Reagent Strips and Conventional Tests for Acid Production from Mannitol and Coagulase Activity of Staphylococcus: a Comparative Study

A total of 244 Staphylococcus strains were tested simultaneously for acid production from mannitol and for coagulase activity with reagent-impregnated paper strips and with their conventional counterparts. Significant correlation was obtained with 97.9% of the strains for mannitol and with 95% for the coagulase test. The paper strip method is a combined test for both mannitol and coagulase tests, thus making it more convenient and simpler than conventional methods. The results are obtained rapidly within 6 hr by the paper strip method. However, as the paper strip method is designed for the aerobic system, the conventional tests were also carried out under aerobic conditions to compare the results.     

Evaluation of Different Assay Systems for Identification of Environmental Aeromonas Strains

Important biochemical reactions in conventional tests were compared with counterpart reactions in two multiple test systems, API-20E (Analytab Products, Plainview, N.Y.) and Aeromonas hydrophila medium, to evaluate their accuracy for the identification of motile Aeromonas spp. isolated from fish. In a total of 49 Aeromonas spp. isolates and 10 A. hydrophila reference strains, false-negative or -positive reactions were detected in the Voges-Proskauer test, indole production, gelatinase activity, production of gas, fermentation of arabinose, and lysine decarboxylase reaction. A good correlation was found, among the three identification systems, for the fermentation of mannitol and inositol as well as for the arginine dihydrolase and ornithine decarboxylase tests. The failure of A. hydrophila medium in the detection of gas indicates that this medium is not entirely suitable for defining aerogenic or anaerogenic strains. From the results of the present study, we consider that of the identification method and taxonomic scheme to be adopted for environmental Aeromonas spp. must be standardized.     

2-methylbutanal, a volatile biomarker, for non-invasive surveillance of Proteus

Pathogen detection needs a paradigm shift from time-consuming conventional microbiological and biochemical tests to much simpler identification methods with higher sensitivity and specificity. In this regard, a simple detection method for frequently isolated nosocomial uropathogen, Proteus spp., was developed using the characteristic volatile 2-methylbutanal released in Luria Bertani broth. The instant reaction of the compound with 5-dimethylaminonaphthalene-1-sulfonylhydrazine (DNSH) has been adapted to develop a sensitive fluorescence assay named “ProteAl” (Prote, “Proteus” & Al, “Aldehyde”). The assay was performed by direct addition of the fluorescence reagent to the culture after 7 h of growth. The distinct green fluorescence by Proteus (other organisms show orange fluorescence) served as the simplest and quicker identification test available for Proteus. In the laboratory, it exhibited 100 % specificity and 100 % sensitivity during testing of 95 strains including standard and known clinical isolates representing frequently encountered uropathogens.     

Quantitative detection of Proteus species by real-time polymerase chain reaction using SYBR Green I

A SYBR Green real-time polymerase chain reaction (PCR) method for rapid detection of Proteus species was developed and evaluated. Of 322 clinical and food samples tested, 75 samples were positive for Proteus species by using conventional PCR and real-time PCR assays. The results were consistent with standard culture methods and the Vitek auto-microbe system, indicating a 100 % specificity obtained by both PCR assays. For the real-time PCR method, the minimum detectable level was 10 colony forming units (CFU) /ml, which was a 10³ multiple higher than the conventional PCR method. Correlation coefficients of standard curves which were constructed using the threshold cycle (Ct) versus copy numbers of Proteus showed good linearity (R ²?=?0.997). In conclusion, several significant advantages such as higher sensitivity and rapidness were observed by using the SYBR Green real-time PCR method for identifying Proteus species.     

THE USE OF GLUCOSE INORGANIC SALTS MEDIA IN THE CLASSIFICATION OF THE COLI-AEROGENES BACTERIA. I. THE METHYL RED AND VOGES-PROSKAUER REACTIONS

SUMMARY: A chemically defined glucose ammonium phosphate medium gave methy red and Voges-Proskauer reactions which were superior to those in glucose peptone phosphate. Optimum condition for both tests require a starting pH of 6·8 and incubation for 3 days at 30°. The medium gave a negative correlation between the two reactions with 34 out of 35 strains that were positive to both tests in the peptone medium. The addition of glutamic acid helped to eliminate doubtful positive M. R. reactions but seemed to suppress acetylmethylcarbinol production by weakly V.-P. positive strains.     

