Increased production of aureolysin and staphopain A is a primary determinant of the reduced virulence of Staphylococcus aureus sarA mutants in osteomyelitis.

We previously demonstrated that mutation of sarA in Staphylococcus aureus limits biofilm formation, cytotoxicity for osteoblasts and osteoclasts, and virulence in osteomyelitis, and that all of these phenotypes can be attributed to the increased production of extracellular proteases. Here we extend these studies to assess the individual importance of these proteases alone and in combination with each other using the methicillin-resistant USA300 strain LAC, the methicillin-susceptible USA200 strain UAMS-1, and isogenic sarA mutants that were also unable to produce aureolysin (Aur), staphopain A (ScpA), staphylococcal serine protease A (subsp.), staphopain B (SspB), and the staphylococcal serine protease-like proteins A-F (SplA-F). Biofilm formation was restored in LAC and UAMS-1 sarA mutants by subsequent mutation of aur and scpA, while mutation of aur had the greatest impact on cytotoxicity to mammalian cells, particularly with conditioned medium (CM) from the more cytotoxic strain LAC. However, SDS-PAGE and western blot analysis of CM confirmed that mutation of sspAB was also required to mimic the phenotype of sarA mutants unable to produce any extracellular proteases. Nevertheless, in a murine model of post-traumatic osteomyelitis, mutation of aur and scpA had the greatest impact on restoring the virulence of LAC and UAMS-1 sarA mutants, with concurrent mutation of sspAB and the spl operon having relatively little effect. These results demonstrate that the increased production of Aur and ScpA in combination with each other is a primary determinant of the reduced virulence of S. aureus sarA mutants in diverse clinical isolates including both methicillin-resistant and methicillin-susceptible strains.IMPORTANCEPrevious work established that SarA plays a primary role in limiting the production of extracellular proteases to prevent them from limiting the abundance of S. aureus virulence factors. Eliminating the production of all 10 extracellular proteases in the methicillin-resistant strain LAC has also been shown to enhance virulence in a murine sepsis model, and this has been attributed to the specific proteases Aur and ScpA. The importance of this work lies in our demonstration that the increased production of these same proteases largely accounts for the decreased virulence of sarA mutants in a murine model of post-traumatic osteomyelitis not only in LAC but also in the methicillin-susceptible human osteomyelitis isolate UAMS-1. This confirms that sarA-mediated repression of Aur and ScpA production plays a critical role in the posttranslational regulation of S. aureus virulence factors in diverse clinical isolates and diverse forms of S. aureus infection.

A Review on Five and Six-Membered Heterocyclic Compounds Targeting the Penicillin-Binding Protein 2 (PBP2A) of Methicillin-Resistant Staphylococcus aureus (MRSA).

Staphylococcus aureus is a common human pathogen. Methicillin-resistant Staphylococcus aureus (MRSA) infections pose significant and challenging therapeutic difficulties. MRSA often acquires the non-native gene PBP2a, which results in reduced susceptibility to ß-lactam antibiotics, thus conferring resistance. PBP2a has a lower affinity for methicillin, allowing bacteria to maintain peptidoglycan biosynthesis, a core component of the bacterial cell wall. Consequently, even in the presence of methicillin or other antibiotics, bacteria can develop resistance. Due to genes responsible for resistance, S. aureus becomes MRSA. The fundamental premise of this resistance mechanism is well-understood. Given the therapeutic concerns posed by resistant microorganisms, there is a legitimate demand for novel antibiotics. This review primarily focuses on PBP2a scaffolds and the various screening approaches used to identify PBP2a inhibitors. The following classes of compounds and their biological activities are discussed: Penicillin, Cephalosporins, Pyrazole-Benzimidazole-based derivatives, Oxadiazole-containing derivatives, non-ß-lactam allosteric inhibitors, 4-(3H)-Quinazolinones, Pyrrolylated chalcone, Bis-2-Oxoazetidinyl macrocycles (ß-lactam antibiotics with 1,3-Bridges), Macrocycle-embedded ß-lactams as novel inhibitors, Pyridine-Coupled Pyrimidinones, novel Naphthalimide corbelled aminothiazoximes, non-covalent inhibitors, Investigational-ß-lactam antibiotics, Carbapenem, novel Benzoxazole derivatives, Pyrazolylpyridine analogues, and other miscellaneous classes of scaffolds for PBP2a. Additionally, we discuss the penicillin-binding protein, a crucial target in the MRSA cell wall. Various aspects of PBP2a, bacterial cell walls, peptidoglycans, different crystal structures of PBP2a, synthetic routes for PBP2a inhibitors, and future perspectives on MRSA inhibitors are also explored.

