Major Methicillin-resistant Staphylococcus Aureus Clones Research Articles

Genomic evolution of ST228 SCCmec-I MRSA 10 years after a major nosocomial outbreak.

In this study, we investigated the genomic changes in a major methicillin-resistant Staphylococcus aureus (MRSA) clone following a significant outbreak at a hospital. Whole-genome sequencing of MRSA isolates was utilized to explore the genomic evolution of post-outbreak MRSA strains. The epidemicity of the clone declined over time, coinciding with the introduction of multimodal infection control measures. A genome-wide association study (GWAS) identified multiple genes significantly associated with either high or low epidemic success, indicating alterations in mobilome, virulence, and defense mechanisms. Random Forest models pinpointed a gene related to fibrinogen binding as the most influential predictor of epidemicity. The decline of the MRSA clone may be attributed to various factors, including the implementation of new infection control measures, single nucleotide polymorphisms accumulation, and the genetic drift of a given clone. This research underscores the complex dynamics of MRSA clones, emphasizing the multifactorial nature of their evolution. The decline in epidemicity seems linked to alterations in the clone's genetic profile, with a probable shift towards decreased virulence and adaptation to long-term carriage. Understanding the genomic basis for the decline of epidemic clones is crucial to develop effective strategies for their surveillance and management, as well as to gain insights into the evolutionary dynamics of pathogen genomes.

Distribution of sasX, qacA/B and mupA genes and determination of genetic relatedness of methicillin-resistant Staphylococcus aureus among clinical isolates and nasal swab samples from the same patients in a hospital in Malaysia.

This study determined the distribution of sasX, qacA/B and mupA genes from methicillin-resistant Staphylococcus aureus (MRSA) isolated from clinical samples and nasal swab samples of the same patients and analysed their genetic relatedness. Polymerase chain reaction was used to detect the presence of sasX, qacA/B and mupA genes from 47 paired MRSA isolates. A paired isolate was defined as one nasal swab (colonising) isolate and clinical isolate that caused infection in the same patient. 22 selected paired isolates were subjected to multilocus sequence typing (MLST). The genetic relatedness among the isolates and association between the putative genes with epidemic sequence types (STs) were investigated. 7 (14.9%, n = 14) paired isolates were positive for the sasX gene. qacA/B genes were positive in 7.4% (n = 7) of the isolates, from three paired isolates and one clinical isolate whose paired colonising isolate was negative. The paired sample of three patients were positive for both genes. The mupA gene was not detected in all the isolates. MLST revealed two epidemic STs, ST22 and ST239, and a novel ST4649. sasX and qacA/B genes were found in ST239 in 29.5% (n = 13) and 13.6% (n = 6) of cases, respectively. Gene co-existence occurred in 13.6% (n = 6) of MRSA ST239 and 2.3% (n = 1) of MRSA ST4649. sasX and qacA/B genes were present in the MRSA isolates, while the mupA gene was undetected. ST22 and ST239 were the major MRSA clones. The circulating MRSA genotypes conferred different virulence and resistance determinants in our healthcare settings.

High prevalence of t895 and t9364 spa types of methicillin-resistant Staphylococcus aureus in a tertiary-care hospital in Mexico: different lineages of clonal complex 5

BackgroundStaphylococcus aureus is a leading cause of broad-spectrum infections both in the community and within healthcare settings. Methicillin-resistant Staphylococcus aureus (MRSA) has become a global public health issue. The aim of this study was to examine the clinical and molecular characteristics of Staphylococcus aureus isolates and to define the population structure and distribution of major MRSA clones isolated in a tertiary-care hospital in Mexico.ResultsFrom April 2017 to April 2018, 191 Staphylococcus aureus isolates were collected. The frequency of MRSA was 26.7%; these strains exhibited resistance to clindamycin (84.3%), erythromycin (86.2%), levofloxacin (80.3%), and ciprofloxacin (86.3%). The majority of MRSA strains harbored the SCCmec type II (76.4%) and t895 (56.8%) and t9364 (11.7%) were the most common spa types in both hospital-associated MRSA and community-associated MRSA isolates. ST5-MRSA-II-t895 (New York /Japan clone) and ST1011-MRSA-II-t9364 (New York /Japan-Mexican Variant clone) were the most frequently identified clones. Furthermore, different lineages of Clonal Complexes 5 (85.4%) and 8 (8.3%) were predominantly identified in this study.ConclusionOur study provides valuable information about the epidemiology of MRSA in a city of the central region of Mexico, and this is the first report on the association between t895 and t9364 spa types and ST5 and ST1011 lineages, respectively. These findings support the importance of permanent surveillance of MRSA aimed to detect the evolutionary changes of the endemic clones and the emergence of new strains.

