Analysis of three phenotyping methods and pulsed-field gel electrophoresis for differentiation of methicillin resistant Staphylococcus aureus isolated from two hospitals in Thailand

Methicillin resistant Staphylococcus aureus (MRSA) is an important hospital and community-acquired pathogen. Rapid and reliable epidemiologic typing is necessary for controlling the spread of MRSA outbreak. The objective of this study was to compare the phenotyping with the genotyping method to differentiate MRSA isolates obtained from the two hospitals in Thailand (central and northeastern). Seventy-four MRSA isolates were randomly collected and confirmed by the presence of mecA gene. Antibiogram, phage typing and enterotoxin production were used for the phenotyping analysis. Pulsed-field gel electrophoresis (PFGE) with Smal digestion of chromosomal DNA was used for the genotyping analysis. We found 17 distinct profiles by the 3 phenotypic typing methods and 18 PFGE types designated as 5 major types (A-E) and 13 subtypes. The most frequent PFGE types and their related subtypes found in both hospitals were A and C, comprising 54 and 27%, respectively. The antibiogram could differentiate 6 different types. All isolates were resistant to the majority of antimicrobial agents tested, but were susceptible to vancomycin and fosfomycin. Ten (13.5%) MRSA isolates produced enterotoxin A. Nontypable phage and phage type 77 were found predominantly in MRSA isolated from the northeast and central hospital, respectively. A significant correlation was found between the phenotyping and the genotyping methods and there was a good correlation between antibiogram and PFGE. Antibiogram typing alone can be used as a useful epidemiological marker for practical purposes. PFGE types A and C were the common endemic MRSA clones in both hospitals in Thailand.     

Pulsed-field gel electrophoresis type and antimicrobial susceptibility of arbekacin mupirocin and teicoplanin resistant methicillin-resistant Staphylococcus aureus

Among the isolates of methicillin resistant Staphylococcus aureus (MRSA) which were isolated at Juntendo University Hospital between 1992 and 1998, there were 2 arbekacin resistant strains, 9 mupirocin resistant strain and 7 teicoplanin resistant strains. For each resistant strain, we studied pulsed-field gel electrophoresis (PFGE) pattern, coagulase type, antimicrobial susceptibility pattern and investigated a distribution of the resistant strains at the hospital wards. Two arbekacin resistant strains showed PFGE type A (A3 and A7, respectively). Nine mupirocin resistant strains were type C1; 3 strains, type A (A1, A4, A8); 3 strains and type D, E, G for 1 one each strain. The antimicrobial susceptibility pattern was not similar to each other and the strains were isolated from different year. It is suggested that these strains were not same origin. Seven teicoplanin resistant strains had PFGE type B (B1; 5 strains, B2/B5; 1 each strain). The antimicrobial susceptibility pattern of the strains was similar to each other and the strains were isolated from the same hospital ward between 1995 and 1997. From the above fact, the resistant strains appear to be hospital strains which have same origin.     

Investigation of infective endocarditis clinical isolates of methicillin resistant Staphylococcus aureus non-responsive to vancomycin

Methicillin resistant Staphylococcus aureus (MRSA) is one of the most important strains which induce hospital and post-operative infection. In cases of infective endocarditis in which VCM was not efficacious, MRSA strains were chronologically isolated at three different times and examined with the following parameters: minimum inhibitory concentration (MIC), fractional inhibitory concentration (FIC) index, Mu 3 agar, population analysis, pulse field gel electropholesis (PFGE). The PFGE banding patterns of the three MRSA isolates were the same, therefore, it was concluded that the same strain of MRSA was selected for reduced susceptibility. A pattern of Mu 3 and Mu 50 was demonstrated under population analysis.     

