Characteristics of the community-genotype sequence type 72 methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates that underlie their persistence in hospitals

Panton-Valentine leukocidin-negative methicillin-resistant Staphylococcus aureus (MRSA) clone ST72, known as a major community-associated MRSA in Korea, has emerged as an important pathogen in hospitals. To understand bacterial properties that underlie transformation of this clone into a nosocomial pathogen, we compared characteristics of the community-genotype ST72 MRSA isolates with those of ST5 and ST239 MRSA, which have been predominant nosocomial MRSA clones in Korea. Several genes associated with adhesion and virulence were absent or rarely found in ST72 isolates. Many ST72 isolates (70.1%) belonged to agr group I, but the agr group of other ST72 isolates could not be determined. As indicated by d-hemolysin production, ST72 isolates expressed fully functional agr, whereas agr dysfunction was observed in ST5 and ST239 isolates. In the biofilm formation assay, no upregulation of biofilm-forming activity of ST72 MRSA was detected. However, ST72 isolates demonstrated persistence under hypotonic and desiccating conditions (survival rates 72.3% and 33.9%, respectively), which was similar to characteristics of ST5 or ST239 isolates. ST72- MRSA isolates showed low virulence, but properties of their functional agr system could facilitate their spread in hospitals. In conclusion, tolerance to stressful environments, e.g., hypotonic and dry conditions, may also contribute to survival of the community-associated MRSA clones in healthcare facilities.     

Molecular characterization of methicillin-resistant Staphylococcus aureus in hospitals in Niigata, Japan: divergence and transmission

The major methicillin-resistant Staphylococcus aureus(MRSA) distributed among hospitals in Japan is New York/Japan clone [multilocus sequence type 5 (ST5), agr type 2 and methicillin resistance locus type (SCC mec) II] which possesses both the toxic shock syndrome toxin 1 gene (tst) and staphylococcal enterotoxin C gene (sec). In this study, we collected 245 MRSA strains from four hospitals during 2001 to 2005 in Niigata, Japan, and analyzed tst and sec genes and SCC mec type among them. A total of 13 strains were further examined for their genotypes, virulence gene patterns and drug resistance. Among the 245 strains four tst sec genes patterns were observed; tst(+) sec(+) strains represented a majority of 86.5% and 9.4% were tst(-) sec(-). SCCmec typing revealed that 91.4% had type II, 4.1% type IV and 4.1% type I. Multilocus sequence typing (MLST) revealed that 10 of the 13 typed strains belonged to clonal complex 5 (7 had ST5 while 3 were single locus variants of ST5) with similar characteristics to the New York/Japan clone and possessed multi-drug resistance with high virulence gene content. The remaining 3 strains were ST8 (n=2) and ST91 (n=1). The ST91 strain had SCC mec IV and seemed to originate in the community, while ST8 strains exhibited SCC mec type I, which is distinct from community type IV. The data suggest that MRSA in hospitals in Niigata now mainly includes the New York/Japan clone (undergoing genomic divergence and clonal expansion) and other minor types (e.g. ST8) as well as the community type.     

Polyclonal non multiresistant Staphylococcus aureus isolates from clinical cases of infection occurring in Palermo, Italy, during a one-year surveillance period

ABSTRACT: 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 beta-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.     

Molecular analysis and antimicrobial susceptibility of methicillin resistant Staphylococcus aureus in one of the hospitals of Tehran University of Medical Sciences: high prevalence of sequence type 239 (ST239) clone

