High genetic diversity among community-associated Staphylococcus aureus in Europe: results from a multicenter study

Background

Several studies have addressed the epidemiology of community-associated Staphylococcus aureus (CA-SA) in Europe; nonetheless, a comprehensive perspective remains unclear. In this study, we aimed to describe the population structure of CA-SA and to shed light on the origin of methicillin-resistant S. aureus (MRSA) in this continent.

Methods and Findings

A total of 568 colonization and infection isolates, comprising both MRSA and methicillin-susceptible S. aureus (MSSA), were recovered in 16 European countries, from community and community-onset infections. The genetic background of isolates was characterized by molecular typing techniques (spa typing, pulsed-field gel electrophoresis and multilocus sequence typing) and the presence of PVL and ACME was tested by PCR. MRSA were further characterized by SCCmec typing. We found that 59% of all isolates were associated with community-associated clones. Most MRSA were related with USA300 (ST8-IVa and variants) (40%), followed by the European clone (ST80-IVc and derivatives) (28%) and the Taiwan clone (ST59-IVa and related clonal types) (15%). A total of 83% of MRSA carried Panton-Valentine leukocidin (PVL) and 14% carried the arginine catabolic mobile element (ACME). Surprisingly, we found a high genetic diversity among MRSA clonal types (ST-SCCmec), Simpson’s index of diversity = 0.852 (0.788–0.916). Specifically, about half of the isolates carried novel associations between genetic background and SCCmec. Analysis by BURP showed that some CA-MSSA and CA-MRSA isolates were highly related, suggesting a probable local acquisition/loss of SCCmec.

Conclusions

Our results imply that CA-MRSA origin, epidemiology and population structure in Europe is very dissimilar from that of USA.     

Prevalence and Trends of Staphylococcus aureus Bacteraemia in Hospitalized Patients in South Africa, 2010 to 2012: Laboratory-Based Surveillance Mapping of Antimicrobial Resistance and Molecular Epidemiology

Introduction

We aimed to obtain an in-depth understanding on recent antimicrobial resistance trends and molecular epidemiology trends of S. aureus bacteraemia (SAB).

Methods

Thirteen academic centres in South Africa were included from June 2010 until July 2012. S. aureus susceptibility testing was performed on the MicroScan Walkaway. Real-time PCR using the LightCycler 480 II was done for mecA and nuc. SCCmec and spa-typing were finalized with conventional PCR. We selected one isolate per common spa type per province for multilocus sequence typing (MLST).

Results

S. aureus from 2709 patients were included, and 1231 (46%) were resistant to methicillin, with a significant decline over the three-year period (p-value = 0.003). Geographical distribution of MRSA was significantly higher in Gauteng compared to the other provinces (P<0.001). Children <5 years were significantly associated with MRSA with higher rates compared to all other age groups (P = 0.01). The most prevalent SCCmec type was SCCmec type III (531 [41%]) followed by type IV (402 [31%]). Spa-typing discovered 47 different spa-types. The five (87%) most common spa-types were t037, t1257, t045, t064 and t012. Based on MLST, the commonest was ST612 clonal complex (CC8) (n = 7) followed by ST5 (CC5) (n = 4), ST36 (CC30) (n = 4) and ST239 (CC8) (n = 3).

Conclusions

MRSA rate is high in South Africa. Majority of the isolates were classified as SCCmec type III (41%) and type IV (31%), which are typically associated with hospital and community- acquired infections, respectively. Overall, this study reveals the presence of a variety of hospital-acquired MRSA clones in South Africa dominance of few clones, spa 037 and 1257. Monitoring trends in resistance and molecular typing is recommended to detect changing epidemiological trends in AMR patterns of SAB.     

