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PubMed1990

Cloning and restriction analysis of DNA conferring new quinolone antimicrobial agent resistance from Staphylococcus aureus and other coagulase-negative Staphylococcus species.

DNA conferring resistance to new quinolone antimicrobial agents (NQR) in clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci (CNS), S. epidermidis and S. haemolyticus, was analyzed after cloning in Escherichia coli. The NQR phenotypes were expressed in E. coli at lower levels. NQR gene(s)-carring HindIII fragments were very similar to each other among S. aureus or CNS, although the S. aureus fragments differed from the CNS fragments in terms of the NQR phenotypes and restriction endonuclease maps. The data suggest the possibility that the NQR genes have disseminated in evolutionarily distinct routes among S. aureus and CNS.

4-Quinolones↗
PubMed1995

Effects of culture conditions on production of type 5 capsular polysaccharide by human and bovine Staphylococcus aureus strains.

Two Staphylococcus aureus strains, the prototype human Reynolds strain and a bovine isolate, were grown in different complex media and in a synthetic medium (D. Taylor and K. T. Holland, J. Appl. Bacteriol. 66:319-329, 1989) and compared for their ability to produce type 5 capsular polysaccharide. Cell-bound and cell-free type 5 capsular polysaccharide were measured by a new one-step competition enzyme-linked immunosorbent assay. The total production and the proportion of cell-bound type 5 capsular polysaccharide were dependent on the nature of the medium, the duration of the culture, and the strain. Both strains produced more type 5 capsular polysaccharide when cultivated in the synthetic medium than when cultivated in complex media. The best yield of type 5 capsular polysaccharide, about 300 micrograms/ml of medium, was obtained with strain Reynolds grown for 48 h with shaking in the synthetic broth containing glucose as a carbon source.

Agar↗
PubMed1992

Lethal and mutational effects of solar and UV radiation on Staphylococcus aureus.

Strains of Staphylococcus aureus, an opportunistic pathogen commonly found on human skin, were exposed to sunlight and UV C radiation, and the lethal and mutational effects measured. Sunlight killed cells with an inactivation constant of 3 x 10(-5) per joule per square metre; UV C was much more lethal, giving an inactivation constant of approximately 0.1 per joule per square metre. Some strains tested showed a sensitivity to sunlight that was dependent on the growth phase of the cells, exponentially growing cells showing a greater sensitivity. Mutational effects of irradiation were measured by the appearance of mutants sensitive to methicillin following irradiation of a multiresistant strain. Mutants appeared at a frequency of 10(-3); this high frequency of mutation in the region of the mec gene has also been observed when multiresistant strains are subjected to nutritional or thermal stress. Mutants showed the same chromosomal alteration (seen in pulse-field gel electrophoresis of SmaI-digested DNA) whether induced by solar or UV C irradiation.

DNA Damage↗
PubMed1987

Electron microscopic demonstration and isolation of ribosomes in mesosomes from Staphylococcus aureus.

Mesosomes of Staphylococcus aureus 209P were observed to be extruded as tubules upon protoplast formation by electron microscopy and isolated under hypertonic conditions to maintain their structural integrity by differential centrifugation followed by sucrose density gradient centrifugation. Isolated mesosomes were composed of long, branched tubules of irregular sizes and they were shortened during purification. Thin sections of isolated mesosomes showed that the mesosomal tubule was surrounded by a triple-layered membrane and contained ribosome-like particles in diameter of about 15 to 20 nm. These particles were isolated from purified mesosomal preparation by disrupting the mesosomal tubule with deoxycholate and Triton X-100 under hypotonic conditions followed by a linear sucrose density gradient centrifugation. Negatively stained preparations of the isolated particles revealed the same appearance as those of the ribosomes isolated from the cytoplasm. The mesosomal particles sedimented at 70S in sucrose gradients in the presence of 10 mM Mg2+, but they were dissociated into two subparticles, 50S and 30S subunits, upon lowering the Mg2+ concentration to 1 mM. These findings indicate that the mesosomal tubule is packed with ribosomes.

Centrifugation, Density Gradient↗
PubMed1993

[Protective effect of a human immunoglobulin preparation on mouse experimental infection of methicillin resistant Staphylococcus aureus].

