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PubMed2006

Role of murF in cell wall biosynthesis: isolation and characterization of a murF conditional mutant of Staphylococcus aureus.

The Staphylococcus aureus murF gene was placed under the control of a promoter inducible by IPTG (isopropyl-beta-d-thiogalactopyranoside). It was demonstrated that murF is an essential gene; it is cotranscribed with ddlA and growth rate, level of beta-lactam antibiotic resistance, and rates of transcription of the mecA and pbpB genes paralleled the rates of transcription of murF. At suboptimal concentrations of the inducer, a UDP-linked muramyl tripeptide accumulated in the cytoplasm in parallel with the decline in the amounts of the normal pentapeptide cell wall precursor. The abnormal tripeptide component incorporated into the cell wall as a monomeric muropeptide, accompanied by a decrease in the oligomerization degree of the peptidoglycan. However, incorporation of the tripeptide into the cell wall was limited to a relatively low threshold value. Further reduction of the amounts of pentapeptide cell wall precursor caused a gradual decrease in the cellular amounts of peptidoglycan, the production of a thinner peripheral cell wall, aberrant septae, and an overall increase in the diameter of the cells. The observations suggest that the role of murF exceeds its primary function in peptidoglycan biosynthesis and may also be involved in the control of cell division.

Cell Wall↗
PubMed1991

Correlation of carotenoid production, decreased membrane fluidity, and resistance to oleic acid killing in Staphylococcus aureus 18Z.

Staphylococcus aureus is susceptible to killing by host-derived fatty acids. Studies were performed to test for a correlation between carotenoid production by S. aureus and protection against oleic acid. Oleic acid killing of cells grown in carotenoid expression medium was determined as the dosage of oleic acid in 2 M NaCl-2 mM EDTA that would kill 20% of the cells in 60 min at 37 degrees C (i.e., the 20% lethal dose). Compared with the wild-type strain (18Z), a carotenoid-deficient mutant strain (18Z-76) and strain 18Z grown in a medium that suppressed carotenoid production both showed increased sensitivity to oleic acid. Spontaneous revertants of strain 18Z-76 that regained the ability to produce carotenoids were as resistant to oleic acid as the wild-type strain. Oleic acid was shown by fluorescence polarization to decrease polarization values. Lower polarization values indicate a more-fluid membrane. To determine whether protection against oleic acid killing might depend on carotenoid stabilization of membranes, fluorescence polarization values were determined for strains showing different levels of carotenoid production. An indirect correlation was found between membrane fluidity and carotenoid production. We were able to conclude that there is a direct correlation between carotenoid production (i.e., cell pigmentation), cell membrane stability, and resistance to oleic acid-induced cell killing.

Carotenoids↗
PubMed2006

Functional reconstitution of SdcS, a Na+-coupled dicarboxylate carrier protein from Staphylococcus aureus.

In Staphylococcus aureus, the transport of dicarboxylates is mediated in part by the Na+-linked carrier protein SdcS. This transporter is a member of the divalent-anion/Na+ symporter (DASS) family, a group that includes the mammalian Na+/dicarboxylate cotransporters NaDC1 and NaDC3. In earlier work, we cloned and expressed SdcS in Escherichia coli and found it to have transport properties similar to those of its eukaryotic counterparts (J. A. Hall and A. M. Pajor, J. Bacteriol. 187:5189-5194, 2005). Here, we report the partial purification and subsequent reconstitution of functional SdcS into liposomes. These proteoliposomes exhibited succinate counterflow activity, as well as Na+ electrochemical-gradient-driven transport. Examination of substrate specificity indicated that the minimal requirement necessary for transport was a four-carbon terminal dicarboxylate backbone and that productive substrate-transporter interaction was sensitive to substitutions at the substrate C-2 and C-3 positions. Further analysis established that SdcS facilitates an electroneutral symport reaction having a 2:1 cation/dicarboxylate ratio. This study represents the first characterization of a reconstituted Na+-coupled DASS family member, thus providing an effective method to evaluate functional, as well as structural, aspects of DASS transporters in a system free of the complexities and constraints associated with native membrane environments.

Bacterial Proteins↗
PubMed2001

Production, purification and some properties of Bac201, a bacteriocin-like inhibitory substance produced by Staphylococcus aureus AB201.

