耐甲氧西林金黄色葡萄球菌耐药性及分子流行病学研究

目的了解耐甲氧西林金黄色葡萄球菌(MRSA)的耐药特点和分子流行病学情况。方法对临床分离的金黄色葡萄球菌应用VITEK-32微生物分析仪及配套GPS118药敏卡测定其对12种抗菌药物的敏感性,头孢西丁纸片扩散法筛选MR-SA。对已筛选的12株MRSA,应用随机扩增多态DNA(RAPD)技术进行基因型别分析。结果 12株MRSA对氨苄西林、苯唑西林、青霉素G和红霉素全部耐药;对氯洁霉素、庆大霉素、左氧氟沙星均出现8株耐药;对万古霉素、利奈唑烷、喹努普汀/达福普汀全部敏感。RAPD将检测菌株分为3个型别(A～C)其中RAPD A型和B型是本院的主要流行菌株。结论 MRSA表现为多重耐药菌,本地区存在两种不同型别的MRSA流行克隆。     

儿童烧伤患者感染MRSA的耐药性及分子流行病学研究

目的：调查儿童烧伤患者耐甲氧西林金黄色葡萄球菌（MRSA）的耐药性及分子流行病学情况,为控制感染提供科学依据。方法通过PCR扩增mecA基因鉴定 MRSA菌株,K-B纸片法检测菌株耐药性,随机扩增多态性DNA（RAPD）对其进行同源性分析。结果从住院烧伤儿童临床标本分离得到的52株金黄色葡萄球菌中 MRSA占44．2％（23/52）,且对多种抗菌药物均耐药。23株MRSA经RAPD分析可分为4型,以Ⅱ型为主。结论儿童烧伤患者分离MRSA菌株具有多重耐药性,应根据药敏试验结果合理选用抗菌药物进行治疗;RAPD技术能为控制感染提供分子流行病学依据。     

实时荧光定量PCR在快速检测耐甲氧西林金黄色葡萄球菌中的应用评估

目的评价实时荧光定量聚合酶链反应(PCR)在快速检测耐甲氧西林金黄色葡萄球菌(MRSA)中的应用。方法采用头孢西丁纸片扩散法和mecA基因PCR检测法,将85株临床分离的金黄色葡萄球菌区分为MRSA和甲氧西林敏感金黄色葡萄球菌(MSSA),并采用实时荧光定量PCR对这些菌株进行检测,评价MRSA检测中实时荧光定量PCR与目前常规检测方法的一致性。结果根据头孢西丁纸片扩散法和mecA基因扩增结果进行分组,85株金黄色葡萄球菌中MRSA组菌株45株,MSSA组菌株40株;实时荧光定量PCR检测结果与上述结果完全一致,符合率为100%。结论实时荧光定量PCR检测MRSA的结果与常规方法一致性好,且具有操作简便、耗时短等特点。作为一种快速检测方法,实时荧光定量PCR能将金黄色葡萄球菌的鉴定和MRSA的筛选结合起来,对于指导临床用药及MRSA医院感染控制具有重要意义。     

耐甲氧西林金黄色葡萄球菌的两种同源性分析方法的比较

目的 比较使用药物敏感试验的表型研究方法与随机扩增多态DNA(RAPD)染色体分析方法对耐甲氧西林金黄色葡萄球菌(MRSA)的同源性分析,探讨使用表型方法研究细菌同源性的可行性.方法 采用WHO推荐纸片扩散法(K-B),以及随机扩增多态DNA(RAPD)技术对同期住院患者临床标本分离的30株MRSA进行同源性分析.结果 从产生的RAPD图谱分析可将30株MRSA分为5个型别,其中I型为22株占73.3%(22/30).采用青霉素、头孢西丁、红霉素、环丙沙星、四环素、庆大霉素、复方新诺明、氯霉素、强力霉素、阿米卡星、左氧氟沙星、阿齐霉素、米诺环素、莫西沙星、亚胺培南、加替沙星、哌拉西林/他唑巴坦、美罗培南等18种抗生素,可将30株MRSA进行同源性分析.结论 RAPD技术及纸片扩散法(K-B)可以很好地用于细菌的同源性分析.     

