Opposing roles of the Staphylococcus aureus virulence regulators, Agr and Sar, in Triton X-100- and penicillin-induced autolysis

The regulation of murein hydrolases is a critical aspect of peptidoglycan growth and metabolism. In the present study, we demonstrate that mutations within the Staphylococcus aureus virulence factor regulatory genes, agr and sar, affect autolysis, resulting in decreased and increased autolysis rates, respectively. Zymographic analyses of these mutant strains suggest that agr and sar exert their effects on autolysis, in part, by modulating murein hydrolase expression and/or activity.     

The femR315 gene from Staphylococcus aureus, the interruption of which results in reduced methicillin resistance, encodes a phosphoglucosamine mutase

The femR315 gene was recently identified by Tn551 insertional mutagenesis as one of the new auxiliary genes, the alteration of which resulted in a drastically reduced methicillin resistance of the Staphylococcus aureus strain COL. femR315 (also known as femD) theoretically encoded a protein of 451 amino acids showing significant amino acid sequence homology with phosphoglucomutases and similar enzymes catalyzing the isomerization of hexoses and hexosamine phosphates (S. Wu, H. de Lencastre, A. Sali, and A. Tomasz, Microb. Drug Resist. 2:277-286, 1996). We describe here the overproduction and purification of the FemR315 protein as well as its identification as the phosphoglucosamine mutase which catalyzes the formation of glucosamine-1-phosphate from glucosamine-6-phosphate, the first step in the reaction sequence leading to the essential peptidoglycan precursor UDP-N-acetylglucosamine. On the basis of these findings, we propose to change the names femR315 and femD to the functionally more appropriate name glmM.     

Molecular characterization of the gene operon of heat shock proteins HSP60 and HSP10 in methicillin-resistant Staphylococcus aureus

Methicillin-resistance of S.aureus (MRSA) was diminished or depressed at 44 degrees C. In order to investigate whether bacterial heat shock response is correlated with methicillin resistance, we examined the inducibility of the heat shock proteins (HSPs) in MRSA, and cloned and sequenced of their genes. A temperature shift from 37 degrees C to 46 degrees C enhanced the production of at least 8 kinds of cytoplasmic proteins as measured with two-dimensional PAGE. The induced protein profile was almost the same as methicillin sensitive S.aureus, and stress conditions due to ethanol, cadmium or low pH. Two of these proteins were HSP60 and HSP10. Their N-terminal amino acid sequences were 79%, and 83%, homologous with thermobacterium PS3, respectively. A positively hybridized 4.2 kbp DNA fragment encoding both proteins was isolated from the chromosomal DNA of MRSA. The resulting sequence revealed two reading frames and showed high homology to those of hsp60 (groEL) and hsp10 (groES) of bacteria (E.coli) and several other species. The genes of HSP60 and HSP10 in S.aureus comprised an operon as in E.coli. The relationship between those HSPs and PBP2' is currently under investigation.     

Examination of methicillin-resistant and methicillin-susceptible Staphylococcus aureus mutants with low-level fluoroquinolone resistance

For Staphylococcus aureus, stepwise mutations result in high-level quinolone resistance. Methicillin-resistant and -susceptible quinolone-resistant, first-step mutants generated in vitro were obtained and found to be no different than those recovered from murine abscesses. Approximately 10% of all first-step mutants were resistant to ethidium bromide, and selected strains had mutations that mapped to flqB. NorA-mediated resistance among first-step mutants may be more prevalent than previously reported.     

Reassessment of the number of auxiliary genes essential for expression of high-level methicillin resistance in Staphylococcus aureus

A new transposon library constructed in the background of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL yielded 70 independent insertional mutants with reduced levels of antibiotic resistance. Restriction analysis with HindIII, EcoRV, EcoRI, and PstI and then Southern hybridization with probes for the transposon and for the femA-femB gene demonstrated that 41 of the 70 Tn551 mutants carried distinct and novel, as yet undescribed insertion sites, all of which were outside of the mecA gene and were also outside the already-characterized auxiliary genes femA, femB, femC, and femD. All previously described Tn551 mutations of this type were in genes located either on SmaI fragment A or SmaI fragment I. In contrast, inserts of the new library were located in 7 of the 16 SmaI chromosomal fragments, fragments A, B, C, D, E, F, and I. In all of the mutants, expression of methicillin resistance became heterogeneous, and the MIC for the majority of cells was reduced (1.5 to 200 micrograms ml-1) from the homogeneous methicillin MIC (1,600 micrograms ml-1) of the parental cells. Although identification of the exact number of genes inactivated through the new set of transposon inserts will require cloning and sequencing, a rough estimate of this number from mapping data suggests a minimum of at least 10 to 12 new genetic determinants, all of which are needed together with femA, femB, femC, and femD for the optimal expression of methicillin resistance.     

Molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) in a university hospital in Italy

The molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in a university hospital in Italy was studied in a five-month period in 1996, during which all S. aureus isolated were collected. All MRSA isolates (95) and a sample of methicillin-susceptible S. aureus (20) were typed with a variety of phenotypic and genotypic methods. Clonal identities were determined by pulsed-field gel electrophoresis (PFGE) of chromosomal SmaI digests and, for MRSA isolates, by probing ClaI digests with a mecA probe and a Tn554 probe. Overall, MRSA represented 32.3% of all isolates, with very high percentages from the intensive care units (adult and neonatal). PFGE after restriction with SmaI resolved genomic DNA of 95 MRSA strains into 26 major PFGE patterns. The use of southern blot hybridization of ClaI genomic digests with mecA and Tn554 allowed us a significant increase in discrimination, differentiating at least 32 different clones. Two major clones, however, each sharing common ClaI-mecA and Tn554 type and PFGE pattern as well as a common resistance phenotype, represented more than 50% of all MRSA isolates. The recovery of these two clones in the majority of the isolates of adult and neonatal intensive care units, respectively, is indicative of typical nosocomial outbreaks and clonal spread. It is concluded that intensive care units are major areas requiring preventative interventions.     

Typing, resistance behavior and occurrence of methicillin-resistant Staphylococcus aureus strains in a surgical intensive care unit

Over a period of three years, the frequency of the appearance of methicillin-resistant S. aureus strains (MRSA) was observed on a surgical intensive care unit. During this above-mentioned period of investigation it came to a heaped occurrence of nosocomial infections on this ICU with altogether 332 S. aureus-stems being isolated from different patient specimen. 204 (61.5%) of these were resistant against methicillin and could be divided into 48 first- and 156 follow-up-isolates. The thereupon accomplished differentiation of the 48 MRSA-first isolates by means of lysotyping and the pioneered GenePath Strain Typing System for a standardized pulsed-field-gel-electrophoresis (PFGE) gave the proof of 7 different MRSA-types. Around 7 different, in part parallel chains of infection on this ICU were observed, which could be led back to different strains. In reference to all analyzed S. aureus, an especially high rate (90%) of MRSA on this ICU could be isolated in taken wound-swabs, followed by 83.3% MRSA at catheter tips and 71,9% in tracheal and bronchial secretion. A consideration of the antibiotic susceptibility yielded, that also gentamicin and the quinolones showed an in-vitro resistance against MRSA, while fosfomycin, fusidic acid, chloramphenicol and trimethoprim/sulfamethoxazole reached positive responding rates between 80 and 100%. On the other hand, presently still 100% of the explored MRSA-strains are susceptible for glycopeptides such as vancomycin and teicoplanin. Because of intensive hospital hygienic measures the number of newly isolated MRSA could be reduced clearly on this ward.     

Spread and control of methicillin resistant Staphylococcus aureus in a department of dermatology

Patients with skin diseases caused a spread of methicillin-resistant Staphylococcus aureus (MRSA) to 17 patients in our Department of Dermatology, because of their heavily scaly skin. Patients with severe dermatosis are regularly treated with dicloxacillin. The resistance of bacteria strain concerned suggests a selection because of the use of dicloxacillin in the Department. The strain is sensitive to gentamicin, which differentiates it from strains imported from abroad. Increased hygienic precautions, isolation of infected patients, staff and management efforts and a close contact with the microbiologists prevented MRSA from spreading to other hospital wards.     

Selection and characterization of strains of Staphylococcus aureus displaying unusual resistance to aminoglycosides

Susceptibility tests with aminoglycosides against Staphylococcus aureus have revealed discrepancies between the minimal inhibitory concentrations and the minimal bactericidal concentrations. To further evaluate these discrepancies, kill curves were performed against a susceptible strain of S. aureus with five different aminoglycosides (amikacin, kanamycin, tobramycin, gentamicin, sisomicin) at concentrations up to 16-fold above the minimal inhibitory concentration. Results revealed the presence of small subpopulations of cells capable of growth within 24 h in concentrations of aminoglycoside up to eightfold above the minimal inhibitory concentration for the parent strain. These subpopulations occurred at a frequency of >/=10(-7) parent cells, were not physiologically different from the susceptible parent strains, and were present in approximately one-half of 30 strains of S. aureus tested. The resistance of these subpopulations was approximately eightfold higher than that of the parent for all five aminoglycosides and was independent of concentration or type of aminoglycoside used to select them. This resistance was not due to extracellular degradation of drug and was stable over eight transfers in drug-free medium, except when selected by gentamicin or sisomicin.     