Molecular Characterization of Methicillin Resistant Staphylococcus aureus in West Bank-Palestine.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a public health threat and a major cause of hospital-acquired and community-acquired infections. This study aimed to investigate the genetic diversity of MRSA isolates from 2015 to 2017 and to characterize the major MRSA clones and anti-biogram trends in Palestine.Methodology: Isolates were obtained from 112 patients admitted to different hospitals of West Bank and East Jerusalem, originating from different clinical sources. Antibiotic susceptibility patterns, staphylococcal chromosomal cassette mec (SCCmec) typing, and Staphylococcus aureus protein A (spa) typing were determined. Also, a panel of toxin genes and virulence factors was studied, including: Panton-Valentine Leukocidin (PVL), ACME-arcA, Toxic Shock Syndrome Toxin-1 (TSST-1), and Exfoliative Toxin A (ETA).Results: Of the 112 confirmed MRSA isolates, 100% were resistant to all β-lactam antibiotics. Resistance rates to other non- β-lactam classes were as the following: 18.8% were resistant to trimethoprim-sulfamethoxazole, 23.2% were resistant to gentamicin, 34.8% to clindamycin, 39.3% to ciprofloxacin, and 63.4% to erythromycin. All MRSA isolates were susceptible to vancomycin (100%). Of all isolates, 32 isolates (28.6%) were multidrug- resistant (MDR). The majority of the isolates were identified as SCCmec type IV (86.6%). The molecular typing identified 29 spa types representing 12 MLST-clonal complexes (CC). The most prevalent spa types were: spa type t386 (CC1)/(12.5%), spa type t044 (CC80)/(10.7%), spa type t008 (CC8)/(10.7%), and spa type t223 (CC22)/(9.8%). PVL toxin gene was detected in (29.5%) of all isolates, while ACME-arcA gene was present in 18.8% of all isolates and 23.2% had the TSST-1 gene. The two most common spa types among the TSST-1positive isolates were the spa type t223 (CC22)/(Gaza clone) and the spa type t021 (CC30)/(South West Pacific clone). All isolates with the spa type t991 were ETA positive (5.4%). USA-300 clone (spa type t008, positive for PVL toxin gene and ACME-arcA genes) was found in nine isolates (8.0%).Conclusions: Our results provide insights into the epidemiology of MRSA strains in Palestine. We report a high diversity of MRSA strains among hospitals in Palestine, with frequent SCCmec type IV carriage. The four prominent clones detected were: t386-IV/ CC1, the European clone (t044/CC80), Gaza clone (t223/CC22), and the USA-300 clone (t008/CC8).

Burden of Rifampicin- and Methicillin-Resistant Staphylococcus aureus in Italy.

