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Relation of mucoid growth of Staphylococcus aureus to clumping factor reaction, morphology in serum-soft agar, and virulence.

The growth characteristics of several strains of Staphylococcus aureus in Brain Heart Infusion and in a modified Staphylococcus Medium No. 110 were compared. In the latter medium all of the strains studied showed an increased mucoid character. Some of the strains studied showed a greater potential to synthesize excess slime layer material than others. The highly mucoid strains grew as diffuse-type colonies in modified Staphylococcus Medium No. 110 serum-soft agar and reacted as though they were negative in the test for clumping factor. These strains were also found to be more virulent when used to challenge normal mice intraperitoneally.

Animals↗

Presence of new mecA and mph(C) variants conferring antibiotic resistance in Staphylococcus spp. isolated from the skin of horses before and after clinic admission.

Because of the frequency of multiple antibiotic resistance, Staphylococcus species often represent a challenge in incisional infections of horses undergoing colic surgery. To investigate the evolution of antibiotic resistance patterns before and after preventative peri- and postoperative penicillin treatment, staphylococci were isolated from skin and wound samples at different times during hospitalization. Most staphylococci were normal skin commensals and belonged to the common coagulase-negative group. In some cases they turned out to be opportunistic pathogens present in wound infections. MICs were determined for 12 antibiotics, and antibiotic resistance genes were detected by microarray. At hospital admission, horses harbored staphylococci that were susceptible to antibiotics or resistant to one group of drugs, mainly due to the presence of new variants of the methicillin and macrolide resistance genes mecA and mph(C), respectively. After 3 days, the percentage of Staphylococcus isolates displaying antibiotic resistance, as well as the number of resistance genes per isolate, increased moderately in hospitalized horses without surgery or penicillin treatment but dramatically in hospitalized horses after colic surgery as well as penicillin treatment. Staphylococcus species displaying multiple resistance were found to harbor mainly genes conferring resistance to beta-lactams (mecA and blaZ), aminoglycosides [str and aac(6')-Ie-aph(2')-Ia], and trimethoprim [dfr(A) and dfr(D)]. Additional genes conferring resistance to macrolides [mph(C), erm(C), and erm(B)], tetracycline [tet(K) and tet(M)], chloramphenicol [cat(pC221) and cat(pC223)], and streptothricin (sat4) appeared in several strains. Hospitalization and preventive penicillin use were shown to act as selection agents for multidrug-resistant commensal staphylococcal flora.

Animals↗

Identification of Staphylococcus species of bovine origin with the API Staph-Ident system.

The API Staph-Ident system was evaluated as a means for identifying the species of bovine strains of staphylococci routinely isolated from quarter-milk samples. The species identity of 314 of 581 (54%) isolates of staphylococci was correctly determined by this method. The API Staph-Ident system was more accurate in correctly identifying Staphylococcus aureus (93.9%) than in correctly identifying non-S. aureus species (41.8%). False identifications of Staphylococcus epidermidis and Staphylococcus hominis were the main reasons for the incorrect identifications of the non-S. aureus species.

Animals↗

Reliability of latex agglutination tests for identification of Staphylococcus aureus resistant to oxacillin.

Commercial latex agglutination tests (LATs) for the simultaneous detection of clumping factor and protein A are gaining increased acceptance as a means of identifying Staphylococcus aureus. We evaluated two LATs (Accu-Staph; Carr-Scarborough, Decatur, Ga.; Staphaurex; Wellcome, Dartford, England) with particular emphasis on their ability to correctly identify oxacillin-resistant S. aureus. We tested 59 oxacillin-resistant S. aureus, 136 oxacillin-susceptible S. aureus, and 92 coagulase-negative staphylococcal strains with the two LATs and with thermonuclease, slide clumping factor, tube coagulase, and protein A hemagglutination tests. Clumping factor and protein A were present in 96.9 and 82.1% of our S. aureus strains, respectively. Accu-Staph correctly identified 92.8% and Staphaurex correctly identified 91.3% of S. aureus strains. No significant difference in LAT positivity rates, presence of clumping factor, or presence of protein A was found between oxacillin-resistant and -susceptible S. aureus. Overall, there were 31 false-negative LATs for 20 S. aureus strains, 14 with Accu-Staph and 17 with Staphaurex. Ninety-five percent of these strains possessed either clumping factor or protein A or both when these factors were determined independently. There were five false-positive LATs for four strains of coagulase-negative staphylococci (three Staphylococcus epidermidis and one Staphylococcus warneri), four with Accu-Staph and one with Staphaurex. Clumping factor was present in one S. warneri strain. Thus, the specificities of Accu-Staph, Staphaurex, and the clumping factor test were 95.6, 98.9, and 98.9%, respectively. Our results indicated that LATs identify oxacillin-resistant and -susceptible S. aureus equally well; however, they offer no greater sensitivity or specificity than the clumping factor test for identification of S. aureus.

