Methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus in retail meat, Detroit, Michigan, USA.

To the Editor: Because methicillin-resistant Staphylococcus aureus (MRSA) has been identified in retail meat worldwide (1-4), the potential exists for its transmission to humans.Of the various meat products surveyed, pork had the highest contamination rate in the United States and Canada (1,2), as did beef in Korea (3) and poultry in the Netherlands (4).The study in Korea also observed MRSA from chicken, which demonstrated sequence type (ST) 692 by multilocus sequence typing (MLST), a type distinct from that isolated in beef and pork.Despite sample size variations, these studies suggested that MRSA contamination in different meat categories can vary by location and that molecular distinction may exist among MRSA isolates in meat of different origin.We collected 289 raw meat samples (156 beef, 76 chicken, and 57 turkey) from 30 grocery stores in Detroit, Michigan, USA, during August 2009-January 2010.Up to 3 presumptive S. aureus colonies per sample were identified by coagulase test and species-specific PCR (1).Antimicrobial drug MICs were determined and interpreted according to Clinical and Laboratory Standards Institute guidelines (5).S. aureus were characterized by pulsed-field gel electrophoresis (PFGE), mecA identification, SCCmec typing, Panton-Valentine leukocidin identification, agr typing, MLST, and spa typing as described (1,6).Sixty-five (22.5%) samples yielded S. aureus: 32 beef (20.5%), 19 chicken (25.0%), and 14 turkey (24.6%) samples.Six samples, consisting of 2 beef (1.3%), 3 chickens (3.9%), and 1 turkey (1.7%), were positive for MRSA as evidenced by the presence of mecA.The overall lower prevalence of S. aureus and MRSA than found in a previous study in the United States (40% and 5%, respectively) (1) might be explained by our exclusion of pork because pork and swine production have been major reservoirs of MRSA (4,7).However, different geographic location

Methicillin resistant Staphylococcus aureus (MRSA) bacteremia has a high rate of morbidity and mortality. Multiple clinical studies have demonstrated improved outcomes when MRSA bacteremia is treated with dual antibiotic therapy that includes a β-lactam antibiotic such as cefazolin. This is a paradox as MRSA should be inherently resistant to this class of antibiotics. We report on a serendipitous observation of a phenotype where MRSA became sensitive to cefazolin when cultured in a physiologic relevant media of fetal bovine serum as well as in synovial fluid. This could be observed across multiple clinical isolates. Expected resistance was maintained when cultured in Muller Hinton Broth (MHB). MRSA β-lactam antibiotic resistance is mediated by PBP2a, a penicillin-binding protein encoded by mecA. We hypothesized that this phenotype of antibiotic sensitivity in physiologic medium was based, in part, on levels of PBP2a expression and post-translational modifications of peptidoglycan wall teichoic acid (WTA). We, therefore, conducted quantitative RT-PCR analysis and Western blotting which demonstrated limited mecA expression PBP2a protein level when cultured in FBS as compared to the clinical microbiology standard MHB, respectively. Whole genome sequencing of loss of function mutants generated through serial passaging in FBS revealed that the clp family of proteins and rpo genes were involved in β-lactam resistance. Cell wall peptidoglycan analysis suggested that WTA glycosylation was altered between β-lactam resistant and sensitive MRSA phenotypes. Together, this suggests that clpP, rpoB, and WTA glycosylation are involved with the β-lactam sensitivity phenotype in MRSA and can be new potential targets for MRSA treatment.

Part of the success of methicillin-resistant Staphylococcus aureus (MRSA) as a pathogen responds to the rapid spread of pandemic lineages with diverse virulence and antimicrobial susceptibility profiles. In Colombia, several healthcare-associated MRSA (HA-MRSA) clones have been found, including the pediatric clone (CC5-ST5-SCC mec IV), the Brazilian clone (CC8-ST239-SCC mec III), and the Chilean/Cordobés clone (CC5-ST5-SCC mec I). Moreover, the community-associated MRSA (CA-MRSA) clone USA300 has been reported as causing hospital-acquired infections. To describe the changes over time in the distribution of MRSA clones from a university hospital in Medellín collected at two time points a decade apart. A total of 398 MRSA strains were analyzed. Of these, 67 strains were collected in 1994, while the remaining 331 strains were collected between 2008 and 2010. Species identification and methicillin resistance were confirmed by detection of nuc and mec A genes, respectively. Molecular characterization included spa typing, SCC mec typing, PFGE and MLST. Analysis of the MRSA strains collected in 1994 revealed that they belonged to a single clone, the CC5-SCC mec IV, whereas among the isolates from 2008-2010, two dominant clones were identified: CC8-SCC mec IVc, which included spa types t008 and t1610 and is closely related to the USA 300 clone, and CC5-SCC mec I ( spa type t149), related to the Chilean clone. The ST5-SCC mec IV clone from 1994 was not detected. This study identifies temporal dynamics in MRSA clone diversity, and highlights the importance of local surveillance and dissemination of results, especially in countries like Colombia where MRSA is prevalent and knowledge regarding its epidemiology is still insufficient.