Methicillin-resistant Staphylococcus aureus (MRSA), particularly the multidrug-resistant clones, is an increasing worldwide problem. The average incidence rate of MRSA in Tehran was found to be over 40%. A total of 140 MRSA isolates obtained from patients attending a teaching hospital in Tehran, from May 2009 to December 2009, were included in this study. The antimicrobial susceptibility profile of MRSA isolates was determined by the agar disk diffusion method. Molecular analysis of MRSA strains was accomplished by Pulsed-Field Gel Electrophoresis (PFGE) and Multi-locus sequence typing (MLST). Detection of mecA gene was used to confirm resistance to methicillin among the MRSA isolates. All the MRSA isolates were susceptible to chloramphenicol, teicoplanin, tigecycline and vancomycin. All MRSAisolates were resistant to oxacillin, whilst 139 strains showed resistance against ciprofloxacin, erythromycin, gentamicin, tetracycline and trimethoprim-sulfamethoxazole. PFGE analysis of all the 140 MRSA isolates produced five distinct pulsotypes designated as pulsotypes A-E. Most of the isolates (n=132) were clustered into pulsotype A. The most prevalent sequence type (ST) was ST 239 (pulsotype A) found in 82% (37/45) of the tested isolates. The second most prevalent type was ST 1238 (pulsotypes B, C and D) found in 15% (7/45) of the isolates. The remaining type, ST 8 (pulsotype E) was found in a single isolate. The results of this study indicated that the MRSA clone ST 239 was a major clone in the selected university hospital of Tehran and that it was widely spread among the different wards as well as all the age groups of patients.     

金黄色葡萄球菌所致肺部感染的耐药特点及其Panton—Valentine杀白细胞素基因的携带状况分析

目的分析金黄色葡萄球菌所致肺部感染的耐药性特点及其Panton—Valentine杀白细胞素基因的携带状况。方法回顾性调查了温州医学院第一附属医院2005年1月至2006年1月医院感染的金黄色葡萄球菌所致肺部感染患者132例,对其体外药敏试验进行分析;并利用多重PCR检测其PVL基因,应用多位点基因序列分型（multilocus sequence typing,MLST）技术对PVL基因阳性的菌株进行序列分型。耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus,MRSA）的SCCmec基因分型采用多重聚合酶链反应。结果致肺部感染的132株金黄色葡萄球菌的耐药现象较为严重,仅对万古霉素、呋喃妥因及复方新诺明等药物的敏感率较高;其中经多重PCR筛选出10株携带PVL基因的金葡菌,全部为MRSA菌株,3株为ST239-SCCⅢ,2株为ST398-SCCmecⅢ,2株为ST398-SCCmecⅣ,ST25-SCCmecⅢ、ST59-SCCmecⅠ和ST88-SCCmecⅢ各1株。结论肺部感染的金黄色葡萄球菌对多种抗生素耐药,呈多重耐药性;其携带PVL基因占一定比例。     

Genomic Heterogeneity of Methicillin Resistant Staphylococcus aureus Associated with Variation in Severity of Illness among Children with Acute Hematogenous Osteomyelitis

Introduction

The association between severity of illness of children with osteomyelitis caused by Methicillin-resistant Staphylococcus aureus (MRSA) and genomic variation of the causative organism has not been previously investigated. The purpose of this study is to assess genomic heterogeneity among MRSA isolates from children with osteomyelitis who have diverse severity of illness.

Materials and Methods

Children with osteomyelitis were prospectively studied between 2010 and 2011. Severity of illness of the affected children was determined from clinical and laboratory parameters. MRSA isolates were analyzed with next generation sequencing (NGS) and optical mapping. Sequence data was used for multi-locus sequence typing (MLST), phylogenetic analysis by maximum likelihood (PAML), and identification of virulence genes and single nucleotide polymorphisms (SNP) relative to reference strains.

Results

The twelve children studied demonstrated severity of illness scores ranging from 0 (mild) to 9 (severe). All isolates were USA300, ST 8, SCC mec IVa MRSA by MLST. The isolates differed from reference strains by 2 insertions (40 Kb each) and 2 deletions (10 and 25 Kb) but had no rearrangements or copy number variations. There was a higher occurrence of virulence genes among study isolates when compared to the reference strains (p = 0.0124). There were an average of 11 nonsynonymous SNPs per strain. PAML demonstrated heterogeneity of study isolates from each other and from the reference strains.

Discussion

Genomic heterogeneity exists among MRSA isolates causing osteomyelitis among children in a single community. These variations may play a role in the pathogenesis of variation in clinical severity among these children.     

