Diversity of Staphylococcus aureus Strains Isolated from Two European Regions with Different Prevalences of Methicillin Resistance

The genodiversity of Staphylococcus aureus isolates from the Nottingham region of the United Kingdom was compared with isolates from the Freiburg region of Germany. The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) isolates was higher in Nottingham than in Freiburg. In patients from Nottingham hospitals, 80% of MRSA isolates were classical epidemic MRSA-15, but genotypic variants of epidemic MRSA-15 comprised 72% of isolates from Nottingham community-based patients. In contrast, MRSA isolates from Freiburg showed greater diversity, but 47% and 23% of isolates, respectively, belonged to two predominant MRSA genotypes found in isolates from both hospitalised and community-based patients. The results suggest that genodiversity becomes increasingly more confined in settings with a higher frequency and longer duration of MRSA prevalence. Electronic Publication     

Typing of Panton-Valentine leukocidin-encoding phages carried by methicillin-susceptible and methicillin-resistant Staphylococcus aureus from Italy

Panton-Valentine leukocidin (PVL) is the hallmark of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) but can also be found in methicillin-susceptible S. aureus (MSSA) sharing pathogenic and epidemiological characteristics of CA-MRSA. PVL is encoded by two co-transcribed genes that are carried by different staphylococcal bacteriophages. We applied an extended PCR-based typing scheme for the identification of two morphological groups (elongated-head group and icosahedral-head group I phages) and specific PVL phage types in S. aureus isolates recovered in Italy. We examined 48 PVL-positive isolates (25 MSSA and 23 MRSA) collected from different hospital laboratories from April 2005 to May 2011. spa typing, multilocus sequence typing and staphylococcal cassette chromosome mec typing were applied to categorize the isolates. Phage typeability was 48.0% in MSSA and 91.3% in MRSA, highlighting the limitation of the PCR typing scheme when applied to PVL-positive MSSA. Five different PVL phages and two variants of a known phage were detected, the most prevalent being ΦSa2usa, recovered in 15 out of 48 (31.2%) isolates, and carried by both MSSA and MRSA belonging to CC8 and CC5. The recently described ΦTCH60 was recovered in four isolates. A PVL phage (ΦSa119) from an ST772 MRSA, that was not detected using the previous typing scheme, was sequenced, and new primers were designed for the identification of the icosahedral-head group II PVL phages present in ST772 and ST59 MRSA. A comprehensive PVL-phage typing can contribute to the understanding of the epidemiology and evolution of PVL-positive MSSA and MRSA.     

Use of Vitek 2 Antimicrobial Susceptibility Profile To Identify mecC in Methicillin-Resistant Staphylococcus aureus

The emergence of mecC methicillin-resistant Staphylococcus aureus (MRSA) poses a diagnostic challenge for clinical microbiology laboratories. Using the Vitek 2 system, we tested a panel of 896 Staphylococcus aureus isolates and found that an oxacillin-sensitive/cefoxitin-resistant profile had a sensitivity of 88.7% and a specificity of 99.5% for the identification of mecC MRSA isolates. The presence of the mecC gene, determined by bacterial whole-genome sequencing, was used as the gold standard. This profile could provide a zero-cost screening method for identification of mecC-positive MRSA strains.     

Genotyping and antimicrobial resistance of Staphylococcus aureus isolates from diseased turkeys

Staphylococcus aureus is a highly versatile pathogen in a large number of domestic animals, including avian species. To gain deeper insight into the epidemiology and diversity of S. aureus associated with articular disease in domestic turkeys, isolates were collected from infected foot joints of turkeys in Brittany (France). A total of 34 isolates were recovered and characterized by means of antimicrobial resistance, staphylococcal protein A typing, macrorestriction pulsed-field gel electrophoresis and micro-array analysis. Thirty isolates were identified as clonal complex (CC) 398 and methicillin-susceptible S. aureus (MSSA), one was identified as a methicillin-resistant S. aureus (MRSA) CC398 isolate, and the remaining were also MSSA and belonged to CC5, CC101, and CC121. Eleven different antimicrobial resistance patterns were detected, with most isolates resistant to penicillin and tetracycline. Based on all typing methods used, the 34 isolates could be divided into 22 different strains. Results on selected isolates, genotyped using microarrays, indicated a high homogeneity among pathogenic MSSA isolates from turkeys. Moreover, all isolates, except the unique MRSA isolate, carried specific φAvβ prophage avian-niche-specific genes, demonstrating the versatility of S. aureus to adapt to the specific ecological poultry niche.     

