preliminary species diagnosis.

Two-hour miniaturized system for detection of enteropathogenic bacteria in stool cultures.

A miniaturized 2-h system for detection of lactose-negative potentially enteropathogenic bacteria on the basis of nine enzymatic tests was evaluated. The system was challenged with 210 strains of lactose-negative and lactose-positive species grown on MacConkey agar. Specific and constant patterns were found for Salmonella, Shigella A-C, Yersinia enterocolitica, Aeromonas spp. and Vibrio cholerae. Shigella sonnei and Hafnia alvei had an identical pattern, likewise Plesiomonas shigelloides, halophilic vibrios and Pseudomonas aeruginosa.

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.