Rifampicin is one of the major drugs used on its own and also in combination to treat numerous infections sustained by methicillin-resistant Staphylococcus aureus (MRSA). In Italy, rifampicin resistance (RIF-R) is increasing in multidrug-resistant-MRSA isolates (16.4%), with respect to Europe (5.7%). In our study, the relationship between clones, rpoB mutations, and susceptibility profiles in 50 RIF-R MRSA isolated from hospitalized patients was evaluated. Antimicrobial susceptibility testing was performed by the broth microdilution method. Isolates were typed by MLST/SCCmec/spa-typing. The rpoB gene was analyzed by PCR and sequence analysis. RIF-R isolates were 60% heterogeneous vancomycin-intermediate S. aureus (hVISA) and 22% daptomycin nonsusceptible and belonged to the major MRSA clones: ST228-SCCmec I (44%), ST8-SCCmec IV (18%), ST239-SCCmec III (16%), ST5-SCCmec II (14%), and ST22-SCCmec IVh (4%). Thirteen diverse RpoB amino acid substitutions were identified. Half of the strains harbored the H481N substitution, conferring low-level resistance. Different single mutations at the equivalent locus (H481D; H481Y) or in other loci, and multiple mutations conferred high-level resistance. In conclusion, this study investigated the nature of RIF-R in Italy among RIF-R-MRSA strains, finding a prevalence of ST228, strongly associated with reduced susceptibility to glycopeptides (hVISA). The spread of RIF-R strains in clinical settings represents a serious threat, due to their complex resistance nature even to new anti-Gram-positive drugs, making these infections particularly difficult to treat.

Small Colony Variants and Triclosan Resistance in Five International Clones of Methicillin Resistant Staphylococcus aureus

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.

Methicillin-Resistant Staphylococcus aureus in the Community in Luanda, Angola: Blurred Boundaries with the Hospital Setting.

Although the nosocomial prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Angola is over 60% and one of the highest in Africa, the extent of MRSA in the community is unknown. To fill this gap, we conducted a hospital-based study in which 158 children attending the emergency ward and ambulatory services of a pediatric hospital in Luanda, the capital of Angola, were screened for S. aureus nasal colonization. Overall, 70 (44.3%) individuals were colonized with S. aureus, of which 20 (28.6%) carried MRSA, resulting in a prevalence of 12.7% (20/158) of MRSA in the population screened. Molecular characterization by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing, and SCCmec typing distributed the isolates into two major MRSA clones and one dominant methicillin-susceptible S. aureus (MSSA) lineage, corresponding to the main clones circulating in hospitals in Luanda. The MRSA isolates mainly belonged to clones A (PFGE type A, spa type t105, ST5-IVa-65%) and B (PFGE B, t3869, ST88-IVa-30%), while MSSA isolates mainly belonged to clone L (PFGE type L, t861, ST508-42%). S. aureus isolates showed resistance to penicillin (96%), rifampin (87%), and trimethoprim-sulfamethoxazole (21%). In conclusion, the prevalence of MRSA among children in the community in Luanda is high and seems to originate from hospitals, warranting continuous monitoring and implementation of additional infection control measures.

Methicillin‐resistant Staphylococcus aureus carriage among veterinary staff and dogs in private veterinary clinics in Hokkaido, Japan

To explore the prevalence and molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) in veterinary medical practices, MRSA carriage was tested among 96 veterinarians (Vets), 70 veterinary technicians (VTs) and 292 dogs with which they had contact at 71 private veterinary clinics (VCs) in Hokkaido, Japan. MRSA isolates were obtained from 22 Vets [22.9%] and 7 VTs [10%]. The prevalence of MRSA among Vets was as high as that found in an academic veterinary hospital in our previous study. In contrast, only two blood donor dogs and one dog with liver disease (1.0%, 3/292) yielded MRSA. All MRSA-positive dogs were reared or treated in different VCs, in each of which at least one veterinary staff member carrying MRSA worked. Sequence types (ST) identified by multilocus sequence typing, spa types, and SCCmec types for canine MRSA isolates (ST5-spa t002-SCCmec II [from two dogs] or ST30-spa t021-SCCmec IV [from a dog]) were concordant with those from veterinary staff members in the same clinics as the MRSA-positive dogs, with which they had potentially had contact. Most MRSA isolates from veterinary staff were the same genotype (SCCmec type II and spa type t002) as a major hospital-acquired MRSA clone in Japan. The remaining MRSA was the same genotypes as domestic and foreign community-associated MRSA. Measures against MRSA infection should be provided in private VCs.