耐甲氧西林金黄色葡萄球菌基因分型和同源性分析

摘要：目的：研究长治医学院附属和平医院2007～2009年耐甲氧西林金黄色葡萄球菌（MRSA）的检出率、耐药率及其同源性。 方法：用纸片扩散法mecA基因扩增、SCCmec分型、spa分型及ERIC-PCR法对该院在2007年1月至2009年12月收集的234株金黄色葡萄球菌分别进行MRSA初筛、MRSA确证试验、基因分型和同源性分析。 结果：3年中该院共分离MRSA菌株73株,MRSA菌株对多种药物的耐药率均>90.0%,对万古霉素的MIC值范围为0.5～2 μg/mL;ERIC-PCR、SCCmec、spa分型结果显示,67株MRSA菌株均为A型SCCmecⅢ-t030(67/73,91.8%),为同一克隆菌株。 结论：该院检出MRSA菌株多为同一克隆菌株,且耐药率较高,要注意及时检测MRSA菌株并且预防医院感染暴发的发生。     

三种检测耐甲氧西林金黄色葡萄球菌方法的比较

目的 比较苯唑西林纸片扩散法、微量琼脂稀释法(MIC)和聚合酶链反应(PCR)法检测甲氧西林耐药金黄色葡萄球菌(MRSA)的差异.方法 用上述3种方法同时检测86株临床分离的金黄色葡萄球菌.结果 3种方法同时检测出53株MRSA和33株甲氧西林敏感金黄色葡萄球菌,一致性为100%.结论 三种方法都适于临床实验室准确检测MRSA,尤其是纸片扩散法和MIC法可作为不同级别临床常规实验室确证MRSA检测方法.     

评价头孢西丁和苯唑西林纸片扩散法测定耐甲氧西林金黄色葡萄球菌

目的评价头孢西丁和苯唑西林纸片扩散法检测耐甲氧西林金黄色葡萄球菌(MRSA)的临床价值.方法用头孢西丁和苯唑西林纸片扩散法检测临床分离的200株金黄色葡葡球菌,以测定PBP2a的MRSA-乳胶凝集试验作为″金标准″.结果92株PBP2a阳性金黄色葡萄球菌中,头孢西丁和苯唑西林纸片扩散法分别检出MRSA 91株和84株;108株PBP2a阴性中,头孢西丁纸片扩散法全部为阴性,苯唑西林纸片扩散法阴性为103株.以PBP2a检测结果为标准,头孢西丁和苯唑西林纸片扩散法检测MRSA的特异性分别为100.0%和95.4%,敏感性分别为98.9%和91.3%.5株苯唑西林纸片扩散法检测为中介的菌株,经PBP2a检测结果证实,3株为MRSA,2株为MSSA.结论头孢西丁比苯唑西林更适合检测MRSA,并能提高测定MRSA的可靠性,是临床实验室常规检测MRSA的首选方法.     

耐甲氧西林金黄色葡萄球菌医院感染爆发流行的实验室调查

目的采用耐甲氧西林金黄色葡萄球菌(MRSA)mecA基因相关高变区(HVR)长度多态性分型了解MRSA的医院感染爆发流行情况。方法选取临床分离的38株MRSA菌株,采用聚合酶链反应(PCR)方法扩增MRSA mecA基因HVR,据扩增片段大小差异进行基因分型。结果根据PCR产物片段大小,38株MRSA被分为A、B、C、D 4个基因型,其中C型23株(60.53%)、A型7株(18.42%)、B型3株(7.89%)、D型2株(5.26%)、未分型3株(7.89%)。C型在临床各科室均有分布,但主要集中于老干部科。MRSA呈严重且多重耐药,故首选抗菌药物为万古霉素、替加环素、奎奴普汀/达福普汀和替考拉宁。结论医院内存在着MRSA菌株的克隆传播,应加强检测,控制医院感染爆发流行。     

三种耐甲氧西林金黄色葡萄球菌检测方法的评估

目的评价PBP2a乳胶凝集法、头孢西丁纸片扩散法和苯唑西林纸片扩散法对检测耐甲氧西林金黄色葡萄球菌(MRSA)的临床应用价值。方法以PCR法扩增mecA基因为金标准,用三种方法对临床分离的105株金黄色葡萄球菌进行检测并作比较。结果105株细菌中有82株mecA基因阳性,头孢西丁纸片扩散法、乳胶凝集法与PCR法结果一致,苯唑西林纸片扩散法与PCR法结果相符合均为98.8%。结论头孢西丁纸片扩散法与PBP2a乳胶凝集法一样比苯唑西林纸片扩散法能更准确检测MRSA,值得推广应用。     