Characterization of Virulence Factors and Genetic Background of Staphylococcus aureus Isolated from Peking University People’s Hospital Between 2005 and 2009

The aim of this study was to investigate both the genetic features of MRSA strains and the occurrence of virulence factors produced by Staphylococcus aureus strains isolated from Peking University People’s Hospital in Beijing, China, between 2005 and 2009. A total of 179 S. aureus strains were isolated, 139 of which were MRSA. The MRSA strains were characterized epidemiologically by SCCmec typing, spa typing and agr typing, then were classified into different genetic groups. The prevalence of genes coding for 14 exotoxins and eight adhesion factors among the S. aureus samples was assessed via polymerase chain reaction. Cluster analysis based on virulence factors-encoding gene content was performed to divide the MRSA isolates into valid clusters. Correspondence analysis was done to analyze the correlation between virulence factors clusters and genetic groups. JCSC1716-agrI-t030 (67.6%), SCCmec-IIIA-agrI-t030 (14.4%), SCCmec-IIIA-agrI-t037 (8.6%) and SCCmecII-agrII-t002 (2.2%) were four predominant MRSA clones. PVL was positive only in MSSA strains, there were at least three superantigenic toxins in our HA-MRSA clones, the prevalence of 16 virulence factors genes (sea, seb, sec, sed, seg, sei, sej, pvl, lukE-lukD, eta, bbp, can, ebp, clfA, fib, fnbB) in MRSA and MSSA was found to be significantly different from MSSA (P < 0.05). Results of correspondence analysis among clusters based on virulence factors genes and groups based on genetic typing illustrated not only the correspondence relationship between groups and clusters overall (P < 0.001), but also the genetic diversity of MRSA strains with respect to virulence factors genes.     

Prospective surveillance of community-onset and healthcare-associated methicillin-resistant Staphylococcus aureus isolated from a university-affiliated hospital in Japan

We conducted a prospective comparative study of community-onset (CO) and healthcare-associated (HA) methicillin-resistant Staphylococcus aureus(MRSA) strains between 2000 and 2001 at Tokyo Women's Medical University Hospital (1,500 beds) in Japan. Of the 172 consecutive MRSA isolates analyzed, 13 (8%) were categorized as CO-MRSA. The mean age of patients with CO-MRSA was significantly younger than that of patients with HA-MRSA. Most CO-MRSA strains were isolated from skin and more likely to be susceptible to erythromycin, clindamycin, tetracycline, levofloxacin, and spectinomycin compared to HA-MRSA isolates. Pulsed-field gel electrophoresis (PFGE) analysis, staphylococcal cassette chromosome mec(SCCmec) typing, and multi-locus sequence typing (MLST) revealed that CO-MRSA strains were divided into the following multi-clones: 3 clone A: II: ST5 (PFGE type: SCCmec type: MLST sequence type); 1 L: II: ST5; 1 H: IV: ST1; 1 I: IV: ST81; 2 D: IV: ST8; 1 B: IV: ST89; 1 B: IV: ST379; and 3 B: IV: ST91. Of the 159 HAMRSA strains, 124 (78%) belonged to a single clone (PFGE clone A: SCCmec type II: tst and sec positive: coagulase type II: multi-drug resistance). Four CO-MRSA strains belonging to PFGE clone B: SCCmec type IV: MLST clonal complex 509 (ST89, 91, 379) had the exfoliative toxin B (etb) genes, but all CO-MRSA and HA-MRSA strains did not possess the Panton-Valentine leukocidin (pvl) genes. These results demonstrate that multiple lineages of CO-MRSA have the potential for dissemination in the community in Japan.     

