Clinical Methicillin-resistant S. Aureus Research Articles

Effects of Subinhibitory Concentrations of Ceftaroline on Methicillin-Resistant Staphylococcus aureus (MRSA) Biofilms.

Ceftaroline (CPT) is a novel cephalosporin with in vitro activity against Staphylococcus aureus. Ceftaroline exhibits a level of binding affinity for PBPs in S. aureus including PBP2a of methicillin-resistant S. aureus (MRSA). The aims of this study were to investigate the morphological, physiological and molecular responses of MRSA clinical strains and MRSA biofilms to sub-MICs (1/4 and 1/16 MIC) of ceftaroline by using transmission, scanning and confocal microscopy. We have also used quantitative Real-Time PCR to study the effect of sub-MICs of ceftaroline on the expression of the staphylococcal icaA, agrA, sarA and sasF genes in MRSA biofilms. In one set of experiments, ceftaroline was able to inhibit biofilm formation in all strains tested at MIC, however, a strain dependent behavior in presence of sub-MICs of ceftaroline was shown. In a second set of experiments, destruction of preformed biofilms by addition of ceftaroline was evaluated. Ceftaroline was able to inhibit biofilm formation at MIC in all strains tested but not at the sub-MICs. Destruction of preformed biofilms was strain dependent because the biofilm formed by a matrix-producing strain was resistant to a challenge with ceftaroline at MIC, whereas in other strains the biofilm was sensitive. At sub-MICs, the impact of ceftaroline on expression of virulence genes was strain-dependent at 1/4 MIC and no correlation between ceftaroline-enhanced biofilm formation and gene regulation was established at 1/16 MIC. Our findings suggest that sub-MICs of ceftaroline enhance bacterial attachment and biofilm formation by some, but not all, MRSA strains and, therefore, stress the importance of maintaining effective bactericidal concentrations of ceftaroline to fight biofilm-MRSA related infections.

Phenotypic and genomic comparisons of highly vancomycin-resistant Staphylococcus aureus strains developed from multiple clinical MRSA strains by in vitro mutagenesis.

The development of vancomycin (VCM) resistance in Staphylococcus aureus threatens global health. Studies of the VCM-resistance mechanism and alternative therapeutic strategies are urgently needed. We mutagenized S. aureus laboratory strains and methicillin-resistant S. aureus (MRSA) with ethyl methanesulfonate, and isolated mutants that exhibited high resistance to VCM (minimum inhibitory concentration = 32 μg/ml). These VCM-resistant strains were sensitive to linezolid and rifampicin, and partly to arbekacin and daptomycin. Beta-lactams had synergistic effects with VCM against these mutants. VCM-resistant strains exhibited a 2-fold increase in the cell wall thickness. Several genes were commonly mutated among the highly VCM-resistant mutants. These findings suggest that MRSA has a potential to develop high VCM resistance with cell wall thickening by the accumulation of mutations.

Clinical MRSA isolates from skin and soft tissue infections show increased in vitro production of phenol soluble modulins

Phenol-soluble modulins (PSMs) are amphipathic, pro-inflammatory proteins secreted by most Staphylococcus aureus isolates. This study tested the hypothesis that invitro PSM production levels are associated with specific clinical phenotypes. 177 methicillin-resistant S. aureus (MRSA) isolates from infective endocarditis (IE), skin and soft tissue infection (SSTI), and hospital-acquired/ventilator-associated pneumonia (HAP) were matched by geographic origin, then genotyped using spa-typing. Invitro PSM production was measured by high performance liquid chromatography/mass spectrometry. Statistical analysis was performed using Chi-squared or Kruskal-Wallis tests as appropriate. Spa type 1 was significantly more common in SSTI isolates (62.7% SSTI; 1.7% IE; 16.9% HAP; p<0.0001) while HAP and IE isolates were more commonly spa type 2 (0% SSTI; 37.3% IE; 40.7% HAP; p<0.0001). USA300 isolates produced the highest levels of PSMs invitro. SSTI isolates produced significantly higher quantities of PSMα1-4, PSMβ1, and δ-toxin than other isolates (p<0.001). These findings persisted when USA300 isolates were excluded from analysis. Increased invitro production of PSMs is associated with an SSTI clinical source. This significant association persisted after exclusion of USA300 genotype isolates from analysis, suggesting that PSMs play a particularly important role in the pathogenesis of SSTI as compared to other infection types.