Proteomic Changes in Methicillin-Resistant Staphylococcus aureus Exposed to Cannabinoids.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen that causes a wide range of infections. Its resistance to ß-lactam antibiotics complicates treatment due to the limited number of antibiotics with activity against MRSA. To investigate development of alternative therapeutics, the mechanisms that mediate antibiotic resistance in MRSA need to be fully understood. In this study, MRSA cells were subjected to antibiotic stress from methicillin in combination with three cannabinoid compounds and analyzed using proteomics to assess the changes in physiology. Subjecting MRSA to nonlethal levels of methicillin resulted in an increased production of penicillin-binding protein 2 (PBP2). Exposure to cannabinoids showed antibiotic activity against MRSA, and differential proteomics revealed reduced levels of proteins involved in the energy production as well as PBP2 when used in combination with methicillin.

A Simple and Rapid Methicillin-Resistant Staphylococcus aureus (MRSA) Screening Test Using a Mannose-Binding Lectin (MBL)-Conjugated Gold Nanoparticle Probe.

Rapid diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is essential for guiding clinical treatment and preventing the spread of MRSA infections. Herein, we present a simple and rapid MRSA screening test based on the aggregation effect of mannose-binding lectin (MBL)-conjugated gold nanoparticles (AuNP), called the MRSA probe. Recombinant MBL protein is a member of the lectin family and part of the innate immune system. It can recognize wall teichoic acid (WTA) on the membrane of MRSA more specifically than that of methicillin-sensitive Staphylococcus aureus (MSSA) under optimized salt conditions. Thus, the MRSA probe can selectively bind to MRSA, and the aggregation of the probes on the surface of the target bacteria can be detected and analyzed by the naked eye within 5 min. To demonstrate the suitability of the method for real-world application, we tested 40 clinical S. aureus isolates (including 20 MRSA specimens) and recorded a sensitivity of 100%. In conclusion, the MRSA probe-based screening test with its excellent sensitivity has the potential for successful application in the microbiology laboratory.

Nontargeted metabolomics reveals differences in the metabolite profiling among methicillin-resistant and methicillin-susceptible Staphylococcus aureus in response to antibiotics.

Staphylococcus aureus (S. aureus) causes infections and can be fatal. In the long-term struggle against antibiotics, S. aureus has acquired resistance toward antibiotics and become more difficult to kill. Metabolomics could directly reflect the responses of S. aureus toward antibiotics, which is effective for studying the resistance mechanism of S. aureus. In this study, based on a nontargeted metabolic figure printing technique, the metabolome of a pair of isogenic methicillin-susceptible and resistant S. aureus strains ATCC25923 (MSSA) and ATCC43300 (MRSA) treated with or without oxacillin was characterized. 7 and 29 significantly changed metabolites in MRSA and MSSA were identified by combined accurate mass and mass fragmentation analysis. Pathway enrichment analysis suggested that DNA repair and flavin biosynthesis are the universal pathways of both MSSA and MRSA under antibiotic stress. MRSA systematically and effectively fights against oxacillin through precise control of energy production, PBP2a substrate biosynthesis and antioxidant function. In contrast, MSSA lacks effective defense pathways against oxacillin. The different metabolome responses of MSSA and MRSA toward antibiotics provide us with new insights into how S. aureus develops antibiotic resistance.

Inhibition of Staphylococcus aureus Biofilm Formation and Virulence Factor Production by Petroselinic Acid and Other Unsaturated C18 Fatty Acids.

