Comparison of different methods for detecting methicillin resistance in MRSA isolates belonging to international lineages commonly isolated in the American continent

The aim of the present paper was to compare different methods for detecting methicillin resistance in Staphylococcus aureus . Among the isolates analyzed, 52 belonged to MRSA international lineages commonly detected in the American continent and 14 to sporadic MRSA clones. Both 30 μg-cefoxitin disk and PBP2a had 100% sensibility/specificity when the low-level heterogeneous isolates were tested and, thus, are highly recommended.     

长沙地区临床分离金黄色葡萄球菌的耐药监测

目的了解长沙地区临床分离金黄色葡萄球菌（以下简称金葡菌）对常用抗菌药物的耐药现状,探讨金黄色葡萄球菌对甲氧西林的耐药水平。方法收集长沙地区11家医院2009年11月至2010年11月临床分离的非重复金葡菌279株,应用Vitek-2全自动微生物分析系统进行鉴定,K-B法检测金葡菌对24种药物的敏感性,产色头孢菌素试验检测β-内酰胺酶以及D试验检测诱导型克林霉素耐药。应用头孢西丁和苯唑西林纸片扩散法筛查耐甲氧西林的金葡菌（MRSA）,琼脂稀释法检测头孢西丁和苯唑西林的最低抑菌浓度（MIC）。结果在被检测的24种药物中,敏感率〉50%的药物为9种,未发现对万古霉素、替考拉宁和利奈唑胺耐药菌株;耐药率〉50%的抗菌药物有11种,其中以青霉素和氨苄西林的耐药率最高（均为97.1%）。MRSA的分离率达54.5%,且对常用的16种抗菌药物的耐药率均显著高于甲氧西林敏感金黄色葡萄球菌（MSSA）。279株金葡菌中,β-内酰胺酶阳性250株（89.6%）;红霉素耐药而克林霉素敏感或中介的30株中,D试验阳性22株（73.3%）。苯唑西林（OXA）和头孢西丁（FOX）MIC范围分别为0.125～〉256μg/mL和2～〉256μg/mL,苯唑西林的MIC50和MIC90分别为128μg/mL和256μg/mL,头孢西丁的MIC50和MIC90分别为64μg/mL和256μg/mL。结论长沙地区临床分离金葡菌对常用抗菌药物呈多重耐药;MRSA不仅分离率高,而且对甲氧西林呈高水平耐药。     

Spa typing of Staphylococcus aureus Isolated from Clinical Specimens from Outpatients in Iraq

Methicillin-resistant Staphylococcus aureus (MRSA) is notorious as a hospital superbug and a problematic pathogen among communities. The incidence of MRSA has substantially increased over time in Iraq. The aim of this study was to determine the prevalence and spa types of MRSA isolates from outpatients or patients upon admission into hospitals. Various biochemical tests identified S. aureus isolates, and then this identification was confirmed by PCR using species-specific 16S rRNA primer pairs. Antibiotic susceptibility was determined against methicillin, oxacillin, and vancomycin using the disk diffusion method. Vancomycin MIC was detected by VITEK 2 compact system. All the identified isolates were screened for the presence of mecA and lukS-PV-lukF-PV genes; 36 of them were subjected to spa typing-based PCR. Out of 290 clinical samples, 65 (22.4%) were S. aureus, of which 62 (95.4%) strains were resistant to oxacillin and methicillin. Except for two isolates, all MRSA isolates were mecA positive. One of the three MSSA isolates was mecA positive. Five strains were resistant to vancomycin. Fourteen (21.5%) isolates were positive for the presence of lukS-PV-lukF-PV genes. Spa typing of 36 S. aureus isolates revealed eleven different spa types, t304 (30.3%), t307 (19.4%), t346 (8.3%), t044 (8.3%), t15595 (8.3%), t386 (5.5%), t5475 (5.5%), t17928 (2.8%), t14870 (2.8%), t021 (2.8%), and t024 (2.8%). These findings could be useful for assessing the genetic relatedness of strains in the region for epidemiological and monitoring purposes, which would be essential to limiting the spread of MRSA.     

