MRSA耐药性特征及分型研究
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摘要
抗菌药物是人类在医药领域取得的伟大成就之一,对人类健康水平的提高和生命安全的保障起到了极其重要的作用。然而,目前国内外抗菌药物滥用的问题十分突出,导致耐药菌急剧增多,其中耐甲氧西林金黄色葡萄球菌(Methicillin- Resistant Staphylococcus aureus, MRSA)是一种引起医院内获得性感染的多重耐药菌。1961年在英国发现了世界首例MRSA,从此MRSA的感染遍布全世界。据美国疾病控制和预防中心(CDC)统计,世界每年约有100,000人感染MRSA,并逐渐扩展到社区。因其携带多种毒素且感染极强,所以与艾滋病、病毒性乙型肝炎并列成为世界三大感染性疾病。
为了解山西省人民医院MRSA感染的现状及耐药特征,以利于临床合理选用抗生素,预防暴发流行,本研究对2005年9月-2008年9月期间从住院病人的标本中分离到的18株无重复的临床分离株进行MRSA表型筛选,采用Kerby-Bauer纸片扩散法测定MRSA对9种抗菌药物的敏感性,并采用PCR法进行杀白细胞毒素(PVL)基因检测；应用多重PCR进行葡萄球菌染色体盒(SCCmec)分型,采用随机扩增多态性DNA(RAPD)技术对其作同源性分析,以调查我院MRSA耐药特征,鉴别基因型,总结暴发流行趋势,为临床及时控制感染提供依据。主要研究结果如下：
一、药物敏感性试验及PVL基因检测结果
本研究中,18株MRSA对青霉素、苯唑西林、头孢西丁、环丙沙星和庆大霉素的耐药率均为100%,对克林霉素和红霉素的耐药率为88.9%,对利福平耐药率为55.6%、中敏率11.1%,没有万古霉素耐药菌株；全部呈多重耐药,多重耐药主要表现为同时耐5种(22.2%)、6种(33.3%)及7种(44.5%)抗菌药物,所有菌株同时对青霉素类、头孢菌素类、喹诺酮类和氨基糖苷类4种抗菌药耐药,其中同时对青霉素类、头孢菌素类、林克酰胺类、大环内酯类、喹诺酮类、氨基糖苷类、安沙霉素类7种药物耐药的为44.5%,是主要的耐药模式。18株MRSA均未检测到PVL基因。
二、SCCmec分型及RAPD同源性分析结果
多重PCR对18株MRSA进行SCCmec分型,发现9株(50%)携带有3种新的SCCmec型／亚型(New1～3)、6株(33.3%)携带ⅢB型SCCmec、3株(16.7%)为未定型菌株。18株MRSA的SCCmec型别具有高度多态性,ⅢB型SCCmec是优势型别。RAPD电泳图谱经NTSYS软件聚类分析,共有A、B两种分型,其中A型又可分为四个亚型：A1、A2、A3和A4。
MRSA耐药谱分型、SCCmec分型和RAPD分型三者结果不完全一致,没有直接的相关性。
MRSA是院内和社区感染的重要病原菌,致病性强,多重耐药现象严重,但迄今为止,未有对山西省MRSA的流行趋势的报道。本文从山西省人民医院收集了18株MRSA,并对其耐药模式和分型进行研究。我院MRSA菌株主要是ⅢB型SCCmec,RAPD分析我院暴发流行菌株是A型,18株MRSA全部是HA-MRSA,未发现CA-MRSA。
The antibiotic is one of the greatest achievements in the medical field, and it plays an extremely important role in the improvement of the human health and guarantee of the life security. However, it is very outstanding at present that antibiotic is abused in domestic and abroad, which results the antibiotic-resistant bacteria to increase sharply. Methicillin-resistant staphylococcus aureus (MRSA) is one of the most important multidrug-resistant bacterium in the hospital. The first MRSA was reported in 1961 in England. Since then, MRSA becomes one of the most significant nosocomial pathogens throughout the world and is capable of causing a wide range of hospital infections. At present a number of different clones of MRSA have arisen in many countries, and there are about 100,000 persons who are infected by MRSA every year in the world, which is reported by Centers for Disease Control and Prevention (CDC), while it also has become increasingly prevalent in community-acquired infections. Because of carrying a variety of toxins, strong infections, MRSA parallels to be the three major infectious diseases with AIDS and viral hepatitis B in the world.
