Coagulase and Deoxyribonuclease Activities of Staphylococci Isolated From Clinical Sources

A total of 504 clinical isolates of the family Micrococcaceae were tested for coagulase, deoxyribonuclease, clumping factor, and phosphatase to determine whether there is a correlation between the results of these tests and the pathogenicity of staphylococci. In the tests for coagulase production, it was found that either human or rabbit plasma could be used with broth cultures, whereas rabbit but not human plasma was satisfactory when microorganisms removed from solid culture medium were used. Deoxyribonuclease production correlated better than the fermentation of mannitol with coagulase production. The use of methyl green, Toluidine Blue O, or acridine orange offered no advantage over the use of HCl for detecting the production of deoxyribonuclease. Neither the presence of clumping factor nor the production of phosphatase correlated well with coagulase production. Strains of staphylococci that did not produce coagulase and deoxyribonuclease were isolated as frequently as, and from a greater variety of clinical sources than, strains which produced these substances. It is concluded that the production of coagulase and deoxyribonuclease are properties of staphylococci which are not necessarily indicative of potential pathogenicity of the organisms for man.     

Characterization of Staphylococci Isolated from Raw Milk

To evaluate the pathogenicity of staphylococci from bovine raw milk, the general characteristics of 775 strains isolated from 798 samples of milk were studied. The coagulase test was performed by use of rabbit plasma. Chromogenesis, mannitol fermentation, and gelatin liquefaction were investigated on Chapman's Medium 110, after 48 hr of incubation. Production of β-hemolysin, which has been considered indicative of pathogenic staphylococci of animal origin, was determined by streaking different strains on sheep blood-agar plates in the presence of a strain of Lancefield group B streptococci. Plates were incubated at 37 C for 24 hr, and strong hemolysis was produced in the zone of interaction of β-hemolysin and some substance liberated by streptococcus (CAMP test). Of 404 strains found to be coagulase-positive, 95.8% exhibited a deep-orange pigment, 76.5% produced β-hemolysin, 91.8% fermented mannitol, and 75% liquefield gelatin. Of 371 strains which gave a negative coagulase test, about 16% fermented mannitol and liquefied gelatin; none of these strains produced β-hemolysin. When results are grouped according to pigmentation and coagulase production, β-hemolysin seems to be developed by pathogenic strains of Staphylococcus aureus only. If suitability of these tests for investigation of pathogenicity is compared, production of β-hemolysin appears to be the most useful one, since no “false positive” results were found. The use of the CAMP test as a simple and rapid technique to determine production of β-hemolysin by pathogenic strains of animal staphylococci during routine bacteriological work is suggested.     

A Comparison of Computer-Based Methods for Estimating the Age of Animals

Russian Journal of Marine Biology - The advantages and disadvantages of statistical methods for estimating the age of animals that lack recording structures are discussed in the article. The...     

Identification of Staphylococci Isolated from Clinical Material

A total of 350 staphylococci isolated from various clinical sources were examined for bound and free coagulase, fermentation of mannitol, and deoxyribonuclease. The economical coagulase-mannitol-agar method of Esber and Faulcomer was found to be suitable for the detection of free coagulase and mannitol fermentation. A significant number of coagulase- and mannitol-negative staphylococci proved to be deoxyribonuclease-positive.     

The Vitamin Requirements of Staphylococci Isolated from Human Skin

The vitamin requirements of 46 strains representing nine species of Staphylococcus isolated from human skin together with nine authentic reference strains of these species were determined using a chemically defined medium. Strains of Staphylococcus saprophyticus and Staphylococcus cohnii were isolated on selective media. All the strains investigated required nicotinic acid and thiamine for growth. Biotin was essential or stimulatory for all coagulase negative strains except one strain of Staphylococcus capitis. Oleic acid substituted for biotin in all cases except with one strain of Staphylococcus haemolyticus. No species pattern of biotin requirement or of the ability of oleic acid to substitute for biotin was apparent. Five out of six strains of Staph. cohnii required pantothenic acid.     

The Application of Miniaturized Methods for the Characterization of Various Organisms Isolated from the Animal Gut

Miniaturized methods for the characterization of streptococci, lactobacilli and aerobic Gram negative rods are described and the results obtained with 461 cultures from the calf rumen and quail gut tabulated. The methods afford a considerable saving in time and materials without concomitant loss of accuracy.     

Comparison of Resubstitution,Data Splitting,the Bootstrap,and the Jackknife as Methods for Estimating Validity Indices of New Marker Tests: A Monte Carlo Study

Estimating the diagnostic efficiency of marker tests on the basis of the training set is an intricate problem of discriminant analysis for which no analytical solution exists. The paper outline the problem, describes various popular estimation procedures and presents the results of computer simulations comparing the estimators with respect to both bias and variance.     

A Medium for the Isolation of Staphylococci from Foodstuffs

S ummary . A liquid medium for the isolation of staphylococci from foodstuffs, containing lithium chloride, potassium tellurite and glycine as inhibitors of contaminants other than cocci, is described. Micrococci are inhibited in the cultures by anaerobic conditions produced by agar seals. With this medium staphylococci are easily isolated even when their numbers in foodstuffs are low.     