Glycopeptide and daptomycin susceptibility trends among clinical isolates of methicillin-resistant Staphylococcus aureus in a tertiary care center in North India.

Increased vancomycin minimum inhibitory concentration (MIC) levels in Staphylococcus aureus and their association with vancomycin treatment failure are well-known problems. Few studies have recognized progressive increases in glycopeptide MIC levels for S. aureus strains in recent years. This study determined glycopeptide and daptomycin susceptibility among methicillin resistant S. aureus (MRSA) strains. A total of 776 clinical isolates of MRSA recovered from 2009 to 2012 were studied for glycopeptide and daptomycin susceptibility using the E-test method. The vancomycin MIC geometric mean (GM) of the MRSA isolates was 0.923, 0.944, 1.134 and 1.294 mg/L in the years 2009, 2010, 2011 and 2012, respectively, and the trend significantly increased over the years (P < 0.0001). Similarly, the teicoplanin MIC GM was 1.47, 1.49, 1.8 and 2.04 mg/L in the years from 2009 to 2012, respectively (P < 0.0001). MIC shifts were not found for daptomycin (P > 0.232). A significant increase in the MIC for glycopeptides was observed among the clinical MRSA isolates at our center over a 4-year period. However, the daptomycin MIC did not increase in the observed MRSA isolates.

Antibiotic-specific differences in the response of Staphylococcus aureus to treatment with antimicrobials combined with manuka honey.

Skin infections caused by antibiotic resistant Staphylococcus aureus are a significant health problem worldwide; often associated with high treatment cost and mortality rate. Complex natural products like New Zealand (NZ) manuka honey have been revisited and studied extensively as an alternative to antibiotics due to their potent broad-spectrum antimicrobial activity, and the inability to isolate honey-resistant S. aureus. Previous studies showing synergistic effects between manuka-type honeys and antibiotics have been demonstrated against the growth of one methicillin-resistant S. aureus (MRSA) strain. We have previously demonstrated strong synergistic activity between NZ manuka-type honey and rifampicin against growth and biofilm formation of multiple S. arueus strains. Here, we have expanded our investigation using multiple S. aureus strains and four different antibiotics commonly used to treat S. aureus-related skin infections: rifampicin, oxacillin, gentamicin, and clindamycin. Using checkerboard microdilution and agar diffusion assays with S. aureus strains including clinical isolates and MRSA we demonstrate that manuka-type honey combined with these four antibiotics frequently produces a synergistic effect. In some cases when synergism was not observed, there was a significant enhancement in antibiotic susceptibility. Some strains that were highly resistant to an antibiotic when present alone become sensitive to clinically achievable concentrations when combined with honey. However, not all of the S. aureus strains tested responded in the same way to these combinational treatments. Our findings support the use of NZ manuka-type honeys in clinical treatment against S. aureus-related infections and extend their potential use as an antibiotic adjuvant in combinational therapy. Our data also suggest that manuka-type honeys may not work as antibiotic adjuvants for all strains of S. aureus, and this may help determine the mechanistic processes behind honey synergy.

Molecular characterization of methicillin-resistant Staphylococcus aureus isolated from tertiary care hospitals.

Methicillin-resistant S. aureus (MRSA) tends to be resistant to multiple antibiotics. Methicillin resistance is conferred by the acquisition of the mecA gene, which is carried by a mobile genetic element called the staphylococcal cassette chromosome mec (SCCmec). There are five major types of SCCmec elements (I-V). The majority of hospital-acquired MRSA (HA-MRSA) strains carry SCCmec types I, II, or III, whereas community-acquired MRSA (CA-MRSA) strains carry SCCmec types IV or V. In addition, Panton-Valentine Leucocidin (PVL) is a gene encoding a powerful cytotoxin that is strongly associated with CA-MRSA strains. The present study was aimed to identify the types of SCCmec and PVL genes among clinical MRSA isolates. This study was conducted in 5 tertiary care hospitals in Makkah city from March to September of 2012. A total of 206 S. aureus clinical isolates were analysed using standard microbiological methods. Multiplex PCR was performed on genomic DNA from MRSA isolates in order to identify the types of SCCmec. In addition, PCR was performed to detect the PVL gene among the isolates. Of the 206 S. aureus isolates, 114 (55.3%) were MRSA, and 100 of the MRSA isolates carried the mecA gene. RESULTS from SCCmec typing revealed that 3% were type I; 9% were type II; 47% were type III, and 29% were type IV. Nineteen per cent of the isolates harboured the PVL gene. Furthermore, there was a statistically significant correlation between the presence of the PVL gene and SCCmec type IV. The virulence of MRSA strains is increasing in both hospital and community settings in Makkah, highlighting the importance of their rapid identification in order to appropriately control infection.