In order to understand the infection status caused by MRSA in the Shanxi Provincial People's Hospital (SPPH), the research was done to detect MRSA in 18 samples from the patients in clinic and hospital which was infected by staphylococcus aureus from September 2005 to September 2008 by cefoxitin disc diffusion method, oxacillin disc diffusion method and mecA Genes PCR method. Kerby- Bauer disk diffusion test was used to detect the susceptibility of MRSA to 9 kinds of antibacterial agents. PCR method was used to dectect PVL gene from the genomic DNA of infection strains. RAPD was used to type the chromosome DNA of MRSA strains. Novel multiplex PCR was used to type Staphylococcal chromosomal cassette mec (SCCmec). This study aims to find out MRSA molecular epidemiology, distinguish genotype and prevail trend in SPPH, and to afford method for controlling infection in clinical. The main results of the research were as follows.
1. The results of antibiotic resistance and detecting PVL
In this study, PVL gene was not detected from the genomic DNA of 18 samples. The rate of resistance to penicillin, oxacillin, cefoxitin, ciprofloxacin and gentamicin were 100% in 18 MRSA strains; Rate of resistance to clindamycin and red neomycin was 88.9%, and the mid-sensitivity rate of Rifampin was 11.1%, the resistant rate of rifampin was 55.6%.100% MRSA strains were susceptible to Vancomycin. All of 18 MRSA strains were multidrug-resistant bacterium, and the resistant model were 5 kinds of antibiotics agents (22.2%),6 kinds (33.3%),7 kinds (44.5%). The majority (44.5%) of the strains were resistant to penicillin, cephalosporins, linklactams, macrolides, quinolones, aminoglycosides, sha-neomycin. All isolates were resistant to penicillins, cephalosporins, fluoroquinolones and aminoglycosides.
2. The SCCmec genotype of MRSA
By using multiplex PCR of the 18 MRSA for SCCmec type, we found nine strains (50%) carrying three kinds of new SCCmec type/subtype (Newl～3),6 strains (33.3%) carryingⅢB SCCmec, while 3 strains (16.7%) were not stereotyped strain. MRSA in this region of the SCCmec types were highly polymorphic, and IIIB SCCmec were the local advantage type. RAPD electrophoresis profiles of cluster was analysed by the NTSYS software. The strains were fall into two types A, B, in which type A can be further divided into four subtypes:A1, A2, A3 and A4.
The results of MRSA resistance profile typing, SCCmec typing and RAPD typing were not completely consistent, suggesting they didn't have direct correlation.
MRSA is one of the most important pathogen in the hospital- and community- acquired. They had the charcteristic of pathogenicity and serious multiple drug resistance, however so far there wasn't report on the popular trend of MRSA in Shanxi Province. In this research, we collected 18 MRSA from the Shanxi Province People's Hospital, and carried a study on their resistance patterns and genotyping. All of 18 MRSA strains were multidrug-resistant bacterium, and the majority of MRSA wereⅢB SCCmec. RAPD analysis of hospital outbreak strains are type A.18 strains of MRSA were all HA-MRSA, while CA-MRSA was not found.