Evaluation of the TPX MRSA assay for the detection of methicillin-resistant Staphylococcus aureus

The aim of this study was to evaluate a new type of assay for the phenotypic detection of methicillin-resistant Staphylococcus aureus (MRSA). The assay is based on a point-of-care compatible two-photon excitation fluorescence detection technology (TPX). A collection of 243 epidemic MRSA isolates was tested in addition to 138 sporadic MRSA and 101 negative control strains. The assay proved to be both sensitive (97.9%) and specific (94.1%) in the identification of MRSA, with adequate positive (98.4%) and negative (92.2%) predictive values. The time required for obtaining a positive test result was less than 14 h for 99.0% of the MRSA true-positive samples. After a test run, the selectively enriched reaction mixtures may be recovered and further studied by molecular or standard phenotypic methods. The main benefits of the TPX methodology include a simple assay procedure, low reagent consumption, and a high-throughput capacity.     

European multicentre evaluation of a commercial system for identification of methicillin-resistantStaphylococcus aureus

A commercial system for the rapid detection of methicillin-resistantStaphylococcus aureus, the BBL Crystal MRSA test (C-MRSA ID; Becton Dickinson, USA), was evaluated prospectively and compared with a polymerase chain reaction test for the presence of themecA gene. Ten European centres tested a total of 676 isolates ofStaphylococcus aureus from blood cultures. The system correctly identified 661 (97.8%) isolates within 4 h. All but threemecA gene-negative isolates (99.4% specificity) yielded a negative C-MRSA ID reaction, and 158 of 170mecA gene-positive isolates were accurately detected (92.9% sensitivity). After repeated testing of discrepant results, sensitivity and specificity increased to 99% and 100%, respectively.     

Latex agglutination for rapid identification of methicillin-resistantStaphylococcus aureus recovered from selective media

The accuracy of combining latex agglutination with selective media for the identification of methicillin-resistantStaphylococcus aureus (MRSA) was determined. Test strains were identified by latex agglutination on blood agar, the heat-stable thermonuclease test and broth microdilution MICs of oxacillin and included 97 MRSA, 56 methicillin-susceptibleStaphylococcus aureus, 52 methicillin resistant, and 49 methicillin-susceptibleStaphylococcus species. Isolates were grown on trypticase-soy agar with 5 % sheep red blood cells (TSAB), Mueller-Hinton agar (MHA), mannitol-salt agar (MSA), and four media designed for the selective growth of MRSA: TSAB with clindamycin and gentamicin, MHA with oxacillin, MSA with oxacillin, and lipovitellin-salt-mannitol agar (LVSM) with 1 µg oxacillin disks applied. The mean sensitivity, specificity, and positive predictive value for the combination of latex agglutination with selective media for the identification of MRSA was 96 %, 99 % and 98 % respectively.     

High prevalence of ST121 in community-associated methicillin-susceptible Staphylococcus aureus lineages responsible for skin and soft tissue infections in Portuguese children

In order to evaluate the incidence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Portugal, we analyzed a collection of 38 S. aureus isolates recovered from 30 children attending the pediatric emergency department of a central hospital in Lisbon due to skin and soft tissue infections. Molecular characterization identified seven clonal lineages among the 35 methicillin-susceptible S. aureus (MSSA) isolates, of which the major lineage PFGE A/t159/ST121 included 63% of the isolates. The three MRSA isolates belonged to the Pediatric clone PFGE D/t535/ST5-IV (n = 2) and to the European CA-MRSA clone PFGE G/t044/ST80-IVc (n = 1). All isolates harbored several virulence factors, namely, leukocidins. Panton–Valentine leukocidin (PVL) was produced by isolates from five MSSA lineages and by the ST80 MRSA. Of interest, this is the first reported isolation of CA-MRSA ST80 in Portugal.     