Staphylococcus aureus is a major human pathogen that secretes several toxins associated with the pathogenesis of sepsis and pneumonia. Its antibiotic resistance is notorious, and its biofilms play a critical role in antibiotic tolerance. We hypothesized fatty acids might inhibit S. aureus biofilm formation and the expressions of its virulence factors. Initially, the antibiofilm activities of 27 fatty acids against a methicillin-sensitive S. aureus strain were investigated. Of the fatty acids tested, three C18 unsaturated fatty acids, that is, petroselinic, vaccenic, and oleic acids at 100 µg/mL, inhibited S. aureus biofilm formation by more than 65% without affecting its planktonic cell growth (MICs were all > 400 µg/mL). Notably, petroselinic acid significantly inhibited biofilm formation of two methicillin-resistant S. aureus strains and two methicillin-sensitive S. aureus strains. In addition, petroselinic acid significantly suppressed the production of three virulence factors, namely, staphyloxanthin, lipase, and α-hemolysin. Transcriptional analysis showed that petroselinic acid repressed the gene expressions of quorum sensing regulator agrA, effector of quorum sensing RNAIII, α-hemolysin hla, nucleases nuc1 and nuc2, and the virulence regulator saeR. Furthermore, petroselinic acid dose-dependently inhibited S. aureus biofilm formation on abiotic surfaces and porcine skin. These findings suggest that fatty acids, particularly petroselinic acid, are potentially useful for controlling biofilm formation by S. aureus. IMPORTANCE Fatty acids with a long carbon chain have recently attracted attention because of their antibiofilm activities against microbes. Here, we report the antibiofilm activities of 27 fatty acids against S. aureus. Of the fatty acids tested, three C18 unsaturated fatty acids (petroselinic, vaccenic, and oleic acids) significantly inhibited biofilm formation by S. aureus. Furthermore, petroselinic acid inhibited the production of several virulence factors in S. aureus. The study also reveals that the action mechanism of petroselinic acid involves repression of quorum-sensing-related and virulence regulator genes. These findings show that natural and nontoxic petroselinic acid has potential use as a treatment for S. aureus infections, including infections by methicillin-resistant S. aureus strains, and in food processing facilities.

[The evaluation of effectiveness of application of automated system of polymerase chain reaction to detect meticillin-resistant and meticillin-sensitive Staphylococcus aureus in clinical material from trauma orthopedic patients].

The article considers the evaluation of effectiveness of application of automated polymerase chain reaction system GeneXpert DX ("Cepheid USA) as compared with bacteriologic method in detection of S. aureus (SA) and meticillin-resistant S. aureus (MRSA) under infection of skin and soft tissues in orthopedic patients. The results of analysis of material from patients got in 2009-2011 using analyzer Vitek-2 to identify microorganisms demonstrated that separation and identification of agent was successful in 70.04% of 2153 examined samples. The representatives of genus of Staphylococcus made up 56% of strains. The percentage of MSRA consisted 29.8% of SA isolates. In 2012, the GeneXpert DX system was used to analyze 50 samples of clinical material. The analysis established full matching of results of detection of SA/MRSA in case of using this method and bacteriological analysis to detect agent. The DNA of SA was detected in 61.5% of patients in whom the application of bacteriological method was unsuccessful in separation and identification of agent. In 25% of patients DNA of MRSA was detected. This occurrence made it possible to begin corresponding therapy and to get clinical effect. The diagnostic using GeneXpert DX system took less than 1.5 hours from moment of availability of sample in laboratory. The application of polymerase chain reaction system GeneXpert DX is an effective additional method to identify SA/MRSA which does not exclude application of bacteriological analysis.

Antibacterial drug leads targeting isoprenoid biosynthesis.

With the rise in resistance to antibiotics such as methicillin, there is a need for new drugs. We report here the discovery and X-ray crystallographic structures of 10 chemically diverse compounds (benzoic, diketo, and phosphonic acids, as well as a bisamidine and a bisamine) that inhibit bacterial undecaprenyl diphosphate synthase, an essential enzyme involved in cell wall biosynthesis. The inhibitors bind to one or more of the four undecaprenyl diphosphate synthase inhibitor binding sites identified previously, with the most active leads binding to site 4, outside the catalytic center. The most potent leads are active against Staphylococcus aureus [minimal inhibitory concentration (MIC)(90) ∼0.25 µg/mL], and one potently synergizes with methicillin (fractional inhibitory concentration index = 0.25) and is protective in a mouse infection model. These results provide numerous leads for antibacterial development and open up the possibility of restoring sensitivity to drugs such as methicillin, using combination therapies.