为了解山西省人民医院MRSA感染的现状及耐药特征,以利于临床合理选用抗生素,预防暴发流行,本研究对2005年9月-2008年9月期间从住院病人的标本中分离到的18株无重复的临床分离株进行MRSA表型筛选,采用Kerby-Bauer纸片扩散法测定MRSA对9种抗菌药物的敏感性,并采用PCR法进行杀白细胞毒素(PVL)基因检测；应用多重PCR进行葡萄球菌染色体盒(SCCmec)分型,采用随机扩增多态性DNA(RAPD)技术对其作同源性分析,以调查我院MRSA耐药特征,鉴别基因型,总结暴发流行趋势,为临床及时控制感染提供依据。主要研究结果如下：
一、药物敏感性试验及PVL基因检测结果
本研究中,18株MRSA对青霉素、苯唑西林、头孢西丁、环丙沙星和庆大霉素的耐药率均为100%,对克林霉素和红霉素的耐药率为88.9%,对利福平耐药率为55.6%、中敏率11.1%,没有万古霉素耐药菌株；全部呈多重耐药,多重耐药主要表现为同时耐5种(22.2%)、6种(33.3%)及7种(44.5%)抗菌药物,所有菌株同时对青霉素类、头孢菌素类、喹诺酮类和氨基糖苷类4种抗菌药耐药,其中同时对青霉素类、头孢菌素类、林克酰胺类、大环内酯类、喹诺酮类、氨基糖苷类、安沙霉素类7种药物耐药的为44.5%,是主要的耐药模式。18株MRSA均未检测到PVL基因。
二、SCCmec分型及RAPD同源性分析结果
多重PCR对18株MRSA进行SCCmec分型,发现9株(50%)携带有3种新的SCCmec型／亚型(New1～3)、6株(33.3%)携带ⅢB型SCCmec、3株(16.7%)为未定型菌株。18株MRSA的SCCmec型别具有高度多态性,ⅢB型SCCmec是优势型别。RAPD电泳图谱经NTSYS软件聚类分析,共有A、B两种分型,其中A型又可分为四个亚型：A1、A2、A3和A4。
MRSA耐药谱分型、SCCmec分型和RAPD分型三者结果不完全一致,没有直接的相关性。
MRSA是院内和社区感染的重要病原菌,致病性强,多重耐药现象严重,但迄今为止,未有对山西省MRSA的流行趋势的报道。本文从山西省人民医院收集了18株MRSA,并对其耐药模式和分型进行研究。我院MRSA菌株主要是ⅢB型SCCmec,RAPD分析我院暴发流行菌株是A型,18株MRSA全部是HA-MRSA,未发现CA-MRSA。
The antibiotic is one of the greatest achievements in the medical field, and it plays an extremely important role in the improvement of the human health and guarantee of the life security. However, it is very outstanding at present that antibiotic is abused in domestic and abroad, which results the antibiotic-resistant bacteria to increase sharply. Methicillin-resistant staphylococcus aureus (MRSA) is one of the most important multidrug-resistant bacterium in the hospital. The first MRSA was reported in 1961 in England. Since then, MRSA becomes one of the most significant nosocomial pathogens throughout the world and is capable of causing a wide range of hospital infections. At present a number of different clones of MRSA have arisen in many countries, and there are about 100,000 persons who are infected by MRSA every year in the world, which is reported by Centers for Disease Control and Prevention (CDC), while it also has become increasingly prevalent in community-acquired infections. Because of carrying a variety of toxins, strong infections, MRSA parallels to be the three major infectious diseases with AIDS and viral hepatitis B in the world.
In order to understand the infection status caused by MRSA in the Shanxi Provincial People's Hospital (SPPH), the research was done to detect MRSA in 18 samples from the patients in clinic and hospital which was infected by staphylococcus aureus from September 2005 to September 2008 by cefoxitin disc diffusion method, oxacillin disc diffusion method and mecA Genes PCR method. Kerby- Bauer disk diffusion test was used to detect the susceptibility of MRSA to 9 kinds of antibacterial agents. PCR method was used to dectect PVL gene from the genomic DNA of infection strains. RAPD was used to type the chromosome DNA of MRSA strains. Novel multiplex PCR was used to type Staphylococcal chromosomal cassette mec (SCCmec). This study aims to find out MRSA molecular epidemiology, distinguish genotype and prevail trend in SPPH, and to afford method for controlling infection in clinical. The main results of the research were as follows.