Staphylococcus aureus AB201, a clinical isolate from wound pus, produced a bacteriocin-like inhibitory substance termed as Bac201, that was inhibitory to Streptococcus agalactiae, Enterococcus faecalis, Acinetobacter calcoaceticus, Neisseria meningitidis and a number of staphylococcal species. It was purified to homogeneity by ammonium sulfate precipitation, gel filtration (BioSil-SEC-125), and reversed-phase high performance liquid chromatography (Vydac C4). The native Bac201 was sized at approximately 170-kDa as determined by GF HPLC. Fraction-I (native Bac201), having antibacterial activity was also examined by transmission electron microscopy and appeared as globular structure showing resemblance with phage-like objects. The purification of Bac201 resulted in 466-fold increase in specific activity and recovery of 0.94% of total antibacterial activity. The purified Bac201 migrated as single band on SDS-PAGE with an estimated molecular mass of about 41-kDa. Bac201 was sensitive to proteolytic enzymes, resistant to heat and organic solvents, and active over a wide range of pH (2.5-10). The amino acid composition revealed a general resemblance with other reported high molecular mass bacteriocins and predominance of glycine (39%), proline (13%) and alanine (8%) residues. Further results showed that Bac201 has a bactericidal effect on sensitive cells which is not produced by either cell lysis or apparent loss of membrane permeability.

Amino Acids↗
PubMed2006

Ciprofloxacin and trimethoprim cause phage induction and virulence modulation in Staphylococcus aureus.

In Staphylococcus aureus strains of human origin, phages which integrate into the chromosomal gene coding for beta-hemolysin (hlb) are widely distributed. Most of them encode accessory virulence determinants such as staphylokinase (sak) or enterotoxins. Here, we analyzed the effects of ciprofloxacin and trimethoprim on phage induction and expression of phage-encoded virulence factors by using isolates from patients with cystic fibrosis for which the induction of hlb-converting phages was demonstrated in vivo (C. Goerke, S. Matias y Papenberg, S. Dasbach, K. Dietz, R. Ziebach, B. C. Kahl, and C. Wolz, J. Infect. Dis. 189:724-734, 2004) as well as a phi13 lysogen of phage-cured strain 8325-4. Treatment of lysogens with subinhibitory concentrations of either antibiotic resulted in (i) delysogenization of strains resembling the isolates picked up after chronic lung infection and (ii) replication of phages in the bacterial host in a dose-dependent manner. Ciprofloxacin treatment resulted in enhanced recA transcription, indicating involvement of the SOS response in phage mobilization. Induction of phi13 was linked to elevated expression of the phage-encoded virulence gene sak, chiefly due to the activation of latent phage promoters. In summary, we could show the induction of hlb-converting phages and a subsequent virulence modulation of the host bacterium by ciprofloxacin and trimethoprim.

Bacterial Toxins↗
PubMed1999

Isolation and partial characterization of Bac201: a plasmid-associated bacteriocin-like inhibitory substance from Staphylococcus aureus AB201.

Staphylococcus aureus AB201, a clinical isolate from wound pus, produced a bacteriocin-like inhibitory substance termed as Bac201 that exhibited a broad-spectrum activity against both gram-positive as well as gram-negative bacteria. Among gram-negative bacteria it was active against Neisseria meningitidis and Acinetobacter calcoaceticus both being gram-negative cocci. Inhibition due to the effect of organic acids, hydrogen peroxide, or bacteriophages was excluded. This inhibitory substance could not be induced or eluted. It was partially purified to 80% saturation by ammonium sulfate precipitation resulting in maximum specific activity of 829 AU/mg (25-fold increase). Proteolytic enzymes rapidly inactivated the antagonistic activity of the partially purified material, whereas glycolytic and lipolytic enzymes had no effect. It remained stable in the presence of mild organic solvents. It could be stored at -20 degrees C without loss of activity, stable at 60 degrees C and 80 degrees C for 30 min, 100 degrees C for 20 min and autoclaving temperature (121 degrees C for 15 min), and exhibited activity within a wide range of pH (2.5-10). Bac201 had an estimated M(r) of 41kDa, as indicated by activity detection after SDS-PAGE. Temperature-mediated (44 degrees C) plasmid curing studies suggested linkage of bacteriocin production to a 4.8 MDa plasmid. The Bac201 was bactericidal rather than bacteriolytic.