Angiotensin-converting enzyme overexpression in mouse myelomonocytic cells augments resistance to Listeria and methicillin-resistant Staphylococcus aureus.

Gene targeting in ES cells was used to substitute control of angiotensin converting enzyme (ACE) expression from the endogenous promoter to the mouse c-fms promoter. The result is an animal model called ACE 10/10 in which ACE is overexpressed by monocytes, macrophages, and other myelomonocytic lineage cells. To study the immune response of these mice to bacterial infection, we challenged them with Listeria monocytogenes or methicillin-resistant Staphylococcus aureus (MRSA). ACE 10/10 mice have a significantly enhanced immune response to both bacteria in vivo and in vitro. For example, 5 days after Listeria infection, the spleen and liver of ACE 10/10 mice had 8.0- and 5.2-fold less bacteria than wild type mice (WT). In a model of MRSA skin infection, ACE 10/10 mice had 50-fold less bacteria than WT mice. Histologic examination showed a prominent infiltrate of ACE-positive mononuclear cells in the skin lesions from ACE 10/10. Increased bacterial resistance in ACE 10/10 is directly due to overexpression of ACE, as it is eliminated by an ACE inhibitor. Critical to increased immunity in ACE 10/10 is the overexpression of iNOS and reactive nitrogen intermediates, as inhibition of iNOS by the inhibitor 1400W eliminated all in vitro and in vivo differences in innate bacterial resistance between ACE 10/10 and WT mice. Increased resistance to MRSA was transferable by bone marrow transplantation. The overexpression of ACE and iNOS by myelomonocytic cells substantially boosts innate immunity and may represent a new means to address serious bacterial infections.

Effect of human plasma on the antimicrobial activity of iclaprim in vitro.

OBJECTIVES: Iclaprim is a novel diaminopyrimidine for which a human plasma binding level of approximately 93% has been reported. The purpose of this study was to evaluate the effect of human plasma on the in vitro activity of iclaprim and to compare it with that of fusidic acid, teicoplanin and vancomycin, antibiotics with protein binding to human plasma of 97%, >90% and 55%, respectively. METHODS: MICs were determined using 40 methicillin-susceptible Staphylococcus aureus (MSSA) and 38 methicillin-resistant S. aureus (MRSA) isolates in Mueller-Hinton broth (MHB) alone or in the presence of 50% human plasma. RESULTS: MICs of iclaprim were not affected by the addition of human plasma. MIC ranges (MIC(90)) for iclaprim against MSSA and MRSA were < or =0.016-0.06 mg/L (MIC(90) 0.06 mg/L) and < or =0.016-0.5 mg/L (MIC(90) 0.06 mg/L), respectively, in MHB and < or =0.016-0.125 mg/L (MIC(90) 0.06 mg/L) and < or =0.016-0.25 mg/L (MIC(90) 0.125 mg/L), respectively, in the presence of human plasma. As expected, the antimicrobial activity of fusidic acid was greatly affected by the presence of human plasma (MIC elevations of 4- to >128-fold), whereas MICs of vancomycin remained unchanged. By contrast, despite the high protein binding, MICs of teicoplanin were only marginally affected by the presence of plasma with an MIC elevation of maximum 8-fold for two strains. CONCLUSIONS: This study demonstrates that human plasma does not affect the MIC of iclaprim in vitro.

High-level (beta)-lactam resistance and cell wall synthesis catalyzed by the mecA homologue of Staphylococcus sciuri introduced into Staphylococcus aureus.