Coagulase↗

Encapsulation of Staphylococcus aureus isolates from mastitic milk: relationship between capsular polysaccharide types 5 and 8 and colony morphology in serum-soft agar, clumping factor, teichoic acid, and protein A.

A total of 193 Staphylococcus aureus isolates from bovine, caprine, and ovine mastitis producing type 5 or 8 capsular polysaccharides were investigated for colony morphology in serum-soft agar and agglutinability by an anti-teichoic acid serum, after cultivation in modified staphylococcus medium no. 110. Also, 40 of these strains were cultivated in brain heart infusion and submitted to clumping factor and protein A detection tests. Considering capsular serotyping as a reference method, diffuse growth in serum-soft agar and inagglutinability by anti-teichoic acid serum identified, respectively, 57.5 and 45% of encapsulated strains cultivated in brain heart infusion and 85.5 and 77.2% of those cultivated in modified staphylococcus medium 110. Consequently, these indirect techniques underestimated encapsulation and were greatly influenced by culture conditions. Whatever the medium used, diffuse colony morphology in serum-soft agar was generally characterized by a masking of teichoic acid and protein A. By contrast, these surface antigens were detected in association with compact morphology; the presence of a thin or discontinuous capsular material could explain this result. Moreover, the masking of teichoic acid and the removal of capsular polysaccharide by washing in saline suggest that type 8 capsular polysaccharide is more abundant and labile than type 5.

Agglutination↗

Case of Staphylococcus schleiferi endocarditis and a simple scheme to identify clumping factor-positive staphylococci.

Staphylococcus schleiferi is a coagulase-negative staphylococcus infrequently reported as a human pathogen. We report a case of prosthetic valve endocarditis attributed to this organism, contrast it to another Staphylococcus species that gives similar clumping factor results (S. lugdunensis), and propose a simple, effective identification scheme for identification of clumping factor-positive staphylococci.

Aged↗

Dissemination of two methicillin-resistant Staphylococcus aureus clones exhibiting negative staphylase reactions in intensive care units.

From December 1997 to March 1998, 25 methicillin-resistant Staphylococcus aureus (MRSA) isolates exhibiting negative Staphylase (Oxoid Ltd., Basingstoke, England) reactions were identified from various clinical specimens from 13 patients in six intensive care units (ICUs) or in wards following a stay in an ICU at the National Taiwan University Hospital. The characteristics of these isolates have not been previously noted in other MRSA isolates from this hospital. Colonies of all these isolates were grown on Trypticase soy agar supplemented with 5% sheep blood and were nonhemolytic and unpigmented. Seven isolates, initially reported as Staphylococcus haemolyticus (5 isolates) and Staphylococcus epidermidis (2 isolates) by the routine identification scheme and with the Vitek GPI system (bioMerieux Vitek, Inc., Hazelwood, Mo.), were subsequently identified as S. aureus by positive tube coagulase tests, standard biochemical reactions, and characteristic cellular fatty acid chromatograms. The antibiotypes obtained by the E test, coagulase types, restriction fragment length polymorphism profiles of the staphylococcal coagulase gene, and random amplified polymorphic DNA patterns generated by arbitrarily primed PCR of the isolates disclosed that two major clones disseminated in the ICUs. Clone 1 (16 isolates) was resistant to clindamycin and was susceptible to trimethoprim-sulfamethoxazole (TMP-SMZ) and was coagulase type II. Clone 2 (eight isolates) was resistant to clindamycin and TMP-SMZ and was coagulase type IV. These two epidemic clones from ICUs are unique and underline the need for caution in identifying MRSA strains with colonial morphologies not of the typical type and with negative Staphylase reactions.

Adult↗

Development and evaluation of a quality-controlled ribosomal sequence database for 16S ribosomal DNA-based identification of Staphylococcus species.