1. The results of antibiotic resistance and detecting PVL
In this study, PVL gene was not detected from the genomic DNA of 18 samples. The rate of resistance to penicillin, oxacillin, cefoxitin, ciprofloxacin and gentamicin were 100% in 18 MRSA strains; Rate of resistance to clindamycin and red neomycin was 88.9%, and the mid-sensitivity rate of Rifampin was 11.1%, the resistant rate of rifampin was 55.6%.100% MRSA strains were susceptible to Vancomycin. All of 18 MRSA strains were multidrug-resistant bacterium, and the resistant model were 5 kinds of antibiotics agents (22.2%),6 kinds (33.3%),7 kinds (44.5%). The majority (44.5%) of the strains were resistant to penicillin, cephalosporins, linklactams, macrolides, quinolones, aminoglycosides, sha-neomycin. All isolates were resistant to penicillins, cephalosporins, fluoroquinolones and aminoglycosides.
2. The SCCmec genotype of MRSA
By using multiplex PCR of the 18 MRSA for SCCmec type, we found nine strains (50%) carrying three kinds of new SCCmec type/subtype (Newl～3),6 strains (33.3%) carryingⅢB SCCmec, while 3 strains (16.7%) were not stereotyped strain. MRSA in this region of the SCCmec types were highly polymorphic, and IIIB SCCmec were the local advantage type. RAPD electrophoresis profiles of cluster was analysed by the NTSYS software. The strains were fall into two types A, B, in which type A can be further divided into four subtypes:A1, A2, A3 and A4.
The results of MRSA resistance profile typing, SCCmec typing and RAPD typing were not completely consistent, suggesting they didn't have direct correlation.
MRSA is one of the most important pathogen in the hospital- and community- acquired. They had the charcteristic of pathogenicity and serious multiple drug resistance, however so far there wasn't report on the popular trend of MRSA in Shanxi Province. In this research, we collected 18 MRSA from the Shanxi Province People's Hospital, and carried a study on their resistance patterns and genotyping. All of 18 MRSA strains were multidrug-resistant bacterium, and the majority of MRSA wereⅢB SCCmec. RAPD analysis of hospital outbreak strains are type A.18 strains of MRSA were all HA-MRSA, while CA-MRSA was not found.
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[1]Duarte C O, Herminia de L. Multiplex PCR strategy for rapid identification of structural types and variants of the mec Elements in methicillin-resistant S. aureus. Antimicrob Agents and Chemother,2002,7,2155-2161.
[2]Poren Hsueh, Leejene Teng, Panchyr Yang, et al. Dissemination of Two Methicillin-Resistant Staphylococcus aureus Clones Exhibiting Negative Staphylase Reactions in Intensive Care Units. J Clin Microbiol,1999,3,504-509.
[3]A. Tambic, E. G. M. Power, H. Talsania, et al. Analysis of an Outbreak of Non-Phage-Typeable Methicillin-Resistant Staphylococcus aureus by Using a Randomly Amplified Polymorphic DNA Assay. J Clin Microbiol,1997,12, 3092-3097.
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[17]Grif K, Dierich MP, Pfaller K, et al. In vitro activity of fosfomycin in combination with various antistaphylococcol substances. J Antimicrob Chemother,2001,2, 209-217.
[18]陈代杰.抗菌药物与细菌耐药.上海,华东理工大学出版社,2001,90-112.
[19]Roberts MC, Sutcliffe J, Courvalin P, et al. Nomenclature for macrolide and macrolide-lincosamide-streptogramin B resistance determinants. Antimicrob Agents Chemother,1999,12,2823-2830.
[20]Vandenesch F, Naimi T, Enright MC, et al. Community-Acquired methicillin-Resistant Staphylococcus aureus Carrying Panton-valentine leukocidin Genes: Worldwide Emergence. Emerg Infect Dis,2003,9,978-984.
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[1]Duarte C O, Herminia de L. Multiplex PCR strategy for rapid identification of structural types and variants of the mec Elements in methicillin-resistant S. aureus. Antimicrob Agents and Chemother,2002,7,2155-2161.