Infectivity-Resistotype-Genotype Clustering of Methicillin-Resistant Staphylococcus aureus Strains in the Central Blacksea Region of Turkey

The increase in the prevalence of epidemic strains of methicillin resistant Staphylococcus aureus (MRSA) in hospitals and community requires special attention of infection control. The aim of this study was to determine the pathogenic phenotype (i.e. infectivity and resistotype) and genotypic characteristics (i.e. PFGE-pulsotyping, SLST-spa typing, MLST-sequence typing, eBURST-clonal complex detection algorithm) of clinical MRSA isolates in the Central Blacksea region of Turkey, in order to understand their short- and long-term epidemiological and evolutionary dynamics, and to investigate any probable presence of a significant clustering. This prospective study included consecutive but non-repetitive 48 MRSA isolates (of them 18 were colonized strains and 30 were causes of nosocomial infection) and seven methicillin-susceptible S.aureus (MSSA, all were isolated from nosocomial infection), collected between December 2006-February 2007 period from hospitalized patients. Identification of the isolates were performed by Vitek-2 automated system (BioMérieux, USA), and in vitro antimicrobial susceptibility testing by broth microdilution method and Vitek-2 automated system. The MRSA isolates found susceptible to erythromycin (n= 10) were further investigated for the presence of ermA gene by the PCR method. All the strains were typed by spa-typing and PFGE-pulsotyping methods. Among the isolates with different spa-types, representatives were selected (3 MRSA, 7 MSSA) and typed with MLST typing method. Among the isolates with different spa-types, representatives with different antimicrobial susceptibility patterns were selected (n= 8), and SCCmec types were determined by the multiplex PCR method. Antimicrobial resistance patterns of the isolates were digitized to get standardized antimicrobial resistance phenotypes. Clustering of MRSA isolates in pattern groups on the basis of discriminatory characteristics, namely infectivity, phenotype and genotype were statistically analyzed with specific inclusion and exclusion criteria. As a result, three different antimicrobial resistance phenotypes were found in MSSA isolates, whereas 13 were identified in MRSA isolates. In MSSA isolates, seven different PFGE-pulsotypes were detected, as compared to 14 pulsotypes in MRSA isolates. Among MRSA isolates, 10 sporadic strains with single PFGE-pulsotypes were detected. All MRSA isolates, with two exceptions (t459, t632), were of t030 spa-type; in the MLST analysis of the representatives of different spa-types (n= 3), a single type of MLST-clonal complex (CC8) and single MLST-sequence type (ST239) were identified. Each of the seven MSSA isolates yielded different spa-types, MLST-clonal complex types and MLST-sequence types (t777-ST5-CC5; t660-ST25-CC5; t153-ST34-CC30; t015-ST45-CC45; t267-ST97-CC97; t377-ST360-CC8; t084-ST15-C15). In the statistical analysis of 38 non-sporadic MRSA isolates, the isolates in Group-13 (n= 16; infectious, resistotype 14, pulsotype 4; antimicrobial resistance score= 24) displayed significant infectivity-phenotype-genotype clustering (p< 0.001). In 27 of the MRSA isolates, decreased susceptibility to teicoplanin (MIC= 4 µg/mL) was detected. Although, global MRSA isolates belonging to MLST-CC8, MLST-ST239, t030 spa-type were usually expected to be resistant to erythromycin, 10 such strains were erythromycin susceptible. However, ermA gene was found in six of these 10 strains, leading to a conclusion that the ermA gene of these isolates might be dysfunctional due to a point mutation or deletion. Selected representatives of MRSA isolates with different antimicrobial susceptibility patterns (n= 8) were detected to be SCCmec type III. In conclusion, S.aureus isolates in the patient population of our hospital representing the Central Blacksea region showed statistically significant clustering in infectivity, antimicrobial resistance phenotype and clonal genotype (p< 0.001). The dominant MRSA clone was ST239 which was one of the five major pandemic MRSA clones. Nosocomial MSSA isolates displayed long-term clonal diversity. This study produced regional evolutionary-epidemiological data that may support further regional, national and international long-term surveillance studies of S.aureus strains.

