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PubMed1997

epr, which encodes glycylglycine endopeptidase resistance, is homologous to femAB and affects serine content of peptidoglycan cross bridges in Staphylococcus capitis and Staphylococcus aureus.

Staphylococcus capitis EPK1 produces a glycylglycine endopeptidase, ALE-1 (M. Sugai, T. Fujiwara, T. Akiyama, M. Ohara, H. Komatsuzawa, S. Inoue, and H. Suginaka, J. Bacteriol. 179:1193-1202, 1997), which hydrolyzes interpeptide pentaglycine chains of cell wall peptidoglycan of S. aureus. Characterizations of the enzyme activity and cloning of ale-1 revealed that ALE-1 is very similar to prolysostaphin produced by S. simulans bv. staphylolyticus. Strain EPK1 is resistant to lysis by ALE-1 and by lysostaphin. A gene that renders the cells resistant to glycylglycine endopeptidase (epr) was found 322 bp upstream of and in the opposite orientation to ale-1. The deduced amino acid sequence of epr showed similarities to FemA and FemB, which have been characterized as factors essential for methicillin resistance of S. aureus. Inactivation of either femA or femB causes decreased resistance to methicillin, increased resistance to lysostaphin, and decreased glycine content in the interpeptide chains of peptidoglycan. Therefore, femAB is suggested to be involved in the addition of glycine to pentapeptide peptidoglycan precursor. S. aureus with epr on a multicopy plasmid had phenotypes similar to those of femAB mutants except that it did not alter resistance level to methicillin. These results suggest that epr and femAB belong to the protein family involved in adding amino acids to the pentapeptide peptidoglycan precursor and that epr is involved in the addition of serine to the pentapeptide.

Acetylglucosaminidase↗

PubMed1988

Problems with rapid agglutination methods for identification of Staphylococcus aureus when Staphylococcus saprophyticus is being tested.

Six rapid agglutination tests for identification of Staphylococcus aureus were evaluated by using 62 strains of S. aureus, 63 strains of S. saprophyticus, and 67 strains of other coagulase-negative staphylococci. S. saprophyticus was responsible for 19 of 26 false-positive results and 20 uninterpretable reactions. Thus, urinary staphylococcal isolates that are positive by rapid agglutination tests may require other confirmatory tests for the identification of possible S. saprophyticus.

Hemagglutination Tests↗

PubMed2000

Genetic and serologic evaluation of capsule production by bovine mammary isolates of Staphylococcus aureus and other Staphylococcus spp. from Europe and the United States.

Bovine mastitis caused by Staphylococcus aureus is responsible for major economic losses to the dairy industry, and more-effective therapeutic or preventive approaches are sorely needed. The predominance of staphylococcal capsular polysaccharide types 5 and 8 among human isolates from many sources is well documented, but there seems to be a greater variation in the distribution of capsular serotypes among isolates from cows. A total of 636 isolates of S. aureus from cases of bovine mastitis in Sweden, Denmark, Finland, Iceland, Ireland, and the United States were investigated for production of capsular polysaccharide types 5 and 8. Approximately half of all the European isolates tested were of serotype 8, although variation among countries and among isolates of clinical and subclinical origin was observed. Sweden had the highest frequency (87%) of serotypeable isolates, and Finland had the lowest (48%). Capsule types 5 and 8 accounted for only 42% of the U.S. isolates tested. A few isolates showed weak reactivity with CP5 antiserum in a colony blot assay, and an enzyme-linked immunosorbent assay inhibition method confirmed that the levels of capsule produced by these strains were <10% of those produced by control strains. Fifty isolates that failed to react with capsular antisera all possessed the genes for production of capsular polysaccharide type 5 or 8. These results underscore the variability in capsule production by bovine isolates of S. aureus from different geographic regions. This information is important for the rational design of a capsule-based vaccine to prevent S. aureus bovine mastitis.

Animals↗

PubMed2001

A Shared noncapsular antigen is responsible for false-positive reactions by Staphylococcus epidermidis in commercial agglutination tests for Staphylococcus aureus.