Comparison of reagent-impregnated paper strips and conventional tests for distinguishing Escherichia from Aerobacter: correlation with colonial morphology

The means for distinguishing Escherichia from Aerobacter (Enterobacter) differ in laboratories and range from complete dependence on colonial reactions on typical gram-negative media to reliance on one or more of the classical indole, methyl red, Voges-Proskauer, citrate (IMViC) parameters. Three colonial types (one prejudged as Escherichia) of lactose-positive rods were catalogued on each of the most commonly used selective media, MacConkey Agar, Endo Agar, and E M B Agar. Each cultural type was presumptively diagnosed and then compared with the expected outcome of individual IMViC tests. The distribution of preliminary identifications was similar from growth patterns on MacConkey Agar and E M B Agar, but it differed markedly from Endo Agar. When organisms initially diagnosed by cultural methods were compared by single IMViC tests, it was found that for each colonial type one of the biochemical parameters was best suited. Thus, for those types initially considered Escherichia, the methyl red or Voges-Proskauer test results agreed most consistently; for other types, the citrate reaction was most satisfactory. In addition, when newly formulated reagent-impregnated paper strip methods for indole, Voges-Proskauer, and citrate were evaluated and compared to the standard methods, agreement was 97% for indole, 90% for Voges-Proskauer, and 95% for Simmons' citrate.     

Rapid Methods for Biochemical Testing of Anaerobic Bacteria

Rapid biochemical tests for nitrate, indole, gelatin, starch, esculin, and o-nitrophenyl-beta-D-galactopyranoside were performed on 112 strains of anaerobic bacteria. All tests were incubated under aerobic conditions, and results were recorded within 4 h. The tests for nitrate, indole, and starch showed a 95% or greater correlation when compared to the standard biochemical tests. Tests for esculin and gelatin showed an agreement of 86 and 77%, respectively. PathoTec test strips for nitrate, indole, esculin, o-nitrophenyl-beta-D-galactopyranoside, Voges-Proskauer, and urease were also tested and showed encouraging results.     

Comparative analysis of bacterial community and antibiotic-resistant strains in different developmental stages of the housefly (Musca domestica)

The housefly (Musca domestica) is an important host for a variety of bacteria, including some pathogenic and antibiotic-resistant strains. To further investigate the relationship between the housefly and the bacteria it harbors, it is necessary to understand the fate of microorganisms during the larval metamorphosis. The major bacterial communities in three developmental stages of the housefly (maggot, pupa, and adult fly) were investigated by a culture-independent method, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR?DGGE) analysis of 16S rRNA genes. The bacteria that were identified using DGGE analysis spanned phyla Proteobacteria, Firmicutes, and Bacteroidetes. Changes in the predominant genera were observed during the housefly development. Bacteroides, Koukoulia, and Schineria were detected in maggots, Neisseria in pupae, and Macrococcus, Lactococcus, and Kurthia in adult flies. Antibiotic-resistant bacteria were screened using a selective medium and tested for antibiotic susceptibility. Most resistant isolates from maggots and pupae were classified as Proteus spp., while those from adult flies were much more diverse and spanned 12 genera. Among 20 tested strains across the three stages, 18 were resistant to at least two antibiotics. Overall, we demonstrated that there are changes in the major bacterial communities and antibiotic-resistant strains as the housefly develops.     

Etude comparee de la detection chimique des proies par Proteus anguinus,cavernicole, et son parent de surface Necturus maculosus (proteidae,urodela)

Do the blind cave salamander Proteus and its epigean relative Necturus use the chemical sense in searching for prey? Proteus shows a significant, and Necturus only a weak, preference for water which passes living prey before entering a choice chamber. Prey which was frozen before the test also gave the same result in both species. However, with this immobilized prey the time for a decision in the test was much longer. The results of ten series of tests demonstrate the importance of chemoreception for prey detection in Proteus. The weaker reactions in Necturus can be explained probably by the different biotope.Six other series of tests have been conducted to show that our results are not influenced by stimuli relating to mechanoreception, thigmotaxis and rheotropism.     

