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Nucleotide sequence of pS194, a streptomycin-resistance plasmid from Staphylococcus aureus.

pS194 is a naturally occurring Staphylococcus aureus plasmid encoding streptomycin resistance. The plasmid has a copy number of about 25 per cell, and belongs to the inc5 incompatibility group. The nucleotide sequence of pS194 has been determined and consists of 4397 base pairs including four open reading frames potentially encoding proteins of greater than 100 amino acids. All four of these reading frames are on the same coding strand. The first reading frame, repE, encodes a 38 kd protein specifically required for pS194 replication. The second open reading frame, str, encodes a 34 kd polypeptide required for streptomycin resistance, probably a streptomycin adenylyltransferase. The third potential polypeptide, rlx, would be 37 kd and is probably required for relaxation complex formation and plasmid mobilization by conjugative plasmids. The fourth, orfD, overlapping the rlx reading frame, is potentially 27 kd, and may also be involved in mobilization.

Amino Acid Sequence↗

Effects of penicillin-binding protein 4 overproduction in Staphylococcus aureus.

The pbp4 gene of a Staphylococcus aureus strain selected stepwise in vitro for growth on increasing concentrations of penicillin and of its susceptible parent strain showed overall identity except in the promoter region. In the mutant a deletion upstream of the pbp4 structural gene removed 90 nucleotides (nt) that were framed by a 12 nt inverted repeat. This deletion occurred in step 4 of the in vitro selection procedure and was paralleled by a significant increase in the penicillin-binding protein 4 (PBP4) production. The in vitro step selected mutant showed a remarkable increase in the cross-linking of the peptidoglycan compared to its parent. This was linked to morphological changes in the appearance of the cells, which were surrounded by a very thick and fuzzy cell wall.

Amino Acid Sequence↗

Vaginal isolates of Staphylococcus aureus associated with toxic shock syndrome.

Staphylococcus aureus has been isolated from vaginal fluids of women with toxic shock syndrome (TSS), a multisystem disease with onset usually during menses. A total of 15 vaginal isolates of S. aureus from TSS patients were compared with 18 vaginal isolates from women without TSS. Phenotypic traits which were significantly more frequent in the TSS group of strains than in the non-TSS group were arsenate resistance, proteolysis of hemoglobin, reduced hemolysis of sheep blood in agar medium, and lack of lethality of culture filtrates for chicken embryos and rabbits. In addition, isoelectric focusing of ethanol extracts of culture filtrates showed differences between the two groups in the occurrence of two proteins. All hemolytic and chicken embryo-lethal strains (3 TSS strains and 14 non-TSS strains) produced an extracellular protein with an isoelectric point of 8.6. In contrast, all TSS strains, but only one-half of non-TSS strains, released a protein with an isoelectric point of 7.0 and an apparent subunit molecular weight of 22,000.

Animals↗

Use of contour-clamped homogeneous electric field (CHEF) electrophoresis to type methicillin-resistant Staphylococcus aureus.

Strains of methicillin-resistant Staphylococcus aureus (MRSA) from Australia and the UK were compared by digesting their chromosomal DNA with the low-frequency-cutting restriction enzyme SmaI and separating the restriction fragment length polymorphisms (RFLPs) by contour-clamped homogeneous electric field (CHEF) electrophoresis. The numbers of restriction fragments produced were in the range 14-17 and the sizes of the bands were 7-700 kb. Generally, the results confirmed previous conclusions based on antimicrobial resistance and plasmid profiles. The earlier MRSA isolates were different from more recent isolates, and the epidemic MRSA from eastern Australia (EA MRSA) was the same as the epidemic MRSA (EMRSA) found in London hospitals. However, contrary to previous results, the EA MRSA did not constitute a homogeneous group. The results showed that comparison of RFLPs by CHEF electrophoresis is a useful technique for studying the epidemiology of MRSA.

Australia↗

[The study of Staphylococcus aureus strain resistant to actinomycin D].