253株金黄色葡萄球菌耐药现状分析

目的 了解医院金黄色葡萄球菌临床分布情况及其对常用抗菌药物的耐药率,为临床合理使用抗菌药物提供依据.方法 回顾分析医院2010年5月至2011年4月检出的金黄色葡萄球菌,采用VITEK-AMS全自动微生物分析仪进行菌种鉴定和药敏分析.结果 共检出金黄色葡萄球菌253株,菌株的主要来源为痰130株(51.4％)、血液39株(15.4％)、创面24株(9.5％);菌株主要科室分布前3位是神内科35株(13.8％)、ICU30( 11.8％)、脑外科26株(10.3％);其中耐甲氧西林金黄色葡萄球菌( MRSA)为165株(65.2％),MRSA对多种抗菌药物耐药率＞70.0％,MSSA为88株(34.8％),对除青霉素、红霉素外的大多数抗菌药物敏感,未发现耐万古霉素菌株.结论 MRSA检出率高,耐药现状严重,应加强对金黄色葡萄球菌耐药性的监测,并根据药敏试验结果合理使用抗菌药物.     

mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌

目的 应用mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌（methicillin resistant staphylococcus aureus,MRSA）。方法 临床分离的70株金黄色葡萄球菌,应用mecA基因PCR扩增法鉴定MRSA,并与苯唑西林纸片扩散法进行比较。结果 70株金黄色葡萄球菌用PCR扩增法和纸片扩散法有6株鉴定有差异,4株。mecA基因阳性而纸片扩散法鉴定为敏感,1株mecA基因阳性纸片扩散法鉴定为临界耐药,1株mecA基因阴性却表现为苯唑西林耐药,2种方法符合率为91．43％。结论 mecA基因PCR扩增法可以准确、快速判定MRSA,特别是对隐匿型或低水平耐药菌株的检出有重要的价值。     

Experimental endocarditis model of methicillin resistant Staphylococcus aureus (MRSA) in rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.     

Resistance heterogeneity in methicillin-resistant Staphylococcus aureus

Abstract A striking feature of methicillin resistance in Staphylococcus aureus is the considerable heterogeneity of expression of resistance by cells in clonal populations: some are sensitive (or almost so), others are highly resistant, and others show intermediate resistance to the antibiotic. Subclones generally are also heterogeneous, suggesting variable inheritance or control of expression of resistance.
The degree of heterogeneity and mean resistance is influenced by environmental parameters: temperature, osmolality, pH, light, anaerobiosis, chelating agents and metal ions, and prior exposure to β-lactam antibiotics.     

Molecular analysis of Staphylococcus aureus isolates associated with staphylococcal food poisoning in South Korea

AIMS: To investigate the molecular epidemiological study of Staphylococcus aureus from staphylococcal food poisoning (SFP) incidents in South Korea. METHODS AND RESULTS: Three hundred and thirty-two strains isolated from ten provinces between June 1999 and January 2002 were characterized by staphylococcal enterotoxin genes, toxic shock syndrome toxin 1 (tst) gene, and exfoliative toxin genes. Toxin genotypes were sea-seh (n=197), sea (n=51), sea-seg-sei (n=14), seg-sei (n=10), seb (n=10), seb-sed-seg-sei-sej (n=3), sea-seg-seh-sei (n=1), sea-seb (n=1), sea-sec (n=1), seg-sei plus eta (n=4), and sea-seg-sei plus tst (n=40). Most of the strains could be classified into three clusters of pulsed-field gel electrophoresis (PFGE) types A and B with coagulase type VII and type E with coagulase type IV. Of the ten sequence types (ST), ST1, ST59, and ST30 were frequently showed by multilocus sequence typing. CONCLUSIONS: The strain belonging to PFGE pattern A with sea-seh gene, coagulase VII, and ST1 was the most epidemic clone of SFP incidents in Korea.     

乳源耐甲氧西林金黄色葡萄球菌icaA/D基因缺失株的构建及其生物被膜形成能力与耐药性分析

[背景]耐甲氧西林金黄色葡萄球菌(Methicillin Resistant Staphylococcus aureus,MRSA)是一种具有多重耐药性的人畜共患病原菌,常引起奶牛乳房炎等疾病.形成生物被膜是MRSA重要的耐药机制之一.研究发现ica操纵子调控的胞间多糖黏附素(Polysaccharide Interc...     