Bacteriocins↗
PubMed2002

Outcome and attributable mortality in critically Ill patients with bacteremia involving methicillin-susceptible and methicillin-resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus bacteremia carries high mortality rates. The clinical impact of methicillin resistance remains controversial: outcome comparisons between patients with bacteremia involving methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) S aureus are difficult to perform because of important differences in severity of illness. METHODS: A retrospective cohort analysis and 2 independent case-control analyses were performed to determine and compare outcomes and attributable mortality rates of MSSA (n = 38) and MRSA bacteremia (n = 47) in critically ill patients. For the case-control studies, matching (1:2 ratio) was based on the APACHE (Acute Physiology and Chronic Health Evaluation) II classification: APACHE II score (+/-1 point) and diagnostic category. RESULTS: Patients with MRSA bacteremia had more acute renal failure and hemodynamic instability than patients with MSSA bacteremia. They had a longer intensive care unit stay and ventilator dependency. Patients with MRSA bacteremia had a higher 30-day mortality rate (53.2% vs 18.4%) and in-hospital mortality rate (63.8% vs 23.7%) (P<.05). Multivariate survival analysis demonstrated acute renal failure, length of mechanical ventilation, age, and methicillin resistance to be independently associated with mortality (P<.05). The attributable mortality rate for MSSA bacteremia was 1.3%: mortality rates for cases and controls were respectively 23.7% and 22.4% (P =.94). The attributable mortality rate for MRSA bacteremia was 23.4%: mortality rates for cases and controls were respectively 63.8% and 40.4% (P =.02). The difference (22.1%) between both attributable mortality rates was significant (95% confidence interval, 8.8%-35.3%). CONCLUSION: In critically ill patients, after accurate adjustment for disease severity and acute illness, we found MRSA bacteremia to have a higher attributable mortality than MSSA bacteremia.

Acute Kidney Injury↗
PubMed1991

Tyrosine phosphorylation is an essential event in the stimulation of B lymphocytes by Staphylococcus aureus Cowan I.

Staphylococcus aureus Cowan I (SAC) is a potent mitogen for purified human B cells. By using Western blotting with antiphosphotyrosine antibodies, we demonstrated that the mitogenic effect of SAC is associated with rapid tyrosine phosphorylation of proteins of 45, 68, 75, 97, and 145 kDa. This tyrosine phosphorylation was detected within 30 s of the addition of SAC; it reached a maximum within 10 min, after which it declined gradually. In contrast to SAC, most soluble anti-IgM antibodies do not induce proliferation of isolated human B cells. As indicated by Western blotting, soluble anti-IgM antibodies induced a similar pattern of tyrosine phosphorylation, with the exception of the 68-kDa protein, which was the most heavily phosphorylated protein in SAC-treated cells. A similar but less intense 68-kDa band was also induced by mitogenic anti-IgM bound to beads. This suggested that tyrosine phosphorylation, especially of p68, may play an important role in B cell mitogenesis. To test this hypothesis, we determined the effect of specific tyrosine kinase inhibitors (tyrphostins) on SAC-induced tyrosine phosphorylation, oncogene expression, and B cell proliferation. The concentration dependencies of inhibition of these processes suggested that they were linked. Nonspecific toxic effects of the tyrphostins were ruled out by the demonstration that the tyrphostins did not alter cell viability and did not inhibit B cell proliferation induced by phorbol esters, which do not induce tyrosine phosphorylation. For maximal inhibition of SAC-induced cell proliferation, the tyrophostins needed to be added before or shortly after addition of SAC. Taken together, these data indicate that tyrosine phosphorylation is an obligatory early signal in B cell proliferation.

Antibodies, Anti-Idiotypic↗
PubMed2001

Staphylococcus aureus bacteriuria and surgical site infections by methicillin-resistant Staphylococcus aureus.

Surgical site infection (SSI) remains an important cause of morbidity among hospitalized patients. We reviewed 421 patients who underwent open urological operations between January 1993 and December 1997 in our institute. Group I consisted of 259 patients who received uncontrolled antimicrobial prophylaxis (AMP) between 1993 and 1995. Group II consisted of 162 patients who received controlled AMP between 1996 and 1997. In group II, penicillins or first to second-generation cephalosporins was used and the duration of use for these agents regulated according to the wound class of each operation. The operations with clean wounds showed the lowest rate of SSI in both groups; the operations with contaminated wounds showed the highest rate of SSI (32.0% in group I and 33.3% in group II). There was no significant difference in the total rates of SSI between the two groups (P=0.216). The most frequently isolated bacterial species was methicillin-resistant Staphylococcus aureus (MRSA), isolated in 73.3% of the cases in group I and in 93.3% in group II. There was no significant difference in the incidence of MRSA isolation between the two groups (P=0.114). The controlled AMP could not lower the incidence of MRSA-induced SSIs. In SSI patients, 22.7% of group I and 35.7% in group II, had MRSA bacteriuria before operation. The prohibition of third-generation cephalosporins and shorter duration of AMP did not reduce the incidence of SSI induced by MRSA because MRSA was not the emerging microorganism but rather a resident in the urological ward. On the other hand, the total incidence of SSI did not increase after regulation of AMP. This finding suggests that older antibacterial agents can prevent infection, except those caused by resistant microorganisms such as MRSA. The effective counter-measure for the prevention of MRSA-induced SSI is needed.