糖尿病足感染中耐甲氧西林葡萄球菌的脉冲场凝胶电泳分型研究

目的研究分离白糖尿病足感染溃疡创面的耐甲氧西林金黄色葡萄球菌（MRSA）和耐甲氧西林表皮葡萄球菌（MRSE）的分子流行病学特点。方法收集2008年1月至2010年6月从天津医科大学代谢病医院388例糖尿病足溃疡住院患者中分离的24株MRSA和18株MRSE菌株,应用脉冲场凝胶电泳方法对菌株进行同源性分析。结果MRSA菌株相似系数为42％～97％,分为11型（A—K）,12株为A型克隆株（其中11株分离于2008年的1至5月）,包含12个亚型;B型2株;c型2株;其余8型各1株。MRSE菌株相似系数为34％～100％,分14型（A—N）,5株A型克隆株中3株A1亚型,A2、A3亚型各1株;其余13型各1株。结论本院糖尿病足病科2008年1至5月分离的MRSA菌株很可能有A型MRSA克隆株的暴发流行,2008年6月至2010年6月呈散发感染;2008年1月至2010年6月分离的MRSE菌株呈散发性,未发现有克隆株暴发流行。     

甲氧西林耐药的金黄色葡萄球菌感染一例并文献复习

目的 通过对1例金黄色葡萄球菌染色体基因盒(SCCmec)Ⅴ型的甲氧西林耐药的金黄色葡萄球菌(MRSA)感染病例的分析,增强对MRSA社区或医院获得性感染的认识.方法 对2008年6月19日北京医院皮肤科收治、呼吸科会诊的1例SCCmec Ⅴ型的MRSA感染病例分离自血及痰标本的3株MRSA菌株进行培养及体外药物敏感试验.应用PCR方法对分离菌株进行甲氧西林耐药性决定因子A(mec A)基因鉴定和杀白细胞素(PVL)基因检测,应用多重PCR方法对其进行SCCmec分型.结果 患者男,73岁,因剥脱性皮炎入院,病程中出现MRSA败血症,继之出现双侧肺炎,最终因感染性休克死亡.药物敏感试验结果显示,该MRSA菌株除对β-内酰胺类抗生素耐药外,对其他抗生素如克林霉素敏感,对莫西沙星、庆大霉素为中介性耐药.分离的3株MRSA均属于SCCmec β型.结论 该患者为医院获得性MRSA感染,但其菌株具有社区获得性MRSA的特点,临床医师应注意鉴别.     

Analysis of methicillin-resistant Staphylococcus aureus isolates by proton magnetic resonance spectroscopy.

OBJECTIVES: To evaluate the stability and(1)H nuclear magnetic resonance ((1)H NMR) differences among bacterial strains, we analysed(1)H NMR spectra for 50 methicillin-resistant Staphylococcus aureus (MRSA) isolates from 42 patients. METHODS:(1)H NMR spectra for 50 MRSA isolates were obtained at 30 degrees C using a JNM-GX 270 NMR spectrometer at a field strength of 6.34 Tesla (270 MHz for(1)H). DNA fingerprints were obtained by pulsed-field gel electrophoresis (PFGE). RESULTS: Each strain had eight to nine specific resonance features in the 0.5-4.5 ppm spectral region. These features were found in all strains, but the intensity of each feature varied between strains. Six resonance features (A-F) were selected for investigation. The relative integrated intensity (mean+/-SD, normalized to feature C = 1) of each feature was: feature A, 0.49+/-0.16; feature B, 3.93+/-0.81; feature D, 0.38+/-0.22; feature E, 1.51+/-0.96; feature F, 2.18+/-0.47. Within-strain reproducibility of feature intensities for A, B, D and F was good (coefficient of variation < 10%) for replicate cultures, analyzed on separate days. Feature E showed poor within-strain reproducibility. Storage at 4 degrees C for 4 months or disintegration of the micro-organisms by ultrasound did not alter(1)H NMR spectra. Two isolates from different patients, but the same hospital, showed indistinguishable NMR spectra, and were also indistinguishable in PFGE. Five strains with distinct PFGE patterns showed differences in NMR spectra outside the range of within-strain variation. CONCLUSIONS: It is possible to analyse the whole molecular structure of MRSA by(1)H NMR. With this technique, we established that there are detectable, reproducible differences in quantitative cell composition between MRSA strains.(1)H NMR spectroscopy appears to have potential as a useful tool for epidemiological typing of bacteria.     