Combination of Alpha-Melanocyte Stimulating Hormone with Conventional Antibiotics against Methicillin Resistant Staphylococcus aureus

Our previous studies revealed that alpha-melanocyte stimulating hormone (α-MSH) is strongly active against Staphylococcus aureus (S. aureus) including methicillin resistant S. aureus (MRSA). Killing due to α-MSH occurred by perturbation of the bacterial membrane. In the present study, we investigated the in vitro synergistic potential of α-MSH with five selected conventional antibiotics viz., oxacillin (OX), ciprofloxacin (CF), tetracycline (TC), gentamicin (GM) and rifampicin (RF) against a clinical MRSA strain which carried a type III staphylococcal cassette chromosome mec (SCCmec) element and belonged to the sequence type (ST) 239. The strain was found to be highly resistant to OX (minimum inhibitory concentration (MIC) = 1024 µg/ml) as well as to other selected antimicrobial agents including α-MSH. The possibility of the existence of intracellular target sites of α-MSH was evaluated by examining the DNA, RNA and protein synthesis pathways. We observed a synergistic potential of α-MSH with GM, CF and TC. Remarkably, the supplementation of α-MSH with GM, CF and TC resulted in ≥64-, 8- and 4-fold reductions in their minimum bactericidal concentrations (MBCs), respectively. Apart from membrane perturbation, in this study we found that α-MSH inhibited ∼53% and ∼47% DNA and protein synthesis, respectively, but not RNA synthesis. Thus, the mechanistic analogy between α-MSH and CF or GM or TC appears to be the reason for the observed synergy between them. In contrast, α-MSH did not act synergistically with RF which may be due to its inability to inhibit RNA synthesis (<10%). Nevertheless, the combination of α-MSH with RF and OX showed an enhanced killing by ∼45% and ∼70%, respectively, perhaps due to the membrane disrupting properties of α-MSH. The synergistic activity of α-MSH with antibiotics is encouraging, and promises to restore the lost potency of discarded antibiotics.

Distinction of Staphylococcal Cassette Chromosome mec type V elements from Staphylococcus aureus ST398

Methicillin resistant S. aureus (MRSA) is a major threat for human health and well-being. In recent years, it has become clear that livestock is a potential reservoir for MRSA, most livestock-associated isolates belonging to the ST398 lineage. Importantly, ST398 strains were also reported as causative agents of severe invasive infections in humans with no evidence for livestock associations. Here we document the sequence of the J1 region of the type V (5C2&5) SCCmec element and its right chromosomal junction in the clinical PVL-positive ST398 MRSA isolate UMCG-M4. Sequence comparisons show that this SCCmec element and related type V elements from other S. aureus isolates share a common core structure, but differ substantially in the so-called J1 region. Additional PCR analyses and typing studies indicate that the J1 region of strain UMCG-M4 is specific for SCCmec elements of PVL-positive ST398 isolates. Lastly, we show that the sequenced right chromosomal junction is invariant in strains of the ST398 lineage.

Comparing micellar, hemolytic, and antibacterial properties of di- and tricarboxyl dendritic amphiphiles