Associations between dru Types and SCCmec Cassettes

Molecular typing is an important tool in the investigation of methicillin resistant Staphylococcus aureus (MRSA) outbreaks and in following the evolution of MRSA. The staphylococcal cassette chromosome mec (SCCmec) contains a hypervariable region with a variable number of 40 bp repeats named direct repeat units (dru). The dru region has been suggested as a supplementary typing method for MRSA and an international nomenclature exists. The purpose of this study was to investigate the diversity and variability of the dru region in a diverse collection of MRSA. We studied 302 MRSA isolates harbouring SCCmec types I to VI. The isolates represented a broad genetic background based on Staphylococcal protein A (spa) typing and multilocus sequence typing (MLST) and included 68 isolates (68 patients) from an outbreak with t024-ST8-IVa and 26 isolates from the same patient. Sequencing identified 53 dru types (dt) in 283 isolates, while eighteen isolates contained no dru repeats and one isolate resisted sequencing. The most common dru type, dt10a, was present in 53% of the sequenced isolates and was found in all SCCmec types, except type II. Seven (10%) of the 68 epidemiologically related patients had isolates with dru type variants indicating that dru typing is not useful as a first line epidemiological typing tool. However, MRSA isolates cultured from a single patient over a three year period exhibited a single dru type. The finding of dt10a in most SCCmec types suggests that dru and mecA originate from the same Staphylococcus species.     

Spread of epidemic MRSA-ST5-IV clone encoding PVL as a major cause of community onset staphylococcal infections in Argentinean children

Background

Community-associated methicillin-resistant Staphylococcus aureus-(CA-MRSA) strains have emerged in Argentina. We investigated the clinical and molecular evolution of community-onset MRSA infections (CO-MRSA) in children of Córdoba, Argentina, 2005–2008. Additionally, data from 2007 were compared with the epidemiology of these infections in other regions of the country.

Methodology/Principal Findings

Two datasets were used: i) lab-based prospective surveillance of CA-MRSA isolates from 3 Córdoba pediatric hospitals-(CBAH1-H3) in 2007–2008 (compared to previously published data of 2005) and ii) a sampling of CO-MRSA from a study involving both, healthcare-associated community-onset-(HACO) infections in children with risk-factors for healthcare-associated infections-(HRFs), and CA-MRSA infections in patients without HRFs detected in multiple centers of Argentina in 2007. Molecular typing was performed on the CA-MRSA-(n: 99) isolates from the CBAH1-H3-dataset and on the HACO-MRSA-(n: 51) and CA-MRSA-(n: 213) isolates from other regions. Between 2005–2008, the annual proportion of CA-MRSA/CA-S. aureus in Córdoba hospitals increased from 25% to 49%, P<0.01. Total CA-MRSA infections increased 3.6 fold-(5.1 to 18.6 cases/100,000 annual-visits, P<0.0001), associated with an important increase of invasive CA-MRSA infections-(8.5 fold). In all regions analyzed, a single genotype prevailed in both CA-MRSA (82%) and HACO-MRSA(57%), which showed pulsed-field-gel electrophoresis-(PFGE)-type-“I”, sequence-type-5-(ST5), SCCmec-type-IVa, spa-t311, and was positive for PVL. The second clone, pulsotype-N/ST30/CC30/SCCmecIVc/t019/PVL⁺, accounted for 11.5% of total CA-MRSA infections. Importantly, the first 4 isolates of Argentina belonging to South American-USA300 clone-(USA300/ST8/CC8/SCCmecIVc/t008/PVL⁺/ACME⁻) were detected. We also demonstrated that a HA-MRSA clone-(pulsotype-C/ST100/CC5) caused 2% and 10% of CA-MRSA and HACO-MRSA infections respectively and was associated with a SCCmec type closely related to SCCmecIV(2B&5).

Conclusions/Significance

The dissemination of epidemic MRSA clone, ST5-IV-PVL⁺ was the main cause of increasing staphylococcal community-onset infections in Argentinean children (2003–2008), conversely to other countries. The predominance of this clone, which has capacity to express the h-VISA phenotype, in healthcare-associated community-onset cases suggests that it has infiltrated into hospital-settings.     

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

Background

The 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 Findings

Between 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/Significance

Public 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.     