Molecular characteristics of Staphylococcus aureus isolated from a major hospital in Lebanon

The occurrence and dissemination of methicillin-resistant Staphylococcus aureus (MRSA) in healthcare settings and the community and its risk of being introduced into hospitals are matters of great concern. The purpose of this study was to conduct a miniaturized epidemiological analysis of S. aureus-associated infections and to characterize the isolates by a variety of molecular typing techniques. Ongoing molecular surveillance is essential to prevent S. aureus strains from becoming endemic in the Lebanese healthcare setting. A total of 132 S. aureus from different clinical specimens were isolated over a 6-month period. Characterization of the isolates was done by detection of the mecA gene, Panton-Valentine leukocidin determinant detection, staphylococcal chromosomal cassette (SCCmec) typing of MRSA, S. aureus protein A (spa) typing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and antibiogram analysis. MRSA represented 30% of the isolates, with PVL being detected in 54% of MRSA and 12% of methicillin-susceptible S. aureus (MSSA). A difference between MRSA and MSSA was observed in the spa types. Clustering SCCmec with MLST identified seven MRSA and 20 MSSA clones, with PVL-positive ST80-MRSA-IV being the dominant clone (7%), while PFGE revealed 32 groups with 80% cutoff similarity. Although the results of this study are based on samples collected from one hospital, the high diversity observed along with the lack of any equivalence in the genetic backgrounds of the major MSSA and MRSA clones, emphasizes the urgent need for standardized surveillance combined with the application of well-validated typing methods to assess the occurrence of MRSA and subsequently to control its spread.

Massive Dissemination of Methicillin ResistantStaphylococcus aureusin Bloodstream Infections in a High MRSA Prevalence Country: Establishment and Diversification of EMRSA-15

Portugal is the European country with the highest prevalence of methicillin resistant Staphylococcus aureus (MRSA), in which EMRSA-15 (ST22-IVh) has been the dominant clone since soon after its introduction in Portuguese hospitals in 2001. In this study, we intend to not only, assess the evolution of the invasive MRSA in Portuguese hospitals, but also to evaluate the invasive methicillin susceptible S. aureus (MSSA) population and the relationship between both populations. In the current study, two major MRSA clones were identified: EMRSA-15 that has been dominant for more than 10 years and accounts for 75% of the MRSA isolates, and ST105-II, a clone related with the New York/Japan clone (ST5-II). In contrast, among MSSA, several clonal backgrounds were identified. Despite of the massive predominance of EMRSA-15 in the last decade, an increase in spa diversity has been observed in the last few years, which suggests a recent and local diversification of this clone. Interestingly, MRSA and MSSA populations with related clonal backgrounds appear to have increased as a result of the dissemination of MRSA to the community environment.

Genotype diversity of methicillin-resistant Staphylococcus aureus in Shenyang, China

The aim of this study was to better understand methicillin-resistant Staphylococcus aureus (MRSA) at the molecular level by investigating the genotypic characteristics and evolutionary patterns of MRSA clones in Shenyang, China. We analyzed the molecular epidemiology of 60 MRSA isolates in Shenyang, China, between 2002 and 2008, using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, and S. aureus protein A (spa) typing. They were examined for their antimicrobial susceptibilities. Among the 60 isolates, ST239 was identified most frequently (34 isolates; 58%), followed by ST5 (20 isolates; 34%). Nine spa types were obtained and 4 PFGE strain families (A, B, C, and D) were resolved. Spa type t030, which corresponded to PFGE genotypes A1, A3, and A4, constituted 45% (27/60) of all isolates; spa type t037, which corresponded to PFGE type A2, accounted for 13% (8/60) of all isolates. These 2 spa genotypes belonged to ST239 and carried SCCmec type III. Isolates genotyped as spa type t002 comprised 27% (16/60) of the study set and included isolates typed as PFGE B1 and B2, ST5, and SCCmec II. Most of MRSA isolates belonging to ST239 were susceptible to trimethoprim-sulfamethoxazole. The minimum inhibitory concentration (MIC50) of vancomycin among MRSA isolates belonging to ST5 (2 mg/l) was higher than that for other isolates (1 mg/l). These data document 2 major epidemic MRSA clones in Shenyang, China: ST239-MRSA-SCCmec type III-t037/t030 and ST5-MRSA-SCCmec type II-t002.

