Comparison of methods for the identification of coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) species identification is still difficult for most clinical laboratories. The scheme proposed by Kloos and Schleifer and modified by Bannerman is the reference method used for the identification of staphylococcal species and subspecies; however, this method is relatively laborious for routine use since it requires the utilization of a large number of biochemical tests. The objective of the present study was to compare four methods, i.e., the reference method, the API Staph system (bioMérieux) and two methods modified from the reference method in our laboratory (simplified method and disk method), in the identification of 100 CNS strains. Compared to the reference method, the simplified method and disk method correctly identified 100 and 99% of the CNS species, respectively, while this rate was 84% for the API Staph system. Inaccurate identification by the API Staph method was observed for Staphylococcus epidermidis (2.2%), S. hominis (25%), S. haemolyticus (37.5%), and S. warneri (47.1%). The simplified method using the simple identification scheme proposed in the present study was found to be efficient for all strains tested, with 100% sensitivity and specificity and proved to be available alternative for the identification of staphylococci, offering, higher reliability and lower cost than the currently available commercial systems. This method would be very useful in clinical microbiology laboratory, especially in places with limited resources.     

The use of PCR to aid in the rapid identification of Vibrio harveyi isolates

AIMS: Vibrio harveyi is an important pathogen, causing potential devastation to marine aquaculture. This organism, however, is extremely difficult to identify because it is phenotypically diverse. Biochemical identification can involve many tests and take weeks to perform. The aim of this work is to develop a PCR that can reduce the number of biochemical tests, and the time taken, to get a definitive identification of this organism. METHODS AND RESULTS: The PCR was developed using 16S rDNA sequences from a number of V. harveyi strains, and other vibrios. The described test gave positive results for all strains of V. harveyi tested. However, some strains of V. alginolyticus also gave positive results and a small number of biochemical tests were required to differentiate between these two species. This indicated that preisolation of the bacteria was needed and therefore the test was not applicable to the testing of mixed populations directly. CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: The duration of identification of this species was significantly reduced from a number of weeks to a few days. Hence, diagnosis of affected animals will be faster and earlier treatment can be administered which may increase the survival rate from vibriosis.     

Evaluation of oxacillin and cefoxitin disks for detection of resistance in coagulase negative staphylococci

Coagulase-negative Staphylococcus spp. was considered nonpathogenic until the emergence of multiresistance and the demonstration of their participation as infectious agents. In Brazil, oxacillin resistance may be present in over 80% of isolates, and the Clinical and Laboratory Standards Institute standardized a disk-diffusion method to predict this resistance in Staphylococcus. The aim of this study was to evaluate the variability among commercial disks of oxacillin (1 microg) and cefoxitin (30 microg) widely used in clinical laboratories of microbiology, compared with mecA gene and minimum inhibitory concentration (MIC) of oxacillin. The use of oxacillin and cefoxitin disks simultaneously allowed the detection of important differences, particularly, in less frequent species such as S. cohnii, S. haemolyticus, S. saprophyticus, and S. sciuri. Disks of cefoxitin of the brand 2 displayed good correlation with the mecA gene (98.7%) and oxacillin MIC (97.8%), while major discrepancies were observed using disks of brand 1. One of the critical points in the diffusion disk test is the quality of the disks: the use of better quality disks associated with molecular methods lead to better results to define the best antibiotic therapy.     

The use of an amino acid analyzer for the rapid identification and quantitative determination of chitosan oligosaccharides.

The oligosaccharides of chitosan, glucosamine through chitohexaose, were separated and quantitated with the use of an amino acid analyzer. The oligosaccharides can be satisfactorilly resolved with a step gradient using two buffers, but resolution is improved with a linear buffer gradient. Using experimentally determined color factors, the method permits the rapid quantitation and identification of small amounts of chitosan oligosaccharides.     

Carbohydrate dissimilation tests in the identification of staphylococci

Reproducible and comparable results were obtained in different media containing different nutrients and indicators when representative Staphylococcus strains were tested for acid production from carbohydrates. Only in tests with melezitose, ribose and turanose many discrepant, weak or delayed positive reactions occurred. In tests with other carbohydrates similarly many weak results were visible after 1 day of incubation, but acid reactions were clear and easy to read after 2 days in phenol red broth and Andrade peptone water media, commonly used in the routine identification of other bacterial genera.Important alkalinization effects were observed after prolonged incubation, but not after short periods up to 24 h in peptone-rich test media. Growth was poor and numbers of acid reactions from carbohydrate breakdown detected did not increase when peptone-free media were used.     