PCR-based procedures for detection and quantification of Staphylococcus aureus and their application in food

AIMS: To evaluate the specificity of nuc targeted primers for PCR detection of Staphylococcus aureus in different food matrices and to establish a RTQ-PCR procedure suitable for the routine detection and quantification of this pathogen in food. METHODS AND RESULTS: Specificity of nuc targeted primers (Pri1-Pri2 and the newly designed RTQ-PCR primers) was tested on a total of 157 strains of genetically confirmed identity, including reference and food isolates. PCR detection on artificially inoculated beef samples by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) showed a sensitivity value around 10(3) CFU g(-1). The two RTQ-PCR systems, incorporating SYBR-Green I and TaqMan, respectively, applied in the present work improved the sensitivity of conventional PCR by lowering the detection level to 10 and 100 cells, respectively. Out of 164 naturally contaminated foods tested for the presence of Staph. aureus, 74 were positive by conventional PCR and 69 by the traditional culture method with a high degree of result agreement between both methodologies (93.3%). CONCLUSIONS: PCR approaches, using nuc targeted primers, have proved specific and combined with growth techniques may improve detection of Staph. aureus in different types of food. SIGNIFICANCE AND IMPACT OF THE STUDY: The SYBR-Green I real-time PCR approach established allows the sensitive, automated and quantitative detection of Staph. aureus for routine analysis at a reasonable cost.     

Comparative Proteomic Profiling of Methicillin‐Susceptible and Resistant Staphylococcus aureus

Staphylococcus aureus is a highly successful human pathogen responsible for a wide range of infections. This study provides insights into the virulence, pathogenicity, and antimicrobial resistance determinants of methicillin‐susceptible and methicillin‐resistant S. aureus (MSSA; MRSA) recovered from non‐healthcare environments. Three environmental MSSA and three environmental MRSA are selected for proteomic profiling using isobaric tag for relative and absolute quantitation tandem mass spectrometry (iTRAQ MS/MS). Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway annotation are applied to interpret the functions of the proteins detected. 792 proteins are identified in MSSA and MRSA. Comparative analysis of MRSA and MSSA reveals that 8 of out 792 proteins are upregulated and 156 are downregulated. Proteins that have differences in abundance are predominantly involved in catalytic and binding activity. Among 164 differently abundant proteins, 29 are involved in pathogenesis, antimicrobial resistance, stress response, mismatch repair, and cell wall synthesis. Twenty‐two proteins associated with pathogenicity including SPA, SBI, CLFA, and DLT are upregulated in MRSA. Moreover, the upregulated pathogenic protein ENTC2 in MSSA is determined to be a super antigen, potentially capable of triggering toxic shock syndrome in the host. Enhanced pathogenicity, antimicrobial resistance, and stress response are observed in MRSA compared to MSSA.     

Lead resistance and sensitivity in Staphylococcus aureus

Abstract Five lead-resistant strains of Staphylococcus aureus were isolated. Plasmid-free lead-sensitive variants were obtained from the three plasmid-bearing strains. Lead-resistant strains tolerated an approximately 600 × higher Pb(NO₃)₂ concentration than lead-sensitive strains. Both types of strains initially bound lead, but only the resistant strains accumulated the metal as an intracellular lead-phosphate.     

232株金黄色葡萄球菌的临床分布特征及耐药性分析

目的了解金黄色葡萄球菌(金葡菌)临床分离株的感染分布特点及耐药现状,以便为临床感染治疗及预防提供帮助。方法收集南昌大学第二附属医院2007年1月至2009年12月临床分离的非重复金葡菌232株。常规方法进行菌株分离,血浆凝固酶、金葡菌单克隆抗体及Vitek-32型仪进行菌株鉴定,纸片扩散法测定菌株对抗菌药物的敏感性,头孢西丁法检测耐甲氧西林金葡菌(MRSA),WHONET5.5软件分析数据。结果下呼吸道标本中分离的金葡菌最多,占44.0%,其次是脓液(20.3%)和血液(18.1%)。232株金葡菌中共检测到MRSA 131株(56.6%),金葡菌对青霉素的耐药率最高(90.0%),对庆大霉素、左氧氟沙星、克林霉素、红霉素和四环素的耐药率均50.0%;耐药率在10.0%~50.0%的有阿米卡星、复方磺胺甲噁唑和夫西地酸;对替考拉宁耐药率非常低(1.3%);未出现耐万古霉素的菌株。结论下呼吸道及皮肤软组织是金葡菌的主要感染部位;金葡菌对临床常用抗菌药物的耐药率近3年来趋于稳定,糖肽类抗菌药物对其仍有非常强的抗菌活性。     