To establish an improved ribosomal gene sequence database as part of the Ribosomal Differentiation of Microorganisms (RIDOM) project and to overcome the drawbacks of phenotypic identification systems and publicly accessible sequence databases, both strands of the 5' end of the 16S ribosomal DNA (rDNA) of 81 type and reference strains comprising all validly described staphylococcal (sub)species were sequenced. Assuming a normal distribution for pairwise distances of all unique staphylococcal sequences and choosing a reporting criterion of > or =98.7% similarity for a "distinct species," a statistical error probability of 1.0% was calculated. To evaluate this database, a 16S rDNA fragment (corresponding to Escherichia coli positions 54 to 510) of 55 clinical Staphylococcus isolates (including those of the small-colony variant phenotype) were sequenced and analyzed by the RIDOM approach. Of these isolates, 54 (98.2%) had a similarity score above the proposed threshold using RIDOM; 48 (87.3%) of the sequences gave a perfect match, whereas 83.6% were found by searching National Center for Biotechnology Information (NCBI) database entries. In contrast to RIDOM, which showed four ambiguities at the species level (mainly concerning Staphylococcus intermedius versus Staphylococcus delphini), the NCBI database search yielded 18 taxon-related ambiguities and showed numerous matches exhibiting redundant or unspecified entries. Comparing molecular results with those of biochemical procedures, ID 32 Staph (bioMerieux, Marcy I'Etoile, France) and VITEK 2 (bioMerieux) failed to identify 13 (23.6%) and 19 (34.5%) isolates, respectively, due to incorrect identification and/or categorization below acceptable values. In contrast to phenotypic methods and the NCBI database, the novel high-quality RIDOM sequence database provides excellent identification of staphylococci, including rarely isolated species and phenotypic variants.

Bacterial Typing Techniques↗

Emergence of methicillin resistant Staphylococcus aureus (MRSA) bacteraemia among children in England and Wales, 1990-2001.

Methicillin resistant Staphylococcus aureus (MRSA) is now a major cause of adult bacteraemia. All reports of Staphylococcus aureus bacteraemia to the Health Protection Agency were analysed from 1990 to 2001. There were 376 cases of MRSA bacteraemia in children <15 years over this time. The proportion of Staphylococcus aureus bacteraemia due to MRSA increased steadily from 0.9% in 1990 to 13% in 2000. The proportion was higher in infants. MRSA bacteraemia is now a serious problem in children in England and Wales. More data on the risk factors for acquisition and spread of MRSA in children are required.

Adolescent↗

Identification of slide coagulase positive, tube coagulase negative Staphylococcus aureus by 16S ribosomal RNA gene sequencing.

AIMS: To ascertain the clinical importance of a strain of slide coagulase positive but tube coagulase negative Staphylococcus species isolated from the blood culture of a 43 year old patient with refractory anaemia with excessive blasts in transformation who had neutropenic fever. METHODS: The isolate was investigated phenotypically by standard biochemical methods using conventional biochemical tests and two commercially available systems, the Vitek (GPI) and API (Staph) systems. Genotypically, the 16S ribosomal RNA (rRNA) gene of the bacteria was amplified by the polymerase chain reaction (PCR) and sequenced. The sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank by multiple sequence alignment. RESULTS: Conventional biochemical tests did not reveal a pattern resembling a known Staphylococcus species. The Vitek system (GPI) showed that it was 94% S. simulans and 3% S. haemolyticus, whereas the API system (Staph) showed that it was 86.8% S. aureus and 5.1% S. warneri. 16S rRNA gene sequencing showed that there was a 0 base difference between the isolate and S. aureus, 28 base difference between the isolate and S. lugdunensis, 39 base difference between the isolate and S. schleiferi, 21 base difference between the isolate and S. haemolyticus, 41 base difference between the isolate and S. simulans, and 23 base difference between the isolate and S. warneri, indicating that the isolate was a strain of S. aureus. Vancomycin was subsequently prescribed and blood cultures taken four days after the start of treatment were negative. CONCLUSIONS: 16S rRNA gene sequencing was useful in ascertaining the clinical importance of the strain of slide coagulase positive but tube coagulase negative Staphylococcus species isolated from blood culture and allowing appropriate management.