Characterization of mutations in the rpoB gene that confer rifampin resistance in Staphylococcus aureus

Mutations in the rifampin resistance-determining (Rif) regions of the rpoB gene of Staphylococcus aureus mutants obtained during therapy or in vitro were analyzed by gene amplification and sequencing. Each of the resistant clinical isolates, including five nonrelated clones and two strains isolated from the same patient, and of the 10 in vitro mutants had a single base pair change that resulted in an amino acid substitution in the beta subunit of RNA polymerase. Eight mutational changes at seven positions were found in cluster I of the central Rif region. Certain substitutions (His481/Tyr and Asp471/Tyr [S. aureus coordinates]) were present in several mutants. Substitutions Gln468/Arg, His481/Tyr, and Arg484/His, which conferred high-level rifampin resistance, were identical or in the same codon as those described in other bacterial genera, whereas Asp550/Gly has not been reported previously. Substitutions at codon 477 conferred high- or low-level resistance, depending on the nature of the new amino acid. The levels of resistance of in vivo and one-step in vitro mutants carrying identical mutations were similar, suggesting that no other resistance mechanism was present in the clinical isolates. On the basis of these data and the population distribution of more than 4,000 clinical S. aureus isolates, we propose /=8 microg/ml as new breakpoints for the clinical categorization of this species relative to rifampin.     

Introduction of the mec element (methicillin resistance) into Staphylococcus aureus alters in vitro functional activities of fibrinogen and fibronectin adhesins

Some methicillin-resistant strains of Staphylococcus aureus are defective in the production of major surface components such as protein A, clumping factor, or other important adhesins to extracellular matrix components which may play a role in bacterial colonization and infection. To evaluate the impact of methicillin resistance (mec) determinants on bacterial adhesion mediated by fibrinogen or fibronectin adhesins, we compared the in vitro attachment of two genetically distinct susceptible strains (NCTC8325 and Newman) to protein-coated surfaces with that of isogenic methicillin-resistant derivatives. All strains containing an intact mec element in their chromosomes were found to be defective in adhesion to fibrinogen and fibronectin immobilized on polymethylmethacrylate coverslips, regardless of the presence or absence of additional mutations in the femA, femB, or femC gene, known to decrease expression of methicillin resistance in S. aureus. Western ligand affinity blotting or immunoblotting of cell wall-associated adhesins revealed similar contents of fibrinogen- or fibronectin-binding proteins in methicillin-resistant strains compared to those of their methicillin-susceptible counterparts. In contrast to methicillin-resistant strains carrying a mec element in their genomes, methicillin-resistant strains constructed in vitro, by introducing the mecA gene on a plasmid, retained their adhesion phenotypes. In conclusion, the chromosomal insertion of the mec element into genetically defined strains of S. aureus impairs the in vitro functional activities of fibrinogen or fibronectin adhesins without altering their production. This effect is unrelated to the activity of the mecA gene.     

Trimethoprim-sulfamethoxazole for the prevention of methicillin-resistant Staphylococcus aureus pneumonia in severely burned patients

This study sought to examine the association between cigarette smoking and serum bilirubin antioxidant concentrations in 715 middle-aged men undergoing coronary angiography. The study involved 153 current smokers, 251 who quit smoking and 311 who never smoked. Serum bilirubin concentrations were divided into the following quartiles; 0.20-0.57, 0.58-0.73, 0.74-0.95 and 0.96-3.26 mg/dl. The percentage of individuals within each quartile were as follows; current smokers (42, 22, 24, 12), former smokers (22, 27, 23, 28), nonsmokers (16, 28, 27, 29). A total of 42% of the current smokers had bilirubin concentrations in the lowest quartile compared to 16% of the nonsmokers. Also, 12% of the current smokers had bilirubin concentrations in the highest quartile compared to 29% in the nonsmoking group. The Mantel-Haenszel chi-square test for association between ordered categorical variables was 30.6 (P < 0.0001). Subdividing the subjects according to maximum percent stenosis on angiography (< 10, 10-49, 50-100%) revealed a significant inverse association between smoking and bilirubin (< 0.01) within each subset. The data shows that smoking is associated with decreased serum bilirubin concentrations. In addition, it supports the hypothesis that cigarette smoking may increase the risk of coronary artery disease by lowering antioxidant concentrations and raising oxidized lipid and lipoprotein concentrations.     