Homologous dicarboxyl dendritic amphiphiles—RCONHC(CH 3)(CH 2CH 2COOH) 2, 4( n); and ROCONHC(CH 3)(CH 2CH 2COOH) 2, 5( n), where R = n-C n H 2 n +1 and n = 13–22 carbon atoms—were synthesized. Critical micelle concentrations (CMCs) in aqueous triethanolamine solutions and at pH 7.4 were measured along with hemolytic activity (effective concentrations, EC 10) in phosphate-buffered saline (PBS). Log CMC showed a linear dependence on chain length ( n); the longest chain in each series had the lowest CMC—in triethanolamine: 4(21), 180 μM and 5(22), 74 μM and at pH 7.4: 4(21), 78 μM and 5(22), 33 μM. These two series, 4( n) and 5( n), and three series of homologous tricarboxyl dendritic amphiphiles—RCONHC(CH 2CH 2COOH) 3, 1( n); ROCONHC(CH 2CH 2COOH) 3, 2( n); RNHCONHC(CH 2CH 2COOH) 3, 3( n), where R = n-C n H 2 n +1 and n = 13–22 carbon atoms—were tested for growth inhibition of Staphylococcus aureus strain ATCC 6358 and methicillin-resistant S. aureus (MRSA) strain ATCC 43330 by microdilution in 0.1-strength brain heart infusion broth (BHIB). Amphiphiles 4(19), 4(21), 5(18), and 5(20) showed the strongest antibacterial activity (2.2–3.4 μg/mL) against S. aureus (vancomycin, MIC = 0.25 μg/mL). These four plus 1(21), 2(20), 2(22), and 3(20) exhibited the strongest antibacterial activity (1.7–6.8 μg/mL) against MRSA (vancomycin, MIC = 0.25 μg/mL). The MICs of these amphiphiles against six clinical MRSA were similar to those against the ATCC strain. In PBS, EC 10s of the most active homologues ranged from 7 to 18 μg/mL and 18 to 220 μg/mL for di- and tricarboxyl dendritic amphiphiles, respectively. To assess the potential safety of using dendritic amphiphiles as drugs, measurements of micellar and hemolytic properties were conducted in the same medium (full-strength BHIB) that was used for antibacterial activity. The CMCs (9–36 μg/mL, ∼18–72 μM) of ten amphiphiles were measured by microdilution (log 2 progression) with dye-covered beads. The EC 10s were similar to those in PBS. The MICs of most amphiphiles (14–72 μg/mL) and vancomycin (1.1–2.2 μg/mL) against both S. aureus and MRSA increased significantly compared to the MICs measured in 0.1-strength BHIB. The one exception, 5(18), had an MIC against S. aureus of 1.1 μg/mL compared to vancomycin (2.2 μg/mL). With CMC (9–18 μg/mL) and EC 10 (16 μg/mL) values higher than the MIC, 5(18) was discovered as a lead for further development.

Characterisation of anti‐Staphylococcus aureus activity of quercetin

SummaryAlthough many antibiotics are available for the treatment of bacterial infections, the emergence and global spread of antibiotic‐resistant bacteria is a community‐wide problem. To overcome this problem, we must explore alternative antimicrobials. This study investigated the antibacterial properties of quercetin, a flavonoid present in vegetables and fruits. Quercetin was tested against gram‐positive and gram‐negative bacteria and was found to exert selective antibacterial activity against Staphylococcus aureus, including methicillin‐resistant S. aureus (MRSA), and Staphylococcus epidermidis. Some clinical MRSA strains showed remarkable susceptibility to quercetin. In combination with antibiotics, such as oxacillin, ampicillin, vancomycin, gentamicin, and erythromycin, quercetin showed markedly enhanced antibacterial activity against MRSA. We also report quercetin‐induced aggregation of S. aureus cells; the morphological changes in these cells, as assessed by electron microscopy; and the colony‐spreading ability of quercetin‐sensitive MRSA, all of which revealed the unique antibacterial properties of quercetin against S. aureus.

Panton-Valentine Leukocidin Positive Methicillin Resistant S. Aureus Infections in Children with Cancer