Anti-Infective Agents, Urinary↗
PubMed2000

Staphylococcus aureus bacteremia in the surgical patient: a prospective analysis of 73 postoperative patients who developed Staphylococcus aureus bacteremia at a tertiary care facility.

BACKGROUND: Staphylococcus aureus is a frequent cause of infection and bacteremia in the postoperative patient. Unfortunately, there have been no prospective studies evaluating these patients, so the incidence of complications, subsequent treatment algorithms, and prognosis remain undefined. The objectives of this prospective study of postoperative Staphylococcus aureus bacteremia (SAB) were to define the primary sources of bacteremia and to identify the common complications of SAB in the postoperative setting. METHODS: A registry was developed into which 309 consecutive adult patients with SAB were prospectively enrolled between September 1994 and December 1996. Seventy-three of these patients (23.6%) developed SAB in the postoperative setting. RESULTS: Analysis of the clinical features of these 73 postoperative patients revealed three important results. First, infective endocarditis is surprisingly common in postoperative patients with SAB and the classical stigmata of endocarditis are often absent. Transesophageal echocardiography was performed in 31 of 73 patients; 10 of these patients (32.3%) met Duke Criteria for definite endocarditis, but only 3 of these patients had vegetations detected by transthoracic echocardiography, and only 2 patients had peripheral stigmata of infective endocarditis. Second, the development of SAB after cardiothoracic surgery was strongly associated with underlying S. aureus mediastinitis. Twenty-one of the 23 patients who developed SAB after median sternotomy had mediastinitis (positive predictive value 91.3%). In many cases, the diagnosis of mediastinitis was not apparent when SAB was detected. Third, complications, relapses, and mortality were high in postoperative patients with SAB. Fourteen of 73 patients (19.2%) developed multiple noncardiac metastatic complications, including metastatic abscesses (5), septic emboli (3), pneumonia or empyema (2), septic arthritis (1), epidural abscess (1), and other metastatic foci (7). Twelve of 73 patients (16.4%) had recurrent staphylococcal infection after treatment of their first episode of SAB, including 8 patients (11.0%) with recurrent bacteremia. Of patients who survived, those with recurrent staphylococcal infection were more likely to have an infected surgical wound than were patients who were cured of infection (p = 0.05). Finally, mortality attributable to SAB (11.0%), and all-cause mortality (21.9%), was high. CONCLUSIONS: SAB in the postoperative setting is often a severe disease with high morbidity and mortality. A thorough diagnostic evaluation is indicated in surgical patients with S. aureus bacteremia to ensure the early detection of metastatic infections such as infective endocarditis and to define foci such as mediastinitis re quiring surgical intervention.

Adult↗
PubMed2001

Whole genome sequencing of meticillin-resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is one of the major causes of community-acquired and hospital-acquired infections. It produces numerous toxins including superantigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Whole genome analysis is a necessary step towards future development of countermeasures against this organism. METHODS: Whole genome sequences of two related S aureus strains (N315 and Mu50) were determined by shot-gun random sequencing. N315 is a meticillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. The open reading frames were identified by use of GAMBLER and GLIMMER programs, and annotation of each was done with a BLAST homology search, motif analysis, and protein localisation prediction. FINDINGS: The Staphylococcus genome was composed of a complex mixture of genes, many of which seem to have been acquired by lateral gene transfer. Most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity islands were identified in the genome: a toxic-shock-syndrome toxin island family, exotoxin islands, and enterotoxin islands. In the latter two pathogenicity islands, clusters of exotoxin and enterotoxin genes were found closely linked with other gene clusters encoding putative pathogenic factors. The analysis also identified 70 candidates for new virulence factors. INTERPRETATION: The remarkable ability of S aureus to acquire useful genes from various organisms was revealed through the observation of genome complexity and evidence of lateral gene transfer. Repeated duplication of genes encoding superantigens explains why S aureus is capable of infecting humans of diverse genetic backgrounds, eliciting severe immune reactions. Investigation of many newly identified gene products, including the 70 putative virulence factors, will greatly improve our understanding of the biology of staphylococci and the processes of infectious diseases caused by S aureus.