Methicillin resistant Staphylococcus aureus (MRSA) selectively increased by extensive chemotherapy is now one of the major causative agents of opportunistic infections in immunocompromised patients. Immunotherapy should be applied because this infection is based on the immunocompromised state of the patients. But this therapy has not been applied because S. aureus is known to have protein A which binds to the Fc portion of IgG and blocks phagocytosis through Fc receptor of phagocytic cells which plays the major role for the killing of infected bacteria in vivo. It should be known that MRSA strains often did not express protein A. Recently isolated MRSA strains were collected from three medical institutions and examined for their expression of protein A. Incidences of protein A-negative strains were different among institutions. It was found that more than a half of the strains from a general hospital which expressed typical heat sensitive methicillin resistance did not express protein A, but that incidences of protein A-negative strains were around 20% in the MRSA strains of a complicated history from a university hospital and a research center. All protein A-negative strains were found to be agglutinated by a commercially available immunoglobulin preparation (Venilon). And this immunoglobulin was proved to have a protective effect for the mouse experimental infections of these protein A-negative MRSA strains.

Animals↗
PubMed1997

The effects of adherence to silicone surfaces on antibiotic susceptibility in Staphylococcus aureus.

Sensitivity of Staphylococcus aureus to the antibiotics tetracycline, benzylpenicillin and vancomycin was found to decrease by 2-10 fold when cells were grown adherent to silicone catheter surfaces. Sensitivity to rifampicin and fusidic acid was not significantly altered in adherent cells. Susceptibility further decreased with increased adherence time prior to antibiotic challenge. The resistance observed was not genotypic, or due to the presence of a specialized subpopulation of bacteria, as it disappeared when the bacteria were removed from the catheter, subcultured and retested. Also, adherent bacteria were found to grow more slowly than bacteria growing planktonically. It is concluded that the decrease in antibiotic susceptibility of adherent bacteria is a function of the physiological status of the individual cells rather than a function of biofilm formation or slime production. The decrease in growth rate of the adherent bacteria is a result of the adherence process rather than a result of nutrient depletion. The decrease in growth rate is implicated, but is not the sole factor, in the decreased antibiotic susceptibility of adherent bacteria.

Anti-Bacterial Agents↗
PubMed1996

Use of the coagulase gene typing method for detection of carriers of methicillin-resistant Staphylococcus aureus.

Strains of Staphylococcus aureus can be typed on the basis of the polymorphism of the coagulase gene. DNA fragments generated after amplification by polymerase chain reaction (PCR) of the variable region of this gene and digested with the restriction enzyme HaeIII can be compared by their restriction fragment length polymorphism (RFLP). Seventy-nine of 86 (91.8%) methicillin-resistant S. aureus (MRSA) strains isolated in various hospitals had a characteristic RFLP pattern. This pattern differed from those of 32 methicillin-susceptible S. aureus (MSSA). Only one MSSA shared with MRSA the same RFLP pattern. After modification, we applied this method to the rapid detection of MRSA from 255 nasal swabs from patients hospitalized in intensive care units. When screened by plating on Chapman agar, 55 of these samples contained MRSA, whereas 40/55 yielded the expected restriction profile after amplification by PCR. No DNA was amplified by PCR in 9/55 samples and restriction profiles were uninterpretable in six cases. When compared to culture, the positive and negative predictive values of the PCR test were 100% and 93%, respectively. The specificity was 100% and the sensitivity was 72.7%. Since control of spread of MRSA strains in hospitals is based in part on rapid isolation of carriers, this method which allows detection of epidemic MRSA in nasal swabs within a day could be helpful, though culture would still be necessary to confirm the result.

Bacterial Typing Techniques↗
PubMed2006

Osteoblast functions on functionalized PMMA-based polymers exhibiting Staphylococcus aureus adhesion inhibition.

Staphylococcus aureus adhesion and osteoblast functions were assessed on functionalized poly(methyl methacrylate)-based terpolymers bearing randomly distributed carboxylate and sulfonate groups. These terpolymers were synthesized by radical polymerization, characterized by nuclear resonance spectroscopy and classified by the ratio R=[COO(-)/COO(-)+SO(3)(-)] in the range 0.5-0.8. Bacterial adhesion study showed that fibronectin-coated terpolymers with R varying from 0.5 to 0.8 exhibited inhibition rate of S. aureus adhesion from 90% to 98% as compared to the adhesion on unfunctionalized poly(methyl methacrylate). In contrast, the adhesion of osteoblasts onto the same functionalized terpolymers was decreased by 20% when compared to the results obtained on poly(methyl methacrylate). While the amount of attached osteoblasts are similar onto all the functionalized terpolymers whatever its R value, the cell proliferation was different and was found to vary with R in the range 0.5-0.8. Osteoblast proliferation, alkaline phosphatase activity and accumulation of calcium in the extracellular matrix of these cells, cultured on the functionalized terpolymers with R equal to 0.7-0.8 were similar to that observed onto non-functionalized poly(methyl methacrylate). In contrast, osteoblast proliferation was inhibited on terpolymers with an R value around 0.6. These results provide evidence that functionalized poly(methyl methacrylate)-based terpolymers with R ratio equaling 0.7-0.8 simultaneously inhibit bacteria adhesion and support osteoblast functions pertinent to new bone formation. These functionalized polymers could, therefore, be used as coating or grafted on orthopedic and dental implants to render them both bone compatible and able to prevent bacterial infection.

