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Protease produced by Staphylococcus aureus precipitates digests of casein in standard methods caseinate agar (SMCA) plate, and the various sizes of A to E precipitation types were produced depending on strains of inoculated Staphylococcus aureus, as previously reported. To evaluate these phenomena for epidemiological use, we investigated the effects of bacterial passage, storage and dosage on zonal pattern and the size of precipitation rings. Methicillin resistant Staphylococcus aureus (MRSA) was found to produce variable types and sizes of precipitation rings depending upon the inoculated strains. After 5, 30 and 50 passages in heart infusion broth (HIB), each of the strains randomly selected from MRSA strains showed that the precipitation pattern of A to E produced the same types and sizes of precipitation rings. The storage effects were assessed by comparing those strains in different storage time. The strain stored for 1 and 3 years at room temperature in HIB contained 0.8% agar produced the same pattern of precipitation rings at that of the freshly isolated one. Finally, the dosage of inoculated bacteria was examined. Overnight broth (HIB) cultures of MRSA were directly inoculated in SMCA plates, and were found to produce almost the same size of precipitation rings (reproducible within 3 mm). These findings indicated that protease digestion on SMCA plate provides a rapid, sensitive, and specific assay that can supplement the currently available modalities for typing of MRSA strains.
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Ninety-nine clinical staphylococcal isolates (58 coagulase-negative Staphylococcus spp. [CoNS] and 41 Staphylococcus aureus isolates) were evaluated for susceptibility to oxacillin. The following susceptibility testing methods, media, and incubation conditions were studied: agar dilution by using Mueller-Hinton (MH) medium (Difco) supplemented with either 0, 2, or 4% NaCl and incubation at 30 or 35 degrees C in ambient air for 24 or 48 h; disk diffusion by using commercially prepared MH medium (Difco) and MH II agar (BBL) and incubation at 35 degrees C in ambient air for 24 or 48 h; and agar screen (spot or swab inoculation) by using commercially prepared agar (Remel) or MH agar (Difco) prepared in-house, each containing 4% NaCl and 6 microg of oxacillin/ml (0.6-microg/ml oxacillin was also studied with MH agar prepared in-house for the agar swab method and CoNS isolates) and incubation at 35 degrees C in ambient air for 24 or 48 h for swab inoculation and at 30 or 35 degrees C in ambient air for 24 or 48 h for spot inoculation. The results for these methods were compared to the results for mecA gene detection by a PCR method. Given the ability to support growth and the results for susceptibility testing (the breakpoint for susceptible isolates was </=2 microg/ml), the best methods for CoNS isolates were (i) agar dilution by using MH medium supplemented with 4% NaCl and incubation at 35 degrees C for 48 h (no growth failures were noted, and sensitivity was 97.6%) and (ii) agar screen (swab inoculation) by using MH medium prepared in-house supplemented with 4% NaCl and containing 0.6 microg oxacillin/ml and incubation at 35 degrees C for 48 h (one isolate that did not carry the mecA gene did not grow, and the sensitivity was 100%). All but one (agar dilution without added NaCl and incubation at 30 degrees C for 48 h) of the methods tested revealed all oxacillin-resistant S. aureus isolates, and no growth failures occurred with any method. If the breakpoint for susceptibility was lowered to </=1 microg/ml for agar dilution methods, more CoNS isolates with oxacillin resistance related to the mecA gene were detected when 0 or 2% NaCl agar supplementation was used. Only one CoNS isolate with mecA gene-associated resistance was not detected by using agar dilution and MH medium supplemented with 4% NaCl with incubation for 48 h. When the breakpoint for susceptibility was decreased 10-fold (from 6.0 to 0.6 microg of oxacillin per ml) for the agar swab screen method, fully 100% of the CoNS isolates that carried the mecA gene were identified.
Methicillin resistance in S. aureus is primarily due to the presence of the mec A gene. However, in addition to mec, the phenotypic expression of methicillin resistance requires the presence of an additional gene(s), fem A which is chromosomally encoded. Previous studies suggest an increase in the biochemical function of fem A gene products due to base substitutions in the region upstream of the fem A gene and in its coding frame. The partial nucleotide sequences of fem A regions in reference and clinical strains of S.aureus were therefore analyzed by PCR-direct solid-phase sequencing and suitable DNA probes. Amplified target DNAs of 251, 330 and 271 bp were resolved on ethidium bromide-stained gels and hybridized with DNA probes conjugated to alkaline phosphatase. In ATCC 12600 strain, a palindromic sequence was conserved in the region upstream of fem A. However, it was destroyed by the occurrence of mutations in other reference, and clinical strains tested regardless of whether they are methicillin-susceptible or resistant. Furthermore, in the coding frame of fem A, two missense mutations were present in MSSA and MRSA without any regularity. These findings suggest that mutations in the fem A region may not be a single factor essential for regulation of methicillin resistance, although fem A probably functions cooperatively with mec A.