Clonal types and antimicrobial resistance profiles of methicillin-resistant Staphylococcus aureus isolates from hospitals in south Brazil

In the present study were evaluated the DNA macrorestriction profile and SCCmec types for nine multi-resistant MRSA selected. Also antimicrobial susceptibility testing by disk diffusion method was evaluated for 68 MRSA isolates against 12 antimicrobial agents. The isolates were recovered from blood culture collected from hospitalized patients in three hospitals of Porto Alegre, Brazil. PFGE and PCR for mecA and SCCmec I, II, III, IV types genes were done on selected nine isolates with susceptibility only to vancomycin, teicoplanin and linezolid. Two clone profiles, with five subtypes, were demonstrated among multi-resistant MRSA analyzed. Eight isolates showed harbor SCCmec type III and one isolate was not typeable. The knowledge of SCCmec type, clone and antimicrobial profiles among S. aureus is essential mainly to prevention and control of dissemination of the antimicrobial resistance.     

Evaluation of different primers for detecting mecA gene by PCR in comparison with phenotypic methods for discrimination of methicillin-resistant Staphylococcus aureus

Detection of the mecA gene by polymerase chain reaction (PCR) is the gold standard for identifying methicillin-resistant Staphylococcus aureus (MRSA). PCR assays, employing MR1-MR2 primers (primer set 1) and MR3-MR4 primers (primer set 2) to generate 154 and 533 bp fragment, respectively, are most widely used for amplification of mecA gene. The purpose of this study was to evaluate the presence of mecA gene in 100 clinical isolates of S. aureus using PCR with the two pairs of primers. The results were compared to the broth dilution MIC method, oxacillin salt screening method (OSS) and oxacillin disk agar diffusion method (ODD). Fifteen of the 100 isolates showed a discrepancy between the mecA primer sets 1 and 2. Three isolates (3%) without the mecA gene showed discrepancies with phenotypic methods. The sensitivity, specificity and positive and negative predictive values for the 154 and 533 bp products of mecA were 79, 85, 83, 81 and 94, 100, 100, 94%, respectively. The results indicated that primer set 2 was more appropriate than primer set 1 for the detection of mecA gene in MRSA. There was a good correlation among the mecA gene detection, ODD and OSS methods. The discrepancy of three isolates between PCR and phenotypic methods should be clarified for other resistant mechanisms.     

2011-2013年河南省鼠伤寒沙门菌耐药与分子分型研究

目的 分析2011-2013年河南省鼠伤寒沙门菌临床分离株的耐药状况与脉冲场凝胶电泳(PFGE)分子分型带型,为以鼠伤寒沙门菌为代表的食源性疾病暴发预警、调查、溯源及公共卫生意义上的抗生素使用策略提供基线与参考数据。方法 根据国际PulseNet 细菌性传染病分子分型监测网络公布的沙门菌PFGE分型技术与美国临床与实验室标准协会(CLSI)沙门菌K-B法药敏测试方案,对2011-2013年分离自我省5个哨点医院的72株鼠伤寒沙门菌进行13种抗生素的药敏测试与PFGE分子分型分析。结果 72株沙门菌对8类13种抗生素均有不同程度的耐药,有65株为多重耐药菌株(90.3%),其中耐3~5种的为6株(8.3%),耐6~8种的为34株(47.2%),耐9~10种的为10株(13.9%)。72株鼠伤寒沙门菌经Xba Ⅰ酶切与脉冲场凝胶电泳后,获得45种带型,每种带型包含菌株数1~9株不等,相似度区间为65.6%~100%。结论 河南省临床分离的鼠伤寒沙门菌耐药状况普遍比较严重,PFGE带型呈现出多样性的同时又具有较显著的优势带型特点,部分带型与其对应的耐药谱具有一定的关联性与相同的聚集性。     