评价头孢西丁和苯唑西林纸片扩散法测定耐甲氧西林金黄色葡萄球菌

目的 评价头孢西丁和苯唑西林纸片扩散法检测耐甲氧两林金黄色葡萄球菌（MRSA）的临床价值。方法用头孢西丁和苯唑西林纸片扩散法检测临床分离的200株金黄色葡葡球菌，以测定PBP2a的MRSA-乳胶凝集试验作为“金标准”。结果92株PBP2a阳性金黄色葡萄球菌巾．头孢西丁和苯唑西林纸片扩散法分别检出MRSA91株和84株：108株PBP2a阴性中，头孢西丁纸片扩散法全部为阴性，苯唑西林纸片扩散法阴性为103株。以PBP2a检测结果为标准．头孢西丁和苯唑西林纸片扩散法检测MRSA的特异性分别为100．0％和95．4％．敏感性分别为98．9％和91.3％。5株苯唑西林纸片扩散法检测为中介的菌株。经PBP2a检测结果证实，3株为MRSA，2株为MSSA。结论头孢西丁比苯唑西林更适合检测MRSA．并能提高测定MRSA的可靠性，是临床实验室常规检测MRSA的首选方法。     

耐甲氧西林金黄色葡萄球菌的耐药性及分子流行病学调查

目的了解MRSA的耐药性及分子流行病学特点。方法采用纸片扩散法检测住院患者分离的160株MRSA的耐药性;采用随机引物(AP)-PCR法对其中的36株MRSA作同源性分析。结果160株MRSA对万古霉素、替考拉宁和复方磺胺甲噁唑的敏感率分别为100%、100%和95%;对红霉素、庆大霉素和左氧氟沙星的耐药率分别为97.5%、87%和59%;左氧氟沙星的中介率为26%。36株MRSA的AP-PCR图谱有10种类型(A～J型),主要型别为A型12株、B型7株及C型6株,ICU的25株,分别为A型10株、B型6株、C型3株、D型3株及F、I、J各1株。结论医院获得性MRSA是多重耐药菌,ICU的MRSA菌株主要基因型为A、B型。     

多重PCR检测MRSA的SCCmec基因分型

目的了解我院MRSA的流行状况。方法收集2005年1—6月65株社区感染MRSA及60株医院感染MRSA，应用多重PCR对MRSA染色体Dlec基因盒（staphylococcal cassette chromosomeSCCmec）分型及杀白细胞毒素（PVI。）基因检测，应用K—B纸片法进行药敏分析。结果125株MRSA的mecA基因阳性，其中SCCmecII型1株，SCCmecⅢ型120株，SCCmecⅣ型3株，未分型1株；未发现携带PVL基因的MRSA。携带SCCmecII型、SCCmecⅢ型的菌株均为多重耐药株，而携带SCCmecⅣ型的菌株除对8内酰胺类药物耐药外，对其他类别的抗菌药敏感。结论本院分离的MRSA以SCCmecⅢ型为主，发现SCCmecIV型CA-MRSA，但不携带PVL基因；携带SCCmecⅡ、SCCmecⅢ的临床分离株耐药严重。     

耐甲氧西林金黄色葡萄球菌快速检测方法的比较

目的 筛选出快速检测耐甲氧西林金黄色葡萄球菌(methicillin resistant Staphylococcous aureus,MRSA)的实验方法.方法 从天津医科大学附属肿瘤医院细菌室菌库中提取源自肿瘤患者感染的44株金黄色葡萄球菌,利用苯唑西林纸片扩散法、头孢西丁纸片扩散法、微量肉汤稀释法、Etest法、青霉素结合蛋白2a(penicillin-binding protein 2a,PBP2a)乳胶凝集法5种表型检测MRSA方法进行检测,并与PCR方法进行比较.结果 PCR法检测mecA基因,确定MRSA 36株;以PCR作为金标准,苯唑西林纸片扩散法、头孢西丁纸片扩散法和苯唑西林微量肉汤稀释法检测出MRSA均为32株,敏感度和特异度均为88.9%和100%;E试验检测出MRSA为33株,敏感度与特异度分别为88.9%、87.5%;PBP2a乳胶凝集法检测出MRSA 29株,敏感度与特异度分别为80.5%、100%.结论 PBP2a乳胶凝集法比上述试验报告周期缩短24 h,是一种快速、简便、特异度好且便于临床试验室推广开展的检测MRSA方法.     

spa typing of methicillin-resistant Staphylococcus aureus isolated from domestic animals and veterinary staff in the UK and Ireland