Background: Staphylococcus aureus is one of the most prevalent organisms isolated from children and adults with cancer. Strains of methicillin resistant S. aureus (MRSA) have emerged in the past decade, that often cause serious infections, in young, otherwise healthy people. Suppurative skin infections and necrotizing pneumonias are some of the diseases most characteristically associated with these new strains. Such MRSA isolates often harbor the Panton-Valentine Leukocidin (PVL) gene, belong to pulsed-field type USA 300, carry the staphylococcal cassette chromosome (SCC) mec IV, and are susceptible to clindamycin. The presence of the PVL gene has been associated with increased virulence and morbidity in these patients. However, the role of these new MRSA strains in causing infections in children and adults with cancer is presently unknown. Studies are needed to determine if the increase in incidence of MRSA infections in the community is paralleled by an increase in MRSA infections in pediatric cancer patients and if infections with these new strains are more virulent in this population.Methods: The present work represents a retrospective study of all children with cancer diagnosed with MRSA infection over an eight year period from January 2000 to December 2007. Medical record review included patient demographics, underlying disease, and antimicrobial susceptibility patterns of MRSA isolates. Genotyping of isolates was performed using PCR for detection of the PVL gene. SCC mec and spa typing was performed on all PVL positive isolates. Logistic regression models were used to study the trend in the increase of MRSA infection over time. Statistical analysis was performed on frequency data between the different categorical variables among the PVL positive and PVL negative MRSA isolates by the Exact Pearson Chi-square test.Results: A total of 98 clinical MRSA isolates were collected from 88 children with cancer from Jan 2000 to Dec 2007. The number of MRSA isolates increased sevenfold from 3 in 2000 to 21 in 2006 (p<0.0001). During the same period there was a significant decrease in the incidence of methicillin-sensitive S. aureus (MSSA) isolates (p<0.0001). Ten of the children had recurrent infection. The increase in MRSA infections was predominantly due to an increase in skin and soft tissue infections (p=0.0004). Children with African American ancestry and inpatients were significantly overrepresented in having MRSA infection as compared to other children with cancer (p<0.0001). Outcome was generally favorable; 33% had fever and only 10 patients (10%) had complications. Resistance to ciprofloxacin, clindamycin, erythromycin, gentamicin and mupirocin was 23%, 20%, 80%, 5%, and 2% respectively. All the isolates were sensitive to trimethoprim-sulfamethoxazole and vancomycin. PVL positive isolates bearing the SCC mec IV element comprised 35 of 98 (36%) of MRSA isolates. 32 of the 35 isolates belonged to the USA 300 type. No child had a recurrent infection with a PVL positive strain. Children infected with PVL positive strains had a significantly higher incidence of skin and soft tissue infections (SSTI, p=0.045), and bacteremia (p=0.045), as compared to children with PVL negative strains. A significantly higher number of the PVL positive isolates were susceptible to clindamycin (p=0.0158) and ciprofloxacin (p=0.0045).Conclusion: This report represents the first description of the changing epidemiology of MRSA infections in children with cancer. The association between PVL positive MRSA infections and bacteremia is a novel finding in this population. PVL positive MRSA infections caused low morbidity and mortality in these immune-suppressed patients. Future studies may determine if patients with colonization from these virulent strains may benefit from preemptive therapy with mupirocin, to eradicate colonization. This approach may reduce the risk of bacteremia in high risk groups such as patients undergoing hematopoietic stem cell transplantation prior to engraftment.

Methicillin-resistant Staphylococcus aureus carriage, infection and transmission in dialysis patients, healthcare workers and their family members

Carriage and subsequent infection with methicillin resistant S. aureus (MRSA) and its transmission between hospital and community settings have not been studied in dialysis patients and their contacts. Surveillance for nasal MRSA carriage and infection among dialysis patients, healthcare workers (HCWs) and their family members in a dialysis centre was prospectively undertaken during three time periods within 1 year. Molecular typing was used to determine epidemiological relationship. Among 1687 samples collected, MRSA colonization rates were 2.41% (2/83) for peritoneal dialysis patients and 2.36% (12/509) for haemodialysis patients. Five (5/14) subjects subsequently had MRSA infection. The clinical MRSA isolates had the same molecular type as the colonized strains of the same person, indicating MRSA colonization preceded clinical infection. Significantly higher MRSA nasal carriage rates were observed among family members of HCWs than family members of dialysis patients (P = 0.0024). Only three major clones were observed. Pulmonary diseases (OR: 4.873, 95% CI: 1.668-14.235), recent admission to a hospital (OR: 2.797, 95% CI: 1.291-6.059) and recent antibiotics usage (OR: 2.319, 95% CI: 1.053-5.104) were also significantly associated with MRSA carriage. Transmission of MRSA among dialysis patients, HCWs and their family members in a dialysis unit could be inferred. Monitoring and eradication of MRSA from patients, HCWs and their family members should be considered to prevent continuous spread between healthcare facilities and the community.