Methicillin resistant Staphylococcus aureus (MRSA) sepsis has a high rate of morbidity and mortality. Multiple clinical studies have demonstrated improved outcomes when MRSA sepsis is treated with dual antibiotic therapy that includes a β-lactam antibiotic such as cefazolin. This is a paradox as MRSA should be inherently resistant to this class of antibiotics. We report a serendipitous observation revealed a phenotype where MRSA became sensitive to cefazolin when cultured in a physiologic relevant media of fetal bovine serum as well as in synovial fluid. This could be observed across multiple clinical isolates. Expected resistance was maintained when cultured in Muller Hinton Broth (MHB). MRSA β-lactam antibiotic resistance is mediated by PBP2a, a penicillin-binding protein encoded by mecA. We hypothesized that this phenotype of antibiotic sensitivity in physiologic medium was based, in part, on levels of PBP2a expression and post-translational modifications of peptidoglycan wall teichoic acid (WTA). We therefore conducted quantitative RT-PCR analysis and Western blotting which demonstrated limited mecA expression in the mRNA level and limited PBP2a protein level when cultured in FBS or synovial fluid as compared to the clinical microbiology standard MHB, respectively. Whole genome sequencing of loss of function mutants generated through serial passaging in FBS revealed that the clp family of proteins and rpo genes were involved in β-lactam resistance. Cell wall peptidoglycan analysis suggested that WTA glycosylation was altered between β-lactam resistant and sensitive MRSA phenotypes. Together, this suggests pathways for clpP, rpoB, and WTA glycosylation can be new potential targets for MRSA treatment.

Genetic diversity of methicillin resistant Staphylococcus aureus strains isolated from burn patients in Iran: ST239-SCCmec III/t037 emerges as the major clone.

Triclosan (2.4.4’ trichloro-2’-hydroxydiphenyl ether) is a broad-spectrum biocide which is also used to decolonise patients with methicillin resistant Staphylococcus aureus (MRSA). Microbial resistance to biocides has recently been reported, so it is important that new products should be tested for resistance that may arise from continued exposure to such agents. In a previous study 232 strains of MRSA isolated during 1997-2000 in 30 Scottish hospitals were tested for triclosan susceptibility; overall the minimum inhibitory concentrations (MIC) of triclosan for these strains ranged from <=0.015 to 4 mg/L. In the present study, resistance to triclosan was examined in five major international MRSA clones [Clonal Complex (CC22, CC30, CC45, CC8 and CC5)] by growing them in brain heart infusion broth in the presence of increasing concentrations of triclosan (0.03mg/L, 0.06 mg/L, 0.125 mg/L, 0.25 mg/L, and 0.5 mg/L ) for up to 67 days. Different MRSA clones showed different degrees of triclosan tolerance. CC22 (EMRSA–15), CC30 (EMRSA-16) and CC5 triclosan-tolerant derivatives showed a significant increase in triclosan MIC when compared to their parents, principally through the appearance of pinpoint-size small colony variants (SCV), as well as colonies of normal or small size. These MRSA SCVs emerged in different clones and at different times of exposure to triclosan. The triclosan MICs of mutants of all colony sizes rose to 4 mg/L in all clones except MRSA111-29 (CC45) which had an MIC 4-8 mg/L. Triclosan-resistant MRSA strains were also able to grow in the presence of higher triclosan concentrations: 1.25 mg/L (CC22), 10 mg/L (CC30), 25 mg/L (CC45), 5 mg/L (CC8) and 25 mg/L (CC5). In addition, six triclosan resistant derivatives from each MRSA clone, together with their parental clone, were examined by antibiogram, polymerase chain reaction (PCR) ribotyping and detailed susceptibility to triclosan in terms of MIC and kill kinetics. Susceptibility to the aminoglycosides kanamycin, neomycin and tobramycin was decreased in four clones, and tetracycline susceptibility increased in one clone. PCR ribotyping confirmed clonally similar to the mutants. Kill kinetics of both parents and their triclosan resistant mutants showed 5-Logs reduction at 0.5 min and 5 min respectively in all five clones. In conclusion, repeated exposure of MRSA to triclosan may result in resistance to this biocide, and to clinically-relevant antimicrobials.