Infection due to Staphylococcus aureus continues to be a source of significant morbidity and mortality. However, its treatment is increasingly complicated by the rising prevalence of resistance to antibiotics. Apart from the two recognized modes of staphylococcal resistance, namely, penicillinase production and intrinsic resistance, Sabath and associates have described a third type in which resistance is manifested by susceptibility to growth inhibition but tolerance to the lethal action of bactericidal agents. The mechanism of tolerance is attributed to a deficiency of autolytic enzyme activity in the part of bacteria, possibly secondary to an inhibition of autolysins in the tolerant staphylococcal strains. These strains are found in patients with infections responding poorly to treatment with cell-wall active antibiotics including vancomycin. Because of its unique mechanism of action and pharmacokinetic properties, rifampin has been reported to be the most active among 65 antistaphylococcal agents tested and have the capacity to kill intraleukocytic staphylococci. We present 2 cases who were cured following the addition of rifampin to previously established regimens. Case 1 was a 40-year-old male who had fever, cough, dyspnea, a right elbow abscess and left leg swelling for 2 weeks prior to admission. Culture of purulent material from the elbow abscess grew staphylococcus aureus. Chest X-ray showed bilateral septic embolism and phleborheography showed partial deep vein occlusion of the left ileofemoral vein. Case 2 was 22-year-old female with fever, chills and cough for 3 weeks. Blood culture grew staphylococcus aureus, and Chest X-ray revealed bilateral septic embolism with pneumonia. Neither of them responded to standard antibiotics which were judged adequate by in vitro sensitivity tests. Clinical cure was later obtained after rifampin was added to the regimens. These results suggest that rifampin may be a useful adjunct in the therapy of staphylococcal infections.
WCK 771 is a broad-spectrum fluoroquinolone with enhanced activity against quinolone-resistant staphylococci. To understand the impact of the target-level interactions of WCK 771 on its antistaphylococcal pharmacodynamic properties, we determined the MICs for genetically defined mutants and studied the mutant prevention concentrations (MPCs), the frequency of mutation, and the cidality against the wild type and double mutants. There was a twofold increase in the MICs of WCK 771 for single gyrA mutants, indicating that DNA gyrase is its primary target. All first- and second-step mutants selected by WCK 771 revealed gyrA and grlA mutations, respectively. The MICs of WCK 771 and clinafloxacin were found to be superior to those of other quinolones against strains with double and triple mutations. WCK 771 was also cidal for high-density double mutants at low concentrations. WCK 771 and clinafloxacin showed narrow mutant selection windows compared to those of the other quinolones. Against a panel of 50 high-level quinolone-resistant clinical isolates of staphylococci (ciprofloxacin MIC > or = 16 microg/ml), the WCK 771 MPCs were < or =2 microg/ml for 68% of the strains and < or =4 microg/ml for 28% of the strains. Our results demonstrate that gyrA is the primary target of WCK 771 and that it has pharmacodynamic properties remarkably different from those of quinolones with dual targets (garenoxacin and moxifloxacin) and topoisomerase IV-specific quinolones (trovafloxacin). WCK 771 displayed an activity profile comparable to that of clinafloxacin, a dual-acting quinolone with a high affinity to DNA gyrase. Overall, the findings signify the key role of DNA gyrase in determining the optimal antistaphylococcal features of quinolones.
Oxacillin-resistant Staphylococcus aureus (ORSA) infection is an important cause of hospital morbidity and mortality. The objective of this study was to identify the main factors associated with death in patients colonized or infected with Staphylococcus aureus in a cancer center. A matched-pair case-control study enrolled all patients infected or colonized with ORSA (cases) admitted to the Hospital do Câncer in Rio de Janeiro from 01/01/1992 to 12/31/1994. A control was defined as a patient hospitalized during the same period as the case-patients and colonized or infected with oxacillin-susceptible Staphylococcus aureus (OSSA). The study enrolled 95 cases and 95 controls. Patient distribution was similar for the two groups (p > or = 0.05) with respect to gender, underlying diseases, hospital transfer, prior infection, age, temperature, heart and respiratory rates, neutrophil count, and duration of hospitalization. Univariate analysis of putative risk factors associated with mortality showed the following significant variables: admission to the intensive care unit (ICU), presence of bacteremia, use of central venous catheter (CVC), ORSA colonization or infection, pneumonia, use of urinary catheter, primary lung infection, prior use of antibiotics, mucositis, and absence of cutaneous abscesses. Multivariate analysis showed a strong association between mortality and the following independent variables: admission to ICU (OR [odds ratio] = 7.2), presence of Staphylococcus bacteremia (OR = 6.8), presence of CVC (OR = 5.3), and isolation of ORSA (OR = 2.7). The study suggests a higher virulence of ORSA in comparison to OSSA in cancer patients.