Comparative Proteomics Analyses Reveal a Potential Biomarker for the Detection of Vancomycin-Intermediate Staphylococcus aureus Strains

Vancomycin-intermediate Staphylococcus aureus (VISA) strains tend to develop during glycopeptide treatment of infections caused by methicillin-resistant S. aureus (MRSA). Rapid and effective detection methods for VISA strains are lacking, and mechanisms of resistance are unclear. Here, global comparative proteomic approaches have been used to identify potential biomarkers of intermediate vancomycin resistance. With the use of high-resolution two-dimensional gels and iTRAQ mass tagging, numerous proteins were found to be differentially expressed between clinical MRSA and VISA isolates of the same multilocus sequence type. One of these, the predicted lytic transglycosylase SAV2095 (SceD-like protein), was selected for further study based on both its high level of induction in Mu50 and its predicted role in modeling the cell wall, which is the target of vancomycin. Relative SAV2095 mRNA expression levels were compared between 25 MRSA and VISA/heterogeneous VISA clinical isolates by real-time RT-PCR. The SAV2095 mRNA was significantly induced in all VISA isolates relative to all MRSA strains ( p < 0.001), and significant induction of SAV2095 was also seen for several potential heterogeneous VISA strains that appear vancomycin-sensitive by standard minimum inhibitory concentration-determining methods. Furthermore, strains selected in vitro for increasing levels of resistance from four unrelated clinical MRSA isolates displayed concomitant increases in levels of SAV2095 expression. Together, these results suggest that SAV2095 expression level could serve as a molecular diagnostic marker for the rapid detection of VISA.

Characterization of Staphylococcus aureus mutants expressing reduced susceptibility to common house-cleaners

To characterize mutants of Staphylococcus aureus expressing reduced susceptibility to house cleaners (HC), assess the impact of the alternative sigma factor SigB on HC susceptibility, and determine the MIC of clinical methicillin-resistant S. aureus (MRSA) to a HC. Susceptibility to HC, HC components, H2O2, vancomycin and oxacillin and physiological parameters were determined for HC-reduced susceptibility (HCRS) mutants, parent strain COL and COLsigB::kan. HCRS mutants selected with three HC expressed reduced susceptibility to multiple HC, HC components, H2O2 and vancomycin. Two unique HCRS mutants also lost the methicillin resistance determinant. In addition, all HCRS mutants exhibited better growth at two temperatures, and one HCRS mutant expressed reduced carotenoid production. COLsigB::kan demonstrated increased susceptibility to all HC and many HC components. sigB operon mutations were not detected in one HCRS mutant background. Of 76 clinical MRSA, 20 exhibited reduced susceptibility to a HC. HCRS mutants demonstrate altered susceptibility to multiple antimicrobials. While sigB is required for full HC resistance, one HCRS mechanism does not involve sigB operon mutations. Clinical MRSA expressing reduced susceptibility to a common HC were detected. This study suggests that HCRS mutants are not protected against, nor selected by, practical HC concentrations.

In vitro activity of linezolid, quinupristin-dalfopristin, vancomycin, teicoplanin, moxifloxacin and mupirocin against methicillin-resistant Staphylococcus aureus: comparative evaluation by the E test and a broth microdilution method

The E test and broth microdilution showed comparable accuracy for the susceptibility testing of methicillin-resistant S. aureus (MRSA). All of the 109 primary clinical MRSA isolates were fully susceptible to the glycopeptides vancomycin and teicoplanin, the oxazolidinone linezolid, and the streptogramin quinupristin-dalfopristin. Nine out of the 109 MRSA isolates (8.3 percent) demonstrated resistance to moxifloxacin and 5 out of the 109 strains (4.6 percent) were resistant to the topical agent mupirocin. Linezolid and quinupristin-dalfopristin may prove useful alternatives for the treatment of patients with MRSA infections. MRSA isolates should be screened for in vitro susceptibility against mupirocin prior to the topical application.

Detection of methicillin-resistant Staphylococcus aureus and simultaneous confirmation by automated nucleic acid extraction and real-time PCR.