Many of the commercial slide agglutination tests for Staphylococcus aureus incorporate antibodies against cell surface antigens associated with methicillin resistance, including capsular polysaccharides and an uncharacterized antigen, serotype 18. These tests are more sensitive than the first-generation agglutination procedures that detected only bound coagulase and protein A, but they suffer from false-positive reactions with some coagulase-negative staphylococci. The aim of this study was to elucidate the mechanism for false-positive agglutination by S. epidermidis in these tests. A group of methicillin-resistant S. aureus (MRSA) isolates, including a serotype 18 strain, that were not detectable in the first-generation tests were found to be of capsular polysaccharide type 8. All of these isolates were deficient in bound coagulase and/or protein A, and they possessed a heat-stable, proteinaceous antigen that was absent from a prototype capsule type 8 strain. Enzyme-linked immunosorbent assay and agarose gel immunodiffusion experiments demonstrated that this proteinaceous antigen was also present on both methicillin-sensitive and methicillin-resistant S. epidermidis clinical isolates. S. epidermidis strains that gave false-positive agglutination test results had a considerably higher level of this antigen than strains that gave the correct negative result. These findings reveal the importance of the careful selection of MRSA strains for raising anti-capsular type 8 antibodies for use in agglutination tests. Strains devoid of the antigen shared with S. epidermidis should be used to eliminate potential cross-reactions with this coagulase-negative coccus.

Agglutination Tests↗

PubMed2005

Antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis clinical isolates from implant orthopedic infections.

In the last decade the rising phenomenon of resistance to most common antibiotic drugs among staphylococcal clinical isolates has been a reason for serious concern and alarm. The present study investigated the prevalence of antibiotic resistance within a large microbial collection including 530 clinical strains of S. aureus and 408 strains of S. epidermidis to a panel of 16 different drugs. All strains were isolated from orthopedic infections, either associated or non-associated with implant materials. Interestingly, our data show that the profile of the prevalence of antibiotic resistance within the two species of pathogens is extremely similar for the vast majority of the drugs screened. The only statistically significant variations in prevalence concerned, in order of relevance, the following 5 out of 16 antibiotics: sulfamethoxazole (in combination with trimethoprim), erythromycin, and, to a lesser extent, oxacillin, imipenem, and clindamycin. In the case of Staphylococcus aureus, the isolates associated to implant materials were found more frequently resistant to all 4 aminoglycosides screened as well as to ciprofloxacin.

Anti-Bacterial Agents↗

PubMed2002

Early Screening of oxacillin-resistant Staphylococcus aureus and Staphylococcus epidermidis from blood culture.

The timely detection of blood-borne pathogens is one of the most important functions of the microbiology laboratory. Recently, methicillin-resistant staphylococci have become the most important pathogens seen by the laboratory. The purpose of this study was to evaluate Staphy agar, a novel screening medium, for the detection methicillin-resistant Staphylococcus aureus, S. epidermidis, or other coagulase-negative staphylococci (CNS) from positive blood cultures showing Gram-positive cocci in clusters. Eighty-six blood cultures that yielded Gram-positive cocci in clusters were included in this study. The organisms were finally identified by the Vitek system, and oxacillin resistance was confirmed by polymerase chain reaction (PCR)-based mecA gene detection. The identification and oxacillin resistance of all S. aureus strains showed complete agreement with the Vitek and PCR results. The presumptive detection of S. epidermidis and other CNS were consistent with the Vitek system in 94.7%, and the screening of oxacillin resistance was consistent with the result of PCR in 92.1% of 38 strains. The Staphy agar method is reliable and rapid for differentiating Gram-positive cocci in clusters in blood and for determining their methicillin resistance.

Bacterial Proteins↗

PubMed1986

[Range of the antigenic specificity of peptidoglycan in Staphylococcus aureus compared to other representatives of the Staphylococcus genus].

In investigations based on the use of a highly sensitive test system permitting the detection of normal human antibodies to S. aureus peptidoglycan, the antigenic relationships between the peptidoglycans of S. aureus and other representatives of the genus Staphylococcus have been studied. Among other staphylococcal species, S. simulans, S. xylosus, S. hyicus, S. cohnii, S. hyicus s. s. chromogenes have been found to possess peptidoglycans most closely related to S. aureus peptidoglycans, while S. warneri and S. epidermidis peptidoglycans have proved to be least closely related to it.