Drug Resistance of Enteric Bacteria IX. Distribution of R Factors in Gram-negative Bacteria from Clinical Sources

Many isolates belonging to the Enterobacteriaceae were collected in 1965 from the inpatients at geographically scattered hospitals in Japan. Among 2,650 Shigella strains examined, 58.4% were found to be drug-resistant; 95.0% of these resistant strains were multiply resistant. Among 434 resistant strains examined, 81% carried R factors that were transferable by cell-to-cell contact. Of 160 isolates of other enteric bacteria, drug-resistant strains included 84.2% of the Escherichia coli, 93.0% of the Klebsiella, and 90.0% of the Proteus cultures. Among these resistant strains, 70.3% of the E. coli, 66.7% of the Klebsiella, and 52.0% of the Proteus were multiply resistant. Of these resistant strains, 84.0% of the E. coli, 88.0% of the Klebsiella, and 50.0% of the Proteus strains carried R factors. These results indicate that R factors are widespread among gram-negative bacteria of clinical significance.     

Accuracy of the disk method for determining antimicrobic susceptibility of common Gram-negative bacilli

A standardized disk susceptibility test was evaluated by comparing results with minimal inhibitory concentrations obtained with agar dilution methods. The agar overlay method was used to test 152 Gram-negative bacilli against eight different antimicrobial agents. One to 3% of the isolates were resistant to an antimicrobic by the MIC method, but appeared to be susceptible by the disk method. Most very major discrepancies involved disk tests withProteus sp., a microorganism notoriously difficult to test reproducibly.Serratia sp. vs. the polymyxins andKlebsiella sp. vs. nitrofurantoin accounted for most other major discrepancies. With other microorganism-drug combinations, the disk test was a reasonably accurate technique for classifying bacteria into resistant or susceptible categories. Gentamicin disk tests were unsatisfactory, but when an intermediate zone category of 13–16 mm was applied, the false susceptible test results were reduced to 2.6%. Intermediate zone sizes were obtained with 6% of the disk tests; most of those isolates were resistant or susceptible but not intermediate in susceptibility. About 11% of the strains had intermediate MICs (5–20% with different drugs), but most of those strains were fully susceptible by the disk technique. *** DIRECT SUPPORT *** A01R4011 00007     

Differentiation of Streptococcus lactis var. maltigenes from Other Lactic Streptococci

Strains of lactic streptococci isolated from samples of raw milk which had developed a malty aroma were subjected to the cultural, physiological, and serological tests commonly employed in the classification of streptococci. None of the strains could be differentiated from Streptococcus lactis by these tests. Resting cells of strains which produced an organoleptically detectable malty aroma when cultured in milk were usually found to possess an active α-ketoacid decarboxylase, indicating the presence of the mechanism responsible for the characteristic aroma production. This decarboxylase activity was either weak or nonexistent in the nonmalty strains, and no activity was detected in known strains of S. lactis, S. cremoris, or S. diacetilactis. The malty strains usually produced higher acidities in milk than did the nonmalty strains, and, in most instances, they developed a granular type of growth sediment in broth, as opposed to a viscid sediment. Many of them gave weakly positive Voges-Proskauer tests in glucose broth with or without added citrate and appeared to be somewhat more resistant to nisin than the nonmalty strains.     

Safety evaluation of probiotic bifidobacteria by analysis of mucin degradation activity and translocation ability

Although probiotic-containing nutrient formulas for infants and toddlers have become very popular, some adverse effects related to translocation of probiotic strains have been reported. We assessed the safety of probiotic bifidobacteria that have been used in clinical investigations and proven to have beneficial effects, by analyzing mucin degradation activity and translocation ability. Mucin degradation activities of three probiotic bifidobacteria strains; Bifidobacterium longum BB536, Bifidobacterium breve M-16V and Bifidobacterium infantis M-63, were evaluated by three in vitro tests comprising growth in liquid medium, SDS-PAGE analysis of degraded mucin residues, and degradation assay in Petri dish. All test strains and control type strains failed to grow in the liquid medium containing mucin as the only carbon source, although good growth was obtained from fecal sample. In the SDS-PAGE analyses of mucin residues and observation of mucinolytic zone in agar plate, the three test strains also showed no mucin degradation activity as the type strains, although fecal sample yielded positive results. In another study, a high dose of B. longum BB536 was administered orally to conventional mice to examine the translocation ability. No translocation into blood, liver, spleen, kidney and mesenteric lymph nodes was observed and no disturbance of epithelial cells and mucosal layer in the ileum, cecum and colon was detected, indicating that the test strain had no translocation ability and induced no damage to intestinal surface. These results resolve the concern about bacterial translocation when using bifidobacteria strains as probiotics, which have been tested in various clinical trials, supporting the continuous use of these probiotic strains without anxiety.     