Major West Indies MRSA Clones in Human Beings: Do They Travel With Their Hosts?

Descriptions of the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) have seldom been produced in the Caribbean, which is a major tourism destination. Using DNA microarrays and spa typing, we characterized 85 MRSA isolates from human skin and soft-tissue infections from five different islands. In the French West Indies (n = 72), the most frequently isolated clones were the same clones that are specifically isolated from mainland France [Lyon (n = 35) and Geraldine (n = 11) clones], whereas the clones that were most frequently isolated from the other islands (n = 13) corresponded with clones that have a worldwide endemic spread [Vienna/Hungarian/Brazilian (n = 5), Panton Valentine leukocidin-positive USA300 (n = 4), New York/Japan (n = 2), and pediatric (n = 1) clones]. The distribution of the major MRSA clones in the French (Guadeloupe and Martinique) and non-French West Indies (Jamaica, Trinidad, and Tobago) is different, and the clones most closely resemble those found in the home countries of the travelers who visit the islands most frequently. The distribution might be affected by tourist migration, which is specific to each island.

Molecular Typing and Virulence Characteristic of Methicillin-Resistant Staphylococcus Aureus Isolates from Pediatric Patients in Bucaramanga, Colombia

Background Staphylococcus aureus is among the most common global nosocomial pathogens. The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) is a public health problem worldwide that causes nosocomial and community infections. The goals of this study were to establish the clonal complexes (CC) of the isolates of MRSA obtained from pediatric patients in a university hospital in Colombia and to investigate its molecular characteristics based on the virulence genes and the genes of staphylococcal toxins and adhesins.MethodsA total of 53 MRSA isolates from pediatric patients with local or systemic infections were collected. The MRSA isolates were typed based on the SCCmec, MLST, spa and agr genes. The molecular characterization included the detection of Panton-Valentine Leukocidin, superantigenic and exfoliative toxins, and adhesin genes. The correlation between the molecular types identified and the profile of virulence factors was determined for all isolates.ResultsFour CC were identified, including CC8, CC5, CC80 and CC78. The ST8-MRSA-IVc-agrI was the predominant clone among the isolates, followed by the ST5-MRSA-I-agrII and ST5-MRSA-IVc-agrII clones. Twelve spa types were identified, of which t10796 and t10799 were new repeat sequences. The isolates were carriers of toxin genes, and hlg (100%), sek (92%) and pvl (88%) were the most frequent. Ten toxin gene profiles were observed, and the most frequent were seq-sek-hlg (22.6%), sek-hlg (22.6%), seb-seq-sek-hlg (18.9%) and seb-sek-hlg (15.1%). The adhesion genes were present in most of the MRSA isolates, including the following: clf-A (89%), clf-B (87%), fnb-A (83%) and ica (83%). The majority of the strains carried SCCmec-IVc and were identified as causing nosocomial infection. No significant association between a molecular type and the virulence factors was found.ConclusionFour major MRSA clone complexes were identified among the isolates. ST8-MRSA-IVc-agrI pvl+ (USA300-LV) was the most frequent, confirming the presence of community-associated MRSA in Colombian hospitals.

Diversity of Methicillin-Resistant Staphylococcus aureus (MRSA) Strains Isolated from Inpatients of 30 Hospitals in Orange County, California

There is a need for a regional assessment of the frequency and diversity of MRSA to determine major circulating clones and the extent to which community and healthcare MRSA reservoirs have mixed. We conducted a prospective cohort study of inpatients in Orange County, California, systematically collecting clinical MRSA isolates from 30 hospitals, to assess MRSA diversity and distribution. All isolates were characterized by spa typing, with selective PFGE and MLST to relate spa types with major MRSA clones. We collected 2,246 MRSA isolates from hospital inpatients. This translated to 91/10,000 inpatients with MRSA and an Orange County population estimate of MRSA inpatient clinical cultures of 86/100,000 people. spa type genetic diversity was heterogeneous between hospitals, and relatively high overall (72%). USA300 (t008/ST8), USA100 (t002/ST5) and a previously reported USA100 variant (t242/ST5) were the dominant clones across all Orange County hospitals, representing 83% of isolates. Fifteen hospitals isolated more t008 (USA300) isolates than t002/242 (USA100) isolates, and 12 hospitals isolated more t242 isolates than t002 isolates. The majority of isolates were imported into hospitals. Community-based infection control strategies may still be helpful in stemming the influx of traditionally community-associated strains, particularly USA300, into the healthcare setting.