OBJECTIVES: Region X of the protein A gene (spa) was sequenced from methicillin-resistant Staphylococcus aureus (MRSA) isolates originating from animals, humans and the environment at veterinary hospitals in the UK and Ireland. MRSA transmission between animals and veterinary staff was assessed on the basis of spa typing, PFGE and epidemiological data. METHODS: MRSA isolates from dogs (n = 27), horses (n = 9), cats (n = 6), staff (n = 22) and environmental surfaces (n = 3) were analysed by PFGE and spa typing. Known contacts between human and animal MRSA carriers were ascertained from the veterinary hospitals. RESULTS: All feline, most canine (96%) and human (82%) isolates showed PFGE profiles that were either indistinguishable (subtype A1) or closely related (subtypes A2-A10) to that of the epidemic clone EMRSA-15 (CC22), whereas most equine isolates (88%) were related to CC8 (types C, D, E and G). spa polymorphism enabled discrimination among MRSA strains assigned to the same PFGE type. Fifteen spa types clustering into two distinct groups were detected, with t032 being the most prevalent (48%). The spa and PFGE types of MRSA isolated from seven staff members were the same as those of strains isolated from infected animals attended by the staff. CONCLUSIONS: Irrespective of geographical origin, MRSA isolated from equine and small animal hospitals generally clustered into two distinct clonal complexes, CC8 and CC22, respectively. The combined use of spa and PFGE typing allowed better discrimination than each method used individually, and provided useful information on MRSA transmission between animal and human individuals.     

Molecular and epidemiological evidence for spread of multiresistant methicillin-susceptible Staphylococcus aureus strains in hospitals

The excision of the staphylococcal chromosomal cassette mec (SCCmec) from methicillin-resistant Staphylococcus aureus (MRSA) strains results in methicillin-susceptible S. aureus (MSSA) strains. In order to determine the proportion and diversity of multidrug-resistant MSSA (MR-MSSA) strains derived from MRSA strains, 247 mecA-negative isolates recovered in 60 French hospitals between 2002 and 2004 were characterized. The spa types of all strains were determined, and a subset of the strains (n = 30) was further genotyped by multilocus sequence typing. The IDI-MRSA assay was used to test the isolates for the presence of the SCCmec element, which was detected in 68% of all isolates analyzed. Molecular analysis of the samples suggested that 92% of the MR-MSSA isolates were derived from MRSA clones of diverse genetic backgrounds, of which the clone of sequence type 8 and SCCmec type IV(A) accounted for most of the samples. High variations in incidence data and differences in the molecular characteristics of the isolates from one hospital to another indicate that the emergence of MR-MSSA resulted from independent SCCmec excisions from epidemic MRSA isolates, as well as the diffusion of methicillin-susceptible strains after the loss of SCCmec. MR-MSSA could constitute a useful model for the study of the respective genetic and environmental factors involved in the dissemination of S. aureus in hospitals.     

头孢西丁纸片扩散法检测耐甲氧西林葡萄球菌

目的以PCR法检测葡萄球菌的mecA基因(mecA基因法)为标准，评价头孢西丁纸片扩散法、苯唑西林纸片扩散法和苯唑西林盐平板法检测葡萄球菌中耐甲氧西林葡萄球菌的灵敏度和特异性。方法PCR扩增葡萄球菌的特异性mecA基因片段，头孢西丁纸片扩散法、苯唑西林纸片扩散法和苯唑西林盐平板法检测葡萄球菌中耐甲氧西林葡萄球菌，药敏试验方法按标准K—B(Kirby-Bauer)法进行。结果在190株临床分离的葡萄球菌中金黄色葡萄球菌(金葡菌)138株，表皮葡萄球菌30株，溶血葡萄球菌22株，经。PCR法检测mecA基因，金葡菌中耐甲氧西林金葡菌(MRSA)和凝固酶阴性葡萄球菌中耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)的发生率分别为81.2％(112／138)、96．1％(50／52)。头孢西丁纸片扩散法检测金葡菌中MRSA和CNS中MRCNS的发生率分别为81.2％(112／138)、94．2％(49／52)，与mecA基因法结果相比较，头孢西丁纸片扩散法检测葡萄球菌中MRS的灵敏度和特异度分别为99．4％(161／162)、100．0％(28／28)，两者符合率为99．5％(189／190)。2种方法所获得的结果经统计学处理，两者差异无显著性。结论头孢西丁纸片扩散法检测耐甲氧西林葡萄球菌具有很高的灵敏度和特异度，适合在临床微生物实验室中进行推广。     