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus Isolated from Australian Veterinarians

This work investigated the molecular epidemiology and antimicrobial resistance of methicillin-resistant Staphylococcus aureus (MRSA) isolated from veterinarians in Australia in 2009. The collection (n = 44) was subjected to extensive molecular typing (MLST, spa, SCCmec, dru, PFGE, virulence and antimicrobial resistance genotyping) and antimicrobial resistance phenotyping by disk diffusion. MRSA was isolated from Australian veterinarians representing various occupational emphases. The isolate collection was dominated by MRSA strains belonging to clonal complex (CC) 8 and multilocus sequence type (ST) 22. CC8 MRSA (ST8-IV [2B], spa t064; and ST612-IV [2B], spa variable,) were strongly associated with equine practice veterinarians (OR = 17.5, 95% CI = 3.3–92.5, P < 0.001) and were often resistant to gentamicin and rifampicin. ST22-IV [2B], spa variable, were strongly associated with companion animal practice veterinarians (OR = 52.5, 95% CI = 5.2–532.7, P < 0.001) and were resistant to ciprofloxacin. A single pig practice veterinarian carried ST398-V [5C2], spa t1451. Equine practice and companion animal practice veterinarians frequently carried multiresistant-CC8 and ST22 MRSA, respectively, whereas only a single swine specialist carried MRSA ST398. The presence of these strains in veterinarians may be associated with specific antimicrobial administration practices in each animal species.     

MRSA Clonal Complex 22 Strains Harboring Toxic Shock Syndrome Toxin (TSST-1) Are Endemic in the Primary Hospital in Gaza,Palestine

Background

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen in both community and healthcare-related settings worldwide. Current knowledge regarding the epidemiology of S. aureus and MRSA in Gaza is based on a single community-based carriage study. Here we describe a cross-sectional analysis of 215 clinical isolates collected from Al-Shifa Hospital in Gaza during 2008 and 2012.

Methods

All isolates were characterized by spa typing, SCCmec typing, and detection of genes encoding Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin (TSST-1). Representative genotypes were also subjected to multilocus sequence typing (MLST). Antibiotic susceptibility testing was performed using VITEK2 and MicroScan.

Results

MRSA represented 56.3% of all S. aureus strains, and increased in frequency from 2008 (54.8%) to 2012 (58.4%). Aside from beta-lactams, resistance was observed to tetracycline, erythromycin, clindamycin, gentamicin, and fluoroquinolones. Molecular typing identified 35 spa types representing 17 MLST clonal complexes (CC), with spa 998 (Ridom t223, CC22) and spa 70 (Ridom t044, CC80) being the most prevalent. SCCmec types I, III, IV, V and VI were identified among MRSA isolates, while type II was not detected. PVL genes (lukF/S-PV) were detected in 40.0% of all isolates, while the TSST-1 gene (tst) was detected in 27.4% of all isolates, with surprisingly high frequency within CC22 (70.4%). Both PVL and TSST-1 genes were found in several isolates from 2012.

Conclusions

Molecular typing of clinical isolates from Gaza hospitals revealed unusually high prevalence of TSST-1 genes among CC22 MRSA, which is noteworthy given a recent community study describing widespread carriage of a CC22 MRSA clone known as the ‘Gaza strain’. While the latter did not address TSST-1, tst-positive spa 998 (Ridom t223) has been detected in several neighboring countries, and described as endemic in an Italian NICU, suggesting international spread of a ‘Middle Eastern variant’ of pandemic CC22 strain EMRSA-15.     

Comparative Genotypes,Staphylococcal Cassette Chromosome mec (SCCmec) Genes and Antimicrobial Resistance amongst Staphylococcus epidermidis and Staphylococcus haemolyticus Isolates from Infections in Humans and Companion Animals