This study identifies temporal dynamics in MRSA clone diversity, and highlights the importance of local surveillance and dissemination of results, especially in countries like Colombia where MRSA is prevalent and knowledge regarding its epidemiology is still insufficient.

Background: The risk factors for methicillin-resistant Staphylococcus aureus (MRSA) colonization can differ between acute-care, subacute-care, and long-term care facilities, but comparative information is lacking. We compared risk factors for MRSA colonization contemporaneously between an acute-care hospital (ACH) and its affiliated intermediate- and long-term care facilities (ILTCFs). Methods: Serial cross-sectional studies were conducted in a 1,600-bed tertiary-care ACH and its 6 affiliated ILTCFs in Singapore, in June–July 2014–2016. Separate nasal, axillary, and groin swabs were taken and cultured for MRSA. MRSA isolates were subject to whole-genome sequencing. Clinical and epidemiological data were obtained from medical records. To account for clustering, multivariable 2-level multinomial logistic regression models were constructed to assess factors associated with colonization of specific MRSA clones, in the ACH and ILTCFs, respectively. Results: In total, 8,873 samples from 2,985 patients in the ACH and 7,172 samples from 2,409 patients and residents in ILTCFs were included in the study. Patients and residents in the ILTCFs (29.7%) were more likely to be colonized with MRSA than patients in the ACH (12.6%) (P < .0001). The predominant MRSA clones were clonal complexes (CC)22 (n = 692, 46.7%) and CC45 (n = 494, 33.4%), contributing to 80% of MRSA isolates. For ACH patients, after adjusting for age, gender, comorbidities, prior exposures to antibiotics and percutaneous devices, presence of wounds, and screening year, prior MRSA carriage in the preceding 12 months was the strongest predictor of colonization with all MRSA clones: CC22 (aOR, 14.71; 95% CI, 6.17–34.48); CC45 (aOR, 7.75; 95% CI, 2.70–22.22); and others (aOR, 22.22; 95% CI, 3.83–125.00). Hospital stay >14 days was also positively associated with colonization with MRSA CC22 (aOR, 2.67; 95% CI, 1.22–5.88), but not the other clones. For ILTCF patients and residents, after adjusting for age, comorbidities, prior exposure to antibiotics, presence of wounds, and screening year, prior MRSA carriage was a significant predictor of colonization with MRSA CC22 (aOR, 2.72; 95% CI, 1.35–5.46), and CC45 (aOR, 2.36; 95 % CI, 1.06–5.24), but not with other clones. Additionally, prior exposure to a percutaneous device and being male were respectively positively associated with colonization by MRSA CC22 (aOR, 2.70; 95% CI, 1.19–6.17) and CC45 (aOR, 2.17; 95% CI, 1.11–4.26). Conclusions: Prior MRSA carriage was a common risk factor for colonization with the predominant MRSA clones in both the ACH and ILTCFs. Hospital stay >14 days and exposure to percutaneous devices were additional risk factors for CC22 colonization in the ACH and ILTCFs, respectively. Pre-emptive contact precautions for prior MRSA-carriers on admission and active screening for long-stayers in the ACH could prevent intra- and interinstitutional MRSA transmission.Funding: NoneDisclosures: None

The purine-derived signaling molecules c-di-AMP and (p)ppGpp control mecA/PBP2a-mediated β-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) raise the possibility that purine availability can control antibiotic susceptibility. Consistent with this, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis, were shown to significantly reduce MRSA β-lactam resistance. In contrast, adenosine (fluxed to ATP) significantly increased oxacillin resistance, whereas inosine (which can be fluxed to ATP and GTP via hypoxanthine) only marginally increased oxacillin susceptibility. Furthermore, mutations that interfere with de novo purine synthesis (pur operon), transport (NupG, PbuG, PbuX) and the salvage pathway (DeoD2, Hpt) increased β-lactam resistance in MRSA strain JE2. Increased resistance of a nupG mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which is required to reduce β-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including nupG and hpt. Guanosine significantly increased cell size and reduced levels of c-di-AMP, while inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on β-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants, suggesting that guanosine-induced β-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides, as β-lactam adjuvants that interfere with the normal activation of c-di-AMP are required for high-level β-lactam resistance in MRSA. IMPORTANCE The clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis. Predominantly, the safest and most effective class of antibiotics are the β-lactams, which are no longer effective against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report that the purine nucleosides guanosine and xanthosine have potent activity as adjuvants that can resensitize MRSA to oxacillin and other β-lactam antibiotics. Mechanistically, exposure of MRSA to these nucleosides significantly reduced the levels of the cyclic dinucleotide c-di-AMP, which is required for β-lactam resistance. Drugs derived from nucleotides are widely used in the treatment of cancer and viral infections highlighting the clinical potential of using purine nucleosides to restore or enhance the therapeutic effectiveness of β-lactams against MRSA and potentially other AMR pathogens.