Genetic relatedness of Enterococcus faecalis isolates with high-level gentamicin resistance from patients with bacteraemia in the south east of Sweden 1994-2001

High-level gentamicin resistant (HLGR) enterococci (Enterococcus faecalis and Enterococcus faecium) have become a substantial nosocomial problem in many countries. In this study, we investigated the prevalence of HLGR enterococci and their genetic relatedness in blood culture isolates from patients with bacteraemia admitted to the 3 hospitals in Osterg?tland, a county in the south east of Sweden, during 1994-2001. 36 of 250 E. faecalis (14%,) and 4 of 106 E. faecium isolates (4%) were shown by PCR to carry the aac(6')-Ie-aph(2")-Ia aminoglycoside modifying gene and these isolates were also classified as HLGR enterococci by the gentamicin antibiotic disk diffusion method. A majority of HLGR E. faecalis isolates (83%) belonged to the same cluster of genetically related isolates, according to the pulsed-field gel electrophoresis (PFGE) patterns, whereas all 4 HLGR E. faecium isolates had unique PFGE patterns. In conclusion, our study showed that in contrast to studies from many other countries, the presence of HLGR enterococci was more common in E. faecalis than in E. faecium and appeared the first time in 1996 and 1999, respectively. Bacteraemia with HLGR enterococci in Osterg?tland was mainly due to the spread of a cluster of related E. faecalis strains.     

猪源耐甲氧西林金黄色葡萄球菌的耐药表型及其SCCmec基因分型研究

目的 了解上海猪场及屠宰场耐甲氧西林金黄色葡萄球菌(MRSA)的流行状况、耐药谱特征及其SCCmec基因分型特点。方法 我们于2011年8月—2012年5月分别在上海市5个规模化猪场和1个屠宰场,采集猪鼻腔拭子样品共计232份。通过7.5%氯化钠肉汤预增菌,显色培养基显色培养分离金黄色葡萄球菌(SA),采用双重PCR方法扩增其中是否含有nuc基因和mecA基因进行MRSA鉴定;以肉汤稀释法进行药物敏感性试验;应用多重PCR方法进行SCCmec基因分型;同时检测杀白细胞素(PVL)基因。结果 共计分离得到139株SA,其中70株MRSA,MRSA的检出率为30.2%(70/232)。基因分型显示均为SCCmecⅣb型,未检测到PVL基因,药敏结果显示MRSA除对利奈唑胺,万古霉素,阿米卡星全部敏感外,对头孢噻呋,庆大霉素,诺氟沙星耐药率分别为74.3%,62.9%和42.9%,对阿奇霉素,红霉素,氟苯尼考,氯霉素,苯唑西林的耐药率均为100%,值得注意的,本研究中分离的全部MRSA都对截短侧耳类药物泰妙菌素、沃尼妙林以及人医新药瑞他帕林表现高水平耐药。结论 结果显示了上海地区猪源MRSA主要流行SCCmecⅣb型,并且具有多重耐药的特点,尤其对截断侧耳类人医新药全部耐药,这也提示我们要防控动物源耐药菌或耐药基因通过食物链或者环境向人类的传播的潜在风险。     

The spread of a mupirocin-resistant/methicillin-resistant Staphylococcus aureus clone in Kuwait hospitals