This study compares the characteristics of Staphylococcus epidermidis (SE) and Staphylococcus haemolyticus (SH) isolates from epidemiologically unrelated infections in humans (Hu) (28 SE-Hu; 8 SH-Hu) and companion animals (CpA) (12 SE-CpA; 13 SH-CpA). All isolates underwent antimicrobial susceptibility testing, multilocus sequence typing and DNA microarray profiling to detect antimicrobial resistance and SCCmec-associated genes. All methicillin-resistant (MR) isolates (33/40 SE, 20/21 SH) underwent dru and mecA allele typing. Isolates were predominantly assigned to sequence types (STs) within a single clonal complex (CC2, SE, 84.8%; CC1, SH, 95.2%). SCCmec IV predominated among MRSE with ST2-MRSE-IVc common to both Hu (40.9%) and CpA (54.5%). Identical mecA alleles and nontypeable dru types (dts) were identified in one ST2-MRSE-IVc Hu and CpA isolate, however, all mecA alleles and 2/4 dts detected among 18 ST2-MRSE-IVc isolates were closely related, sharing >96.5% DNA sequence homology. Although only one ST-SCCmec type combination (ST1 with a non-typeable [NT] SCCmec NT9 [class C mec and ccrB4]) was common to four MRSH-Hu and one MRSH-CpA, all MRSH isolates were closely related based on similar STs, SCCmec genes (V/V_T or components thereof), mecA alleles and dts. Overall, 39.6% of MR isolates harbored NT SCCmec elements, and ACME was more common amongst MRSE and CpA isolates. Multidrug resistance (MDR) was detected among 96.7% of isolates but they differed in the prevalence of specific macrolide, aminoglycoside and trimethoprim resistance genes amongst SE and SH isolates. Ciprofloxacin, rifampicin, chloramphenicol [fexA, cat-pC221], tetracycline [tet(K)], aminoglycosides [aadD, aphA3] and fusidic acid [fusB] resistance was significantly more common amongst CpA isolates. SE and SH isolates causing infections in Hu and CpA hosts belong predominantly to STs within a single lineage, harboring similar but variable SCCmec genes, mecA alleles and dts. Host and staphylococcal species-specific characteristics were identified in relation to antimicrobial resistance genes and phenotypes, SCCmec and ACME.     

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.     

Livestock-Associated Methicillin Resistant and Methicillin Susceptible Staphylococcus aureus Sequence Type (CC)1 in European Farmed Animals: High Genetic Relatedness of Isolates from Italian Cattle Herds and Humans

Methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type (ST)1, Clonal Complex(CC)1, SCCmec V is one of the major Livestock-Associated (LA-) lineages in pig farming industry in Italy and is associated with pigs in other European countries. Recently, it has been increasingly detected in Italian dairy cattle herds. The aim of this study was to analyse the differences between ST1 MRSA and methicillin-susceptible S. aureus (MSSA) from cattle and pig herds in Italy and Europe and human isolates. Sixty-tree animal isolates from different holdings and 20 human isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing, SCCmec typing, and by micro-array analysis for several virulence, antimicrobial resistance, and strain/host-specific marker genes. Three major PFGE clusters were detected. The bovine isolates shared a high (≥90% to 100%) similarity with human isolates and carried the same SCCmec type IVa. They often showed genetic features typical of human adaptation or present in human-associated CC1: Immune evasion cluster (IEC) genes sak and scn, or sea; sat and aphA3-mediated aminoglycoside resistance. Contrary, typical markers of porcine origin in Italy and Spain, like erm(A) mediated macrolide-lincosamide-streptograminB, and of vga(A)-mediated pleuromutilin resistance were always absent in human and bovine isolates. Most of ST(CC)1 MRSA from dairy cattle were multidrug-resistant and contained virulence and immunomodulatory genes associated with full capability of colonizing humans. As such, these strains may represent a greater human hazard than the porcine strains. The zoonotic capacity of CC1 LA-MRSA from livestock must be taken seriously and measures should be implemented at farm-level to prevent spill-over.     

Distribution of Staphylococcal Cassette Chromosome mec Types and Correlation with Comorbidity and Infection Type in Patients with MRSA Bacteremia

Background

Molecular epidemiological definitions that are based on staphylococcal cassette chromosome mec (SCCmec) typing and phylogenetic analysis of methicillin-resistant Staphylococcus aureus (MRSA) isolates are considered a reliable way to distinguish between healthcare-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA). However, there is little information regarding the clinical features and outcomes of bacteremia patients with MRSA carrying different SCCmec types.

Methods

From January 1 through December 31, 2006, we recorded the demographic data and outcomes of 159 consecutive adult MRSA bacteremia patients from whom isolates for SCCmec analysis were collected. All participants were patients at a tertiary care center in Taiwan.

Principal Findings

The following SCCmec types were identified in MRSA isolates: 30 SCCmec II (18.9%), 87 SCCmec III (54.7%), 22 SCCmec IV (13.8%), and 20 SCCmec V (12.6%). The time from admission to the first MRSA-positive blood culture for patients infected with isolates with the SCCmec III element (mean/median, 50.7/26 days) was significantly longer than for patients infected with isolates carrying SCCmec IV or V (mean/median, 6.7/3 days for SCCmec IV; 11.1/10.5 days for SCCmec V) (P<0.05). In univariate analysis, community onset, soft tissue infection, and deep-seated infection were predictors for SCCmec IV/V. In multivariate analysis, length of stay before index culture, diabetes mellitus, and being bedridden were independent risk factors associated with SCCmec II/III.