Animals↗
PubMed1998

The role of environmental factors in the regulation of virulence-determinant expression in Staphylococcus aureus 8325-4.

Staphylococcus aureus is a major human pathogen, which produces a variety of virulence determinants. To study environmental regulation of virulence-determinant production, several transcriptional reporter gene fusions were constructed. Chromosomal fusions were made with the staphylococcal accessory regulator (sarA), alpha-haemolysin (hla), surface protein A (spa) and toxic-shock syndrome toxin-1 (tst) genes. The effect of many different environmental conditions on the expression of the fusions was examined. Expression of hla, tst and spa was strongly repressed in the presence of sodium chloride (1 M) or sucrose (20 mM), but sarA was relatively unaffected. The global regulator of expression of virulence-determinant genes, agr (accessory gene regulator) was not involved in the salt or sucrose repression. Novobiocin, a DNA gyrase inhibitor, did not significantly increase the expression of tst in wild-type or agr backgrounds and failed to relieve the salt suppression. Expression of tst was strongly stimulated in several low-metal environments, independently of agr, whilst spa levels were significantly reduced by EGTA. The complex, interactive role of environmental factors in the control of expression of the virulence determinants is discussed.

Artificial Gene Fusion↗
PubMed2005

Roles for fibrinogen, immunoglobulin and complement in platelet activation promoted by Staphylococcus aureus clumping factor A.

Staphylococcus aureus is an important cause of infective endocarditis (IE) in patients without a history of prior heart valve damage. The ability to stimulate the activation of resting platelets and their subsequent aggregation is regarded as an important virulence factor of bacteria that cause IE. Clumping factor A is the dominant surface protein responsible for platelet activation by S. aureus cells in the stationary phase of growth. This study used Lactococcus lactis as a surrogate host to study the mechanism of ClfA-promoted platelet activation. Expression of ClfA from a nisin-inducible promoter demonstrated that a minimum level of surface-expressed ClfA was required. Using platelets that were purified from plasma, the requirement for both bound fibrinogen and immunoglobulin was demonstrated. The immunoglobulin G (IgG) requirement is consistent with the potent inhibition of platelet activation by a monoclonal antibody specific for the platelet FcgammaRIIa receptor. Furthermore the IgG must contain antibodies specific for the ClfA A domain. A model is proposed whereby bacterial cells armed with a sufficient number of surface-bound fibrinogen molecules can engage resting platelet glycoprotein GPIIb/IIIa, aided by bound IgG molecules, which encourages the clustering of FcgammaRIIa receptors. This can trigger activation of signal transduction leading to activation of GPIIb/IIIa and aggregation of platelets. In addition, analysis of a mutant of ClfA totally lacking the ability to bind fibrinogen revealed a second, although less efficient, mechanism of platelet activation. The fibrinogen-independent pathway required IgG and complement deposition to trigger platelet aggregation.

Blood Platelets↗
PubMed1981

Energy-dependent efflux of cadmium coded by a plasmid resistance determinant in Staphylococcus aureus.

Resistance of Staphylococcus aureus strain 17810R to Cd2+ appears to be due to a plasmid-coded Cd2+ efflux system. Complete efflux of Cd2+ after transfer of preloaded cells into Cd2+-free medium occurred in the resistant strain 17810R, but not in the plasmidless derivative strain 17810S. Net efflux was blocked by 2,4-dinitrophenol, N,N,-dicyclohexylcarbodiimide (DCCD), and incubation at 4 degrees C. The inhibition of Cd2+ efflux by DCCD paralleled a stimulation of net uptake in the resistant cells by this agent. Cd2+ efflux by the resistant strain was accompanied by a reversal of inhibition of respiration, whereas in the sensitive strain, inhibition of respiration was not reversed after transfer to Cd2+-free medium. Net Cd2+ uptake by strain 17810R was inhibited by p-chloromercuribenzoate. In Cd2+ contrast, Cd2+ uptake by the plasmidless strain 17810S was affected neither by p-chloromercuribenzoate nor by DCCD when added alone, but was blocked by a combination of these two agents. Valinomycin had no effect on the reduced Cd2+ uptake by the resistant strain, whereas nigericin stimulated uptake to values comparable to those of the untreated sensitive cells. With sensitive cells, valinomycin reduced Cd2+ uptake by about 50%, whereas nigericin was without effect. A possible mechanism of Cd2+ movements in both strains is discussed.