Staphylococcus aureus strains, resistant to actinomycin D (AMD) and to gramicidin S (GS) were selected by S. aureus 209P passing on the media containing the above mentioned drugs. Strain R80 resistant to AMD and strain R9 resistant to GS and AMD and described before didn't perform enzyme inactivation of AMD. Cells of both strains had diminished ability to bind exogenous AMD. Electron microscopy investigation revealed that cells of R80 strain had thickened cell walls and they are characterized by more electron density then cells of R9 strain and of parent strain. Adaption to AMD and GS influenced also on functions of some staphylococcal surface proteins--the activity of endogenous coagulase (clumping factor) was found only in R9 strain. Exogenous coagulase was present in all the strains, but development of resistant to AMD and GS diminished this enzyme activity. It is concluded that development of resistance to AMD and GS causes substantial changes in staphylococcal cell wall, but the type of these changes differ.

Anti-Bacterial Agents↗

[Evaluation of cefoxitin and cefotaxime screening plates for the detection of methicillin-resistance in Staphylococcus aureus].

Detection of methicillin-resistant Staphylococcus aureus (MRSA) isolates represents a serious problem to low and media level microbiology labs. In this work cefoxitin (FOX) and cefotaxime (CTX) screen plates (AS) (8-16 microg/ml) with and without 4% of NaCl were evaluated to detect MRSA. Sensitivity, specificity, positive and negative predictive values were determined. The AS oxacillin and the agglutination test MRSA-Screen Latex for the detection of PLP2a were used as reference methods for the evaluation of the different studied screening plates. The 100% (94 strains) PLP2a positive were detected as MRSA with FOX (8 microg/ml), and CTX (8 microg/ml with 4% NaCl) AS. The advantage of FOX AS (8 microg/ml) is that it does not need the addition of NaCl, and CTX AS (8 microg/ml with 4% NaCl) is that cefotaxime is an antimicrobial easily accessible in our country.

Anti-Bacterial Agents↗

Structure of linkage region between ribitol teichoic acid and peptidoglycan in cell walls of Staphylococcus aureus H.

The cell walls of Staphylococcus aureus H were found to contain mannosamine in an amount (28.1 nmol/mg) comparable to the content of muramic acid 6-phosphate. The acidic polymer fraction obtained by heating the cell walls at pH 2.5 was shown to contain mannosamine and glycerol in addition to the components of ribitol teichoic acid. Mild alkali treatment of this polymer fraction followed by gel filtration resulted in separation of a disaccharide N-acetylmannosaminyl(1 leads to 4)N-acetylglucosamine and the ribitol teichoic acid moiety that contained glycerol. Smith degradation of the reduction product from the same polymer fraction gave a fragment characterized as (1,2-ethylene-diol phosphate)-(glycerol phosphate)3-N-acetylmannosaminyl(1 leads to 4)N-acetylxylosaminitol. Thus, the ribitol teichoic acid chain in the cell walls is probably linked to peptidoglycan through a linkage unit, (glycerol phosphate)3-N-acetylmannosaminyl(1 leads to 4)N-acetylglucosamine.

Carbohydrate Conformation↗

Bifactorial versus monofactorial molecular status of Staphylococcus aureus gamma-toxin.

The extracellular Staphylococcus aureus gamma-toxin (hemolysin) released by the Smith 5R strain has been purified (M(r) 38 kDa, pl 9.55). We established that this cytolysin is a single polypeptide fully lytic on rabbit erythrocytes. In contrast, this toxin alone was unable to lyse other cells and was required to act jointly with an accessory 58 kDa protein released by the same strain. This protein, named sensitizing protein (SP), was required in order to damage the cytoplasmic membranes of other red blood cells including human erythrocytes as well as that of other eukaryotic cells (Jurkat and Hep-2). The lytic process can be referred to as conditional synergistic or cooperative lysis. gamma-toxin, and SP were also found able to disrupt phospholipid/cholesterol containing liposomes. We demonstrated that a minor membrane phospholipid, phosphatidylinositol, is crucial for gamma-toxin binding to cells and/or channel formation through membrane lipid bilayer.

Bacterial Proteins↗

Purification and properties of a novel lipase from Staphylococcus aureus 226.