Conclusions

These findings are in agreement with previous studies of the genetic characteristics of CA-MRSA. MRSA bacteremia with SCCmec II/III isolates occurred more among patients with serious comorbidities and prolonged hospitalization. Community onset, skin and soft tissue infection, and deep-seated infection best predicted SCCmec IV/V MRSA bacteremia.     

Molecular and Clinical Characteristics of Clonal Complex 59 Methicillin-Resistant Staphylococcus aureus Infections in Mainland China

Detailed molecular analyses of Clonal Complex 59 (CC59) methicillin-resistant Staphylococcus aureus (MRSA) isolates from children in seven major cities across Mainland China were examined. A total of 110 CC59 isolates from invasive and non-invasive diseases were analyzed by multilocus sequence typing (MLST), Staphylococcus cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) typing and pulsed-field gel electrophoresis (PFGE). Antibiotics susceptibilities, carriage of plasmids and 42 virulence genes and the expression of virulence factors were examined. ST59 (101/110, 91.8%) was the predominant sequence type (ST), while single locus variants (SLVs) belonging to ST338 (8/110, 7.3%) and ST375 (1/110, 0.9%) were obtained. Three SCCmec types were found, namely type III (2.7%), type IV (74.5%) and type V (22.7%). Seven spa types including t437, which accounted for 87.3%, were determined. Thirteen PFGE types were obtained. PFGE types A and B were the major types totally accounting for 81.8%. The dominant clone was ST59-t437-IVa (65.5%), followed by ST59-t437-V (14.5%). The positive rate of luks-PV and lukF-PV PVL encoding (pvl) gene was 55.5%. Plasmids were detected in 83.6% (92/110) of the strains. The plasmid size ranging from 23.4 kb to 50 kb was most prevalent which accounted for 83.7% (77/92). A significantly lower expression of hla was found in ST59-t437-IVa compared with ST59-t437-V. Among the 110 cases, 61.8% of the patients were less than 1 year old. A total of 90 cases (81.8%) were community-associated (CA) infections whereas 20 cases (18.2%) were hospital-associated (HA) infections. Out of the 110 patients, 36.4% (40/110) were diagnosed with invasive infectious diseases in which ST59-t437-IVa accounted for 67.5% (27/40). In brief, ST59-t437-IVa was proved as the dominant clone in CC59 MRSA strains. The carriage rate of pvl gene was high. CC59 MRSA could result in CA and HA infections. The majortiy of MRSA infection children were in young age.     

Nasal Carriage of Methicillin-Resistant Staphylococcus aureus among Pediatricians in Taiwan

Background

Health care workers (HCWs) are at the interface between hospitals and communities. The survey for methicillin-resistant Staphylococcus aureus (MRSA) carriage among HCWs has mostly been conducted to investigate outbreaks or endemics. Community-associated MRSA are prevalent among children in Taiwan. We conducted this study to better understand the carriage rate of MRSA among pediatricians in non-outbreak situations in Taiwan,.

Methods

A total of 220 pediatricians from Taiwan who attended the annual meeting of Taiwan Pediatric Association in April, 2010 were recruited to participate in this study and were sampled from the nares for the detection of MRSA by polymerase chain reaction (PCR) and further by culture. The following molecular analyses were performed, including pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), typing of staphylococcal cassette chromosome mec (SCCmec) and the presence of Panton-Valentine leukocidin (PVL) genes.

Results

MRSA was detected from 15 attendees (6.8%) by PCR. MRSA-colonized attendees had a significantly lower rate (0.041) of working in the medical center, while borderline significantly higher rate of working in the Regional Hospital (p=0.056), than those without MRSA colonization. From those 15 samples, 12 MRSA isolates were identified by culture and molecularly characterized. Three PFGE patterns, two sequence types (ST 59, ST 508), and two SCCmec types (IV and V_T) were identified, respectively. Five isolates, including three carrying SCCmec types V_T, were PVL-positive. All 12 isolates were susceptible to vancomycin, teicoplanin, linezolid, fusidic acid, trimethoprim/sulfamethoxazole, and doxycyclin, and resistant to penicillin.