Biological Transport, Active↗
PubMed1977

A new type of penicillin resistance of Staphylococcus aureus.

Penicillin--"tolerant" Staphylococcus aureus strains are resistant to the lethal action of penicillins, but are inhibited by normal (low) concentrations. They are deficient in autolytic enzyme activity which appears to be necessary for bacteriolysis and the lethal action of penicillins. This "deficiency" is caused by a large excess of an inhibitor of autolysin. Seven such tolerant strains have been isolated from blood, bone, or sputum of patients who responded poorly to penicillin treatment of endocarditis, osteomyelitis, or staphylococcal pneumonia. These isolates were of different phage-types, and most showed cross-tolerance to the killing action of cephalosporins or vancomycin, antibiotics to which they were sensitive (inhibited). They were killed at normal rates by gentamicin, cycloserine, and rifampicin. Population analysis indicated that the proportion of tolerant organisms within a resistant strain is 7% or less; their ability to inhibit autolytic activity within their own and neighbouring cells appears to account for the net decreased autolytic activity of the entire strain; 44% of the bacteraemic strains studied showed penicillin tolerance. Tolerance is thus a common, clinically important form of penicillin resistance, that differs from previously described forms of pencillin resistance, that due to beta-lactamase, and that due to "intrinsic" (e.g., methicillin resistance) mechanisms.

Adult↗
PubMed1991

Mechanisms of clinical resistance to fluoroquinolones in Staphylococcus aureus.

Mechanisms of Staphylococcus aureus resistance to fluoroquinolones were characterized. Subunit A and B proteins of DNA gyrase were partially purified from fluoroquinolone-susceptible strain SA113 and resistant isolate MS16405, which was 250- to 1,000-fold less susceptible to fluoroquinolones such as ciprofloxacin, norfloxacin, ofloxacin, temafloxacin, and sparfloxacin than SA113 was. The supercoiling activity of the gyrase from SA113 was inhibited by the fluoroquinolones, and the 50% inhibitory concentrations of the drugs correlated well with their MICs. In contrast, the gyrase from MS16405 was insensitive to inhibition of supercoiling by all of the quinolones tested, even at 800 micrograms/ml. Combinations of heterologous gyrase subunits showed that subunit A from MS16405 conferred fluoroquinolone resistance, suggesting that an alteration in gyrase subunit A is a cause of the fluoroquinolone resistance in MS16405. Uptake of hydrophilic fluoroquinolones such as ciprofloxacin and norfloxacin by MS16405 was significantly lower than that by SA113. Furthermore, this difference was abolished by the addition of an energy inhibitor, carbonyl cyanide m-chlorophenylhydrazone, suggesting that an alteration in an energy-dependent process, such as an active efflux of hydrophilic quinolones, may lead to decreased drug uptake and hence to increased resistance to fluoroquinolones in MS16405. These findings suggest that the fluoroquinolone resistance in MS16405 is due mainly to an alteration in subunit A of DNA gyrase and may also be associated with an alteration in the drug uptake process.

4-Quinolones↗
PubMed1999

Risk factors for hospital-acquired Staphylococcus aureus bacteremia.

BACKGROUND: Staphylococcus aureus bacteremia (SAB) acquired in hospitals continues to be a frequent and serious complication to hospitalization, and no previous case-control studies dealing with risk factors of this severe disease are available. METHODS: Based on a 1-year prospective analysis, the data from all patients with hospital-acquired SAB admitted to 4 hospitals in Copenhagen County, Denmark, from May 1, 1994, through April 30, 1995, were evaluated. Eighty-five patients with hospital-acquired SAB were matched to 85 control patients with a similar primary diagnosis at admission (matched controls). Of these, 62 patients with hospital-acquired SAB were compared with 118 other patients with a similar time of admission, who were randomly selected with no clinical evidence of SAB (unmatched controls). RESULTS: The incidence of hospital-acquired SAB was 0.71 per 1000 hospital admissions. The presence of a central venous catheter (odds ratio, 6.9; 95% confidence interval [CI], 2.8-17.0), anemia (odds ratio, 3.3; 95% CI, 1.4-7.6), and hyponatremia (odds ratio, 3.3; 95% CI, 1.5-7.0) was significantly associated with hospital-acquired SAB in a conditional and a usual logistic regression analysis. Nasal carriage was not an independent risk factor, but nasal carriers among patients in surgery (odds ratio, 4.0; 95% CI, 1.3-13.0) had a significantly higher risk for hospital-acquired SAB compared with matched and unmatched controls. The presence of hospital-acquired SAB increased the mortality rate 2.4-fold (95% CI, 1.1-5.2). CONCLUSIONS: The presence of a central venous catheter is an important risk factor, and hyponatremia and anemia are associated with the development of hospital-acquired SAB. Furthermore, hospital-acquired SAB in itself increases mortality.