Molecular tracing of the emergence, adaptation, and transmission of hospital-associated methicillin-resistant Staphylococcus aureus.

Hospital-associated infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a global health burden dominated by a small number of bacterial clones. The pandemic EMRSA-16 clone (ST36-II) has been widespread in UK hospitals for 20 y, but its evolutionary origin and the molecular basis for its hospital association are unclear. We carried out a Bayesian phylogenetic reconstruction on the basis of the genome sequences of 87 S. aureus isolates including 60 EMRSA-16 and 27 additional clonal complex 30 (CC30) isolates, collected from patients in three continents over a 53-y period. The three major pandemic clones to originate from the CC30 lineage, including phage type 80/81, Southwest Pacific, and EMRSA-16, shared a most recent common ancestor that existed over 100 y ago, whereas the hospital-associated EMRSA-16 clone is estimated to have emerged about 35 y ago. Our CC30 genome-wide analysis revealed striking molecular correlates of hospital- or community-associated pandemics represented by mobile genetic elements and nonsynonymous mutations affecting antibiotic resistance and virulence. Importantly, phylogeographic analysis indicates that EMRSA-16 spread within the United Kingdom by transmission from hospitals in large population centers in London and Glasgow to regional health-care settings, implicating patient referrals as an important cause of nationwide transmission. Taken together, the high-resolution phylogenomic approach used resulted in a unique understanding of the emergence and transmission of a major MRSA clone and provided molecular correlates of its hospital adaptation. Similar approaches for hospital-associated clones of other bacterial pathogens may inform appropriate measures for controlling their intra- and interhospital spread.

Methicillin Resistance Reduces the Virulence of Healthcare-Associated Methicillin-Resistant Staphylococcus aureus by Interfering With the agr Quorum Sensing System

The difficulty in successfully treating infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has led to them being referred to as highly virulent or pathogenic. In our study of one of the major healthcare-associated MRSA (HA-MRSA) clones, we show that expression of the gene responsible for conferring methicillin resistance (mecA) is also directly responsible for reducing the ability of HA-MRSA to secrete cytolytic toxins. We show that resistance to methicillin induces changes in the cell wall, which affects the bacteria's agr quorum sensing system. This leads to reduced toxin expression and, as a consequence, reduced virulence in a murine model of sepsis. This diminished capacity to cause infection may explain the inability of HA-MRSA to move into the community and help us understand the recent emergence of community-associated MRSA (CA-MRSA). CA-MRSA typically express less penicillin-binding protein 2a (encoded by mecA), allowing them to maintain full virulence and succeed in the community environment.

High prevalence of EMRSA-15 in Portuguese public buses: a worrisome finding.

