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PubMed1996

Comparison of the beta-toxins from Staphylococcus aureus and Staphylococcus intermedius.

The beta-toxins produced by Staphylococcus aureus and Staphylococcus intermedius were purified to homogeneity from culture supernatants. Although the toxin from S. aureus has been throughly studied, less is known about its unique counterpart from S. intermedius. This is the first reported purification and analysis of the S. intermedius beta-toxin. Both toxins have similar enzymatic properties, belong to the class of neutral sphingomyelinases C, and have a high specificity for sphingomyelin. They also hydrolyze lysophosphatidylcholine at a much slower rate, but have no activity toward phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine. The kinetic parameters determined for both proteins (apparent Km 1.4 mM, Vmax 100 mmol/min/microg protein) are identical. Despite these similarities, the size and amino acid composition of the two beta-toxins differ. Molecular mass values, determined by electrophoresis and gel filtration, indicate that the both enzymes are single polypeptides. The decrease in sphingomyelinase activity of S. aureus beta-toxin upon pretreatment with dithiothreitol (DTT) indicates the presence of a disulfide bond in the protein. In contrast, DTT has no effect on the enzymatic activity of S. intermedius beta-toxin. This observation is consistent with the absence of detectable cysteine residue in the protein. N-terminal amino acid sequences determined for the first 19 residues of both beta-toxins also differ, only nine of the first 19 residues are identical. Further evidence that the two proteins differ was obtained by immunological analysis which demonstrated crossreactivity but a lack of identity.

Amino Acid Sequence↗

PubMedDate not supplied

[Antibacterial activity of clindamycin and lincomycin in broth, serum, and in combination with polymorphonuclear leukocytes against Staphylococcus aureus and Staphylococcus epidermidis].

We investigated the antibacterial activity of clindamycin and lincomycin at 1/4 X minimum inhibitory concentration (MIC), 1 X MIC and 4 X MIC against a serum-resistant Staphylococcus aureus and a serum-resistant Staphylococcus epidermidis strain in broth, in serum with and without the presence of leukocytes and in Hank's medium in combination with leukocytes alone. Against both test strains, lincomycin in broth and serum was similarly effective, whereas against S. aureus clindamycin in broth was somewhat more active. In the combined test mixture of serum with leukocytes, even a 1/4 X MIC of clindamycin or lincomycin markedly improved leukocyte killing of S. aureus, whereas both compounds could not further enhance the marked leukocyte killing of S. epidermidis, even at inhibitory concentrations. In Hank's medium with leukocytes alone, clindamycin and lincomycin had at the most only a bacteriostatic effect against both test strains.

Blood↗

PubMed1992

In vitro activity of sparfloxacin and three other fluoroquinolones against methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis.

Fluoroquinolones are variably active against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE). The purpose of this study was to test the in vitro susceptibility of 50 isolates each of MRSA and MRSE to four of the new fluoroquinolones--sparfloxacin, ciprofloxacin, ofloxacin and norfloxacin--and to see if resistance could be induced in five susceptible strains of each species by serial passages with increasing concentrations of drug. A standard microdilution technique was used to determine minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of each drug for each isolate. Agar dilution plates were also prepared containing concentrations of drug varying from one-half of the reported MIC to 128 times the reported MIC, and microorganisms persisting were serially passaged. Initially, 98% of the strains of MRSA were susceptible to the fluoroquinolones. MBCs were essentially identical to MICs. Similarly, 96% of the strains of MRSE were susceptible. Following exposure to increasing concentrations of each fluoroquinolone, resistance appeared to emerge less rapidly to sparfloxacin and ofloxacin than to norfloxacin and ciprofloxacin.

Anti-Infective Agents↗

PubMed1992

Effect of subinhibitory concentrations of cefamandole and cefuroxime on adherence of Staphylococcus aureus and Staphylococcus epidermidis to polystyrene culture plates.

The ability of cefamandole and cefuroxime to inhibit adherence of staphylococci to polystyrene culture plates was tested in an in vitro assay using eight strains each of Staphylococcus aureus and Staphylococcus epidermidis. The results indicated that subinhibitory concentrations of cefamandole and cefuroxime altered the adherence ability of both staphylococcal species, inhibition of adherence being more marked in the presence of cefamandole. It may be important to consider antiadherence properties in association with bactericidal activity when selecting agents for antibiotic prophylaxis.