Clonal heterogeneity, distribution, and pathogenicity of methicillin-resistantStaphylococcus aureus

Four thousand eighty-eightStaphylococcus aureus isolates obtained from patients hospitalised in a university clinic and four community hospitals over a period of one year were screened for methicillin resistance. A resistance rate of 5% was detected among initial isolates. Distribution of methicillin-resistantStaphylococcus aureus (MRSA) and methicillin-sensitiveStaphylococcus aureus showed an increased prevalence of MRSA in clinically significant specimens such as blood, central venous catheter tips, bronchial secretions, and wound secretions. Typing of 110 MRSA strains (initial isolates) by macrorestriction analysis of chromosomal DNA revealed 26 different genotypes that could be divided into five epidemic and 21 sporadic strains. More than 50% of all isolates belonged to one type that was confirmed to be closely related to the southern-German epidemic strain. Production of virulence factors such as enterotoxin A-D and toxic shock syndrome-toxin 1 among MRSA strains (initial isolates) occurred in ten of 26 different MRSA types. A strong correlation between genotype and toxin production was demonstrated.     

Methicillin-ResistantStaphylococcus aureus in Europe

In order to obtain pan-European data on methicillin-resistantStaphylococcus aureus (MRSA), 43 laboratories from ten European countries each screened 200 consecutiveStaphylococcus aureus isolates for methicillin resistance. Only one isolate per patient was permitted. All participants used a uniform oxacillin-supplemented screening plate. MRSA isolates were sent to Munich for reconfirmation and further susceptibility testing. Phage typing of the MRSA strains was performed in Denmark. Of the 7,333Staphylococcus aureus strains screened, 936 (12.8%) were methicillin resistant. The proportion of MRSA in the various European countries ranged from <1% in Scandinavia to > 30% in Spain, France and Italy. Rates of resistance to the non-glycopeptide antibiotics were lowest for rifampin and highest for ciprofloxacin. Sixty percent of the methicillin-resistant strains originated from patients in surgical and medical departments, with wounds being the most common isolation source. MRSA was found more frequently in intensive care patients. Only 13% of the strains were non-typable, and 76% of the isolates belonged to phage group III. For each area phage typing detected one or a few dominating (epidemic) types, but 46% of the strains did not belong to these types; the MRSA population is thus a mixture of epidemic and non-epidemic strains. MRSA seems to be a growing problem, especially in southern Europe, where incidence and rates of antibiotic resistance are alarmingly high.     

Detection of borderline oxacillin-resistantStaphylococcus aureus and differentiation from methicillin-resistant strains

Eighty-eightStaphylococcus aureus clinical isolates meeting criteria for borderline oxacillin resistance (intermediate susceptibility or resistance to oxacillin but susceptibility to amoxicillin/clavulanic acid upon disk diffusion testing) were studied to determine optimal test techniques and conditions for differentiating borderline oxacillin-resistantStaphylococcus aureus (BORSA) from methicillin-resistantStaphylococcus aureus (MRSA). Further testing revealed three distinct resistance patterns: 61 strains (69 %) consistently met BORSA criteria and had average beta-lactamase levels five- to six-fold higher than oxacillin-susceptible controls; 11 strains (13 %) were markedly heteroresistant MRSA with delayed appearance of resistant colonies leading to spurious susceptibility to amoxicillin/clavulanic acid; 16 strains (18 %) appeared to be oxacillin-susceptible on repetitive testing. Under conditions used to elicit intrinsic methicillin resistance inStaphylococcus aureus, a large percentage of BORSA appeared resistant to amoxicillin/clavulanic acid. This clearly shows that BORSA may be misidentified as MRSA while heteroresistant MRSA may appear to be BORSA. It is concluded that amoxicillin/clavulanic acid zone sizes should be measured after a full 24 hours of incubation, that susceptibility testing ofStaphylococcus aureus under certain environmental conditions should be interpreted with caution, and that MIC testing is the most reliable technique for differentiating these two resistance patterns inStaphylococcus aureus.     