Antibodies, Bacterial↗

PubMed1989

[Adhesion capacity and surface properties of Staphylococcus epidermidis and Staphylococcus saprophyticus].

We have comparatively studied the adherence of Staphylococcus epidermidis and S. saprophyticus to epithelial cells of a continuous cellular line of urinary origin (MDCK). The adherence of S. saprophyticus was significantly higher than that of S. epidermidis and this phenomenon was not related to the bacterial surface hydrophobicity or the ability of producing slime in vitro. The pre-incubation of the bacteria with sub-inhibitory concentrations of vancomycin, teicoplanin, amikacin, clindamycin, and roxitromycin did not modify the adherence ability of the microorganisms. Of the antimicrobial agents evaluated, only clindamycin significantly agents evaluated, only clindamycin, significantly inhibited the ability of producing slime of S. saprophyticus strains.

Animals↗

PubMed1978

[Transfer of drug resistance plasmids from Staphylococcus epidermidis to Staphylococcus aureus in mixed culture].

A possibility of transfer of chloramphenicol-resistance and penicillin-resistance plasmids from 4 different donor S. epidermidis strains to 2 S. aureus strains was demonstrated. Chloramphenocol-resistance plasmid was found in S. epidermidis 1065/77 which was not expressed in this strain but could be transferred to and was expressed in Staphylococcus aureus strains. Penicillin-resistance and chloramphenicol-resistance plasmids were transferred simultaneously in 40 per cent of the colonies.

Chloramphenicol↗

PubMed1981

[Protein A-activity of Staphylococcus hyicus in comparison to protein A of Staphylococcus aureus (author's transl)].

Protein A (PA)-Activity was demonstrated in 44 (93.6%) of 47 Staphylococcus hyicus-cultures. PA from S. hyicus, as well as PA from S. aureus, could be released by extraction with concentrated formic acid or by treatment of the staphylococci with lysostaphin. PA was also demonstrable in the culture medium. Purification of PA from S. hyicus and S. aureus could be achieved by affinity-chromatography on IgG-Sepharose. In Ouchterlony-tests both PA-preparations gave single lines of identity with normal sera from man, pig, dog and guinea pig (Fig. 1). Immunoelectrophoretic analysis indicated a significantly faster migration towards the anode for PA from S. hyicus than PA from S. aureus (Fig. 2). Isoelectric focusing revealed maximal activity for PA from culture supernatant of S. hyicus about pH 4.3 and for that of S. aureus at pH 5.0 (Fig 3). Gelchromatographic experiments indicated a lower molecular weight for PA from S. hyicus than for PA from S. aureus (Fig. 4).

Animals↗

PubMed2001

In vitro adherence and accumulation of Staphylococcus epidermidis RP 62 A and Staphylococcus epidermidis M7 on four different bone cements.

Bacterial resistance of Staphylococcus epidermidis, a serious pathogen of implant-related infections, to antibiotics is related to the production of a glycocalyx slime that impairs antibiotic access and the killing by host defense mechanisms. In vitro studies of different bone cements containing antibiotics, developed for the prevention of biomaterial-associated infection, could not always demonstrate complete eradication of biomaterial-adherent bacteria. We have investigated four different bone cements in regard to bacterial accumulation of a slime-producing strain RP 62 A and its isogenic mutant M7 lacking the ability to produce exopolysaccharide slime using a bacterial adhesion assay and modified Kirby-Bauer technique. A significant effect of exopolysaccharide production for the accumulation on bone cement could be demonstrated. The gentamicin/clindamycin bone cement was the only tested biomaterial that produced a large zone of bacterial inhibition in the inoculated area adjacent to the biomaterial. The bacterial adhesion was not reduced significantly and there was no correlation between zones of inhibition on blood agar plates and the quantitative adhesion assay. The clinical efficacy of the gentamicin/clindamycin bone cement must be proven in vivo.

Analysis of Variance↗

PubMed1992

Surface hydrophobicity of Staphylococcus intermedius and Staphylococcus hyicus.