Conclusion/significance

Around seven percent of pediatricians in Taiwan harbored CA-MRSA in their nares.     

Characterization of Staphylococcus aureus from Distinct Geographic Locations in China: An Increasing Prevalence of spa-t030 and SCCmec Type III

Staphylococcus aureus belongs to one of the most common bacteria causing healthcare and community associated infections in China, but their molecular characterization has not been well studied. From May 2011 to June 2012, a total of 322 non-duplicate S. aureus isolates were consecutively collected from seven tertiary care hospitals in seven cities with distinct geographical locations in China, including 171 methicillin sensitive S. aureus (MSSA) and 151 MRSA isolates. All isolates were characterized by spa typing. The presence of virulence genes was tested by PCR. MRSA were further characterized by SCCmec typing. Seventy four and 16 spa types were identified among 168 MSSA and 150 MRSA, respectively. One spa type t030 accounted for 80.1% of all MRSA isolates, which was higher than previously reported, while spa-t037 accounted for only 4.0% of all MRSA isolates. The first six spa types (t309, t189, t034, t377, t078 and t091) accounted for about one third of all MSSA isolates. 121 of 151 MRSA isolates (80.1%) were identified as SCCmec type III. pvl gene was found in 32 MSSA (18.7%) and 5 MRSA (3.3%) isolates, with ST22-MSSA-t309 as the most commonly identified strain. Compared with non-epidemic MRSA clones, epidemic MRSA clones (corresponding to ST239) exhibited a lower susceptibility to rifampin, ciprofloxacin, gentamicin and trimethoprim-sulfamethoxazole, a higher prevalence of sea gene and a lower prevalence of seb, sec, seg, sei and tst genes. The increasing prevalence of multidrug resistant spa-t030 MRSA represents a major public health problem in China.     

Susceptibility of important Gram-negative pathogens to tigecycline and other antibiotics in Latin America between 2004 and 2010

Background

The evolving epidemiology of methicillin resistant Staphylococcus aureus (MRSA) is characterized by the emergence of infections caused by non multiresistant MRSA carrying staphylococcal chromosomal cassette (SCC)mec IV or V in the healthcare settings. A molecular epidemiological analysis of non multiresistant MRSA isolates from four acute general hospitals was performed in Palermo, Italy, during a one year period.

Methods

For the purpose of the study, MRSA isolates were defined as non multiresistant when they were susceptible to at least three classes of non ??-lactam antibiotics. Seventy-five isolates were submitted to antimicrobial susceptibility testing, multilocus sequence typing (MLST) and polymerase chain reaction (PCR) for SCCmec, accessory gene regulator (agr) groups, arginine catabolic mobile element (ACME) and Panton Valentine leukocidin (PVL) toxin genes. For epidemiological typing, Multiple-Locus Variable-Number Tandem Repeat Fingerprinting (MLVF) was performed on all isolates and pulsed field gel electrophoresis (PFGE) on ST8 isolates.

Results

Non multiresistant MRSA isolates were isolated from all hospitals. Resistances to ciprofloxacin, macrolides and tetracycline were the most prevalent. MLST attributed 46 isolates with ST22, 13 with ST8, eight with ST1, three with ST50 and three with ST398. SCCmec type IV was found in all isolates. PVL was detected in one ST22 isolate. All isolates tested negative for the ACME element. MLVF identified 31 different patterns, some subtype clusters ranging in size between two and 22 isolates. The closely related PFGE patterns of the ST8 isolates differed from USA300.

Conclusions

A polyclonal circulation of non multiresistant MRSA along with blurring of boundaries between healthcare associated (HA)-MRSA and community associated (CA)-MRSA appear to be occurring in our epidemiological setting. A better understanding of spread of MRSA with the support of molecular typing can provide invaluable information in the epidemiological, microbiological and clinical fields.     