Genotypes and toxin gene profiles of Staphylococcus aureus clinical isolates from China

A total of 108 S. aureus isolates from 16 major hospitals located in 14 different provinces in China were characterized for the profiles of 18 staphylococcal enterotoxin (SE) genes, 3 exfoliatin genes (eta, etb and etd), and the toxic shock syndrome toxin gene (tsst) by PCR. The genomic diversity of each isolate was also evaluated by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and accessory gene regulator (agr) typing. Of these strains, 90.7% (98/108) harbored toxin genes, in which tsst was the most prevalent toxin gene (48.1%), followed by sea (44.4%), sek (42.6%) and seq (40.7%). The see and etb genes were not found in any of the isolates tested. Because of high-frequency transfer of toxin gene-containing mobile genetic elements between S. aureus strains, a total of 47 different toxin gene combinations were detected, including a complete egc cluster in 19 isolates, co-occurrence of sea, sek and seq in 38 strains, and sec and sel together in 11 strains. Genetic typing by PFGE grouped all the strains into 25 clusters based on 80% similarity. MLST revealed 25 sequence types (ST) which were assigned into 16 clonal complexes (CCs) including 2 new singletons. Among these, 11 new and 6 known STs were first reported in the S. aureus strains from China. Overall, the genotyping results showed high genetic diversity of the strains regardless of their geographical distributions, and no strong correlation between genetic background and toxin genotypes of the strains. For genotyping S. aureus, PFGE appears to be more discriminatory than MLST. However, toxin gene typing combined with PFGE or MLST could increase the discriminatory power of genotyping S. aureus strains.     

天津地区气单胞菌分离株的鉴定与多位点序列分型

[目的]研究气单胞菌菌株分类情况,并分析其致病性.[方法]采集环境样品和鱼类标本,分离并鉴定气单胞菌菌株,并运用多位点序列分型(Multilocus sequence typing,MLST)方法进行分类研究,利用PCR和测序方法分析毒力基因Aera、Hly、Aha1、GCAT和Nuc的分布.[结果]通过对分离菌株的16S rRNA基因进行分析,确认属于4种不同气单胞菌的7个分离株.发现所有菌株至少有1种毒力基因阳性,其中3株具有4种毒力基因.药物敏感实验显示,6株分离株对3种或3种以上抗菌素具有多重耐药性.最后,对看家基因gyrB、groL、gltA、metG、ppsA和recA进行分析,与MLST数据库中的等位基因序列比对,发现7株分离株均为新的不同的序列型(Sequence type,ST).[结论]气单胞菌具有较高的遗传多样性.     

Comparative genotypic and phenotypic characterisation of methicillin-resistant Staphylococcus aureus ST398 isolated from animals and humans

The high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) ST398 among pigs in certain European countries and North America and its occurrence in other animal species raises a question concerning the molecular mechanisms mediating the success of this lineage. In this study a panel of S. aureus strains belonging to sequence type (ST) 5 (n = 4), ST8 (n = 5), ST15 (n = 5), ST22 (n = 8), clonal complex (CC) 30 (n = 8), CC97 (n = 8), CC130 (n = 4), CC151 (n = 4) and ST398 (n = 18) were screened by DNA microarray and PCR for the carriage of virulence and antimicrobial resistance genes. Isolates belonging to the same sequence type/clonal complex (ST/CC) were found to share similar virulence gene profiles. The ST398 lineage displayed the lowest content of virulence genes, which consisted mainly of genes detected among the majority or all of the analysed lineages. All MRSA ST398 isolates lacked accessory virulence genes that were detected in other ST/CC. In contrast to virulence genotype, the antimicrobial resistance genes profiles varied between isolates belonging to the same ST/CC and profile similarities could be observed for isolates from different lineages. MRSA ST398 isolates in particular displayed significant diversity and high content of antimicrobial resistance genes. This was comparable with certain MRSA belonging to other sequence types particularly the equine MRSA ST8. The apparent lack of significant virulence genes among MRSA ST398 strains, demonstrates that the lineage features a unique genetic background but no ST398-specific virulence markers could be identified.     