Animals↗
PubMedDate not supplied

[Presence of a monoclonal component IgG lambda in a 17-day-old newborn with omphalitis caused by Staphylococcus aureus].

Monoclonal components are usually found in elderly patients. They are rarely noted in children, both because of their slight presence, and because the very low concentration makes it difficult to single them out. This report is about the identification of a transitory IgG lambda monoclonal component in a 17 day old baby suffering from aureus Staphylococcus. Electrophoresis was carried out with automatic equipment on cellulose acetate; immunofixation with a manual method on agarose gel; the quantitative doses of the immunoglobulins, reactive protein C and alpha 1-acid glycoprotein with nephelometric technique, the erythrocyte sedimentation rate with automatic equipment. The importance of singling out monoclonal components in children must be emphasized, inasmuch as it may be a sign of an immunological disorder, usually benign and transitory, but sometimes extremely serious.

Electrophoresis↗
PubMed1992

Mobilization of recombinant plasmids from Staphylococcus aureus into coagulase negative Staphylococcus species.

pC221, a small nonconjugative staphylococcal plasmid, can be mobilized between staphylococci by pG01, a larger conjugative plasmid. pC221 carries the two transacting genes, mobA and mobB, which are needed for its mobilization. The products of these genes create a site-specific single-stranded nick (mobA) and then facilitate DNA transfer (mobB). Several useful Escherichia coli-staphylococcal shuttle plasmids containing the cloned single-stranded nick site were created and successfully mobilized into Staphylococcus aureus and two coagulase-negative staphylococci, S. epidermidis and S. saprophyticus, by providing mob genes (pC221) and conjugative transfer genes (pG01) in trans in the donor. These vectors may offer a genetic system for the introduction of recombinant plasmids into coagulase negative staphylococci.

Bacterial Proteins↗
PubMed2005

Evaluation of two new chromogenic media, CHROMagar MRSA and S. aureus ID, for identifying Staphylococcus aureus and screening methicillin-resistant S. aureus.

Thirty-nine methicillin-resistant Staphylococcus aureus (MRSA) isolates with diverse genetic backgrounds and two reference strains were correctly identified as S. aureus on CHROMagar MRSA and S. aureus ID media. Growth inhibition on CHROMagar MRSA was noted. A combination of cefoxitin disk and S. aureus ID was found suitable for rapid MRSA screening.

Cefoxitin↗
PubMed1999

Characteristics in adherence of streptococci and Staphylococcus aureus isolated from various infective skin lesions: serum IgA decreases adherence of Streptococcus pyogenes but not Staphylococcus aureus.

We characterized adherence of streptococci and Staphylococcus aureus strains isolated from various infective skin lesions in terms of hydrophobicity, negative charge, tube adherence, slime production, and influence on adherence to coverslips by plasma and serum immunoglobulins. High hydrophobicity was more frequently observed in Streptococcus pyogenes strains than in Streptococcus agalactiae strains (P < 0.01) and S. aureus strains (P < 0.001) and slime production was more frequently observed in S. agalactiae strains than in S. pyogenes strains (P < 0.05). Serum IgA decreased adherence to coverslips of S. pyogenes strains but not that of S. aureus strains.

Bacterial Adhesion↗
PubMed2003

Comparative in vitro activity of linezolid and five other antimicrobials against nosocomial isolates of methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis and vancomycin-resistant Enterococcus faecium.

The activity of linezolid in comparison to vancomycin, teicoplanin, oxacillin, clindamycin and gentamicin was tested against 60 strains of methicillin-resistant Staphylococcus aureus, 60 strains of methicillin-resistant Staphylococcus epidermidis isolated from patients with nosocomial infections and 24 strains of vancomycin-resistant Enterococcus faecium isolated from feces of hospitalized patients. Minimum Inhibitory Concentrations (MICs) were determined by the Epsilometer test method. All tested strains were sensitive to linezolid and specifically all methicillin-resistant S. aureus had MIC range 0.25-3.00, MIC50 = 0.75, MIC90 = 1.5, all methicillin-resistant S. epidermidis had MIC range 0.125-1.5, MIC50 = 0.5, MIC90 = 1 and all vancomycin-resistant E. faecium had MIC range 0.5-1.5, MIC50 = 1, MIC90 = 1. Linezolid is the first of a novel antimicrobial class, the oxazolidinones, which is a promising treatment for serious Gram-positive infections, including multiresistant strains.

Acetamides↗