A Livestock-Associated,Multidrug-Resistant,Methicillin-Resistant Staphylococcus aureus Clonal Complex 97 Lineage Spreading in Dairy Cattle and Pigs in Italy

Pandemic methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 97 (CC97) lineages originated from livestock-to-human host jumps. In recent years, CC97 has become one of the major MRSA lineages detected in Italian farmed animals. The aim of this study was to characterize and analyze differences in MRSA and methicillin-susceptible S. aureus (MSSA) mainly of swine and bovine origins. Forty-seven CC97 isolates, 35 MRSA isolates, and 6 MSSA isolates from different Italian pig and cattle holdings; 5 pig MRSA isolates from Germany; and 1 human MSSA isolate from Spain were characterized by macrorestriction pulsed-field gel electrophoresis (PFGE) analysis, multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial resistance pattern analysis. Virulence and resistance genes were investigated by PCR and microarray analysis. Most of the isolates were of SCCmec type V (SCCmec V), except for two German MRSA isolates (SCCmec III). Five main clusters were identified by PFGE, with the German isolates (clusters I and II) showing 60.5% similarity with the Italian isolates, most of which (68.1%) grouped into cluster V. All CC97 isolates were Panton-Valentine leukocidin (PVL) negative, and a few (n = 7) tested positive for sak or scn. All MRSA isolates were multidrug resistant (MDR), and the main features were erm(B)- or erm(C)-mediated (n = 18) macrolide-lincosamide-streptogramin B resistance, vga(A)-mediated (n = 37) pleuromutilin resistance, fluoroquinolone resistance (n = 33), tet(K) in 32/37 tet(M)-positive isolates, and blaZ in almost all MRSA isolates. Few host-associated differences were detected among CC97 MRSA isolates: their extensive MDR nature in both pigs and dairy cattle may be a consequence of a spillback from pigs of a MRSA lineage that originated in cattle as MSSA and needs further investigation. Measures should be implemented at the farm level to prevent spillover to humans in intensive farming areas.     

Cost-Effectiveness and Efficacy of spa,SCCmec, and PVL Genotyping of Methicillin-Resistant Staphylococcus aureus as Compared to Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) is a valuable molecular typing assay used for methicillin-resistant Staphylococcus aureus (MRSA) surveillance and genotyping. However, there are several limitations associated with PFGE. In Alberta, Canada, the significant increase in the number of MRSA isolates submitted to the Provincial Laboratory for Public Health (ProvLab) for PFGE typing led to the need for an alternative genotyping method. In this study, we describe the transition from PFGE to Staphylococcus protein A (spa), Staphylococcal cassette chromosome (SCCmec), and Panton-Valentine leukocidin (PVL) typing. A total of 1915 clinical MRSA isolates collected from 2005 to 2009 were used to develop and validate an algorithm for assigning PFGE epidemic types using spa, SCCmec, and PVL typing and the resulting data was used to populate a new Alberta MRSA typing database. An additional 12620 clinical MRSA isolates collected from 2010 to 2012 as part of ongoing routine molecular testing at ProvLab were characterized using the new typing algorithm and the Alberta MRSA typing database. Switching to spa, SCCmec, and PVL from PFGE typing substantially reduced hands-on and turn-around times while maintaining historical PFGE epidemic type designations. This led to an approximate $77,000 reduction in costs from 2010 to 2012. PFGE typing is still required for a small subset of MRSA isolates that have spa types that are rare, novel, or associated with more than one PFGE epidemic type.     

Methicillin Resistance Transfer from Staphylocccus epidermidis to Methicillin-Susceptible Staphylococcus aureus in a Patient during Antibiotic Therapy

Background

The mecA gene, encoding methicillin resistance in staphylococci, is located on a mobile genetic element called Staphylococcal Cassette Chromosome mec (SCCmec). Horizontal, interspecies transfer of this element could be an important factor in the dissemination of methicillin-resistant S. aureus (MRSA). Previously, we reported the isolation of a closely related methicillin-susceptible Staphylococcus aureus (MSSA), MRSA and potential SCCmec donor Staphylococcus epidermidis isolate from the same patient. Based on fingerprint techniques we hypothesized that the S. epidermidis had transferred SCCmec to the MSSA to become MRSA. The aim of this study was to show that these isolates form an isogenic pair and that interspecies horizontal SCCmec transfer occurred.