Survey of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> from neonates and the environment in the NICU

Methicillin-resistant Staphylococcus aureus (MRSA) is a risk factor of nosocomial infection with compromised hosts including neonates. Currently, the prevalence of MRSA carriers among children is increasing in Japan. There are some reports of nosocomial infection caused by MRSA in a pediatric ward or neonatal intensive care unit (NICU). During 6 months (from January 2001 to June 2001), 37 MRSA strains were isolated from 37 neonates who were admitted in the NICU and 52 MRSA were strains isolated from NICU environments. We performed DNA typing of MRSA using an arbitrary primed-PCR method on these isolates. Thirty-seven clinical isolates were classified into four types (A type, 14; B type, 4; C type, 4; D type, 3; and others, 12). The A-type strains of MRSA continued to be isolated for more than 6 months. In the NICU environment, the detection rate of the A-type MRSA was 23.1% (12/52). The A-type strains were frequently isolated from environments around patients. The A-type strains of MRSA were prevalent in the NICU, probably due to nosocomial infection. Although none of the neonatal patients developed severe MRSA infection, the same genotype strains were persistently isolated during a period of more than 6 months from patients and the NICU environment around patients. Environmental control around neonatal patients is important to prevent nosocomial infection in the NICU.     

Molecular characterization of methicillin-resistant Staphylococcus aureus strains from pet animals and their relationship to human isolates

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) isolates from pet animals were characterized and compared with human isolates from clonal complexes most prevalent in Central Europe. METHODS: S. aureus isolates were investigated for their in vitro susceptibility to antimicrobial agents by broth microdilution. Resistance genes and the Panton-Valentine leucocidin gene lukF-lukS were identified by PCR. All isolates were characterized by SmaI macrorestriction analysis and spa typing to assess their genomic relationships. Representative isolates were additionally analysed by multilocus sequence typing and PCR-directed SCCmec typing. RESULTS: All pet isolates carried the resistance genes mecA and erm(C) and proved to be resistant to beta-lactams and MLS(B) antibiotics. In addition, all isolates were resistant to fluoroquinolones. None of the pet isolates carried the Panton-Valentine leucocidin gene lukF-lukS. Macrorestriction analysis revealed that the pet MRSA isolates exhibited four closely related SmaI fragment patterns. Moreover, all isolates revealed spa type t032. Further analysis of representatives of the different PFGE types revealed the presence of multilocus sequence type ST22 in combination with a type IV SCCmec element. Thus, molecular typing results were similar for pet strains and human ST22 reference strains. CONCLUSIONS: Based on their strain characteristics, the MRSA isolates from pets investigated in this study closely resembled ST22 MRSA isolates which are widely disseminated in German hospitals. The results of this study indicate that cross-transmission of MRSA between pet animals and humans might have occurred.     

Distribution of capsular and surface polysaccharide serotypes of Staphylococcus aureus

Because of its ability to cause serious and fatal infections, Staphylococcus aureus remains one of the most feared microorganisms. Methicillin-resistant S. aureus (MRSA) has long been a common pathogen in healthcare facilities, but within the past decade, it has emerged as a problematic pathogen in the community setting as well. The severe consequences of infection heighten the importance of prevention. To analyze the potential applicability of a putative S. aureus polysaccharide conjugate vaccine, we tested 714 German methicillin-susceptible S. aureus (MSSA) and MRSA strains for their capsular and surface polysaccharide serotype by slide agglutination with specific antibodies (anti-T5-DT, anti-T8-DT, anti-336-rEPA). The strain serotypes were confirmed by immunodiffusion using lysostaphin-digested cell lysates. Regarding MRSA strains representing 86 unique spa types and thus covering >90% of MRSA spa types registered, 39 (45.3%) were type 5, 36 (41.9%) were type 8, and 11 (12.8%) were type 336. Of particular interest, type 336 was the second most common serotype among MRSA isolates collected from 10 different laboratories (40 isolates per site) covering university hospitals, general hospitals, and clinics throughout Germany. Type 8-positive strains were more prevalent among isolates recovered from anterior nares of patients who did not subsequently develop S. aureus bacteremia compared with those who became bacteremic with this pathogen. In conclusion, the addition of the newly described type 336 to a capsular polysaccharide-protein conjugate vaccine could extend the coverage substantially and would include virtually all MSSA and MRSA strains currently circulating in Germany.