High-level mupirocin- and methicillin-resistant Staphylococcus aureus (MRSA) isolated from five hospitals in Kuwait were studied by pulsed-field gel electrophoresis (PFGE) to determine their relatedness to one another and to high-level mupirocin-resistant MRSA isolated previously in a Burns Unit. The genetic location of mupirocin resistance determinant was also determined. All of the isolates were resistant to gentamicin, kanamycin, streptomycin, tetracycline and cadmium, and contained plasmids of 38, 26 and 2.8 kb. Two isolates contained additional 4.4-kb plasmids. Transfer experiments demonstrated that the 38-kb plasmid encoded high-level mupirocin resistance and the 4.4-kb plasmid encoded chloramphenicol resistance. PFGE typing of representative isolates from the five hospitals demonstrated that the majority of them had identical or closely related pulsed-field patterns suggesting that they had a common origin. However, they differed from high-level mupirocin-resistant MRSA isolated previously in the Burns Unit in their resistance and pulsed-field patterns. Whereas the majority of the previous isolates were susceptible to ciprofloxacin and resistant to trimethoprim and chloramphenicol, the majority of the current isolates were susceptible to trimethoprim and chloramphenicol, and resistant to ciprofloxacin. Only one of the current isolates had identical pulsed-field pattern to the majority of isolates obtained previously in the Burns Unit. The results suggested that a previously dominant clone of high-level mupirocin-resistant MRSA has been replaced in the Burns Unit by a new clone, which also spread in four other hospitals.     

Decrease in the incidence of mupirocin resistance among methicillin-resistant Staphylococcus aureus in carriers from an intensive care unit

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a serious nosocomial problem, globally distributed. Decolonization with mupirocin can be used to control its dissemination. OBJECTIVE: To determine the incidence of mupirocin resistance among MRSA carriers from an intensive care unit. METHODS: We obtained 2723 nasal swabs during 3 years. Resistance to methicillin and mupirocin were verified (agar diffusion and the E test) and confirmed by polymerase chain reaction (PCR) (mecA for methicillin; ileS-2 and mupA for mupirocin). Plasmid-curing procedure and pulsed-field gel electrophoresis (PFGE) were employed in isolates exhibiting high resistance to mupirocin (HR-Mup) and in other selected organisms. RESULTS: The overall incidence of HR-Mup among MRSA carriers during the studied period was 4.84% (8/165); however, the incidence decreased from 13.04% (6/46) in the first year to 3.5% (2/57) in the second year and was 0% in the last year (P = .02). LR-Mup, in contrast, increased significantly (P = .01). CONCLUSION: Plasmid-curing procedure showed the plasmid location of genes responsible for HR-Mup. PFGE demonstrated that most MRSA, including the isolates with HR-Mup, were genetically related. The decline in HR-Mup may be attributable to the plasmid location of genes (ileS-2/mupA) and to the fact that all patients colonized with HR-Mup MRSA died or were discharged in a relatively short period of time.     

Usefulness of the restriction-modification test plus staphylococcal cassette chromosome mec types and Panton-Valentine leukocidin encoding phages to identify Staphylococcus aureus methicillin-resistant clones

We studied the usefulness of the restriction-modification (RM) test, staphylococcal cassette chromosome (SCC) mec types, and Panton-Valentine leukocidin (PVL)-encoding phages to identify Staphylococcus aureus methicillin-resistant lineages and to differentiate clones that belong to the same lineage. A total of 108 methicillin-resistant S. aureus (MRSA) strains were characterized by pulsed-field gel electrophoresis (PFGE)--multi-locus sequence typing (MLST)--spa-typing. The RM correctly identified the lineages CC5, CC8, CC30, and CC398, but not CC25 and CC72. The SCCmec and RM combined analysis allowed differentiation between MLST types within the same lineage. Only 5 MRSA strains were PVL-positive. Four PVL-positive USA300 isolates carried elongated-head type PVL-encoding phages, while the sequence type (ST)-30 strain carried an icosahedral-head phage. The combination of the RM test method, SCCmec types, and PVL phage identification could be useful for MRSA typing purposes.     