Bacterial Adhesion↗

PubMed2006

Polysaccharide intercellular adhesin or protein factors in biofilm accumulation of Staphylococcus epidermidis and Staphylococcus aureus isolated from prosthetic hip and knee joint infections.

Nosocomial staphylococcal foreign-body infections related to biofilm formation are a serious threat, demanding new therapeutic and preventive strategies. As the use of biofilm-associated factors as vaccines is critically restricted by their prevalence in natural staphylococcal populations we studied the distribution of genes involved in biofilm formation, the biofilm phenotype and production of polysaccharide intercellular adhesin (PIA) in clonally independent Staphylococcus aureus and Staphylococcus epidermidis strains isolated from prosthetic joint infections after total hip or total knee arthroplasty. Biofilm formation was detected in all S. aureus and 69.2% of S. epidermidis strains. Importantly, 27% of biofilm-positive S. epidermidis produced PIA-independent biofilms, in part mediated by the accumulation associated protein (Aap). Protein-dependent biofilms were exclusively found in S. epidermidis strains from total hip arthroplasty (THA). In S. aureus PIA and proteins act cooperatively in biofilm formation regardless of the infection site. PIA and protein factors like Aap are of differential importance for the pathogenesis of S. epidermidis in prosthetic joint infections (PJI) after THA and total knee arthroplasty (TKA), implicating that icaADBC cannot serve as a general virulence marker in this species. In S. aureus biofilm formation proteins are of overall importance and future work should focus on the identification of functionally active molecules.

Adhesins, Bacterial↗

PubMed2004

Prevalence of mupirocin resistance in clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis: results of the Antimicrobial Resistance Surveillance Study of the Paul-Ehrlich-Society for Chemotherapy, 2001.

A multicentre surveillance study comprising 26 laboratories located in Austria, Germany, and Switzerland was carried out in November 2001. A total of 787 isolates of Staphylococcus aureus and 456 isolates of Staphylococcus epidermidis mainly recovered from hospitalised patients, were tested. MICs for mupirocin were determined using the broth microdilution procedure. Breakpoints were < or = 4 mg/l (susceptible), 8-256 mg/l (low-level resistance) and > or = 512 mg/l (high-level resistance). Rates of low- and high-level resistances were 2.9 and 0.9% in S. aureus, and 9.4 and 3.3% in S. epidermidis, respectively. Mupirocin resistance was almost exclusively observed in oxacillin-resistant isolates of S. aureus (MRSA) and S. epidermidis (MRSE). High-level mupirocin resistance was detected in 3.1 and 4.5% of MRSA and MRSE, respectively.

Anti-Bacterial Agents↗

PubMed2004

Mechanisms of biofilm formation in Staphylococcus epidermidis and Staphylococcus aureus: functional molecules, regulatory circuits, and adaptive responses.

Biomaterial-associated infections, most frequently caused by Staphylococcus epidermidis and Staphylococcus aureus, are of increasing importance in modern medicine. Regularly, antimicrobial therapy fails without removal of the implanted device. The most important factor in the pathogenesis of biomaterial-associated staphylococcal infections is the formation of adherent, multilayered bacterial biofilms. In this review, recent insights regarding factors functional in biofilm formation of S. epidermidis, their role in pathogenesis, and regulation of their expression are presented. Similarly, in S. aureus the biofilm mode of growth affects gene expression and the overall metabolic status. Experimental approaches for analysis of differential expression of genes involved in these adaptive responses and evolving patterns of gene expression are discussed.

Adaptation, Physiological↗

PubMed2005

Genomic characterization of oxacillin-resistant Staphylococcus epidermidis and Staphylococcus haemolyticus isolated from Brazilian medical centres.

Studies on the genetic diversity of oxacillin-resistant coagulase-negative staphylococcal (CNS) isolates are important for the control and prevention of infections. The present study evaluated the clonal diversity of oxacillin-resistant Staphylococcus epidermidis (ORSE) and Staphylococcus haemolyticus (ORSH) strains, isolated from patients in nine Brazilian medical centres by using pulsed-field gel electrophoresis (PFGE) after digestion of bacterial DNA using SmaI. PFGE analysis of ORSE (N=44) and ORSH (N=25) strains showed the presence of 29 restriction profiles clustered in 16 PFGE types, and 21 distinct profiles in 15 PFGE types, respectively, indicating a large genetic diversity among isolates of both of these species. Among the ORSE isolates, 23 (52%) strains belonged to two predominant PFGE types (named A and B), which were observed in most of the hospitals assessed, indicating the spread of these PFGE types in hospitals located in Rio de Janeiro. The spread of PFGE types of ORSH was also detected in some of the hospitals investigated. The results show that PFGE is a suitable tool for epidemiological studies of oxacillin-resistant CNS, and can be used as a basis for infection control procedures for these multiresistant organisms.