Staphylococcus aureus isolate, WBG1022, was resistant to penicillin, kanamycin, neomycin, streptomycin, chloramphenicol, trimethoprim, cadmium, and ethidium bromide and harbored plasmids of 34.5, 24.5, 4.4, 3.2, and 2.6 kilobases. The plasmids were transferred in mixed-culture transfer and conjugation experiments. No resistance phenotype was associated with the 2.6-kb plasmid. The 3.2-kb and 4.4-kb plasmids encoded chloramphenicol and streptomycin resistance respectively. The 24.5-kb plasmid, pWBG626, encoded joint resistance to penicillin, kanamycin, neomycin, and ethidium bromide. Resistance to trimethoprim and cadmium were chromosomal. The 34.5-kb plasmid, pWBG661, had no resistance phenotype but was found to be conjugative. It also mobilized the 4.4-kb and 24.5-kb plasmids in WBG1022. Restriction endonuclease analysis of pWBG661 with EcoRI, ClaI, PvuII, and BglII restriction enzymes demonstrated that pWBG661 was identical to two previously isolated S. aureus conjugative plasmids, pWBG620 and pWBG637, that also lack resistance phenotypes.
Pathogenesis of Staphylococcus aureus, an opportunistic human pathogen, is complex and involves many virulence factors including an array of surface proteins (adhesins) that promote bacterial interactions with extracellular matrix components. A better understanding of these interactions can be achieved by studying the expression of membrane and cell wall associated proteins using a proteome analysis approach. To accomplish this, our goal here was to construct a reference map of membrane and cell wall associated proteins for S. aureus. Various lytic and solubilization methods have been tested to identify a suitable methodology for detection of these proteins in two-dimensional electrophoresis (2DE). Results demonstrate that cell lysis with lysostaphin, which lyses staphylococcal peptidoglycan, followed by solubilization with urea, thiourea, amidosulfobetaine 14 (ASB 14) and dithiothreitol (DTT) is an effective method, yielding a sample comprising proteins of wide molecular ranges and isoelectric points with minimum contamination from cytosolic proteins. Mass spectrometric analysis was employed to identify the membrane and cell surface proteins present in the sample and consequently an initial proteomic map of membrane and cell wall associated proteins for S. aureus is presented.
Purified Staphylococcus aureus metalloproteinase contains trace amounts of a serine proteinase which rapidly degrades the metalloproteinase when EDTA is present. However, no degradation occurs when Ca2+ is added or if the serine proteinase is removed by immunoaffinity chromatography. Selective chelation of Zn2+ by o-phenanthroline, which reversibly inactivates the metalloproteinase, does not result in the degradation of the apometalloproteinase, even with excess of serine proteinase. These data are interpreted as follows: EDTA chelates enzyme-bound Ca2+ and Zn2+, causing irreversible inactivation as well as a conformational change in the metal-free protein. This allows proteolysis by the contaminating serine proteinase and explains why the metalloproteinase purified from serine proteinase-deficient strains of S. aureus was previously thought to be stable to autolysis.
Most Staphylococcus aureus strains associated with toxic shock syndrome and producing toxic shock syndrome toxin 1 (TSST-1) require tryptophan because of a genetic defect in tryptophan biosynthesis. The association between TSST-1 production and tryptophan auxotrophy was not correlated with the phage type, the colonization site, or the disease status of the patient from whom the isolate came. Protoplast fusion and transformation mapping located the genetic determinant of TSST-1 production (tst) very close to the trp operon in such strains and very close to tyrB in a Trp+ TSST-1+ strain. Southern blot hybridization of ClaI-restricted chromosomal DNA with a tst-specific probe revealed a common homologous segment in all of the Trp+ strains with tst linked to tyrB. These results confirmed that the tst determinant in Trp- strains is located at one site, whereas in Trp+ TSST-1+ strains the determinant is located elsewhere on the S. aureus chromosome. It is suggested that the TSST-1 determinant is associated with the insertion of a transposonlike segment into several sites on the S. aureus chromosome.