Epidemiological analysis of Salmonella enteritidis isolates using pulsed-field gel electrophoresis and bacteriophage typing over the period of April 2000 to March 2003 in Gifu Prefecture

We examined a total of 151 Salmonella enterica serovar Enteritidis strains isolated in Gifu Prefecture during the period from April 2000 to March 2003 by using bacteriophage typing and pulsed-field gel electrophoresis (PFGE). Bacteriophage typing classified them into twelve phage types (PT) and RDNC (reacted but did not conform). The predominant phage type was PT47 (34.4%) followed by PT1 (21.9%), PT4 (16.6%) and RDNC (11.3%). XbaI- and BlnI-digested PFGE analyses identified 17 and 44 PFGE patterns, respectively, indicating that PFGE with BlnI had more discriminating power than that with XbaI. Combination of the phage types and PFGE types of BlnI could make 53 subtypes. Some isolates with the same phage type were subdivided into different PFGE types, but those with PT47 were not. PT47 isolates were derived from sporadic patients with gastroenteritis, food poisoning outbreaks and healthy carriers through the years. This suggests that PT47 is highly clonal and disseminates over our prefecture.     

The evolution of methicillin resistance in Staphylococcus aureus: similarity of genetic backgrounds in historically early methicillin-susceptible and -resistant isolates and contemporary epidemic clones

The key genetic component of methicillin resistance, the mecA determinant, is not native to Staphylococcus aureus. Thus, the evolution of methicillin-resistant S. aureus (MRSA) must have begun with the acquisition of the mecA determinant from an unknown heterologous source some time before the first reported appearance of MRSA isolates in clinical specimens in the U.K. and Denmark (in the early 1960s). We compared the genetic backgrounds and phenotypes of a group of methicillin-susceptible S. aureus (MSSA) isolates to the properties of MRSA strains isolated in Denmark and the U.K. during the same time period, and also to the genetic profiles of contemporary epidemic clones of MRSA. All early MRSA isolates resembled a large group of the early MSSA blood isolates in phenotypic and genetic properties, including phage group, antibiotype (resistance to penicillin, streptomycin, and tetracycline), pulsed-field gel electrophoresis pattern, and spaA type and multilocus sequence type, strongly suggesting that the early MSSA examined here represented the progeny of a strain that served as one of the first S. aureus recipients of the methicillin-resistance determinant in Europe. The genetic background of this group of early MSSA isolates was also very similar to that of the widely disseminated contemporary "Iberian clone" of MRSA, suggesting that genetic determinants present in early MSSA and essential for some aspects of the epidemicity and/or virulence of these strains may have been retained by this highly successful contemporary MRSA lineage.     

Prevalence of methicillin-resistant Staphylococcus aureus among university students in Thailand

We studied the prevalence of methicillin sensitive Staphylococcus aureus (MSSA) and methicillin resistant Staphylococcus aureus (MRSA) nasal colonization among healthy young Thai adults. MSSA nasal colonization was found in 30 of 200 subjects (15%). The prevalence of MRSAnasal carriage was 1% (2 of 200) detected by cefoxitin/oxacillin disk diffusion and oxacillin salt screening methods. These carriers were associated with health care risk factors. The two MRSA isolates were mecA positive, SCCmec type II. All S. aureus isolates were tested for antibiotic resistance. Their resistance rates to penicillin, erythromycin, clindamycin, oxacillin and cefoxitin were 96.7, 26.7, 26.7, 6.7 and 6.7%, respectively. All MSSA and MRSA isolates were susceptible to gentamicin, chloramphenicol, trimethoprim/sulfamethoxazole, rifampicin, linezolid, fusidic acid, mupirocin, ciprofloxacin and vancomycin. The results of this first study of MRSA nasal colonization among healthy young Thai adults suggests MRSA is present in the Thai community.     