A close homologue of mecA, the determinant of broad-spectrum beta-lactam resistance in Staphylococcus aureus was recently identified as a native gene in the animal commensal species Staphylococcus sciuri. Introduction of the mecA homologue from a methicillin-resistant strain of S. sciuri into a susceptible strain of S. aureus caused an increase in drug resistance and allowed continued growth and cell wall synthesis of the bacteria in the presence of high concentrations of antibiotic. We determined the muropeptide composition of the S. sciuri cell wall by using a combination of high-performance liquid chromatography, mass spectrometric analysis, and Edman degradation. Several major differences between the cell walls of S. aureus and S. sciuri were noted. The pentapeptide branches in S. sciuri were composed of one alanine and four glycine residues in contrast to the pentaglycine units in S. aureus. The S. sciuri wall but not the wall of S. aureus contained tri- and tetrapeptide units, suggesting the presence of dd- and ld-carboxypeptidase activity. Most interestingly, S. aureus carrying the S. sciuri mecA and growing in methicillin-containing medium produced a cell wall typical of S. aureus and not S. sciuri, in spite of the fact that wall synthesis under these conditions had an absolute dependence on the heterologous S. sciuri gene product. The protein product of the S. sciuri mecA can efficiently participate in cell wall biosynthesis and build a cell wall using the cell wall precursors characteristic of the S. aureus host.

Effect of certain bioactive plant extracts on clinical isolates of beta-lactamase producing methicillin resistant Staphylococcus aureus.

Ethanolic extracts and some fractions from 10 Indian medicinal plants, known for antibacterial activity, were investigated for their ability to inhibit clinical isolates of beta-lactamase producing methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA). Synergistic interaction of plant extracts with certain antibiotics was also evaluated. The MRSA test strains were found to be multi-drug resistant and also exhibited high level of resistance to common beta-lactam antibiotics. These strains produced beta-lactamases, which hydrolyze one or other beta-lactam antibiotics, tested. The extract of the plants from Camellia sinensis (leaves), Delonix regia (flowers), Holarrhena antidysenterica (bark), Lawsonia inermis (leaves), Punica granatum (rind), Terminalia chebula (fruits) and Terminalia belerica (fruits) showed a broad-spectrum of antibacterial activity with an inhibition zone size of 11 mm to 27 mm, against all the test bacteria. The extracts from the leaves of Ocimum sanctum showed better activity against the three MRSA strains. On the other hand, extracts from Allium sativum (bulb) and Citrus sinensis (rind) exhibited little or no activity, against MRSA strains. The antibacterial potency of crude extracts was determined in terms of minimum inhibitory concentration (MIC) by the tube dilution method. MIC values, of the plant extracts, ranged from 1.3 to 8.2 mg/ml, against the test bacteria. Further, the extracts from Punica granatum and Delonix regia were fractionated in benzene, acetone and methanol. Antibacterial activity was observed in acetone as well as in the methanol fractions. In vitro synergistic interaction of crude extracts from Camellia sinensis, Lawsonia inermis, Punica granatum, Terminalia chebula and Terminalia belerica was detected with tetracycline. Moreover, the extract from Camellia sinensis also showed synergism with ampicillin.TLC of the above extracts revealed the presence of major phytocompounds, like alkaloids, glycosides, flavonoids, phenols and saponins. TLC-bioautography indicated phenols and flavonoids as major active compounds.

Increase in methicillin-resistant Staphylococcus aureus acquisition rate and change in pathogen pattern associated with an outbreak of severe acute respiratory syndrome.

BACKGROUND: An outbreak of severe acute respiratory syndrome (SARS) occurred in our 22-bed intensive care unit (ICU; Prince of Wales Hospital, Hong Kong, HKSAR, China) from 12 March to 31 May 2003, when only patients with SARS were admitted. This period was characterized by the upgrading of infection control precautions, which included the wearing of gloves and gowns all the time, an extensive use of steroids, and a change in antibiotic prescribing practices. The pattern of endemic pathogenic organisms, the rates of acquisition of methicillin-resistant Staphylococcus aureus (MRSA), and the rates of ventilator-associated pneumonia (VAP) were compared with those of the pre-SARS and post-SARS periods. METHODS: Data on pathogenic isolates were obtained from the microbiology department (Prince of Wales Hospital). Data on MRSA acquisition and VAP rates were collected prospectively. MRSA screening was performed for all ICU patients. A case of MRSA carriage was defined as an instance in which MRSA was recovered from any site in a patient, and cases were classified as imported or ICU-acquired if the first MRSA isolate was recovered within 72 h of ICU admission or after 72 h in the ICU, respectively. RESULTS: During the SARS period in the ICU, there was an increase in the rate of isolation of MRSA and Stenotrophomonas and Candida species but a disappearance of Pseudomonas and Klebsiella species. The MRSA acquisition rate was also increased: it was 3.53% (3.53 cases per 100 admissions) during the pre-SARS period, 25.30% during the SARS period, and 2.21% during the post-SARS period (P<.001). The VAP rate was high, at 36.5 episodes per 1000 ventilator-days, and 47% of episodes were caused by MRSA. CONCLUSIONS: A SARS outbreak in the ICU led to changes in the pathogen pattern and the MRSA acquisition rate. The data suggest that MRSA cross-transmission may be increased if gloves and gowns are worn all the time.