Bacterial Proteins↗

PubMed2005

A comparison of adherence by four strains of Staphylococcus intermedius and Staphylococcus hominis to canine corneocytes collected from normal dogs and dogs suffering from atopic dermatitis.

The aim of this study was to compare the adherence of four strains of Staphylococcus intermedius and a single strain of Staphylococcus hominis to corneocytes from both normal dogs and dogs suffering from atopic dermatitis. Cells from the skin surface, corneocytes, were collected from 10 normal dogs and 10 dogs suffering from atopic dermatitis. Four strains of S. intermedius, three isolated from canine pyoderma skin lesions (strains A, B and C), and one isolated form from canine synovial membrane sample from a case of septic arthritis (strain D) were compared. S. hominis, which is not normally associated with canine disease, was also evaluated for its ability to adhere to canine corneocytes. S. hominis did not adhere to canine corneocytes. All four strains of S. intermedius adhered well to canine corneocytes collected from both normal and atopic dogs. All strains of S. intermedius showed statistically greater adherence to corneocytes collected from atopic dogs compared with those collected from normal dogs. It was concluded that the adherence assay employed here showed that S. hominis does not adhere to canine corneocytes, S. intermedius adheres preferentially to atopic corneocytes.

Animals↗

PubMed2006

Biofilm-forming ability profiling of Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates.

Biofilm-forming ability has been increasingly recognized as an important virulence factor in Staphylococci, facilitating their persistence in the host, evading its defences and allowing bacterial survival at high antimicrobial concentrations. Staphylococcus aureus remains a major pathogen of chronic mastitis, but in the last years Staphylococcus epidermidis has emerged as a relevant mastitis pathogen. The present work aimed at the evaluation of the biofilm-forming ability of Staphylococci field isolates from bovine subclinical mastitis and at the development of a fluorescent in situ hybridisation (FISH) protocol that would allow the direct observation of biofilm formation in milk samples. The analysis of phenotypic expression in Congo Red Agar (CRA) and by FISH, showed that 37.5% of the S. aureus isolates produced biofilm, while by optical density measurement only 18.75% isolates revealed this phenotype. The results showed a fair agreement according to the kappa coefficient test (kappa = 0.259). Regarding S. epidermidis mastitis isolates, 37.5% revealed the ability to produce biofilm, but only four isolates were positive by all methods. This agreement was moderate (kappa = 0.467). The application of FISH to artificially contaminated milk samples allowed the direct observation of biofilm production by 37.5% isolates, showing total agreement with the CRA results. This method better mimics the in vivo conditions, especially in terms of the presence of calcium and iron, which in high concentrations, respectively, are known to inhibit or induce biofilm production.

Animals↗

PubMed2003

Lack of biofilm contribution to bacterial colonisation in an experimental model of foreign body infection by Staphylococcus aureus and Staphylococcus epidermidis.

The contribution of in vivo biofilm-forming potential of Staphylococcus aureus and Staphylococcus epidermidis was studied in an experimental model of foreign body infections. Increasing inocula (from 10(2) to 10(7) organisms) of ica-positive strains of S. aureus and S. epidermidis and their ica-negative isogenic mutants (the ica locus codes for a major polysaccharide component of biofilm) were injected into subcutaneously implanted tissue cages in guinea pigs. Surprisingly, bacterial counts and time-course of tissue cage infection by ica-positive strains of S. aureus or S. epidermidis were equivalent to those of their respective ica-negative mutants, in the locally infected fluids and on tissue-cage-inserted plastic coverslips.

Animals↗

PubMed1996

Selection of subpopulations resistnat to amikacin and netilmicin of gentamicin-resistant clinical strains of Staphylococcus aureus and Staphylococcus epidermidis.