Molecular Relatedness of Methicillin-Resistant S. aureus Isolates from Staff, Environment and Pets at University Veterinary Hospital in Malaysia

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a problem in veterinary medicine and is no longer considered as a mere nosocomial pathogen. We studied the occurrence of MRSA in veterinary personnel, cats and dogs and the environmental premises in University Veterinary Hospital (UVH). We found the prevalence of MRSA as follows: UVH 2/28 (7.1%) staff, 8/100 (8%) of the pets [5/50 (10%) of the dogs and 3/50 (6%) of the cats)], and 9/28 (4.5%) of the environmental samples. Antibiotic sensitivity tests (AST) show multi-resistance characteristics of the MRSA and the minimum inhibitory concentration (MIC) values for the isolates ranged from 1.5 μg to >256 μg/ml. Molecular typing by using multi-locus sequence typing (MLST), staphylococcal protein A typing (spa typing) and pulsed-field gel electrophoresis (PFGE) was conducted and the results from MLST indicated that an isolate from a veterinary personnel (PG21), typed as ST1241 belonged to the same clonal complex (CC) as the two isolates from two dogs (DG16 and DG20), both being typed as ST59. The PFGE results revealed that the two isolates from two veterinary personnel, PG21 and PG16 belonged to closely related MRSA strains with isolates from dog (DG36) and from environmental surface (EV100) respectively. The fact that PFGE revealed close similarity between isolates from humans, a dog and environmental surfaces indicates the possibility for either of them to be the source of MRSA and the potential routes and risks of spread.     

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.     

199株猪链球菌临床分离株血清型、毒力基因及多位点序列分型分析

【背景】猪链球菌（Streptococcus suis,SS）血清型、基因型众多,毒力因子复杂。【目的】了解SS临床分离株血清型、毒力基因分布、分子分型特征及其之间的相关性。【方法】针对199株SS临床分离株,应用PCR技术进行血清分型和毒力基因检测,采用多位点序列分型方法（multilocus sequence typing,MLST）进行基因分型,并分析SS血清型、毒力基因型和序列型（sequence type,ST型）的流行特点及其关联性。【结果】199株SS临床分离株分属于16种血清型（1、2、3、4、6、7、8、9、10、12、15、16、21、24、29和30型）,主要以2、4、3型为主,分别占26.13%（52/199）、14.57%（29/199）和12.06%（24/199）,未定型（NT）菌株占21.61%（43/199）。共鉴定出72种ST型,其中ST1、ST94、ST117、ST7、ST28和ST87为主要ST型,分别占12.56%（25/199）、11.56%（23/199）、9.56%（19/199）、9.04%（18/199）、6.03%（12/199）和3.01%（6/199）,另有24种新发现的ST型（ST1224—ST1227,ST1229—ST1235,ST1241—ST1242,ST1300—ST1310）;分为12个克隆群（cloning complexes,CC）和32个单个ST型。199株SS分离株中毒力基因fbps的检出率最高,为96.98%（193/199）;共有19种毒力基因型,其中66株（33.17%）epf-/mrp-/sly-/gapdh+/fbps+/orf2+型SS为优势毒力基因型。【结论】近年来SS的优势血清型为2、4和3型;ST型具有明显的遗传异质性,种内分化程度较高且与ST型存在一定交叉性;毒力基因分布情况存在差异,毒力基因型呈现多样化。本研究对SS临床分离株的流行特征进行探究,为猪SS病诊断、治疗和制定防控措施提供科学依据。     

Comparative analysis of whole genome structure of <Emphasis Type="Italic">Streptococcus suis</Emphasis> using whole genome PCR scanning

An outbreak associated with Streptococcus suis infection in humans emerged in Sichuan province, China in 2005. The outbreak is atypical for the apparent large number of human cases, high fatality rate and geographical spread. To determine whether the bacterium has changed, we compared both human and animal isolates from the Sichuan outbreak with those collected previously within China and in other countries using whole genome PCR scanning (WGPScaning) comparative sequencing of several known virulence factor genes and multilocus sequence typing (MLST) analysis. WGPScanning analysis showed that all primer pairs yielded PCR products of the expected sizes in all four strains tested. The nucleotide sequences of all the detected virulence factor genes are identical in the four strains and MLST results showed that the four isolates studied and reference strain all belonged to the ST1 complex. No new genetic changes were found in the genome structure of the isolates from this Sichuan outbreak.     

