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Patterns of extracellular proline-specific endopeptidases in Legionella and Flavobacterium spp. demonstrated by use of chromogenic peptides.

Some Legionella strains possess a strong extracellular proline-specific endopeptidase (PSE) activity. Using an enlarged selection of chromogenic peptides representing a variety of N-terminal amino-acids binding to a -prolyl-proline, paranitroanilide chain, PSE activity of Legionella and Flavobacterium strains was examined. Differences in PSE activity emphasized the importance of the chemical structure at the nonchromogenic end of the peptide substrates. There seem to be distinct patterns of N-terminal specificity of PSE in the two bacterial groups.

Chromogenic Compounds↗

Rapid confirmatory identification of Neisseria gonorrhoeae with lectins and chromogenic substrates.

A group of five tests utilizing wheat germ and soybean lectins and chromogenic substrates (orthonitrophenyl-beta-D-galactopyranoside, gamma-glutamyl-beta-naphthylamide, and prolyl-beta-naphthylamide derivatives) was used as a rapid (30-min) method for the identification of Neisseria gonorrhoeae. The rapid method agreed with Minitek test results for all 126 N. gonorrhoeae isolates and all 39 nongonococcal isolates tested. Soybean lectin was useful for the identification of rare strains (4 of 126) of N. gonorrhoeae which are not agglutinated by wheat germ lectin. The chromogenic substrates differentiate N. gonorrhoeae from Neisseria meningitidis, Neisseria lactamica, and other Neisseria species which may grow on Thayer-Martin or other selective media.

Agglutination Tests↗

Evaluation of the RIM-N, Gonochek II, and Phadebact systems for the identification of pathogenic Neisseria spp. and Branhamella catarrhalis.

Methods for identifying Neisseria spp. include conventional and modified carbohydrate degradation procedures, chromogenic enzyme substrate tests, and immunologic coagglutination tests for Neisseria gonorrhoeae. In this study, we evaluated the abilities of the RIM-N carbohydrate degradation system (American MicroScan, Campbell, Calif.), the Gonochek II enzymatic identification system (Du Pont Co., Wilmington, Del.), and the Phadebact Gonococcus coagglutination test (Pharmacia Diagnostics, Piscataway, N.J.) to identify pathogenic Neisseria spp. and Branhamella catarrhalis. Both stock strains and clinical isolates, including 176 N. gonorrhoeae, 173 Neisseria meningitidis, 48 Neisseria lactamica, and 12 B. catarrhalis strains, were tested. The RIM-N identified 98% of the gonococci, 99% of the meningococci, 94% of the N. lactamica strains, and 100% of the B. catarrhalis strains within 1 h. The Gonochek II system identified 99% of the gonococci, 97% of the meningococci, 100% of the N. lactamica strains, and 100% of the B. catarrhalis strains within 30 min. Phadebact coagglutination provided clearly positive results for only 77% of the N. gonorrhoeae strains, producing negative or equivocal results with 23% of the strains. The RIM-N and Gonocheck II tests generally produced clear-cut reactions. An additional advantage of the Gonocheck II system was the small inoculum required for the performance of the test compared with the other systems, thus allowing the identification of N. gonorrhoeae directly from the primary isolation medium.

Agglutination Tests↗

Chromogenic Limulus amoebocyte lysate assay for rapid detection of gram-negative bacteriuria.

A chromogenic Limulus amoebocyte lysate assay was evaluated as a rapid screening test for the detection of clinically significant gram-negative bacteriuria. The development of a distinctive yellow color after the addition of chromogenic substrate to the Limulus amoebocyte lysate-urine reaction mixture was used to measure greater than or equal to 10(5) gram-negative bacteria per ml. A total of 324 urine specimens were assayed, with 68 gram-negative urinary tract infections identified as defined by quantitative urine colony counts of greater than or equal to 10(5) bacteria per ml. Of these, 68 and 67 of 68 were detected by the chromogenic Limulus amoebocyte lysate assay at urine dilutions of 1:10 and 1:20, respectively. Nine false-positive chromogenic Limulus amoebocyte lysate assay results were observed at both urine dilutions and in the same specimens. At a urine dilution of 1:10, sensitivity and specificity were 100 and 96.6%, respectively, with predictive values of 100% for a negative test and 88.3% for a positive test. At a urine dilution of 1:20, sensitivity and specificity were 98.6 and 96.6%, respectively; predictive values were 99.6% for a negative test and 88.3% for a positive test. These data suggest that chromogenic Limulus amoebocyte lysate assay of urine has potential usefulness as a rapid, reliable, and easily performed and interpreted screening test for the diagnosis of clinically significant gram-negative bacteriuria.