Joint Healthcare Infection Society (HIS) and Infection Prevention Society (IPS) guidelines for the prevention and control of meticillin-resistant Staphylococcus aureus (MRSA) in healthcare facilities

Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolates from regional hospitals in Trinidad and Tobago

Sir, Triclosan (2,4,4′-trichloro-2′-hydroxydiphenyl ether) is a broad-spectrum biocide that has been used for more than 30 years and can be found in a variety of household items, including toothpastes, cosmetics, antiseptic soaps, carpets, toys, floor cleaners, dishwashing detergents and even chopsticks.1,2 More recently it has been used in the clinical setting as part of a regimen for decolonizing patients with methicillinresistant Staphylococcus aureus (MRSA).1,3 Triclosan acts on a range of cytoplasmic membrane and intracellular target sites. Increased resistance to triclosan has recently been attributed to an alteration in the fabI gene coding for enoyl reductase.1 Although patient non-compliance with the decolonizing regimen may be responsible for failure to eradicate MRSA, it is also possible that increased resistance to triclosan may contribute. In a recent study of a small number of clinical MRSA isolates, a triclosan MIC range of 0.025–1 mg/L was reported,3 whereas in an earlier study, MICs of 2–4 mg/L were reported for MRSA.4 We studied 232 clinical MRSA isolates referred to the Scottish MRSA Reference Laboratory from 30 Scottish hospitals between 1997 and 2000. The isolates were characterized by phenotypic and genotypic methods and included 14 different clones,5 the majority of which were EMRSA-15 (72) and EMRSA-16 (73). Triclosan (Irgasan, Ciba, UK) MIC determination was based on NCCLS methodology.6 Briefly, 107 cfu/mL were seeded on to Mueller–Hinton-II agar (Beckton Dickinson, UK) containing triclosan (0.015– 64 mg/L) and incubated at 37°C for 48 h. S. aureus (NCTC 12973) and Staphylococcus hominis (NCTC 11320) were used as low and high controls, respectively. For all tested MRSA, the triclosan MIC50 was 0.03 mg/L and MIC90 was 0.06 mg/L (range ≤0.015–4 mg/L). EMRSA-15 and EMRSA-16, which account for 70% and 26% of Scottish MRSA, respectively, both had a triclosan MIC50 of 0.03 mg/L but MIC90 of 0.06 mg/L (≤0.015–0.25 mg/L) and 0.03 mg/L (≤0.015–0.25 mg/L), respectively. Three SMRSA-99 (Scottish MRSA type 99) isolates, also known as the Iberian clone, two SMRSA-117 isolates and five sporadic isolates had triclosan MICs of 1–4 mg/L. This study does not support the contention that the widespread use of triclosan in various products will select for resistance in MRSA. It is significant that the two dominant UK epidemic strains (EMRSA-15 and EMRSA-16) were particularly susceptible to triclosan and this should encourage its use in decolonization regimes. However, our finding that some MRSA clones may have reduced susceptibility to triclosan highlights the need to be aware of such MRSA clones.

Virtually all methicillin-resistant Staphylococcus aureus (MRSA) infections in the largest Portuguese teaching hospital are caused by two internationally spread multiresistant strains: the ‘Iberian’ and the ‘Brazilian’ clones of MRSA

In September 2001 4 patients in the neonatal intensivecare unit (NICU) were culture-positive for methicillinresistant Staphylococcus aureus (MRSA) and this prompted an epidemiologic investigation. Initial screening of all 28 babies and 236 healthcare workers (HCWs) in the NICU demonstrated MRSA colonization of 11 additional infants and 6 HCWs. An enhanced infection control program was introduced. This included weekly nasal surveillance cultures of all NICU patients, cohorting and strict contact isolation of MRSA-positive babies, and introduction of a waterless hand hygiene gel. Designated HCWs cared for MRSA-positive babies. All staff underwent intensive reeducation. Molecular typing of the MRSA isolates revealed 3 MRSA clones (A, B, and G) in neonates and 6 MRSA clones (A-F) in HCWs. Transmission of the initial MRSA clone A was controlled 3 weeks after starting weekly surveillance cultures. Typing of subsequent MRSA isolates was suggestive of repeated reintroduction of different MRSA clones from the adjacent adult hospital. Further spread of these MRSA clones was controlled within 1 to 5 weeks. This experience demonstrates the benefit of active surveillance cultures and molecular typing as part of a comprehensive program to manage the spread of MRSA and limit infections from this important hospital pathogen.