Effect of different combinations of sparfloxacin, oxacillin, and fosfomycin against methicillin-resistant staphylococci

The in vitro activity of combinations of sparfloxacin/oxacillin, sparfloxacin/fosfomycin, and oxacillin/fosfomycin was investigated against 16 methicillin-resistantStaphylococcus aureus (MRSA) isolates and 12 methicillin-resistantStaphylococcus epidermidis (MRSE) isolates moderately resistant to sparfloxacin. Synergic interactions were observed more frequently against MRSE than against MRSA strains. The most effective combination on both species was fosfomycin plus oxacillin, synergistic against ten of 16 MRSA and eight of 12 MRSE strains.     

Variation and persistence of methicillin-resistantStaphylococcus aureus strains among individual patients over extended periods of time

To determine the strain variation and persistence among isolates of methicillin-resistantStaphylococcus aureus (MRSA) cultured from patients with colonization over extended time spans, pulsed-field gel electrophoresis was used to analyze the isolates from 47 patients for whom at least twomecA-positive isolates collected a minimum of six months apart were available. For 22 (47 %) patients, the isolates represented multiple distinct strains ofStaphylococcus aureus, while 20 (43 %) patients had only a single strain detected; five (11 %) patients had similar, genetically related isolates. MRSA were frequently associated with mucocutaneous abnormalities; 29 (62 %) patients had focal cutaneous defects, and ten (21 %) had chronic dermatitis. Multiple strains of MRSA were detected more frequently than single strains among patients in whom the initial focus of MRSA resolved clinically and another mucocutaneous defect subsequently developed compared to patients with clinically persistent foci (11/15 versus 9/23, respectively; p=0.05, Fisher's exact test). Among the 21 patients in this series for whom isolates cultured within a two-month time span were available, there were seven (33 %) patients with multiple strains of MRSA, including one patient with polyclonal bacteremia. In summary, patients with long-term MRSA colonization often have several different strains of MRSA, which typically change over time in association with removal or resolution of a colonized focus and the recurrence of mucocutaneous defects.     

MRSA carriage in a tertiary governmental hospital in Thailand: emphasis on prevalence and molecular epidemiology

We investigated prevalence and risk factors for methicillin-resistant Staphylococcus aureus (MRSA) in a case-control study performed in a 900-bed tertiary governmental healthcare facility in Bangkok, Thailand. Multivariate unconditional logistic regression was used to identify risk profiles for MRSA carriage. Phage typing, pulsed-field gel electrophoresis (PFGE), polymorphisms of the coa and spa genes, hypervariable region (HVR) of SCCmec, multi-locus sequence typing (MLST), and identification of ST30/ST8 mosaic chromosome by heteroduplex-polymerase chain reaction (heteroduplex-PCR) were used to demonstrate a clonal relationship. Fifty-seven of 619 in-patients (9.2%) were positive for MRSA. Risk factors were being male, long admission, low modified McCabe score, history of MRSA infection, and use of broad spectrum cephalosporin. Molecular typing results indicated close relatedness among MRSA isolates. Successful epidemic subtypes were recovered from many different wards. However, all subtypes with different multi-locus sequence types were single locus variants (SLVs) of ST239. Heteroduplex-PCR gave two positive bands from ST8/ST30 mosaic chromosomal structures in all SLVs indicating all isolates were of the ST239 origin. The burden of MRSA nosocomial infections is high in the governmental tertiary hospital. The sole ST239 and its SLVs identified in this hospital is striking and calls for better policy for infection control and prevention.     