Methodology/Results

Whole genome sequencing of both isolates was performed and for the MSSA gaps were closed by conventional sequencing. The SCCmec of the S. epidermidis was also sequenced by conventional methods. The results show no difference in nucleotide sequence between the two isolates except for the presence of SCCmec in the MRSA. The SCCmec of the S. epidermidis and the MRSA are identical except for a single nucleotide in the ccrB gene, which results in a valine to alanine substitution. The main difference with the closely related EMRSA-16 is the presence of SaPI2 encoding toxic shock syndrome toxin and exfoliative toxin A in the MSSA-MRSA pair. No transfer of SCCmec from the S. epidermidis to the MSSA could be demonstrated in vitro.

Conclusion

The MSSA and MRSA form an isogenic pair except for SCCmec. This strongly supports our hypothesis that the MRSA was derived from the MSSA by interspecies horizontal transfer of SCCmec from S. epidermidis O7.1.     

Population Structure of a Hybrid Clonal Group of Methicillin-Resistant Staphylococcus aureus,ST239-MRSA-III

The methicillin-resistant Staphylococcus aureus (MRSA) clonal group known as ST239-MRSA-III is notable for its hybrid origin and for causing sustained hospital epidemics worldwide since the late 1970s. We studied the population structure of this MRSA clonal group using a sample of 111 isolates that were collected over 34 years from 29 countries. Genetic variation was assessed using typing methods and novel ascertainment methods, resulting in approximately 15 kb of sequence from 32 loci for all isolates. A single most parsimonious tree, free of homoplasy, partitioned 28 haplotypes into geographically-associated clades, including prominent European, Asian, and South American clades. The rate of evolution was estimated to be approximately 100× faster than standard estimates for bacteria, and dated the most recent common ancestor of these isolates to the mid-20th century. Associations were discovered between the ST239 phylogeny and the ccrB and dru loci of the methicillin resistance genetic element, SCCmec type III, but not with the accessory components of the element that are targeted by multiplex PCR subtyping tools. In summary, the evolutionary history of ST239 can be characterized by rapid clonal diversification that has left strong evidence of geographic and temporal population structure. SCCmec type III has remained linked to the ST239 chromosome during clonal diversification, but it has undergone homoplasious losses of accessory components. These results provide a population genetics framework for the precise identification of emerging ST239 variants, and invite a re-evaluation of the markers used for subtyping SCCmec.     

Molecular Characterization of Staphylococcus aureus from Patients with Surgical Site Infections at Mulago Hospital in Kampala,Uganda

Background

The prevalence of Methicillin resistant Staphylococcus aureus (MRSA) is progressively increasing globally with significant regional variation. Understanding the Staphylococcus aureus lineages is crucial in controlling nosocomial infections. Recent studies on S. aureus in Uganda have revealed an escalating burden of MRSA. However, the S. aureus genotypes circulating among patients are not known. Here, we report S. aureus lineages circulating in patients with surgical site infections (SSI) at Mulago National hospital, Kampala, Uganda.

Methods

A cross-sectional study involving 314 patients with SSI at Mulago National Hospital was conducted from September 2011 to April 2012. Pus swabs from the patients’ SSI were processed using standard microbiological procedures. Methicillin sensitive Staphylococcus aureus (MSSA) and MRSA were identified using phenotypic tests and confirmed by PCR-detection of the nuc and mecA genes, respectively. SCCmec genotypes were determined among MRSA isolates using multiplex PCR. Furthermore, to determine lineages, spa sequence based-genotyping was performed on all S. aureus isolates.

Results

Of the 314 patients with SSI, S. aureus accounted for 20.4% (64/314), of which 37.5% (24/64) were MRSA. The predominant SCCmec types were type V (33.3%, 8/24) and type I (16.7%, 4/24). The predominant spa lineages were t645 (17.2%, 11/64) and t4353 (15.6%, 10/64), and these were found to be clonally circulating in all the surgical wards. On the other hand, lineages t064, t355, and t4609 were confined to the obstetrics and gynecology wards. A new spa type (t10277) was identified from MSSA isolate. On multivariate logistic regression analysis, cancer and inducible clindamycin resistance remained as independent predictors of MRSA-SSI.

Conclusion

SCCmec types I and V are the most prevalent MRSA mecA types from the patients’ SSI. The predominant spa lineages (t645 and t4353) are clonally circulating in all the surgical wards, calling for strengthening of infection control practices at Mulago National Hospital.