Adult↗

Acetic, propionic, and oleic acid as the possible factors influencing the predominant residence of some species of Propionibacterium and coagulase-negative Staphylococcus on normal human skin.

The minimum inhibitory concentration (MIC) of acetic and propionic acid for resident bacteria on normal human skin, such as Propionibacterium acnes and Staphylococcus epidermidis, was 25 mg/mL or more at any pH tested (pH 5.5-6.8). While the MIC of these acids for most of the transient bacteria was markedly decreased by lowering the pH of the media and at pH 5.5, the mean pH value of the normal human skin, the MIC was 6.25 mg/mL or less. The MIC of oleic acid for some strains of Gram-positive transient bacteria of Streptococcus, Micrococcus, or Bacillus was 100 micrograms/mL or less at all pH's tested. Staphylococcus aureus was resistant to this acid at pH 6.8, but became as sensitive as Streptococcus when the pH was lowered. The growth of P. acnes, the most predominant resident bacterium, was enhanced markedly and reached a maximum level at 6.25 mg/mL of propionic acid, 12.5 mg/mL of acetic acid, and 50-100 micrograms/mL of oleic acid. On the basis of these results, we presumed that acetic, propionic, and oleic acids are factors influencing the predominant residence of some species of Propionibacterium and coagulase-negative Staphylococcus on normal human skin.

Acetates↗

Staphylococcus lugdunensis: an emerging cause of ventriculoperitoneal shunt infections.

Staphylococcus lugdunensis, a coagulase-negative staphylococcus first described in 1988, has gained recognition as an organism with considerable pathogenic capability in adults. In contrast to the indolent presentation characteristic of other coagulase-negative staphylococci, S. lugdunensis infections resemble the aggressive behavior of Staphylococcus aureus. Although the organism has been isolated from a wide variety of infections in adults, it is a very rare cause of pediatric infections. We describe the first two pediatric patients who developed ventriculoperitoneal shunt infections caused by S. lugdunensis. These cases suggest that coagulase-negative staphylococci should be identified to the species level and that, if S. lugdunensis is identified, greater morbidity compared to that associated with other coagulase-negative staphylococcal shunt infections should be anticipated. A longer course of therapy is recommended for S. lugdunensis infections.

Adolescent↗

Investigation of beta-Lactamases in clinical isolates of Staphylococcus aureus for further explanation of borderline methicillin resistance.

BACKGROUND: Borderline methicillin resistance in Staphylococcus aureus is due to beta-lactamase overproduction and/or specific methicillinases. METHODS: beta-Lactamase activity in culture supernatants and in cytoplasmic membrane fractions was estimated by bioassay and by SDS-PAGE combined with nitrocefin assay. RESULTS: During the investigation of borderline methicillin-resistant Staphylococcus aureus (BORSA) strains VU94 and 822 two beta-lactamases were detected in the membranes, with molecular weights of 13 and 30 kDa. The latter could be found in the culture supernatants, too. In the presence of globomycin, this enzyme disappeared from the membrane, and the oxacillin-hydrolyzing activity of the membrane decreased to the level of susceptible strains. Both beta-lactamases were detected in the methicillin-resistant Staphylococcus aureus strain studied, but the susceptible strains possessed only the first enzyme. CONCLUSIONS: The 30-kDa beta-lactamase proved to be a methicillinase, and it can be one of the main causes of the borderline phenotype of BORSA strains. The other enzyme is one of the smallest beta-lactamases published to date.

Humans↗

The role of methicillin-resistant Staphylococcus aureus in orthopaedic implant surgery.

The problems presented by methicillin-resistant Staphylococcus aureus (MRSA) must be accommodated in both prophylactic and treatment regimens for orthopaedic implant surgery. The rationale of pre-admission nasal swabbing in directing prophylaxis for orthopaedic patients is discussed. The potential advantage of nasal mupirocin for Staphylococcus aureus and MRSA carriers is described. Methicillin-resistant Staphylococcus epidermidis is commented upon as another hazard in orthopaedics. Criteria for choosing glycopeptides in the treatment of implant infections are discussed, and need to be defined in orthopaedic units. Treatment regimens are briefly described.

Antibiotic Prophylaxis↗

A generic approach for the design of whole-genome oligoarrays, validated for genomotyping, deletion mapping and gene expression analysis on Staphylococcus aureus.