BackgroundThe nosocomial prevalence of methicillin resistant Staphylococcus aureus (MRSA) in Portugal remains one of the highest in Europe and is currently around 50%. Transmission of S. aureus, including MRSA, occurs principally by direct human-to-human skin contact. However, S. aureus can survive for long periods on inanimate objects, which may represent an important reservoir for dissemination as well.Methodology/Principal FindingsBetween May 2009 and February 2010, handrails of 85 public urban buses circulating in Oporto, Portugal, were screened for the occurrence of MRSA. Twenty-two (26%) buses showed MRSA contamination. The molecular characterization of a total of 55 MRSA, by pulsed-field gel electrophoresis (PFGE), staphylococcal cassette chromosome (SCC) mec typing, spa typing, and multilocus sequence typing (MLST), clustered the isolates into three clonal types. However, the overwhelming majority (n = 50; 91%) of the isolates belonged to a single clone (PFGE A, spa types t747, t032, t025 or t020, ST22, SCCmec type IVh) that exhibits the characteristics of the pandemic EMRSA-15, currently the major lineage circulating in Portuguese hospitals, namely in the Oporto region. Two additional clones were found but in much lower numbers: (i) PFGE B, ST5, spa type t002, SCCmec IVa (n = 3), and (ii) PFGE C, spa type t008, ST8, SCCmec IVa (n = 2). None of the 55 isolates was PVL positive.Conclusions/SignificancePublic buses in Oporto seem to be an important reservoir of MRSA of nosocomial origin, providing evidence that the major hospital-associated MRSA clone in Portugal is escaping from the primary ecological niche of hospitals to the community environment. Infection control measures are urgently warranted to limit the spread of EMRSA-15 to the general population and future studies are required to assess the eventual increase of MRSA in the Portuguese community, which so far remains low.

Usefulness of double locus sequence typing (DLST) for regional and international epidemiological surveillance of methicilin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infections worldwide. To differentiate reliably among S. aureus isolates, we recently developed double locus sequence typing (DLST) based on the analysis of partial sequences of clfB and spa genes. In the present study, we evaluated the usefulness of DLST for epidemiological investigations of MRSA by routinely typing 1242 strains isolated in Western Switzerland. Additionally, particular local and international collections were typed by pulsed field gel electrophoresis (PFGE) and DLST to check the compatibility of DLST with the results obtained by PFGE, and for international comparisons. Using DLST, we identified the major MRSA clones of Western Switzerland, and demonstrated the close relationship between local and international clones. The congruence of 88% between the major PFGE and DLST clones indicated that our results obtained by DLST were compatible with earlier results obtained by PFGE. DLST could thus easily be incorporated in a routine surveillance procedure. In addition, the unambiguous definition of DLST types makes this method more suitable than PFGE for long-term epidemiological surveillance. Finally, the comparison of the results obtained by DLST, multilocus sequence typing, PFGE, Staphylococcal cassette chromosome mec typing and the detection of Panton-Valentine leukocidin genes indicated that no typing scheme should be used on its own. It is only the combination of data from different methods that gives the best chance of describing precisely the epidemiology and phylogeny of MRSA.

Tracking methicillin-resistant Staphylococcus aureus clones during a 5-year period (1998 to 2002) in a Spanish hospital.

Three hundred seventy-five consecutive methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates recovered between 1998 and 2002 at the Nuestra Señora de Candelaria University Hospital in Tenerife, Spain, were analyzed by molecular fingerprinting techniques to determine MRSA clonal types and their prevalence over time. After determining antibiotic susceptibility, we used SmaI-digested genomic DNA separated by pulsed-field gel electrophoresis (PFGE) to characterize MRSA isolates and to establish PFGE types. Additionally, several selected isolates representative of each major PFGE type were tested by multilocus sequence typing (MLST) and by a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome mec (SCCmec), generating the corresponding sequence type (ST)-SCCmec types. Results of PFGE, supported by those of MLST and SCCmec typing, allowed us to identify six MRSA clones within the five major PFGE types and document temporal shifts in the prevalence of these MRSA clones from 1998 to 2002. Four of the clones were the pandemic "Iberian" (designated ST247-MRSA-IA), EMRSA-15 (ST22-MRSA-IV), EMRSA-16 (ST36-MRSA-II), and the so-called pediatric (ST5-MRSA-IV) clones, while the other two (ST125-MRSA-IVA and ST146-MRSA-IVA) clones could be derived from the pediatric one. The most striking temporal shift in the dominance of MRSA clones was the replacement of the multidrug-resistant and highly epidemic Iberian clone by the so-called British EMRSA-16 clone during the 5-year surveillance period. Our results are in accordance with previously stated findings showing the worldwide hospital dominance of relatively few pandemic and presumably virulent MRSA clones. We report for the first time the detection in Spain of the British EMRSA-15 and pediatric clones, as well as the abrupt replacement of the Iberian by the EMRSA-16 as the major MRSA clone.