Surface hydrophobicity of 90 Staphylococcus intermedius and 55 S hyicus isolates was evaluated using the hexadecane adherence assay and the ammonium sulphate salt aggregation test. A strongly positive hydrocarbon adherence in the hexadecane adherence assay was demonstrated in 11 per cent of the S intermedius isolates and 7 per cent of the S hyicus isolates. Bacterial aggregation in 1.6 M, or less, ammonium sulphate was observed in 28 per cent of the S intermedius isolates and 37 per cent of the S hyicus isolates. There was no statistical correlation between the two assays. The adherence of both bacterial species to hexadecane was eliminated when the cells were first treated with pronase and trypsin, while it was mildly enhanced by prior heat treatment (60 degrees C and 95 degrees C for up to three hours). In contrast, aggregation of S intermedius in ammonium sulphate was not influenced by trypsin pretreatment, and aggregation of both bacterial species was diminished, or eliminated, with pronase or prior 95 degrees C heat treatment. Surface hydrophobicity, as measured in both assays, appeared to have no relationship with growth patterns in serum soft agar or production of slime. Similarly, the presence or absence of substantial surface receptor activity to fibrinogen, fibronectin or IgG did not appear to be related to surface hydrophobicity.

Agglutination Tests↗

PubMed1983

Construction and characterization of plasmid vectors for cloning in Staphylococcus aureus and Staphylococcus carnosus.

Several plasmid vectors for cloning in Staphylococcus aureus and S. carnosus have been constructed and characterized. The chimeric plasmids are composed of parts of the following parental plasmids: The chloramphenicol-resistance plasmid, pC194, the tetracycline-resistance plasmid, pMK148, and the erythromycin-resistance plasmid, pE12. All the chimeric plasmids confer two selectable antibiotic-resistance markers on host cells. Insertional inactivation of the various antibiotic-resistance markers occurred at the BclI site of pE12, and the Sau96- or AvaII-site of pMK148; only a slight inactivation of the chloramphenicol-resistance marker occurred at the HaeIII-site of pC194. The chimeric plasmids pCT20 and pCE10 are both stable in S. aureus and S. carnosus. In addition, the hybrid plasmids of pCT20 and pCE10, containing lambda-DNA fragments in various restriction sites between 0.4 and 1.2 kb, are stably maintained. The inserted lambda-DNA fragments appear unchanged.

Chloramphenicol↗

PubMed1996

Identification and ribotyping of Staphylococcus xylosus and Staphylococcus equorum strains isolated from goat milk and cheese.

Twenty-five strains of staphylococci isolated from goat milk and cheese were identified as belonging to the Staphylococcus xylosus/equorum group using the ID 32 Staph system (bioMérieux, Marcy-L'Etoile, France). This system, however, was not able to discriminate between these two species for 19 of the strains tested. Ribotyping was performed on these 25 strains, as well as on three reference strains of each of these two species. Hybridization membranes were scanned and analyzed using the Taxotron software package (Taxolab, Institut Pasteur, Paris, France). A dendrogram representation showed that ribotypes were distributed in two clear-cut clusters corresponding to S. equorum (21 strains) and S. xylosus (four strains).

Animals↗

PubMed1986

Cloning and expression in Escherichia coli and Staphylococcus aureus of the beta-lysin determinant from Staphylococcus aureus: evidence that bacteriophage conversion of beta-lysin activity is caused by insertional inactivation of the beta-lysin determinant.

The beta-lysin determinant (Hlb) from Staphylococcus aureus CN6708 was cloned in Escherichia coli K-12 using the bacteriophage replacement vector lambda L47.1. The Hlb determinant was localised to a 1250 base pair DNA sequence by cloning fragments from a Hlb+ recombinant phage into the plasmid vectors pACYC184 and pBR322 in E. coli K-12, and by the subsequent construction and analysis of several sub-clones, in vitro deletion and Tn5 insertion mutations. E. coli cells harbouring Hlb+ plasmids expressed readily detectable levels of beta-lysin and sphingomyelinase activity, which were located in the cytoplasm. Two polypeptides of molecular weight 38,000 and 33,000 which were encoded by the Hlb determinant were detected in E. coli minicells, but only the 33,000 dalton protein was detected in immunoblotting experiments with specific anti-beta-lysin serum. Hybridisation analysis with probes made from the cloned Hlb determinant and from DNA of the staphylokinase-converting phage phi 13, indicated that bacteriophage conversion of S. aureus to loss of beta-lysin activity is due to insertion of phi 13 DNA into or adjacent to the beta-lysin determinant. A shuttle plasmid was used to transfer the cloned Hlb determinant into a beta-lysin negative strain of S. aureus where the wild-type chromosomal determinant was inactivated by lysogenic conversion. Beta-lysin activity was readily detected in supernatants of S. aureus harbouring the cloned determinant.