First Confirmation of Integron-Bearing Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus </Emphasis><Emphasis Type="Italic">aureus</Emphasis>

Six methicillin-resistant Staphylococcus aureus MRSA strains from two nosocomial infection cases described in a previous study [15], of which two occurred in March and the other four in May, 2005, were found to possess one copy of class 1 integron with aadA2 gene cassette located on chromosomes by Southern hybridization. Polymerase chain reaction (PCR) detection of mecA and pvl, SCCmec typing, multilocus sequence typing (MLST), spaA typing and coa typing were also performed. The results revealed 6 MRSA fell into the ST239-MRSA-III group (clonal complex 8), with the spaA type GKAOMQ and coa type HIJKL, whereas the pvl locus was not detected. DNA fingerprinting analysis by random amplified polymorphic DNA-PCR using three different assays were also performed, and all strains exhibited identical patterns, indicating that they were clonally related and might be mainly due to a specific clone in the hospital. This was the first time, to our knowledge, that class 1 integron-bearing MRSA (I-MRSA), simultaneously carrying two mobile genetic elements was confirmed: class 1 integron and SCCmec.     

Assignment of Staphylococcus aureus isolates to clonal complexes based on microarray analysis and pattern recognition

A DNA microarray was designed for the rapid genotyping of Staphylococcus aureus. It covers 185 distinct genes and about 300 alleles thereof, including species-specific controls, accessory gene regulator (agr) alleles, genes encoding virulence factors, and microbial surface components recognizing adhesive matrix molecules, capsule type-specific genes, as well as resistance determinants. It was used to examine 100 clinical isolates and reference strains. Relationships of leukocidin and ssl/set (staphylococcal superantigen-like or exotoxin-like) genes were reviewed considering these experimental results as well as published sequences. A good correlation of overall hybridization pattern and multilocus sequence typing was found. Analysis of hybridization profiles thus allowed not only to assess virulence and drug resistance, but also to assign isolates to strains and to clonal complexes. Hybridization data were used to construct a split network tree and to analyse relationships between strains. Allelic variations of a number of genes indicate a division of S. aureus into three major branches that are not in accordance to agr group or capsule-type affiliations. Additionally, there are some isolated lineages, such as ST75, ST93, or ST152. These strains produce aberrant hybridization profiles, indicating that only a part of the gene pool of S. aureus has been investigated yet.     

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.     

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.     

Multilocus Sequence Typing and Virulence Profiles in Uropathogenic Escherichia coli Isolated from Cats in the United States

The population structure, virulence, and antimicrobial resistance of uropathogenic E. coli (UPEC) from cats are rarely characterized. The aim of this study was to compare and characterize the UPEC isolated from cats in four geographic regions of USA in terms of their multilocus sequence typing (MLST), virulence profiles, clinical signs, antimicrobial resistance and phylogenetic grouping. The results showed that a total of 74 E. coli isolates were typed to 40 sequence types with 10 being novel. The most frequent phylogenetic group was B2 (n = 57). The most frequent sequence types were ST73 (n = 12) and ST83 (n = 6), ST73 was represented by four multidrug resistant (MDR) and eight non-multidrug resistant (SDR) isolates, and ST83 were significantly more likely to exhibit no drug resistant (NDR) isolates carrying the highest number of virulence genes. Additionally, MDR isolates were more diverse, and followed by SDR and NDR isolates in regards to the distribution of the STs. afa/draBC was the most prevalent among the 29 virulence-associated genes. Linking virulence profile and antimicrobial resistance, the majority of virulence-associated genes tested were more prevalent in NDR isolates, and followed by SDR and MDR isolates. Twenty (50%) MLST types in this study have previously been associated with human isolates, suggesting that these STs are potentially zoonotic. Our data enhanced the understanding of E. coli population structure and virulence association from cats. The diverse and various combinations of virulence-associated genes implied that the infection control may be challenging.