Conjugative trimethoprim resistance in Staphylococcus aureus

A multiply resistant Staphylococcus aureus isolate, WBG7410, harbours plasmids of 38, 26, 2.8, 2.4 and 1.9 kb and transfers trimethoprim and kanamycin resistance at high frequencies by conjugation. The transconjugants contained the 38-kb plasmid, pWBG707, and the 2.8-kb plasmid. Plasmid pWBG707 was shown to encode trimethoprim resistance, was conjugative and mobilised at high frequencies the 2.8-kb plasmid which presumably encodes kanamycin resistance. Plasmid pWBG707 was isolated mostly in the open circular form and analysis with EcoRI restriction endonuclease suggests that pWBG707 is a new conjugative plasmid distinct from the other conjugative plasmids reported in S. aureus.     

Comparison of three immunological methods for detecting staphylococcal enterotoxins from food

AIMS: Immunologically based assays for the detection of staphylococcal enterotoxins are numerous. These techniques include radio immunosorbent assays and enzyme-linked immunosorbent assays (ELISA), some of which are available as commercial kits. The purpose of this study was to compare the performances of three commercial immunoassays. METHODS AND RESULTS: Two automated detection systems, VIDAS SET bioMèrieux, VIDAS SET2 bioMérieux and an ELISA method, TRANSIA PLATE Staphylococcal Enterotoxins Diffchamb were compared for detecting different quantities of purified staphylococcal enterotoxins (A, B, C2, D and E) added to food. CONCLUSIONS: VIDAS SET2 had a greater specificity (100%) and sensitivity than VIDAS SET and TRANSIA PLATE Staphylococcal Enterotoxins. More precisely, VIDAS SET2 could detect <0.5 ng g(-1) of toxins A and B, <1 ng g(-1) of toxins C2 and E and 1 ng g(-1) of toxins D and E. SIGNIFICANCE AND IMPACT OF THE STUDY: Because staphylococcal food poisoning (resulting from ingestion of low levels of staphylococcal enterotoxins) is one of the most common forms of foodborne illness there is a need for specific and sensitive methods for detecting these enterotoxins. VIDAS SET2 appears to be suitable for detecting staphylococcal enterotoxins from food.     

Impact of sar and agr on methicillin resistance in Staphylococcus aureus

Abstract The global regulators agr and sar control expression of cell wall and extracellular proteins. Inactivation of either sar and/or agr in a typical heterogeneously methicillin-resistant Staphylococcus aureus resulted in a small but reproducible decrease in the number of cells in the subpopulation expressing high methicillin resistance. The amount of low affinity penicillin-binding protein PBP2', the prerequisite for methicillin resistance, was apparently not affected, however, a reduction in PBP1 and PBP3 production was observed, suggesting that these resident PBPs of the cells might be involved somehow together with PBP2' in high level methicillin resistance.     

74例金黄色葡萄球菌乳腺炎的耐药性分析

目的分析哺乳期乳房脓肿患者的金黄色葡萄球菌的耐药情况,指导临床合理使用抗菌药物。方法将厦门市妇幼保健院乳腺门诊及病房2009年2月至2011年1月抽取的128例哺乳期乳房脓肿患者的乳腺脓液接种于哥伦比亚血平板,置35℃培养箱,过夜孵育18~24 h。革兰阳性球菌,触酶阳性,血浆凝固酶试验(+),必要时采用ATB-Expression细菌鉴定系统确认。用K-B纸片法检测其对苯唑西林等10种抗菌药物的敏感度。结果 128例检出细菌86例,阳性率67.2%,其中检出74株金黄色葡萄球菌(86.0%),其对青霉素、红霉素和克林霉素有较强的耐药性,耐药率分别为94.6%、64.9%和59.5%;耐苯唑西林19株(25.7%),对复方新诺明、磷霉素和庆大霉素较为敏感,对万古霉素、替考拉宁和左旋氧氟沙星100%敏感。结论哺乳期乳房脓肿致病菌主要为金黄色葡萄球菌,耐甲氧西林金黄色葡萄球菌(MRSA)占25.7%,其耐药情况不容忽视,青霉素类、大环内酯类等耐药率较高不能作为经验用药。建议临床使用抗菌药物前及时送检乳腺脓液细菌培养,以药敏结果指导临床合理经济用药。