Staphylococcus aureus is a major cause of infections in both hospitals and communities, and is exhibiting increasing resistance to methicillin (methicillin-resistant S. aureus, MRSA) and related beta-lactams. MRSA is usually considered a nosocomial pathogen, but increasingly it is acquired in the community. In Taiwan, MRSA was colonized in a substantial proportion of healthy children and accounted for 25% to 75% of childhood community-acquired (CA) S. aureus infections. From the preliminary data, the isolates of sequence type (ST) 59 by multilocus sequence typing method appeared to be the major clone of CA-MRSA in northern Taiwan. Compared with those reported from the US and other countries, CA-MRSA isolates in Taiwan did not always harbor type IV staphylococcal cassette chromosome (SCCmec) and were resistant to multiple non-beta-lactam antibiotics, including clindamycin and macrolides. Molecular evidence suggested transmission of the community strain of MRSA into the hospital setting, and that the community strain had became a health care-associated pathogen. The treatment of putative CA S. aureus infection should be stratified according to the severity and the disease entity.
Growth of Staphylococcus aureus in the presence of salicylate leads to reduced ciprofloxacin and ethidium accumulation and increased resistance to ethidium. Salicylate induced reduction in ciprofloxacin accumulation is energy-independent while salicylate induced alterations in ethidium accumulation and efflux is proton motive force-dependent. NorA is an intrinsic multidrug efflux pump that contributes to intrinsic levels of fluoroquinolone and ethidium resistance in S. aureus. The NorA inhibitor reserpine did not dramatically affect the ability of salicylate to induce increased ciprofloxacin and ethidium resistance. Inactivation of norA did not alter the ability of salicylate to induce increased ciprofloxacin and ethidium resistance levels and a reduction in ciprofloxacin accumulation. These data demonstrate that NorA is not absolutely required for the salicylate-inducible multidrug resistance mechanism of S. aureus.
BACKGROUND: Staphylococcus aureus has become the leading cause of endocarditis in most published series, and nosocomial acquisition is becoming more frequent. Previous studies involved community acquired methicillin-sensitive S aureus (MSSA), but recently, methicillin-resistant S aureus(MRSA) infection has increased. This may reflect the growing presence of this microorganism in clinical practice. Few data exist comparing the relative rates of endocarditis with MSSA and MRSA bacteremia. The purpose of this study was to compare these rates in a bacteremic population referred for diagnostic echocardiography. METHODS: Since July 1999, the demographic and clinical information of 104 consecutive patients with at least 2 blood cultures with positive results for S aureus who were referred for echocardiography to be evaluated for endocarditis at Grady Memorial Hospital (Atlanta, Ga) have been entered into a database. This database has further been restricted to patients who have undergone either a transesophageal echocardiogram or a transthoracic echocardiogram. RESULTS: Of the 104 patients with S aureus bacteremia, 53 had an infection of MSSA and 51 had an infection of MRSA. There were 33 patients (31.7%) with echocardiographically confirmed endocarditis, 23 patients (43.4%) in the MSSA group versus 10 patients (19.6%) in the MRSA group (P <.009). Community-acquired MSSA bacteremia was the cause of most of the community-acquired S aureus endocarditis (20 patients [87%] vs 3 patients [30%], P =.004), and the nosocomial-acquired MRSA bacteremia was the cause of most of the nosocomial-acquired S aureus endocarditis (3 patients [13%] vs 7 patients [70%], P =.0001). CONCLUSION: Our study confirms that S aureus bacteremia is associated with high rates of endocarditis. MSSA bacteremia is associated with higher rates of endocarditis than MRSA. Community MSSA is the cause of most of the community endocarditis, whereas nosocomial MRSA is the cause of most of the MRSA endocarditis. Patients with S aureus bacteremia should be aggressively evaluated for endocarditis.
Emergence of Staphylococcus aureus small colony variants (SCVs) has been associated with antibiotic use, particularly with trimethoprim-sulfamethoxazole (TMP-SMZ). In this study, 634 specimens of 125 patients with AIDS were prospectively screened for S. aureus with normal phenotype and for S. aureus SCVs. Charts of these patients were reviewed for previous prophylaxis with TMP-SMZ often used as long-term pneumocystosis prophylaxis. Thirty-seven patients (29.6%) harbored S. aureus in their anterior nares, three of these patients (8.1%) had S. aureus SCVs. Interestingly, TMP-SMZ does not appear to select for S. aureus SCVs in nasal swabs of these patients.
When Staphylococcus aureus is repeatedly positive in blood cultures even under effective antibiotics therapy (vancomycin, teicoplanin, or rifampin), computed tomography scan and sonography should be performed early to exclude mycotic aneurysm of the deeply seated arteries, especially in patients with abdominal aortic calcification. Before 1990, the most common causative organism of suprarenal aortic mycotic aneurysm was Salmonella; since 1990, it has been gram-positive cocci (i.e., Streptococcus and Staphylococcus) rather than gram-negative bacilli (i.e., Salmonella), possibly because of the more invasive procedures performed in clinical settings, but this hypothesis needs further investigation.