Recently we have found several strains of Staphylococcus aureus and Staphylococcus epidermidis, which in spite of containing aminoglycoside-modifying enzymes (AMEs) remained susceptible to antibiotics such as netilmicin (NET) and amikacin (AN). Assuming an interest in this agent from a clinical point of view, the aim of this study was to determine if these strains became resistant after prolonged contact with such antibiotics. We found that minimal inhibitory concentrations (MICs) of the bacterial strains not only increased when using these two agents, but also when using other aminoglycosides such as gentamicin (GM), tobramycin (TM), amikacin (AN) and isepamicin (ISE). In order to see the effect of prolonged use of NET on enzyme production, three strains containing AMEs were selected and we could observe an increase in the enzyme levels after successive passages through media containing NET.

Amikacin↗

PubMed1988

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme IIImtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme IImtl of Escherichia coli.

Enzyme IIImtl is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, we report the isolation of IIImtl from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of IIImtl with [32P]PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase Glu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp- Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which we assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the IIImtl proteins was found to be 15,000. We have also determined the N-terminal sequence of both proteins. Comparison of the IIImtl peptide sequences and the C-terminal part of the enzyme IImtl of Escherichia coli reveals considerable sequence homology, which supports the suggestion that IImtl of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II. In particular, the homology of the active-center peptide of IIImtl of S. aureus and S. carnosus with the enzyme IImtl of E. coli allows one to predict the N-3 histidine phosphorylation site within the E. coli enzyme.

Amino Acid Sequence↗

PubMed1998

Substrate specificity of Staphylococcus hyicus lipase and Staphylococcus aureus lipase as studied by in vivo chimeragenesis.

Staphylococcus hyicus lipase (SHL) and Staphylococcus aureus lipase (SAL) are highly homologous enzymes, yet they show remarkable differences in their biochemical characteristics. SHL displays a high phospholipase activity, hydrolyses neutral lipids, and has no chain length preference, whereas SAL only degrades short-chain fatty acid esters. To identify the regions in the primary sequence of SHL responsible for phospholipase activity and chain length selectivity, a set of histidine-tagged SAL/SHL chimeras was generated by in vivo recombination in Escherichia coli. Several classes of chimeric enzymes were identified on the basis of restriction site analysis. All chimeras were well-expressed as active enzymes. They were characterized for their specific activities on both phospholipids and p-nitrophenyl esters of various chain lengths. Phospholipase activity appeared to be determined by three regions, all located in the C-terminal domain of SHL. Testing of the enzymatic activity of the chimeras toward p-nitrophenyl esters showed that chain length selectivity is defined by elements within the region of residues 180-253. Moreover, also residues along the stretch 275-358 contribute to the binding of acyl chains. Interestingly, several chimeras were even more active than the parent enzymes on long-chain p-nitrophenyl esters.

Amino Acid Sequence↗

PubMed2003

In vitro studies of the pharmacodynamics of teicoplanin against Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecium.

OBJECTIVE: To investigate the basic pharmacodynamic properties of teicoplanin in vitro for Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecium. METHODS: The following experiments were performed: (1) bacterial killing by teicoplanin at different concentrations; (2) bacterial killing by teicoplanin at 8 x MIC against the same strains with inocula of 5 x 10(3), 5 x 10(5) and 5 x 10(7) CFU/mL; (3) studies of the postantibiotic effect (PAE) and the postantibiotic sub-MIC effect (PASME) of teicoplanin; (4) studies of the killing by teicoplanin in an in vitro kinetic model following exposure to simulated human serum pharmacokinetic concentrations (6 mg/kg OD at steady state). RESULTS: Concentration-dependent killing was noted against S. epidermidis, with a > 4 log10 difference in CFUs between 2 x MIC and 64 x MIC at 24 h. Also, against S. aureus there was slight concentration-dependent killing, which, however, did not reach 2 log10 CFU/mL. Teicoplanin exerted a similar killing rate at all inocula for S. epidermidis, except for slower initial killing up to 6 h at the highest inoculum. In contrast, overall slower killing at all inocula was seen for S. aureus, where an inoculum effect was noted at the highest inoculum. For E. faecium, only a bacteriostatic effect was noted at all concentrations and inocula. No or very short PAEs were noted for the investigated strains. However, when the strains in the postantibiotic phase were exposed to 0.1, 0.2 and 0.3 x MIC of teicoplanin (PASME), substantial prolongation of the PAEs was seen. Although no significant killing was achieved in our kinetic model for any of the strains, regrowth of S. epidermidis was noted first after 8 h, despite a T > MIC24 of only 5% (1.2 h), illustrating the long post-MIC effect for this strain. For S. aureus, T > MIC was 38%, and regrowth occurred later than for S. epidermidis. Neither killing nor regrowth was seen for E. faecium with a T > MIC24 of 27%. CONCLUSION: Teicoplanin exerted a concentration-dependent bactericidal effect against S. epidermidis, a less notable one against S. aureus, and a bacteriostatic effect against E. faecium. A reduced killing rate with increasing inocula was seen for S. aureus and also for S. epidermidis at the highest inoculum. No or very short PAEs were noted for the investigated strains, but were substantially prolonged with the addition of subinhibitory concentrations. When human pharmacokinetics was simulated (6 mg/kg OD at steady state) in the kinetic model, no net bactericidal effect was noted for any of the strains at 24 h.