Characterization of Novel Phages Isolated in Coagulase-Negative Staphylococci Reveals Evolutionary Relationships with Staphylococcus aureus Phages

Despite increasing interest in coagulase-negative staphylococci (CoNS), little information is available about their bacteriophages. We isolated and sequenced three novel temperate Siphoviridae phages (StB12, StB27, and StB20) from the CoNS Staphylococcus hominis and S. capitis species. The genome sizes are around 40 kb, and open reading frames (ORFs) are arranged in functional modules encoding lysogeny, DNA metabolism, morphology, and cell lysis. Bioinformatics analysis allowed us to assign a potential function to half of the predicted proteins. Structural elements were further identified by proteomic analysis of phage particles, and DNA-packaging mechanisms were determined. Interestingly, the three phages show identical integration sites within their host genomes. In addition to this experimental characterization, we propose a novel classification based on the analysis of 85 phage and prophage genomes, including 15 originating from CoNS. Our analysis established 9 distinct clusters and revealed close relationships between S. aureus and CoNS phages. Genes involved in DNA metabolism and lysis and potentially in phage-host interaction appear to be widespread, while structural genes tend to be cluster specific. Our findings support the notion of a possible reciprocal exchange of genes between phages originating from S. aureus and CoNS, which may be of crucial importance for pathogenesis in staphylococci.     

A Comparison of Aggregate P-Value Methods and Multivariate Statistics for Self-Contained Tests of Metabolic Pathway Analysis

For pathway analysis of genomic data, the most common methods involve combining p-values from individual statistical tests. However, there are several multivariate statistical methods that can be used to test whether a pathway has changed. Because of the large number of variables and pathway sizes in genomics data, some of these statistics cannot be computed. However, in metabolomics data, the number of variables and pathway sizes are typically much smaller, making such computations feasible. Of particular interest is being able to detect changes in pathways that may not be detected for the individual variables. We compare the performance of both the p-value methods and multivariate statistics for self-contained tests with an extensive simulation study and a human metabolomics study. Permutation tests, rather than asymptotic results are used to assess the statistical significance of the pathways. Furthermore, both one and two-sided alternatives hypotheses are examined. From the human metabolomic study, many pathways were statistically significant, although the majority of the individual variables in the pathway were not. Overall, the p-value methods perform at least as well as the multivariate statistics for these scenarios.     

Cytotoxic and Pathogenic Properties of Klebsiella oxytoca Isolated from Laboratory Animals

Klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. Studies suggest that in humans K. oxytoca exerts its pathogenicity in part through a cytotoxin. However, cytotoxin production in animal isolates of K. oxytoca and its pathogenic properties have not been characterized. Furthermore, neither the identity of the toxin nor a complete repertoire of genes involved in K. oxytoca pathogenesis have been fully elucidated. Here, we showed that several animal isolates of K. oxytoca, including the clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on HEp-2 and HeLa cells, indicating the ability to produce cytotoxin. Cytotoxin production appears to be regulated by the environment, and soy based product was found to have a strong toxin induction property. The toxin was identified, by liquid chromatography-mass spectrometry and NMR spectroscopy, as low molecular weight heat labile benzodiazepine, tilivalline, previously shown to cause cytotoxicity in several cell lines, including mouse L1210 leukemic cells. Genome sequencing and analyses of a cytotoxin positive K. oxytoca strain isolated from an abscess of a mouse, identified genes previously shown to promote pathogenesis in other enteric bacterial pathogens including ecotin, several genes encoding for type IV and type VI secretion systems, and proteins that show sequence similarity to known bacterial toxins including cholera toxin. To our knowledge, these results demonstrate for the first time, that animal isolates of K. oxytoca, produces a cytotoxin, and that cytotoxin production is under strict environmental regulation. We also confirmed tilivalline as the cytotoxin present in animal K. oxytoca strains. These findings, along with the discovery of a repertoire of genes with virulence potential, provide important insights into the pathogenesis of K. oxytoca. As a novel diagnostic tool, tilivalline may serve as a biomarker for K oxytoca-induced cytotoxicity in humans and animals through detection in various samples from food to diseased samples using LC-MS/MS. Induction of K. oxytoca cytotoxin by consumption of soy may be in part involved in the pathogenesis of gastrointestinal disease.     

Comparison of Different Agar Diffusion Methods for the Detection of Antimicrobial Residues in Slaughter Animals

The different agar diffusion methods were compared using antibiotic and sulphonamide-impregnated filter-paper discs and the kidneys of healthy and emergency-slaughtered pigs and cows after slaughter. No method used seemed to be sensitive to all antimicrobial drugs preimpregnated onto discs. Tetracycline yielded a greater zone of inhibition at pH 6 than at pH 8 and aminoglycosides, erythromycin, polymyxin B and lin cornycin at pH 8 than at pH 6. It therefore seems necessary to use different pHs (6 and 8). The addition of trimethoprim to the medium is necessary for the detection of sulphonamides. Bacillus subtilis BGA used as the test organism was more sensitive to sulphonamides on the “Test agar for the inhibitor test” containing trimethoprim than on the “Iso-Sensitest agar” also containing trimethoprim. The addition of trimethoprim to “Test agar for the inhibitor test” is recommended at pH 8 but not at pH 6 because false-positive cases (with inhibition zones > 2 mm) were observed at pH 6 with trimethoprim on the kidneys of healthy pigs.