Comparative analysis of antibiotic and antimicrobial biocide susceptibility data in clinical isolates of methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa between 1989 and 2000.

AIMS: To analyse population minimum inhibitory concentrations (MICs) data from clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa for changes over a 10-year period and to look for correlations between the antimicrobials tested. METHODS AND RESULTS: Data from the MIC study of 256 clinical isolates of Staph. aureus [169 methicillin-sensitive Staph. aureus (MSSA), 87 methicillin-resistant Staph. aureus (MRSA)] and 111 clinical isolates of Ps. aeruginosa against eight antimicrobial biocides and several clinically relevant antibiotics was analysed using anova, Spearman-Rho correlation and principal component analysis. Comparisons suggest that alterations in the mean susceptibility of Staph. aureus to antimicrobial biocides have occurred between 1989 and 2000, but that these changes were mirrored in MSSA and MRSA suggests that methicillin resistance has little to do with these changes. Between 1989 and 2000 a sub-population of MRSA has acquired a higher resistance to biocides, but this has not altered the antibiotic susceptibility of that group. In both Staph. aureus and Ps. aeruginosa several correlations (both positive and negative) between antibiotics and antimicrobial biocides were found. CONCLUSIONS: From the analyses of these clinical isolates it is very difficult to support a hypothesis that increased biocide resistance is a cause of increased antibiotic resistance either in Staph. aureus or in Ps. aeruginosa. SIGNIFICANCE AND IMPACT OF THE STUDY: The observation of negative correlations between antibiotics and biocides may be a useful reason for the continued use of biocides promoting hygiene in the hospital environment.

Risk factors for postoperative mediastinitis due to methicillin-resistant Staphylococcus aureus.

Risk factors for developing postoperative mediastinitis (POM) due to methicillin-resistant Staphylococcus aureus (MRSA) were analyzed in a case-case control study of patients who underwent median sternotomy during the period from 1994 through 2000. Three patient groups were studied. The first consisted of 64 patients with POM due to MRSA; the second consisted of 79 patients with POM due to methicillin-susceptible S. aureus (MSSA); and the third consisted of 80 uninfected control patients. In multivariable analysis, patients who were diabetic (adjusted OR, 2.86; 95% CI, 1.22-6.70), female (OR, 2.70; 95% CI, 1.25-5.88), and >70 years old (OR, 3.43; 95% CI, 1.53-7.71) were more likely to develop POM due to MRSA. In contrast, the only independent risk factor associated with POM due to MSSA was obesity (OR, 2.49; 95% CI, 1.25-4.96). Antimicrobial prophylaxis consisted primarily of cephalosporin antibiotics (administered to 97% of the patients). Changes in perioperative antimicrobial prophylaxis, in addition to other interventions, should be considered for prevention of POM due to MRSA in targeted, high-risk populations.

Epidemiological comparison of true methicillin-resistant and methicillin-susceptible coagulase-negative staphylococcal bacteremia at hospital admission.

We performed 2 case-control studies among 108 patients with true coagulase-negative staphylococcal (CoNS) bacteremia diagnosed within 48 h of hospital admission. Seventy-nine patients (73%) with methicillin-resistant (MR) CoNS bacteremia and 29 patients (27%) with methicillin-susceptible (MS) CoNS bacteremia were compared with 79 randomly selected control patients without CoNS bacteremia. The presence of a central venous catheter was an independent risk factor for both MR- and MS-CoNS bacteremia (P<.001). Patients with MR-CoNS bacteremia were more frequently admitted from health care facilities (P=.02), more likely to have had previous MR Staphylococcus aureus infection or colonization (P=.02), and more likely to have received antibiotics in the previous 30 days (P=.02). The probability that bacteremia was caused by an MR strain, rather than an MS strain, was 62% among patients admitted from the community and 84% among patients admitted from health care facilities. This study identified epidemiological characteristics that can be used to distinguish between MR- and MS-CoNS bacteremia and found high rates of methicillin resistance among CoNS isolates recovered from patients admitted from the community, as well as from health care facilities.