BACKGROUND: DNA microarray technology is widely used to determine the expression levels of thousands of genes in a single experiment, for a broad range of organisms. Optimal design of immobilized nucleic acids has a direct impact on the reliability of microarray results. However, despite small genome size and complexity, prokaryotic organisms are not frequently studied to validate selected bioinformatics approaches. Relying on parameters shown to affect the hybridization of nucleic acids, we designed freely available software and validated experimentally its performance on the bacterial pathogen Staphylococcus aureus. RESULTS: We describe an efficient procedure for selecting 40-60 mer oligonucleotide probes combining optimal thermodynamic properties with high target specificity, suitable for genomic studies of microbial species. The algorithm for filtering probes from extensive oligonucleotides libraries fitting standard thermodynamic criteria includes positional information of predicted target-probe binding regions. This algorithm efficiently selected probes recognizing homologous gene targets across three different sequenced genomes of Staphylococcus aureus. BLAST analysis of the final selection of 5,427 probes yielded >97%, 93%, and 81% of Staphylococcus aureus genome coverage in strains N315, Mu50, and COL, respectively. A manufactured oligoarray including a subset of control Escherichia coli probes was validated for applications in the fields of comparative genomics and molecular epidemiology, mapping of deletion mutations and transcription profiling. CONCLUSION: This generic chip-design process merging sequence information from several related genomes improves genome coverage even in conserved regions.

Algorithms↗

Molecular cloning and sequence analysis of the dnaA gene of Staphylococcus aureus.

The dnaA gene of Staphylococcus aureus has now been cloned and sequenced. The deduced amino acid sequence of Staphylococcus aureus DnaA protein is 62% and 39% identical to those of the DnaA protein from Bacillus subtilis and Escherichia coli, respectively. Expression of the dnaA gene of Staphylococcus aureus in Escherichia coli caused lethality in an oriC-dependent manner.

Amino Acid Sequence↗

[Study of macrolide, lincosamide, and streptogramin B antibiotics resistance in Staphylococcus aureus].

Macrolide antibiotics (Mac) consist of a 12- to 16-membered lactone ring combined with a sugar moiety, and they inhibit protein synthesis via binding to 23S ribosomal RNA in bacteria. The 14- and 16-membered Mac are used for treating infectious diseases caused by Gram-positive and other bacteria; e.g., Haemophilus influenzae, Bordetella pertussis, Legionella pneumophila, Campylobacter, Treponema pallidum and Mycoplasma. Resistance to macrolide, lincosamide, and streptogramin-B (MLS) antibiotics in staphylococci is known to have the following mechanisms: 1) alteration of the target on ribosome due to dimethylation of a specific adenine residue in the 23S ribosomal RNA by the product of the erm gene, and consequently a decrease in binding of MLS antibiotics; 2) inactivation of streptogramin-B (STG-B) and lincosamide by the products of the sbh (encoding streptogramin B hydrolase) and linA' (encoding 3-lincomycin 4-clindamycin O-nucleotidyltransferase) genes, respectively; and 3) active efflux of Mac and STG-B antibiotics determined by the msrA and msrB genes in Staphylococcus epidermidis and Staphylococcus xylosus, respectively, both of which appear to act as an ATP-dependent efflux pump. I have shown that Staphylococcus aureus 8325(pEP2104) exhibits inducible resistance to PMS (partial macrolide and streptogramin B)-antibiotics [the 14-membered macrolides, erythromycin (EM), and oleandomycin (OL), and the 16-membered macrolide mycinamicin (MCM) and STG-B]. The sequence of the N-terminal amino acid residues of a 63 kDa protein (MsrSA) that appeared in the membrane of PMS-resistant strains was identical to that of an MsrA polypeptide related to enhanced efflux of [14C]EM. Ribosomes from PMS-resistant strains showed a similar affinity for EM to those from the PMS-sensitive host strain NCTC8325, and no inactivation of EM by 8325(pEP2104) was observed. In the present study, I showed the DNA sequence of the msrSA region on the constitutive PMS-resistant plasmid pMC38, PMS-inducible resistant plasmid pEP2104 and PMS-sensitive mutant plasmid pSP6, and the region that is essential for inducible expression in PMS resistance. In addition, I investigated the relationship between PMS resistance and intracellular accumulation of EM.

ATP-Binding Cassette Transporters↗