Survey of Selected Filamentous Fungi for Voges-Proskauer Reactions

Of 16 filamentous fungi surveyed for Voges-Proskauer reactants, 81.2% were found to be positive. The total group fell into three classes: those producing considerable acetylmethylcarbinol (AMC), those producing a small amount, and those producing no AMC. Oidium lactis had a highly significant effect upon lowering the amount of Voges-Proskauer reactants produced by Rhizopus nigricans.     

Identification of environmental Serratia plymuthica strains with the new combo panels type 1S

Automated systems are required when numerous samples need to be processed, offering both high through put and test of a multiple simultaneously. This study was performed to compare the MicroScan WalkAway automated identification system in conjunction with the new MicroScan Combo Neg Panels Type 1S with conventional biochemical methods for identifying ten environmental Serratia plymuthica strains. High correlation between both methods were observed for all the 21 tests evaluated, and the MicroScan system was found capable of correctly identifying all S. plymuthica strains tested. In all tests, the percentage of correlation was 100%, except in raffinose test (91%).     

Comparison of Agar Dilution and Broth Microdilution Methods of Anaerobic Antimicrobial Susceptibility Testing using Several Veterinary Antibiotics against Clostridium perfringens Strains Originating from Porcine and Avian Sources

A broth microdilution and a reference agar dilution method was used to determine minimal inhibitory concentrations (MICs) of five veterinary antimicrobials when tested against 96 animal-derived and six American Type Culture Collection (ATCC) strains of Clostridium perfringens. These antimicrobials [bacitracin methylenedisalicylate (bacitracin-MD), tylosin, virginiamycin, erthromycin and tetracycline] are approved for use in animal feed at different levels for growth enhancement, control, and treatment of a variety of enteric diseases. For bacitracin-MD, MICs were higher using the broth microdilution method when compared to the agar dilution method. The two methods had the lowest agreement when using bacitracin-MD compared to the method agreements of other antimicrobials tested (only 34.3% of the C. perfringens tested within ±1 doubling dilution). Tylosin MICs were lower by the broth microdilution method but had better agreement between methods with 75.5% of the C. perfringens tested within ±1 doubling dilution. Good correlation between methods was found for virginiamycin, tetracycline, and erythromycin with 85.3, 76.5, 81.4% of the C. perfringens tested within ±1 doubling dilution, respectively. Differences in susceptibility to individual antimicrobials were found among the avian and porcine strains by both methods. For avian strains, bacitracin-MD, tylosin, and erthromycin MIC₉₀values had differences of at least four doubling dilutions between methods. There were biases toward higher bacitracin-MD and lower tylosin and erythromycin MIC₉₀values using the broth microdilution method. MIC₉₀values against porcine and ATCC strains were more comparable between methods for all five antimicrobials than those generated against avian strains but the biases were still present. Most animal-derived strains were inhibited by approved livestock feed levels of the antimicrobials. Caution should be used when evaluating the potential effectiveness of feed-based antimicrobials against C. perfringens when results are generated using an in vitro test that may not be in agreement with the reference agar dilution method.     

Isolation of Phenylalanine-Negative Proteus vulgaris

Three phenylalanine-negative Proteus vulgaris strains were isolated from three different sources. The significance of these Proteus strains has not been fully recognized.     

Confirmatory identification of Neisseria gonorrhoeae by slide coagglutination

Neisseria gonorrhoeae was identified by the Phadebact gonococcus test, a rapid slide coagglutination technique, and the results obtained were compared with those obtained by conventional methods (Gram stain morphology, oxidase reaction, and carbohydrate utilization tests) for the confirmatory identification of gonococci. Of 308 clinical isolates examined, the coagglutination procedure correctly identified 97.8% of the isolates tested as N. gonorrhoeae and 93.9% of other bacteria as not N. gonorrhoeae. The coagglutination procedure also identified 29 laboratory strains correctly as not N. gonorrhoeae. The slide coagglutination test is easy to perform and offers a valuable alternative to other techniques for the confirmatory identification of N. gonorrhoeae.     