Bacteriuria↗

Improved utility of Gonoscreen, a Limulus amoebocyte lysate assay, in the evaluation of urethral discharges in men.

The Limulus amoebocyte lysate (LAL) assay was improved for the rapid evaluation of exudative urethritis in males. Two hundred men with various quantities of urethral discharge were evaluated. Pyrogen-free Dacron swabs were used for sample collection, and a chromogenic substrate was used for visible endpoint determination after a 10-min incubation. Of the 200 patients studied, 57 (29%) had minimal urethral discharges (less than 15 microliter) and could not be evaluated with Gonoscreen (Mallinckrodt, Inc., St. Louis, Mo.), an LAL test kit for the evaluation of urethritis which involves gentle aspiration for sample collection. The improved LAL assay had a sensitivity and specificity of 95 and 97%, respectively, and an overall accuracy to predict culture results of 96%. These results were not statistically different from Gram-stained smears read by experienced microscopists or from culture results (P greater than 0.05). Prevalence of gonorrhea in the study population was 40%, and the positive predictive value of the LAL assay was 95%. Thus, the use of swabs facilitated sample collection and increased by 29% the number of patients which could be evaluated with the LAL assay. In addition, the use of a chromogenic substrate reduced incubation time by 67% (30 to 10 min) and provided an objective color endpoint.

Chromogenic Compounds↗

Preliminary evaluation of a rapid colorimetric method for identification of pathogenic Neisseria.

A rapid colorimetric method for the identification of pathogenic Neisseria (Identicult-Neisseria; Scott Laboratories, Inc.) based on beta-galactosidase, gamma-glutamylaminopeptidase, and gamma-prolylaminopeptidase is described. All 82 clinical isolates of Neisseria gonorrhoeae, 9 clinical isolates of N. meningitidis, and 5 clinical isolates of N. lactamica were correctly determined to the species level, as were 4 isolates of Branhamella catarrhalis. Reactions were prompt and easily interpreted. The system should be extremely useful in clinical laboratories.

Chromogenic Compounds↗

Evaluation of eight methods for identification of pathogenic Neisseria species: Neisseria-Kwik, RIM-N, Gonobio-Test, Minitek, Gonochek II, GonoGen, Phadebact Monoclonal GC OMNI Test, and Syva MicroTrak Test.

The performance of eight methods in identifying Neisseria species, particularly N. gonorrhoeae, was evaluated. These methods included four rapid carbohydrate utilization tests (Gonobio-Test, Neisseria-Kwik, RIM-N, and Minitek); the Gonochek II, a test which is based on the utilization of chromogenic substrates; and three monoclonal antibody tests (Syva MicroTrak, GonoGen, and Phadebact Monoclonal GC OMNI Test). In all, 182 isolates comprised in six species of Neisseria as well as Branhamella catarrhalis and Moraxella sp. were tested. Cystine-tryptic digest agar supplemented with sugars was included for reference purposes. In the carbohydrate utilization tests, the sensitivity and specificity of the Neisseria-Kwik and Minitek tests for the identification of N. gonorrhoeae were 100%. This compared with sensitivities and specificities, respectively, of 100 and 99.1% for the Gonobio-Test and 99.1 and 100% for cystine-tryptic digest agar sugars and the RIM-N test. The sensitivity and specificity of the Gonochek II test were 99.0 and 86.7%, respectively. Although most test kits did not claim to identify all Neisseria species, in several cases isolates of N. subflava were misidentified or could be misinterpreted as N. gonorrhoeae or N. meningitidis. With the monoclonal reagents, the Syva MicroTrak system was 100% sensitive and 100% specific. The GonoGen test was both 99.1% sensitive and specific, while the Phadebact Monoclonal GC OMNI Test was 99.1% sensitive but 91.2% specific. With this latter test, cross-reactions were observed with strains of B. catarrhalis, N. cinerea, and N. lactamica.