A molecular assay for the simultaneous detection of a Staphylococcus aureus-specific gene and the mecA gene, responsible for the resistance to methicillin in staphylococci, was evaluated. The assay included an automated DNA extraction protocol conducted with a MagNA Pure instrument and real-time PCR conducted with a LightCycler instrument. The performance and robustness of the assay were evaluated for a suspension of methicillin-resistant S. aureus (MRSA) strain with a turbidity equivalent to a McFarland standard of 0.5, which was found to be the ideal working concentration. The specificity of the new molecular assay was tested with a panel of 30 gram-negative and gram-positive bacterial strains other than MRSA. No cross-reactivity was observed. In a clinical study, 109 isolates of MRSA were investigated. All clinical MRSA isolates gave positive results for the S. aureus-specific genomic target, and all but one were positive for the mecA gene. In conclusion, the new molecular assay was found to be quick, robust, and laborsaving, and it proved to be suitable for a routine molecular diagnostic laboratory.

Population-based community prevalence of methicillin-resistant Staphylococcus aureus in the urban poor of San Francisco.

The study objective was to determine the prevalence and risk factors for nasal colonization with Staphylococcus aureus and methicillin resistance among the urban poor and to compare antibiotic resistance and genetic similarity to concurrently collected clinical isolates of methicillin-resistant S. aureus (MRSA). A population-based community sample of 833 homeless and marginally housed adults were cultured and compared with 363 clinical isolates of MRSA; 22.8% of the urban poor were colonized with S. aureus. Of S. aureus isolates, 12.0% were methicillin resistant. Overall prevalence of MRSA was 2.8%. Significant multivariate risk factors for MRSA were injection drug use (odds ratio [OR], 9.7), prior endocarditis (OR, 4.1), and prior hospitalization within 1 year (OR, 2.4). Resistance to antimicrobials other than beta-lactams was uncommon. Only 2 individuals (0.24%) with MRSA had no known risk factors. A total of 22 of 23 community MRSA genotypically matched clinical MRSA isolates, with 15 of 23 isolates identical to MRSA clones endemic among hospitalized patients.

Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin

Since the discovery of the vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50 (minimum inhibitory concentration [MIC] 8 mg/L), there has been concern about the potential spread of such strains throughout Japanese hospitals. Two important questions need to be answered: (1) what is the prevalence of VRSA, and (2) by what mechanism does vancomycin resistance occur. The vancomycin susceptibilities of three methicillin-resistant S aureus (MRSA) strains (Mu50, Mu3, and H1) and the methicillin-susceptible S aureus type strain FDA209P were compared by MIC determinations and population analysis. Mu3 (MIC 3 mg/L) was isolated from the sputum of a patient with pneumonia after surgery who had failed vancomycin therapy. H1 (MIC 2 mg/L), which is a representative vancomycin-susceptible MRSA strain, was isolated from a patient with pneumonia who responded favourably to vancomycin therapy. Subclones of Mu3 with increased resistance against vancomycin were selected with serial concentrations of vancomycin and their MICs were determined. The prevalence of VRSA and Mu3-like strains in Japanese hospitals was estimated by population analysis from 1149 clinical MRSA isolates obtained from 203 hospitals throughout Japan. The genetic traits of the Mu3 and Mu50 strains were compared with clonotypes of MRSA from around the world. Mu3 and Mu50 had an identical pulsed-field gel electrophoresis banding pattern. When grown in a drug-free medium, Mu3 produced subpopulation of cells with varying degrees of vancomycin resistance, thus demonstrating natural heterogeneity, or variability, in susceptibility to vancomycin. In the presence of vancomycin, Mu3 produced subclones with resistance roughly proportional to the concentrations of vancomycin used. Selection of Mu3 with 8 mg/L or more of vancomycin gave rise to subclones with vancomycin resistance equal to that of Mu50 (MIC 8 mg/L) at a frequency of 1/1,000,000. During screening of Japanese MRSA strains, no strain of VRSA additional to Mu50 was found. The prevalence of MRSA isolates heterogeneously resistant to vancomycin was 20% in Juntendo University Hospital, 9.3% in the other seven university hospitals, and 1.3% in non-university hospitals or clinics. Heterogeneously resistant VRSA is a preliminary stage that allows development into VRSA upon exposure to vancomycin. Heterogeneously resistant VRSA was found in hospitals throughout Japan. This finding could explain, at least partly, the frequent therapeutic failure of MRSA infection with vancomycin in Japan.