Antimicrobial Cationic Peptides↗

PubMed2003

Lactoferrin against Staphylococcus aureus Mastitis. Lactoferrin alone or in combination with penicillin G on bovine polymorphonuclear function and mammary epithelial cells colonisation by Staphylococcus aureus.

Antibiotics should combine good antibacterial activity and the capacity to work in association with the host defence system. In this study, we have investigated the effects of bovine lactoferrin alone or in combination with penicillin G on the phagocytic activity of bovine polymorphonuclear leukocytes against Staphylococcus aureus. We have shown that susceptibility of S. aureus to phagocytosis was decreased in the presence of penicillin in the medium. In a kinetic study, lactoferrin alone did not affect phagocytosis but, when used with penicillin, it reversed the negative effect of this antibiotic on phagocytosis. In addition, in an epithelial invasion assay, lactoferrin alone or in combination with penicillin reduced the invasion of mammary epithelial cells in culture by S. aureus. Lactating female CD-1 mice were infected by intra-mammary delivery of a virulent penicillin-susceptible S. aureus strain and were then randomly assigned to treatments according to a 2 x 2 factorial design. In this mouse mastitis model, 2 days of systemic treatments with lactoferrin and/or penicillin did not lead to a total clearance of infection by S. aureus, but bacterial number was significantly reduced by treatments with lactoferrin or penicillin. These data suggest that bovine lactoferrin, alone or in combination with penicillin G, enhances S. aureus susceptibility to immuno-defense mechanisms, which can be beneficial in the treatment of S. aureus infections.

Animals↗

PubMed1999

Characteristics in adherence of streptococci and Staphylococcus aureus isolated from various infective skin lesions: serum IgA decreases adherence of Streptococcus pyogenes but not Staphylococcus aureus.

We characterized adherence of streptococci and Staphylococcus aureus strains isolated from various infective skin lesions in terms of hydrophobicity, negative charge, tube adherence, slime production, and influence on adherence to coverslips by plasma and serum immunoglobulins. High hydrophobicity was more frequently observed in Streptococcus pyogenes strains than in Streptococcus agalactiae strains (P < 0.01) and S. aureus strains (P < 0.001) and slime production was more frequently observed in S. agalactiae strains than in S. pyogenes strains (P < 0.05). Serum IgA decreased adherence to coverslips of S. pyogenes strains but not that of S. aureus strains.

Bacterial Adhesion↗

PubMed1999

Molecular characterization of femA from Staphylococcus hominis and Staphylococcus saprophyticus, and femA-based discrimination of staphylococcal species.

The femA gene encodes a protein precursor which plays a role in peptidoglycan biosynthesis in Staphylococcus aureus and is also considered as a factor influencing the level of methicillin resistance. A femA homologous gene was recently characterized in S. epidermidis, entailing the possibility of femA phylogenetic conservation in staphylococcal species. Accordingly, we assessed the presence of femA homologous genes in S. hominis and S. saprophyticus. Strategy for identification relied upon alignment of S. aureus and D. epidermidis femA sequences and upon identification of potentially conserved regions. Amplifications of portions of the femA genes were performed under permissive annealing conditions, by using several sets of primers designed to match the consensus regions. DNA sequencing of overlapping PCR fragments led to the characterization of the entire femA genes of S. hominis and S. saprophyticus, and provided more precise information on the femA start codon for all five species. The genomic organization of all these femA genes appeared highly conserved, with alternance of homologous and variable regions. On this basis, a consensus sequence of the femA gene was defined and interspecies variations were exploited to design strategies for staphylococci species-specific identification, including multiplex PCR amplification and a reverse hybridization assay.

Amino Acid Sequence↗