Anti-Bacterial Agents↗

PubMed1992

Frequency and transferability of trimethoprim and sulfonamide resistance in methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis.

A total of 374 Staphylococcus aureus and 126 Staphylococcus epidermidis strains from 14 countries were studied for their resistance to methicillin, trimethoprim (Tp) and sulfonamides (Su), alone and combined (TpSu). The frequency of resistance to Tp, Su and TpSu was much higher in methicillin-resistant S. epidermidis (MRSE) than in methicillin-resistant S. aureus (MRSA). Considerable differences, however, existed in isolates from different countries. Resistance to Tp, Su or TpSu in MRSA was low or absent in isolates from Switzerland, Spain, Japan, Mexico, Argentina and Chile, but high in isolates from Germany and Brazil. High level Tp resistance mostly resided on large plasmids. It could be transferred in 17 out of 97 strains. Su resistance was never cotransferred. Strains cured of their large Tp resistance plasmids remained Su-resistant, which suggests a chromosomal location of Su resistance.

Anti-Bacterial Agents↗

PubMed2001

Pharmacodynamics of moxifloxacin and levofloxacin against Staphylococcus aureus and Staphylococcus epidermidis in an in vitro pharmacodynamic model.

An in vitro pharmacokinetic model was used to compare the pharmacodynamics of moxifloxacin and levofloxacin against 3 Staphylococcus aureus and 3 Staphylococcus epidermidis strains. Logarithmic-phase cultures were inoculated into the peripheral compartment of hollow-fiber cartridges and exposed to the peak serum concentrations achieved in humans with oral doses of moxifloxacin (400 mg) and levofloxacin (500 mg). Drugs were added at 0 and 24 h, elimination kinetics were simulated, and changes in viable bacterial counts were evaluated over the course of 36 h. Moxifloxacin was bactericidal against all 6 staphylococci (times to 99.9% kill, 1-3 h). Against most strains, bacterial killing continued through 36 h, with total kills exceeding 5.5 logs. Levofloxacin was bactericidal against 5 of the strains, with similar times to 99.9% kill. In contrast to moxifloxacin, however, resistant subpopulations emerged in 4 strains during therapy with levofloxacin, and this could have important implications for treatment of staphylococcal infections. These in vitro observations warrant the clinical evaluation of moxifloxacin in the treatment of staphylococcal infections.

Anti-Infective Agents↗

PubMed2004

Activity of Staphylococcus epidermidis phenol-soluble modulin peptides expressed in Staphylococcus carnosus.

Staphylococcus epidermidis releases a group of peptides termed phenol-soluble modulin (PSM) that stimulate macrophages. The structure of 3 peptides (PSM alpha, PSM beta, and PSM gamma ) have been described. We report a fourth peptide (PSM delta ), which is a 23mer with the structure fMSIVSTIIEVVKTIVDIVKKFKK. The gene for each of the 4 peptides was introduced singly into Staphylococcus carnosus, and the PSM-like activity of culture medium and bacterial extract were significantly greater than those of the parent strain. PSM peptides from each of the S. carnosus-expressing strains were purified and analyzed by liquid chromatography-mass spectrometry. The products, which appeared to form aggregates, were active in the activation of human immunodeficiency virus type 1 long-terminal repeat and the production of tumor necrosis factor- alpha by the macrophage cell line THP-1. These findings suggest that PSM peptides are responsible, in part, for the modulin-like activity of staphylococci and may contribute to the development of severe staphylococcal sepsis.

Amino Acid Sequence↗