Agglutination Tests↗

Enzymatic characterization of Pseudomonas cepacia by API ZYM profile.

The enzymatic activities of 53 strains of Pseudomonas cepacia were determined by using the API ZYM system. Strong alkaline phosphatase, acid phosphatase, butyrate esterase, caprylate esterase, myristate lipase, leucine arylamidase, and phosphoamidase activities were consistently detected in all strains. Weak activities were observed for valine arylamidase, beta-glucosidase, and N-acetyl-beta-glucosaminidase. No activities could be demonstrated for cystine arylamidase, trypsin, chymotrypsin, alpha-galactosidase, beta-galactosidase, beta-glucuronidase, alpha-glucosidase, alpha-mannosidase, and alpha-fucosidase. Enzymatic activities of pseudomonads may provide useful information about their pathogenesis and information for identification of Pseudomonas species.

Chromogenic Compounds↗

Rapid characterization of oral and nonoral pigmented Bacteroides species with the ATB Anaerobes ID system.

The ATB Anaerobes ID system (API SYSTEM, La Balme Les Grottes, France) was evaluated for its ability to differentiate between species of the pigmented Bacteroides group. This identification system is based on the degradation of chromogenic substrates in combination with sugar fermentation reactions. The results showed that the ATB system can be useful for differentiation between the 10 pigmented Bacteroides species. However, additional tests may be necessary.

Bacteriological Techniques↗

Enzymatic extraction and spectral analysis of the chromophore from cell walls of nutritionally deficient streptococci.

We demonstrated by spectral analysis a method to enzymatically cleave the chromophore precursor from nutritionally deficient streptococcal cell walls by treatment with lysozyme. The peak absorbances without and with lysozyme pretreatment were 511.4 +/- 2.02 nm and 513.1 +/- 0.69 nm, respectively. Extraction yields varied among strains and were found to be growth phase dependent. A secondary peak of absorbance (mean of 477 nm) was found in only five of eight strains. The chromophore at a neutral pH undergoes a reaction with phenol consistent with that of a furan, indicating its carbohydrate composition.

Cell Wall↗

Single-step, chromogenic Limulus amebocyte lysate assay for endotoxin.

A new reagent for the chromogenic Limulus amebocyte lysate (LAL) assay is described. LAL was formulated for optimal performance in either an endpoint procedure or a kinetic procedure with the chromogenic substrate, buffer, and LAL components colyophilized as a single reagent. The kinetic chromogenic method required an incubating microplate reader coupled to a computer for collection and analysis of data. The kinetic method had a longer incubation time than the endpoint method and spanned a range of over 3 orders of magnitude compared with the 1-order-of-magnitude range of the endpoint assay. The kinetic method was less subject to operator error, since readings were continuous and automatic. The endpoint test was more operator intensive, requiring both addition of acetic acid to stop the reaction and transfer of the sample to the reading device. A single-step chromogenic reagent was also prepared without lyophilization by mixing reconstituted gel clot LAL with a buffer and a chromogenic substrate. The reagent prepared in this manner performed as well as the colyophilized agent.

Animals↗

Comparison of the staphylocoagulase activities of Staphylococcus aureus and Staphylococcus intermedius on Chromozym-TH.