Isolation of Oxalic acid tolerating fungi and decipherization of its potential to control Sclerotinia sclerotiorum through oxalate oxidase like protein

Oxalic acid plays major role in the pathogenesis by Sclerotinia sclerotiorum; it lowers the pH of nearby environment and creates the favorable condition for the infection. In this study we examined the degradation of oxalic acid through oxalate oxidase and biocontrol of Sclerotinia sclerotiorum. A survey was conducted to collect the rhizospheric soil samples from Indo-Gangetic Plains of India to isolate the efficient fungal strains able to tolerate oxalic acid. A total of 120 fungal strains were isolated from root adhering soils of different vegetable crops. Out of 120 strains a total of 80 isolates were able to grow at 10?mM of oxalic acid whereas only 15 isolates were grow at 50?mM of oxalic acid concentration. Then we examined the antagonistic activity of the 15 isolates against Sclerotinia sclerotiorum. These strains potentially inhibit the growth of the test pathogen. A total of three potential strains and two standard cultures of fungi were tested for the oxalate oxidase activity. Strains S7 showed the maximum degradation of oxalic acid (23?%) after 60?min of incubation with fungal extract having oxalate oxidase activity. Microscopic observation and ITS (internally transcribed spacers) sequencing categorized the potential fungal strains into the Aspergillus, Fusarium and Trichoderma. Trichoderma sp. are well studied biocontrol agent and interestingly we also found the oxalate oxidase type activity in these strains which further strengthens the potentiality of these biocontrol agents.     

Enterotoxigenic Bacteria in Food and Water from an Ethiopian Community

Food and water samples from an Ethiopian community were screened for the presence of enterotoxin-producing bacteria. Using the Chinese hamster ovary cell assay, 40 of 213 isolates (18.8%) produced heat-labile (LT) enterotoxin. These LT-producing isolates comprised 33 of 177 (18.6%) strains from 24 of 68 food samples (35.3%) and 7 of 36 (19.4%) isolates of 4 of 17 water samples (23.5%). One LT-producing strain each of Salmonella emek and of Shigella dysenteriae was found. Three pseudomonads, all LT producers, produced heat-stable enterotoxin as gauged by the suckling mouse test. Two strains of LT-enterotoxigenic Escherichia coli O68 were found in water samples. No enterotoxigenic E. coli were isolated from food samples, but 13 of the LT-producing strains were Enterobacter, Klebsiella, Serratia, and Proteus species, and 7 food samples yielded more than one species of enterotoxigenic bacterium. Of the enterotoxigenic isolates from food, 15 were oxidase-positive strains of the genera Aeromonas, Pseudomonas, Achromobacter, Flavobacterium, and Vibrio. LT-enterotoxigenic Enterobacter, Acinetobacter, Klebsiella, Proteus, Providencia, and Serratia species represented 20 of the food and water isolates. Culture supernatant fluids of representative strains of oxidase-positive and oxidase-negative species giving positive reactions in Chinese hamster ovary cell tests induced fluid accumulation in rabbit ileal loops. Eight of the food samples and two of the water samples contained more than one isolate or species of enterotoxigenic bacterium. The stability of the LT production by oxidase-positive bacteria and non-E. coli strains was assessed by the rabbit skin and adrenal cell tests after 9 months and 1 year of storage, respectively, in Trypticase soy broth with glycerol at −70°C. Only 33% of the oxidase-positive strains were still LT enterotoxigenic. Of the oxidase-negative strains, 50 and 33% were LT producing at 9 months and 1 year, respectively. None of the E. coli isolates, both enterotoxigenic and nonenterotoxigenic, possessed K88, K99, or colonization factor antigen. The survey demonstrates the presence in food and water of enterotoxigenic bacteria of the same species as those isolated from cases of infantile diarrhea in the same community, although a correlation between these sources and infantile diarrhea remains to be established.