Molecular typing of methicillin-resistant Staphylococcus aureus in a university teaching hospital

Plasmid analysis and pulsed-field gel electrophoresis (PFGE) were used to study the epidemiologic relationship among methicillin-resistant Staphylococcus aureus (MRSA) strains isolated at Tokyo Medical and Dental University Hospital. We found that 263 of 276 MRSA isolates had plasmids, which could be classified into 30 different patterns according to the number and plasmid molecular weight. Strains which harboured a single plasmid of approximately 13.4 Mds in molecular weight were the most numerous (55.7% of the isolates). These strains were isolated from 14 of 17 hospital wards. The largest number of strains with this plasmid pattern (33 strains) were isolated from a single ward. PFGE typing was then performed to further confirm the relationships among these 33 strains. The PFGE banding patterns of these strains were highly similar. The antibiogram profiles of these strains were also correlated with the PFGE pattern. Thus, the results suggest that these strains are epidemiologically related and spread throughout the ward. Combined plasmid analysis and PFGE were effective for discriminating the various MRSA isolates.     

Molecular epidemiological analysis of Mycobacterium kansasii isolates

PURPOSE: To make molecular epidemiological analysis of Mycobacterium kansasii (M. kansasii) isolates. METHODS: We examined 174 M. kansasii isolates from clinical samples of patients at National Hospital Organization Kinki-chuo Chest Medical Center from June 1, 2002 to August 31, 2005 by polymerase chain reaction (PCR) -restriction analysis (PRA) of the heat shock protein (hsp) 65 gene (hsp65-PRA), sequencing (ITS, 16S-23S internal transcribed spacer, and hsp65 for discrepant case between hsp65-PRA and ITS sequence), pulsed-field gel electrophoresis (PFGE), and restriction fragment length polymorphism (RFLP) with the major polymorphic tandem repeat (MPTR) probe and the IS1652 probe of genomic DNA. RESULTS: Of the 174 M. kansasii isolates, 170 strains were classified as M. kansasii type I using hsp65-PRA, while two isolates belonged to type II and one each isolate to type IIb and VI, respectively. Although the ITS sequence of these isolates also identified the same region of polymorphism by hsp 65-PRA, only type II b might be revealed atypical type II, a transitional type from typical type II to intermediate type I by hsp65 sequence. The polymorphic patterns by RFLPs with MPTR and IS1652 probe were shown specific for each homogeneous cluster by hsp 65-PRA. In addition, 159 isolates were recognized the same common pattern A by PFGE analysis. In contrast, the rest 15 isolates revealed significant polymorphism within 11 isolates of type I, and 4 isolates among type II, IIb, and VI. DISCUSSION: We verified the M. kansasii genotype I was predominant, with the same pattern of major worldwide type regions, and reflected a very tight clonal structure. Type I was furthermore indicated recognition of subtypes by PFGE analysis.     

Evaluation of polymerase chain reaction, conventional and MRSA screen latex agglutination methods for detection of methicillin-resistant, -borderline and -susceptible Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA), is difficult and expensive to treat, therefore early screening is essential. Several phenotypic and genotypic methods are used to detect MRSA; however, the method of choice remains problematic. We have evaluated four phenotypic methods, broth microdilution (MIC), oxacillin disk agar diffusion (ODD), oxacillin screening salt agar (OSS), and a new rapid phenotypic (MRSA screen latex agglutination, MSLA) with the genotypic gold standard of PCR mecA detection to determine the most appropriate method for routine laboratory use. We randomly collected 203 S. aureus isolates from patients and carriers at two hospitals in Thailand. Using MIC method, three sub-groups were differentiated from among these isolates, namely MRSA (106 isolates), borderline-resistant S. aureus (BRSA) (65 isolates), and methicillin-susceptible S. aureus (MSSA)(32 isolates). A total of 10 methicillin-resistant S. epidermidis (MRSE) isolates were also included. The sensitivity and specificity of MIC, ODD, OSS, and MSLA were 99 and 96, 100 and 97, 100 and 97, and 100 and 100%, respectively. Our study indicated that ODD is still appropriate for routine laboratory. MSLA had the highest sensitivity and specificity and is rapid but expensive, so is the most appropriate method for emergency cases. MIC method was better for BRSA detection and OSS method was more appropriate for screening clinical specimens and carriers.