The staphylocoagulases of Staphylococcus intermedius (39 strains from clinical samples from dogs and 1 strain from a pigeon) and Staphylococcus aureus (40 strains from nine different animal species) were compared by using the chromogenic methods of Engels et al. (W. Engels, M. Kemps, and C. P. A. van Boven, J. Clin. Microbiol. 14:496-500, 1981). S. intermedius produced staphylocoagulase which resembled that of S. aureus in its rate and method of action on prothrombin, but S. intermedius produced it in lesser amounts. Therefore, chromogenic substrate tests such as Chromozym-TH (Boehringer GmbH, Mannheim, Federal Republic of Germany) (as described by Engels et al.) can be used for the detection of staphylocoagulase in both of these species. However, to detect accurately the presence of S. intermedius staphylocoagulase by this method, preconcentration of the extracellular proteins or an extension of the reaction time of the test would be required. The method described by Engels et al. was designed specifically for clinical laboratories that handle human samples. Under those circumstances the method could be regarded as specific, enabling an identification of S. aureus only. However, as both S. aureus and S. intermedius give positive results in this test, in veterinary diagnostic laboratories, heavy reliance must not be placed on this test for determination of organisms to the species level.

Animals↗

Application of colorimetric microdilution plate hybridization for rapid genetic identification of 22 Mycobacterium species.

Quantitative microdilution plate hybridization was used to identify 22 Mycobacterium species. DNAs of clinical strains were rapidly extracted and labeled with photoreactive biotin. Labeled DNAs were distributed into wells of a microdilution plate in which reference DNAs had been immobilized. After 2 h of hybridization, hybridized DNAs were quantitatively detected with peroxidase-conjugated streptavidin and the substrate, tetramethylbenzidine. This method could differentiate among 20 of the 22 Mycobacterium species tested. The type strains of Mycobacterium tuberculosis and M. bovis were genetically highly related and could not be differentiated by this method. Of 194 biochemically identified human clinical strains, 178 (90%) were genetically identified within 3 h of the small-scale DNA extraction.

Aminosalicylic Acid↗

Fluorogenic substrates for differentiation of gram-negative nonfermentative and oxidase-positive fermentative bacteria.

A total of 803 strains of gram-negative nonfermentative and oxidase-positive fermentative bacteria (38 taxa) were investigated for their ability to hydrolyze 53 different fluorogenic 4-methylumbelliferyl- and beta-naphthylamide-linked substrates within 6 h of incubation. The hydrolysis of 16 fluorogenic substrates showed high separation index values among the tested taxa, was reproducible, and showed good agreement with data in the literature. In combination with other biochemical tests (like carbon substrate utilization tests and classical biochemical tests), hydrolysis profiles can improve the differentiation of gram-negative nonfermentative and oxidase-positive fermentative bacteria.

Chromogenic Compounds↗

CHROMagar Candida, a new differential isolation medium for presumptive identification of clinically important Candida species.

CHROMagar Candida is a novel, differential culture medium that is claimed to facilitate the isolation and presumptive identification of some clinically important yeast species. We evaluated the use of this medium with 726 yeast isolates, including 82 isolated directly on the medium from clinical material. After 2 days of incubation at 37 degrees C, 285 C. albicans isolates gave distinctive green colonies that were not seen with any of 441 other yeast isolates representing 21 different species. A total of 54 C. tropicalis isolates also developed distinctive dark blue-gray colonies with a halo of dark brownish purple in the surrounding agar. C. krusei isolates (n = 43) also formed highly characteristic rough, spreading colonies with pale pink centers and a white edge that was otherwise encountered only rarely with isolates of C. norvegensis. Trichosporon spp. (n = 34) formed small, pale colonies that became larger and characteristically rough with prolonged incubation. Most of the other 310 yeasts studied formed colonies with a color that ranged from white to pink to purple with a brownish tint. The only exceptions were found among isolates identified as Geotrichum sp. or Pichia sp., some of which formed colonies with a gray to blue color and which in two instances formed a green pigment or a dark halo in the agar. The specificity and sensitivity of the new medium for the presumptive identification of C. albicans, C. krusei, and C. tropicalis exceeded 99% for all three species. A blinded reading test involving four personnel and 57 yeast isolates representing nine clinically important species confirmed that colonial appearance after 48 h of incubation on CHROMagar Candida afforded the correct presumptive recognition of C. albicans, C. tropicalis, C, krusei, and Trichosporon spp. None of nine bacterial isolates grew on CHROMagar Candida within 72 h, and bacteria (Escherichia coli) grew from only 4 of 104 vaginal, 100 oral, and 99 anorectal swabs. The new medium supported the growth of 19 of 23 dermatophyte fungi tested and 41 of 43 other molds representing a broad range of fungal pathogens and contaminants. In parallel cultures of 348 clinical specimens set up on Sabourand agar and CHROMagar Candida, both media grew yeasts in the same 78 instances. CHROMagar Candida is recommended as a useful isolation medium capable of the presumptive identification of the yeast species most commonly isolated from clinical material and facilitating recognition of mixed yeast cultures.