The use of virtual screening and differential scanning fluorimetry for the rapid identification of fragments active against MEK1

We report the analysis of an in-house fragment screening campaign for the oncology target MEK1. The application of virtual screening (VS) as a primary fragment screening approach, followed by biophysical validation using differential screening fluorimetry (DSF), with resultant binding mode determination by X-ray crystallography (X-ray), is presented as the most time and cost-effective combination of in silico and in vitro methods to identify fragments. We demonstrate the effectiveness of the VS–DSF workflow for the early identification of fragments to both ‘jump-start’ the drug discovery project and to complement biochemical screening data.     

High-density DNA probe arrays for identification of staphylococci to the species level

Rapid and accurate identification and speciation of staphylococci clinical isolates is important for predicting medical pathology. We evaluated the ability of a high-density DNA probe array based on 16S rDNA sequences to identify Staphylococcus species. Correct identification was observed for 185 out of the 201 strains (92%). Of the 33 tested species, the array was able to correctly identify 30 of them. The total time required for identification of 4 isolates was 5 h. Such a tool represents a powerful method for routine microbiological diagnostic as well as for epidemiological studies.     

Serological identification of enterotoxigenic staphylococci from cheese

Single and double gel-diffusion techniques were employed to examine serologically coagulase-positive staphylococci from cheese for enterotoxigenicity. Supernatant fluid from sac cultures was examined for enterotoxins A and B. The results indicated that 9 of 155 cultures from market cheese and 7 of 77 cultures from food-poisoning cheese produced enterotoxin A, and that none of the cultures produced detectable levels of enterotoxin B. Results of serological tests were confirmed by intravenous injection of cats.     

Development of a rapid identification method for potato cytoplasm and its use for evaluating Japanese collections

The cytoplasm of potatoes, characterized by the presence of T-type chloroplast DNA and β-type mitochondrial DNA, is sensitive to nuclear chromosomal genes that contribute to various types of male sterility. Past breeding efforts with various potato varieties have resulted in several different cytoplasms other than T/β. Varieties with Solanum stoloniferum-derived cytoplasm (W/γ) show complete male sterility, while those with S. demissum-derived cytoplasm (W/α) produce abundant, but non-functional pollen. Thus, identification of cytoplasmic types is important for designing efficient mating combinations. To date, only T-type chloroplast DNA can be accurately identified by a PCR marker. Here, we report a rapid identification technique by multiplex PCR, followed by restriction digestion with BamHI in one reaction tube, and propose a new nomenclature for potato cytoplasm types (T, D, P, A, M, and W). Using this new technique, our collections of 748 genotypes, including 84 Japanese named varieties, 378 breeding lines and 26 landraces, and 260 foreign varieties and breeding lines, were grouped into cytoplasm types: T (73.9?%), D (17.4?%), P (4.5?%), A (1.5?%), M (0.3?%), and W (2.4?%). The utility of this marker system for breeding is discussed.     

Experimental use of a new surface acoustic wave sensor for the rapid identification of bacteria and yeasts

AIMS: Use of an electronic nose (zNose(TM)) to discriminate between volatile organic molecules delivered during bacterial/fungal growth on agar and in broth media. METHODS AND RESULTS: Cultures of bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli) and yeasts (two Candida albicans strains) were grown on agar and in broth media and incubated for 24 h at 37 degrees C. Headspace samples from microbial cultures were analysed by the zNose(TM), a fast gas chromatography-surface acoustic wave detector. Olfactory images of volatile production patterns were observed to be different for the various species tested after 24 h. Moreover, some strains (two K. pneumoniae, two C. albicans) did not show changes in volatile production patterns within our species. CONCLUSIONS: Our experiments demonstrate that the electronic nose system can recognize volatile production patterns of pathogens at species level. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results, although preliminary, promise exciting challenges for microbial diagnostics.