Molecular characterization and antibiotic resistance of clinical isolates of methicillin‐resistant Staphylococcus aureus obtained from Southeast of Iran (Kerman)

Staphylococcus aureus infections, particularly infections caused by methicillin‐resistant S. aureus (MRSA) strains, are emerging as a major public health problem. The aim of this study was to determine the prevalence of MRSA, antibiotic resistance profile and staphylococcal cassette chromosome mec (SCCmec) type of MRSA isolates obtained from clinical samples. Totally, 162 S. aureus isolates were obtained from clinical samples at three university hospitals in Kerman, Iran from March 2011 to February 2012. All isolates were identified as S. aureus by phenotypic methods and confirmed by PCR amplification of the nuc gene . MRSA isolates were screened by phenotypic tests and confirmed by presence of mecA gene. The minimum inhibitory concentrations (MICs) of the MRSA isolates against antibacterial agents were determined by E‐test. All isolates were analyzed by PCR for the presence of mecA and pvl genes. SCCmec typing of MRSA isolates was performed by multiplex PCR assay. Strain typing was carried out with REP‐PCR. Using mecA gene PCR and phenotypic methods, 56.8% of the isolates were identified as MRSA. All MRSA isolates were susceptible to vancomycin and linezolid. The sensitivity of MRSA isolates to trimethoprim‐sulfamethoxazole, clindamycin, ciprofloxacin, gentamicin, and erythromycin was 70.66, 66.53, 42.4, 38.05, and 29.35%, respectively. The most frequent SCCmec types were type III (48.31%) followed by type V (19.1%), type I (16.85%), and type IV (3.37%). The pvl gene was detected in 3.08% of isolates (two MRSA and three MSSA isolates). REP‐PCR typing divided the 92 MRSA isolates into 10 distinct clusters. Our results indicate that vancomycin and linezolid are the most effective antibacterial agents against MRSA isolates and SCCmec type III is predominant in MRSA strains in this area.     

Comparison of three chromogenic media and enrichment broth media for the detection of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> from mucocutaneous screening specimens

This study compares the performance of three chromogenic culture agar plates, chromID MRSA, MRSA-Screen and MRSA-Select, by challenging with a collection of Staphylococcus aureus strains and screening samples obtained from hospitalised patients. All chromogenic media showed excellent sensitivity (>95%) and specificity after 18 h on the methicillin-resistant Staphylococcus aureus (MRSA) collection strains, but the specificity of MRSA-Screen decreased markedly after 42 h. Sixty-eight of 1,002 screening specimens yielded MRSA on at least one medium. The sensitivity of all media to detecting MRSA after 18 h was <50%, but this increased to 75% (chromID MRSA), 81% (MRSA-Screen) and 72% (MRSA-Select) after 42 h and 85% after enrichment and plating on the same media. The specificity at 18 h was excellent, but was significantly lower for MRSA-Screen after 42 h and enrichment. In conclusion, all media showed equivalent sensitivities after 18 h of incubation and performed better when enriched before inoculation. MRSA-Screen was more sensitive but less specific than the two other media after 42 h of incubation.     

Incidence of community‐acquired methicillin‐resistant Staphylococcus aureus carrying Pantone‐Valentine leucocidin gene at a referral hospital in United Arab Emirates