A novel lipase of Staphylococcus aureus 226 was purified by ammonium sulfate precipitation followed by successive chromatographies on hydroxylapatite, Sephadex G-200 and G-150. This method gave 385-fold purification of the enzyme from the culture medium in a yield of 25%. The purified enzyme appeared homogeneous on polyacrylamide gel electrophoresis and isoelectric focusing. The purified lipase hydrolyzed all the triacylglycerols and 1-monoacylglycerols tested, showing maximal activity with trilaurin and 1-monoolein. Furthermore, 2-monoolein was also found to be one of the best substrates for the lipase. The optimum temperature of the purified enzyme was 60 degrees C with triolein. The molecular weight of the enzyme was reduced in the presence of sodium deoxycholate and was estimated as 34,000 by gel filtration and its isoelectric point as 9.7. Mg2+ and Ca2+ ions enhanced the enzymatic reaction, whereas Mn2+ ions were inhibitory. p-Chloromercuribenzoate, iodoacetamide, and N-ethylmaleimide did not inhibit the enzyme.

Ammonium Sulfate↗

In vitro activity of lysostaphin, mupirocin, and tea tree oil against clinical methicillin-resistant Staphylococcus aureus.

Colonization of methicillin-resistant Staphylococcus aureus (MRSA) commonly leads to infection by the same strain. We examined the activity of lysostaphin, mupirocin, and tea tree oil against clinical MRSA (n = 98) isolates. MIC(50) (range) were as follows: lysostaphin, 0.125 mg/L (0.125-0.25); mupirocin, 0.5 mg/L (0.19-1024); tea tree oil, 1024 mg/L (512-2048). High- and low-level mupirocin resistance was noted in 9.2% of our MRSA isolates. Time kill results indicate MRSA activity at 24 h was lysostaphin = gentamicin = vancomycin (P mupirocin > tea tree oil (P >or= .05). Checkerboard testing indicated a synergistic relationship between lysostaphin and mupirocin in combination with gentamicin. Antagonism was observed with the combination of vancomycin and tea tree oil; time kill studies confirmed this result. Decolonization options are limited and resistance to mupirocin exists. Lysostaphin and tea tree oil may offer additional therapeutic options for the decolonization of MRSA where current treatment alternatives are limited.

Anti-Bacterial Agents↗

Effects of antibiotic combinations on methicillin-resistant Staphylococcus aureus in vitro.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has become an important infectious disease problem. Vancomycin is the only antimicrobial agent documented to be effective in the treatment of MRSA infection. Under vancomycin treatment there is still a moderate percentage of treatment failures, especially in severe MRSA infections. In searching for better treatment regimens, we evaluated the effects of several antibiotics in combination with vancomycin or fosfomycin against MRSA isolates by the checkerboard titration method. We found that the combinations of vancomycin with imipenem and fosfomycin with cefazolin had a synergistic effect against 97.1% and 94.3% of 70 tested strains, respectively. Rifampicin, gentamicin, cefazolin, or cefamandole in combination with vancomycin, and cefmetazole in combination with fosfomycin did not have such good results. They were synergistic against far fewer of the tested strains, 0%, 3.3%, 50.0%, 30.0% and 64.3%, respectively. For confirmation, further studies in vivo of the effectiveness are advisable. It is likely that we can improve the treatment of severe MRSA infections by those combination regimens which are highly synergistic against MRSA.

Drug Therapy, Combination↗

Antibiotic-resistant Staphylococcus aureus in dermatology and burn wards.

Staphylococcus aureus isolated between 1967 and 1975 from the nose and skin lesions of patients in dermatology wards and from the burns of patients in a burns unit in Birmingham showed a high incidence of multiple antibiotic-resistant strains in both environments. Over 20% of the strains isolated from patients on admission to the dermatology wards were multiresistant. Resistance to benzylpenicillin, tetracycline, erythromycin, and fusidic acid was common in the dermatology wards; a smaller proportion of strains were resistant to lincomycin, and few (since 1972 none) were resistant to methicillin; resistance to novobiocin and chloramphenicol was uncommon. In the burns unit, fusidic acid resistance was uncommon, but resistance to benzylpenicillin, tetracycline, erythromycin, novobiocin, neomycin, methicilin, and lincomycin was common. Several of the antibiotics to which resistance was common in the burns unit were rarely if ever used there; strains resistant to these antibiotics probably remained common in the ward through the frequent use of other antibiotics, especially erythromycin, to which these strains were also resistant.