An autobacteriographic study on distribution and localization of methicillin-resistant Staphylococcus aureus in the immunosuppressed mice

Cyclophosphamide (CY, 250 mg/kg) was intraperitoneally administered to mice. Four days after, a rifampicin-resistant strain of methicillin-resistant Staphylococcus aureus (MRSA, S. aureus 1-6 RFPr) was intravenously inoculated at the level of 10(7) cfu/mouse. Distribution and localization of the inoculated organism were chronologically investigated by means of whole body autobacteriography. CY (100 mg/kg) was consecutively administered for 4 days following the inoculation. As a result, dense colonies of the organism were detected from many organs and tissues, that is, the liver spleen, gastrointestinal tract, kidneys, urinary bladder and bone (bone marrow) on the day after the inoculation. Following 3 days after the inoculation, the distribution and localization in CY-treated mice remained substantially unchanged and some animals died. It is demonstrated that in an experimental mouse model of MRSA infectious disease under immunosuppressed condition, the inoculated organism can stand still and proliferate not only in the gastrointestinal tract but also in the urinary tract and lymphhemopoietic organs.     

A profile of methicillin resistant Staphylococcus aureus infection in the burn center of the Sultanate of Oman

A retrospective study of patterns of infection in 168 patients admitted during 1995 and 1996 in the burns-unit of Khoula hospital at Muscat, Oman was performed. Out of 819 isolates positive for pathogenic bacterial culture, there were 326 (39.8%) isolates positive for methicillin resistant Staphylococcus aureus (MRSA) infection. Incidence of MRSA infection was marginally more than that of Pseudomonas aeroginosa. The proportion of patients developing MRSA infection sometime or the other during their burns-unit stay ranging from 1 to 112 days rose from 48% in 1995 to 52.7% in 1996. No sophisticated tests were done to identify the MRSA strain but study of the antibiograms of each MRSA positive isolate showed very similar patterns of sensitivity to different antibiotics. This suggests the source of infection to be common and in all probability 'noscomial', since all patients acquired MRSA infection in the hospital. The susceptibility of MRSA to ciprofloxacin, cotrimoxazole and fucidin was 76, 51 and 37% of isolates in 1995, and 59, 44 and 26% in 1996 were susceptible to these drugs. Vancomycin was the antibiotic to which most MRSA cultures were susceptible, but partial resistance was reported due to very low susceptibility observed in 1.4% of the isolates in 1995 and 1.1% of the isolates in 1996. The control measures being practiced in the burns-unit of Khoula Hospital, especially mechanical cleaning and chemical disinfection of all surfaces, are discussed in detail. This paper emphasizes the need for preventive measures against MRSA infection in the burns-unit.     

Association of mutations in grlA and gyrA topoisomerase genes with resistance to ciprofloxacin in epidemic and sporadic isolates of methicillin-resistant Staphylococcus aureus

The types of topoisomerase alterations in genomically diverse epidemic and sporadic strains of methicillin- and fluoroquinolone-resistant Staphylococcus aureus isolated from European hospitals between 1984 and 1994 were characterized. Convergent dual mutations in gyrA (codon 83, 84, or 88) and grlA (codon 79 and/or 80) were found in all strains exhibiting high-level resistance to ciprofloxacin (MIC, 16 to > or = 128 microg/ml). In some epidemic strains, the resistant phenotype and genotype appeared in the 1990s and persisted thereafter.     

Quinolone resistance mutations in the GrlB protein of Staphylococcus aureus

Two altered GrlB proteins (one with an Asp-432-->Asn alteration and one with an Asn-470-->Asp alteration) of Staphylococcus aureus were purified as fusion proteins to maltose-binding protein. The 50% inhibitory concentrations of levofloxacin were 14 and 3.4 microg/ml against topoisomerase IV containing GrlB proteins with alterations at positions 432 and 470, respectively. These results suggest that the alteration of Asp to Asn at position 432 may be responsible for quinolone resistance.     

Clonal analysis and identification of epidemic strains of methicillin-resistant Staphylococcus aureus by antibiotyping and determination of protein A gene and coagulase gene polymorphisms

Forty-three methicillin-resistant Staphylococcus aureus (MRSA) isolates with known genetic and epidemiological relatedness and different degrees of transmission were analyzed by antibiotyping, protein A gene polymorphism analysis, and coagulase gene polymorphism analysis. The three typing systems were evaluated for their performance and convenience to define clones and to discriminate between epidemic MRSA (EMRSA) and sporadic MRSA (SMRSA). Antibiotyping and AluI restriction fragment length polymorphism analysis of the coagulase gene were able to define clones in the same way as DNA macrorestriction analysis (SmaI). However, both techniques presented disadvantages, making neither of them useful as a single typing method. Protein A gene polymorphism analysis appeared to be of no value for clonal analysis. None of the three typing methods was able to differentiate between EMRSA and SMRSA.