Adolescent↗
PubMed1993

Nasal carriers of Staphylococcus aureus among healthy individuals.

Staphylococcus aureus and Staphylococcus spp. were isolated from healthy students and their drug resistance was investigated. S. aureus was isolated from 17 of 70 persons (24.3%). Fourteen strains of 22 isolates of S. aureus were ampicillin-resistant. Two strains each were cefmetazole-resistant and gentamicin-resistant. None of the strains was found to be methicillin-resistant. Compared with the strains isolated from the hospital ward environment, S. aureus in healthy individuals was relatively sensitive to antibiotics.

Adult↗
PubMed2001

Immunization with Staphylococcus aureus lysate incorporated into microspheres.

Antibiotics are of limited value against Staphylococcus aureus due to development of resistant strains, scar tissue formation, and blockage of ducts due to inflammation. Though macrophages are the predominant cell type in the mammary gland, they are primarily scavenger cells and are not effective against bacteria entering the gland. Neutrophil phagocytosis is the bovine's primary defense against S. aureus mastitis. Attempts to develop vaccines that enhance neutrophil phagocytosis by stimulating production of opsonizing antibodies to S. aureus have met with limited success because of the low immunogenicity of the exopolysaccharide capsule surrounding S. aureus. Staphylococcus aureus can also adhere to and penetrate epithelial tissue. This study was conducted to determine whether lysates of S. aureus encapsulated in biodegradable microspheres would increase the production of opsonizing antibodies to capsule and block adherence. Four groups of four cows each were injected with 1 ml of the respective treatment in the area of the supramammary lymph node and 1 ml in the hip muscle. The treatments were: lysate in NaCl, lysate in Freund's incomplete adjuvant (FICA), lysate in microspheres in NaCl, and lysate in microspheres in FICA. Antigen in microspheres produced a similar antibody response to antigen emulsified in FICA, but to a lesser magnitude. Antigen in microspheres produced antibodies that were more opsonic for neutrophils at 20 and 52 wk postimmunization and inhibited S. aureus adherence to mammary epithelium. Ability to control antigen release and presentation, and the benefit of a single injection for long-term immunity using microspheres warrants additional studies.

Animals↗
PubMed2006

[A study on the mechanism of drug resistance in Staphylococcus aureus].

OBJECTIVE: To inquire into the mechanism of drug resistance in Staphylococcus aureus. METHODS: A total of 198 strains of Staphylococcus aureus were isolated from the samples sent to the Clinical Laboratory of Microbiology,West China Hospital. The resistance of Staphylococcus aureus to Methicillin was assayed with agar dilution. Staphylococcus aureus mecA gene was measured by PCR assay and beta-lactamase was detected by Nitrocephin. RESULTS: The rate of resistance to methicillin was 64.65% in 198 strains of Staphylococcus aureus; 118 strains of methicillin-resistant staphylococcus aureus(MRSA) were found to have high level resistance in 128 MRSA;10 strains of MRSA were found to have low level resistance; 41(58.57%) strains of methicillin-sensitive Staphylococcus aureus (MSSA) expressed beta-lactamase; 2 Staphylococcus aureus had mecA among them; 67 Staphylococcus aureus expressed beta-lactamase in high level resistance, 63(53.39%)Staphylococcus aureus expressed beta-lactamase in high level resistance, among them, 5 Staphylococcus aureus had mecA; 40.00% MRSA expressed beta-lactamase in low level resistance, 55 MRSA did not express beta-lactamase in high level resistance, which had all mecA; 9 Staphylococcus aureus did not express beta-lactamase in low level resistance, among them, 5 Staphylococcus aureus had mecA. The difference in expression of beta-lactamase was statistically significant between MSSA and MRSA; MRSA(53.39%) was lower than MSSA (58.57%); the other differences were not significant. The difference in having mecA was statistically significant between MRSA(having high resistant level and no expression of beta-lactamase) and the others; MRSA had higher mecA than did the others. CONCLUSION: The resistance in Staphylococcus aureus mainly involved two mechanisms: the expression of beta-lactamase and the expression of mecA.

Bacterial Proteins↗