Anti-Bacterial Agents↗

Effect of farnesol on Staphylococcus aureus biofilm formation and antimicrobial susceptibility.

Staphylococcus aureus is among the leading pathogens causing bloodstream infections able to form biofilms on host tissue and indwelling medical devices and to persist and cause disease. Infections caused by S. aureus are becoming more difficult to treat because of increasing resistance to antibiotics. In a biofilm environment particularly, microbes exhibit enhanced resistance to antimicrobial agents. Recently, farnesol was described as a quorum-sensing molecule with possible antimicrobial properties. In this study, the effect of farnesol on methicillin-resistant and -susceptible strains of S. aureus was investigated. With viability assays, biofilm formation assessment, and ethidium bromide uptake testing, farnesol was shown to inhibit biofilm formation and compromise cell membrane integrity. The ability of farnesol to sensitize S. aureus to antimicrobials was assessed by agar disk diffusion and broth microdilution methods. For both strains of staphylococci, farnesol was only able to reverse resistance at a high concentration (150 microM). However, it was very successful at enhancing the antimicrobial efficacy of all of the antibiotics to which the strains were somewhat susceptible. Therefore, synergy testing of farnesol and gentamicin was performed with static biofilms exposed to various concentrations of both agents. Plate counts of harvested biofilm cells at 0, 4, and 24 h posttreatment indicated that the combined effect of gentamicin at 2.5 times the MIC and farnesol at 100 microM (22 microg/ml) was able to reduce bacterial populations by more than 2 log units, demonstrating synergy between the two antimicrobial agents. This observed sensitization of resistant strains to antimicrobials and the observed synergistic effect with gentamicin indicate a potential application for farnesol as an adjuvant therapeutic agent for the prevention of biofilm-related infections and promotion of drug resistance reversal.

Biofilms↗

Methicillin resistant Staphylococcus aureus.

The prevalence of methicillin resistant Staphylococcus aureus (MRSA) at the General Hospital, Port-of-Spain, between June 1995 and May 1996 was determined. The MRSA prevalence rate was 4.6% of all S aureus isolates, with all but one nosocomially acquired. 15 isolates were associated with infections, and three were colonizing strains. 17 of the 18 patients with MRSA had received antibiotics previously, including 13 who had received multiple antibiotics. Skin and soft tissue were the sites of infection and colonization in 12 cases; and surgical wards and the Intensive Care Unit (ICU) accounted for 16 MRSA isolates. All isolates were sensitive to vancomycin and all but one were resistant to gentamicin. MRSA occurred sporadically in a wide distribution of wards and physicians' services, although the isolation of three strains from the ICU and three strains from a surgical ward were temporally related. Only one of two deaths was attributable to MRSA. Control of the spread of MRSA in this hospital must include the reinforcement of the appropriate use of antibiotics, hand washing and appropriate isolation of patients in the surgical and intensive care wards.

Anti-Bacterial Agents↗

Slime formation as a marker of serious infection with methicillin-resistant Staphylococcus aureus.

Strains of methicillin-resistant Staphylococcus aureus (MRSA) are potential pathogens in patients undergoing major surgery and in patients who are immunocompromised. Some MRSA produce a viscous extracellular slime that may interfere with immune function. In this study, slime formation by MRSA was examined in cases of nosocomial infection by this pathogen at a single hospital. The antibacterial resistance, phage and enterotoxin characteristics of strains isolated from patients with fatal infections were determined. MRSA strains were classified as those associated with fatal infection; causing progressive infection; causing superficial infection; and those isolated from the nasal cavity of the health-care professionals as a control group. The incidence of slime formation was highest in the MRSA associated with fatal infection. The incidence of slime formation in the control group was significantly lower than that in the other groups. Results suggest that slime formation by MRSA may be associated with a more severe infection. Rapid identification of slime-forming MRSA may facilitate the initiation of appropriate treatment and improve the patient's prognosis.