Community‐acquired methicillin‐resistant Staphylococcus aureus (CA‐MRSA) is an emerging pathogen in hospitalized patients worldwide. The present study was undertaken to identify CA‐MRSA in hospitalized patients in a 350‐bed tertiary care hospital in Sharjah, UAE over a 2‐year period from January 2011 to December 2012. CA‐MRSA was defined based on identification within first 48 h of admission in the hospital. Staphylococcal cassette chromosome (SCC) mec typing of the CA‐MRSA isolates was carried out by multiplex polymerase chain reaction (PCR). Detection of PVL and mecA genes was done by PCR using the GenoType^® MRSA test system (Hain Lifescience). Patient's clinical data and antimicrobial susceptibility pattern of the CA‐MRSA isolates were also evaluated. Fifty seven of the 187 MRSA isolates were identified as CA‐MRSA. All the CA‐MRSA strains in our study belonged to SCCmecIV type and were positive for both PVL and mecA genes. The patients with CA‐MRSA infections were young (median age, 32 years) and the majority of infections involved the skin and soft tissue (36%). Antimicrobial susceptibility pattern of the CA‐MRSA isolates showed a better susceptibility profile to the non‐beta‐lactam antimicrobials with the exception of ciprofloxacin having 28% resistance. This study evidently strengthens the recent observation of an increase in CA‐MRSA emergence among hospitalized patients in the UAE.     

Molecular genetic characterization of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates recovered from Moscow clinics

Methicillin-resistant Staphylococcus aureus (MRSA) is the causative agent of nosocomial infections observed worldwide. The goal of this work was to study the genetic variations in MRSA isolates recovered from Moscow clinics and compare various methods of molecular typing (multiplex PCR, SNP genotyping based on the determination of single-nucleotide polymorphisms). A total of 62 epidemiologically unrelated hospital-acquired MRSA isolates were studied. A previously described multiplex PCR assay was utilized for the molecular typing of staphylococcal cassette chromosome mec (SCCmec). SNP genotyping that targets the seven sequences in five housekeeping genes (arcC162, arcC210, aroE132, gmk123, tpi241, tpi243, and yqiL333) was employed. Primer extension was used to screen single nucleotide variants followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Multilocus sequence typing (MLST) was used as a reference assay. Most MRSA isolates (93.6%) were assigned to the clonal complex (CC) 8 disseminated worldwide. Three MRSA isolates (4.8%) proved to belong to CC1. The following correlation was established in this study between SCCmec cassette types and sequence types (STs): ST8-MRSA carried SCCmec type IV and ST239-MRSA carried type-III a SCCmec. Four SNP groups associated with certain SCCmec types were also identified. The developed SNP genotyping assay aided by MALDI-MS enables the rapid genotyping of S. aureus isolates according to their clonal types.     

Dissemination of Two Methicillin-Resistant Staphylococcus aureus Clones Exhibiting Negative Staphylase Reactions in Intensive Care Units

From December 1997 to March 1998, 25 methicillin-resistant Staphylococcus aureus (MRSA) isolates exhibiting negative Staphylase (Oxoid Ltd., Basingstoke, England) reactions were identified from various clinical specimens from 13 patients in six intensive care units (ICUs) or in wards following a stay in an ICU at the National Taiwan University Hospital. The characteristics of these isolates have not been previously noted in other MRSA isolates from this hospital. Colonies of all these isolates were grown on Trypticase soy agar supplemented with 5% sheep blood and were nonhemolytic and unpigmented. Seven isolates, initially reported as Staphylococcus haemolyticus (5 isolates) and Staphylococcus epidermidis (2 isolates) by the routine identification scheme and with the Vitek GPI system (bioMerieux Vitek, Inc., Hazelwood, Mo.), were subsequently identified as S. aureus by positive tube coagulase tests, standard biochemical reactions, and characteristic cellular fatty acid chromatograms. The antibiotypes obtained by the E test, coagulase types, restriction fragment length polymorphism profiles of the staphylococcal coagulase gene, and random amplified polymorphic DNA patterns generated by arbitrarily primed PCR of the isolates disclosed that two major clones disseminated in the ICUs. Clone 1 (16 isolates) was resistant to clindamycin and was susceptible to trimethoprim-sulfamethoxazole (TMP-SMZ) and was coagulase type II. Clone 2 (eight isolates) was resistant to clindamycin and TMP-SMZ and was coagulase type IV. These two epidemic clones from ICUs are unique and underline the need for caution in identifying MRSA strains with colonial morphologies not of the typical type and with negative Staphylase reactions.