Agar↗

Evaluation of two BBL Crystal systems for identification of some clinically important gram-negative bacteria.

The BBL Crystal system (Becton Dickinson Microbiology Systems, Cockeysville, Md.) is a miniaturized bacterial identification method employing modified conventional and chromogenic substrates. Two products are currently available, the Rapid Stool/Enteric ID Kit and the Enteric/Nonfermenter ID Kit, each comprising thirty tests. We report an evaluation of both systems (using database version 1.1 for both) in the identification of 51 gram-negative taxa likely to be encountered commonly in the clinical laboratory. In all, 266 strains were tested in the Enteric/Nonfermenter ID Kit, and these represented 36 taxa of the family Enterobacteriaceae (188 strains), 5 oxidase-positive fermentative taxa (26 strains), and 10 nonfermentative taxa (52 strains). The majority of these same strains (203 of 266) were also tested in the Rapid Stool/Enteric ID Kit. The Enteric/Nonfermenter ID Kit performed as follows: Enterobacteriaceae, 93% correct, 6% not identified, and 1% incorrect; oxidase-positive fermenters, 88, 12, and 0%, respectively; and nonfermenters, 100% correct, although several only to the genus or group level. The Rapid Stool/Enteric ID Kit gave the following results: Enterobacteriaceae, 91% correct, 7% not identified, and 2% incorrect; oxidase-positive fermenters, 80, 13, and 7%, respectively (but results were based on only 15 strains); and nonfermenters, 100% correct (but results were based on only 11 strains). We found the systems extremely easy and rapid to use, and for the Enteric/Nonfermenter ID Kit an identification rate of 100% in 40 of 51 taxa was achieved, with corresponding figures of 29 of 39 taxa for the Rapid Stool/Enteric ID Kit.

Bacteriological Techniques↗

Evaluation of new medium with chromogenic substrates for members of the family Enterobacteriaceae in urine samples.

A new medium containing 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide cyclohexylammonium salt (Glu agar) for Escherichia coli and a new medium containing 5-bromo-3-indolyl-beta-D-galactoside (Gal agar) for beta-galactosidase-positive members of the family Enterobacteriaceae were compared with MacConkey agar in a diagnostic trial with 3,562 urine specimens. The isolation rates of E. coli and beta-galactosidase-positive Enterobacteriaceae were increased 8.4 and 19.5%, respectively. The sensitivities and specificities of Glu agar and Gal agar were 98.5 and 100% and 99.2 and 99.5%, respectively.

Bacterial Typing Techniques↗

Evaluation of API An-IDENT and RapID ANA II systems for identification of Actinomyces species from clinical specimens.

We compared the accuracy of the An-IDENT system (bioMerieux Vitek, Inc., Hazelwood, Mo.) and the RapID ANA II system (Innovative Diagnostic Systems, Norcross, Ga.) with that of conventional biochemical tests for the identification of 85 strains of Actinomyces species. In our hands, the overall accuracy of the An-IDENT was 59% and that of the RapID ANA II was 24%. The error rate for the An-IDENT was 18% and that for the RapID ANA II was 38%. The results of this study suggest that although the An-IDENT was more accurate than the RapID ANA II (P < 0.005), neither system, in our hands, was able to identify Actinomyces species with an acceptable degree of accuracy. It is recommended that suspected Actinomyces isolates be identified by conventional testing.

Actinomyces↗