Biomarkers↗

Molecular correlation between in vitro and in vivo activity of beta-lactam and beta-lactamase inhibitor combinations against methicillin-resistant Staphylococcus aureus.

Beta-Lactam resistance in Staphylococcus aureus is associated with beta-lactamase production, with the presence of a new penicillin binding protein (PBP) called PBP2a, with reduced affinity for beta-lactam antibiotics, and with modifications of normal PBPs. We have studied these mechanisms of resistance, in vivo and in vitro, for several beta-lactam antibiotics against both beta-lactamase-producing and non-producing methicillin-resistant S. aureus organisms (MRSA). Our results showed that all tested agents inhibited binding of labeled penicillin G to many PBPs. The combination of cefoperazone and sulbactam was the best combination, and it inhibited radiolabeled penicillin G binding to PBP2a at a lower concentration than that needed for cefoperazone alone. In vivo, the regimen of cefoperazone plus sulbactam was also more effective than cefoperazone alone. For beta-lactamase-negative strains this correlated with an increased binding affinity of cefoperazone plus sulbactam to PBP2a and PBP4. The improved efficacy of cefoperazone plus sulbactam versus cefoperazone with a beta-lactamase producing strain was closely related to cefoperazone hydrolysis by beta-lactamase that was inhibited by sulbactam. This study demonstrates that there is more than one effect of beta-lactamase inhibitors when they are combined with beta-lactam antimicrobial agents, and also that there may be a role for these agents in therapy for MRSA infections.

Animals↗

A new staphylococcal enterotoxin, enterotoxin F, associated with toxic-shock-syndrome Staphylococcus aureus isolates.

612 (93.8%) of 65 Staphylococcus aureus strains isolated from 65 patients with toxic-shock syndrome (TSS) produced an enterotoxin-like protein, tentatively identified as staphylococcal enterotoxin F (SEF). One of the other strains produced staphylococcal enterotoxin B and another exterotoxin C. In two blind studies all 34 TSS-associated S. aureau strains examined and 3 (11.5%) of 26 control S. aureau strains produced SEF. 2 of the latter strains were isolated from the vaginas of women who had no history of TSS. SEF was purified, and specific antibodies to it were prepared. Only 4 (4.6%) of 87 S. aureau strains from other sources were found to produce SEF. 5 (17.2%) of 29 TSS patients whose acute sera were available had anti-SEF antibody present in titres of greater than or equal to 1:100 as determined by radioimmunoassay, compared with 44 (78.6%) of 56 controls--demonstrating a greater serosusceptibility of TSS patients to SEF. It is suggested that staphylococcal enterotoxin, particularly SEF, may be a cause of the signs and symptoms of TSS.

Antibodies↗

Cross-reactive antibodies prevent the lethal effects of Staphylococcus aureus superantigens.

The exotoxins produced by Staphylococcus aureus, staphylococcal enterotoxins (SE) A-E and toxic shock syndrome toxin (TSST)-1, which are associated with serious diseases, including food poisoning and toxic shock syndrome, are termed superantigens (SAgs). To examine whether common antigenic epitopes were present and whether vaccination with 1 bacterial SAg could protect against challenge with a different SE or TSST-1, mice were vaccinated with SEA, SEB, SEC1, or TSST-1 individually or in combination. Mice injected with a single toxin developed high antibody titers against other SAgs. Marked improvement in survival was observed when immunized mice were challenged with a heterologous toxin. Mice vaccinated with a mixture of toxins were fully protected against 1 or multiple toxin challenges, indicating no interference effects of multivalent vaccinations. More importantly, higher titers were found against each SAg with the multivalent vaccination than with injection with a single SAg. Thus, immunizations with 1 SAg can induce cross-protective antibodies to heterologous SAgs, and multicomponent vaccination can enhance antibody responses against each bacterial SAg.

Animals↗