Development and validation of a molecular beacon probe-based real-time polymerase chain reaction assay for rapid detection of methicillin resistance in Staphylococcus aureus.

CONTEXT: A rapid, real-time, duplex, fluorescent molecular beacon probe-based polymerase chain reaction (PCR) assay was recently developed for the detection of methicillin-resistant Staphylococcus aureus. OBJECTIVE: To describe the development and validation of this unique assay. DESIGN: Prospective laboratory analysis. SETTING: Urban health region/centralized diagnostic microbiology laboratory. BACTERIAL STRAINS: One hundred eighty-one previously characterized clinical and American Type Culture Collection isolates, including 50 strains each of methicillin-resistant and methicillin-sensitive S aureus, plus 50 strains of coagulase-negative staphylococci and 31 nonstaphylococcal isolates to ensure assay specificity. INTERVENTION: Assays were performed on purified genomic DNA extracted from growing bacterial colonies. Two sets of oligonucleotide primers were used to specifically amplify the mecA and nuc genes, followed by detection of amplicons using fluorophore-labeled molecular beacon probes. Assays were performed on the Mx4000 Multiplex Quantitative PCR System (Stratagene Inc, La Jolla, Calif). MAIN OUTCOME MEASURES: (1) Assay sensitivity and specificity, and (2) analytical sensitivity. RESULTS: The assay demonstrated 100% sensitivity and 100% specificity, and accurately characterized isolates as methicillin-resistant S aureus, methicillin-sensitive S aureus, or methicillin-resistant coagulase-negative staphylococci, with test results available in 2.5 hours. The analytical sensitivity of the assay was determined to be between 6 and 60 genomic equivalents. CONCLUSIONS: This assay is rapid, accurate, easy to perform, and is compatible with other real-time PCR instruments, making it a suitable alternative to conventional PCR methodologies.

The carboxyl terminus of peptidoglycan stem peptides is a determinant for methicillin resistance in Staphylococcus aureus.

A mecA-containing Staphylococcus aureus strain was grown in the presence of high concentrations of D-serine, D-threonine, and D-phenylalanine. These growth conditions resulted in the replacement of the carboxyl-terminal (fifth) D-alanine residue of peptidoglycan stem peptides with the D-amino acid present in the growth medium and a reduced ability to grow in the presence of methicillin. The most dramatic effect was seen with D-serine. With 32 mM D-serine, strains that had been able to grow in the presence of 800 micro g of methicillin per ml were only able to grow in the presence of less than 50 micro g/ml. The results also suggest that in S. aureus vancomycin resistance mediated through the incorporation of precursors not terminating in D-alanyl-D-alanine would be mutually exclusive with expression of mecA-mediated methicillin resistance.

Poor value of pulsed-field gel electrophoresis to investigate long-term scale epidemiology of methicillin-resistant Staphylococcus aureus.

Pulsed-field gel electrophoresis (PFGE) is widely used for epidemic investigations of methicillin-resistant Staphylococcus aureus (MRSA). In the present study, we evaluated its use in a long-term epidemiological setting (years to few decades, country to continent level). The clustering obtained from PFGE patterns after SmaI digestion of the DNA of 20 strains was compared to that obtained using a phylogenetic typing method (multiprimer RAPD). The results showed that the analysis of small PFGE bands (10-85kb) correlates better with multiprimer RAPD than the analysis of large PFGE bands (>85-700kb), suggesting that the analysis of small bands would be more suitable for the investigation of long-term epidemiological setting. However, given the technical difficulties to obtain a good resolution of these bands and the putative presence of plasmids among them, PFGE does not appear to be a method of choice for the long-term epidemiology analysis of MRSA.