[2]Poren Hsueh, Leejene Teng, Panchyr Yang, et al. Dissemination of Two Methicillin-Resistant Staphylococcus aureus Clones Exhibiting Negative Staphylase Reactions in Intensive Care Units. J Clin Microbiol,1999,3,504-509.
[3]A. Tambic, E. G. M. Power, H. Talsania, et al. Analysis of an Outbreak of Non-Phage-Typeable Methicillin-Resistant Staphylococcus aureus by Using a Randomly Amplified Polymorphic DNA Assay. J Clin Microbiol,1997,12, 3092-3097.
[4]Alex Van Belkum, Jan Kluytmans, Willem Van Leeuwen, et al. Multicenter Evaluation of Arbitrarily Primed PCR for Typing of Staphylococcus aureus Strains. J Clin Microbiol,1995,6,1537-1547.
[5]Deurenberg RH, Vink C,Kalenic S, et al. The molecular evolution of methicillin-resistant Staphylococcus aureus. Clin Microbiol Infect,2007,13,222-235.
[6]Hanssen AM, Ericson Sollid JU. SCCmec in staphylococci:genes on the move. FEMS Immunol Med Microbiol,2006,46,8-20.
[7]Ito T, Okuna K, Ma XX, et al. Insights on antibiotic resistance of Staphylococcus aureus from it whole genome:genomic island SCC. Drug Resistance Updates,2003, 1,41-52.
[8]欧阳范献,卜平凤,黄惠琴,等.MRSA的7种新SCCmec型别及其抗药特性.微生物学,2007,2,201-207.
[9]Hall RM, Brookes DE, Stokes HW. Site-specifeic insertion of gene into integrons:role of the 59-base element and determination of the recombination crossover point. Mol Microbiol,1991,8,1941-1959.
[10]Dslsgaard A, Forslund A, Tam NV, et al. Cholera in Vietnam:changes in genotypes and emergence of class 1 integrons containing aminoglycoside resistance gene cassettes in Vibrio cholerae 01 strains isolated from 1979 to 1996. J Clin Microbiol, 1999,4,734-741.
[11]Nesvera J, Hochmannova J, Patek M. An integron of class 1 is present on the plasmid pCG4 from gram-positive bacterium Corynebacterium glutamicum. FEMS Microbiol Lett,1998,2,391-395.
[12]Clark NC, Olsvik O, Swenson JM, et al. Detection of a streptomycin adenylyltransferase gene(aadA) in Enterococcus faecalis. Antimicrob Agents Chemother,1999,1,157-160.
[13]Tenover FC, Arbeit R, Archer G, et al. Comparison of traditional and molecular methods of typing staphylococcus aureus. J Clin Microbiol,1994,1,407-415.
[14]Noskin GA. Methicillin-resistant Staphylococcus aurerus and vancomycin-resistant Enterococci:emerging problems and new prospedts for management. Ann Acad Med Singapore,2001,3,320.
[15]徐德兴,王露霞,韦莉萍.金黄色葡萄球菌随机引物扩增DNA分型与药敏结果分析.中华医院感染学杂志,2002,6,391-392.
[16]Katayama Y, Ito T,Hiramatsu K. A new class of genetic element,staphylococcus cassette chromosome mec,encodes methicillin resistance in Staphylococcus aureus. Antimicrob Agents Chemother,2000,6,1549-1555.
[1]Fey PD, Ssid-Salim B, Rupp ME, et al. Comparative molecular analysis of community- or hospital-acquired methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother,2003,1,196-203.
[2]姚春艳,府伟灵.葡萄球菌医院感染的耐药性分析.中华医院感染学杂志,2004,14,104-106.
[3]熊艳.耐甲氧西林金黄色葡萄球菌耐药现状及检测方法的研究.武汉,华中科技大学,2006.
[4]陈洪武,周学军,王丹君.神经外科手术医院感染及相关因素的临床分析.临床医学,2006,2,31-33.