聚合酶链反应检测耐甲氧西林金黄色葡萄球菌mecA基因

建立聚合酶链反应（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，ＰＣＲ）检测耐甲氧西林金黄色葡萄球菌（ｍｅｔｈｉｃｉｌｌｉｎ－ｒｅｓｉｓｔａｎｔＳ．ａｕｒｅｕｓ，ＭＲＳＡ）ｍｅｃＡ基因的技术。四种ＤＮＡ提取方法检测灵敏度依次为超声裂解法（５×１０５ＣＦＵ／ｍｌ）、溶菌酶表面活性剂法（５×１０６ＣＦＵ／ｍｌ），表面活性剂法（１×１０７ＣＦＵ／ｍｌ）和直接煮沸法（１×１０８ＣＦＵ／ｍｌ），直接煮沸法具有简便性。引物的正链位于１８１～２００、负链位于３１１～３３０，序列分别为５＇－ＧＡＡＡＴＧＡＣＴＧＡＡＣＧＴＣＣＧＡＴ，５＇－ＧＣＧＡＴＣＡＡＴＧＴＴＡＣＣＧＴＡＧＴ，其扩增产物长度为１５０ｂｐ。五种常见菌证明本法具有较高特异性。苯唑青霉素ＭＩＣ水平与ｍｅｃＡ基因之间具有很好的相关性，ＭＩＣ≥４μｇ／ｍｌ的２２株菌均检出ｍｅｃＡ基因，提示苯唑青霉素常规检测ＭＲＳＡ的可行性。ＰＣＲ方法对于隐匿型耐药株的检出具有重要价值。     

耐甲氧西林金黄色葡萄球菌耐药性、mecA及blaTEM基因检测

目的了解临床分离的耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)耐药性、mecA及blaTEM基因存在状况.方法采用ATB Staph药敏试验板微量肉汤法对40株MRSA进行15种抗菌药物敏感性测定,采用聚合酶链反应(PCR)技术检测mecA和blaTEM基因.结果所有菌株对万古霉素、替考拉宁、呋西地酸和奎奴普丁-达福普汀均敏感,对青霉素、庆大霉素、红霉素、克林霉素、四环素、诺氟沙星和左旋沙星均耐药,对利福平、呋喃妥因、复方新诺明和米诺四环素的耐药率分别为5.0%、2.5%、97.5%和97.5%.mecA和blaTEM基因阳性率分别100.0%和37.5%.结论临床分离的MRSA多重耐药性十分严重;金黄色葡萄球菌对甲氧西林耐药均由mecA基因介导,blaTEM基因阳性率较高;治疗MRSA引起的感染,应以药敏试验结果为依据.     

菌液直接进行聚合酶链反应快速检测葡萄球菌mecA基因的研究

耐甲氧西林葡萄球菌 (MRS)因其对 β内酰胺类、氨基糖苷类、氟喹诺酮类等药物的多重耐药而成为危害严重的医院感染病原菌之一 ,不适当的治疗常常造成病情的延误以及病原菌在病房内的流行 ,这就需要临床微生物室快速准确地检测出MRS。应用聚合酶链反应 (PCR)直接检测mecA耐药基因 ,具有高度的特异性和灵敏度且报告时间较快[1]。以往报道的PCR方法都要求先提取细菌染色体DNA做模板 ,我们在实验中发现不进行标本的预处理 ,直接以菌液进行PCR扩增 ,亦可得到满意结果。一、材料和方法1.菌株 :2 15株葡萄球菌多数为本院 2 0 0 1～ 2 0 0 2…     

耐甲氧西林金黄色葡萄球菌耐药性、mecA及6laTEM基因检测

目的 了解临床分离的耐甲氧西林金黄色葡萄球菌（methicinin-resistant Staphylococcu8all—reus，MRSA）耐药性、mecA及6laTEM基因存在状况。方法采用ATBStaph药敏试验板微量肉汤法对40株MRSA进行15种抗菌药物敏感性测定，采用聚合酶链反应（PCR）技术检测mecA和6laTEM基因。结果 所有菌株对万古霉素、替考拉宁、呋西地酸和奎奴普丁-达福普汀均敏感。对青霉素、庆大霉素、红霉素、克林霉素、四环素、诺氟沙星和左旋沙星均耐药，对利福平、呋喃妥因、复方新诺明和米诺四环素的耐药率分别为5．0％、2．5％、97．5％和97．5％。mecA和blaTEM基因阳性率分别100．0％和37．5％。结论 临床分离的MRSA多重耐药性十分严重；金黄色葡萄球菌对甲氧西林耐药均由mecA基因介导．blaTEM基因阳性率较高；治疗MRSA引起的感染。应以药敏试验结果为依据。     

错配PCR对耐甲氧西林金黄色葡萄球菌 mecI基因点突变的分析

目的建立用于检测耐甲氧西林金黄色葡萄球菌（MRSA）上mecI常见突变位点的错配聚合酶链反应（PCR）,并用该方法分析上海地区分离MRSA中mecI G→T突变的发生情况。方法用头孢西丁纸片扩散法、mecA基因PCR法检测MRSA,用琼脂稀释法检测细菌的最低抑菌浓度（MIC）,用PCR检测mecI基因,建立错配PCR并用20株mecI基因和mec启动子区分析清楚的菌株对该方法进行评价,用错配PCR分析111株MRSA中mecI基因的突变情况。结果111株mecI阳性的MRSA中,mecI43位突变的5株,mecI202位突变的102株,另有4株MRSA的mecI43位和202位碱基均无突变。结论就其简便易行和准确性而言,错配PCR可用于检测mecI上已知的突变。     

耐甲氧西林金黄色葡萄球菌：耐药特点和快速检测

耐甲氧西林金黄色葡萄球菌（MRSA）是引进医院和社区获得性感染的主要原菌之一,由于其质性和多重耐药,导致临床检测,治疗的困难,MRSA耐药分子机理,检测方法及防止新的耐药菌出现一直是这一领域研究特点,本文对MRSA耐药特点和快速检测方法的研究进展作一综述。     

错配PCR对耐甲氧西林金黄色葡萄球菌mecI基因点突变的分析

目的建立用于检测耐甲氧西林金黄色葡萄球菌(MRSA)上mecI常见突变位点的错配聚合酶链反应(PCR),并用该方法分析上海地区分离MRSA中mecI G→T突变的发生情况。方法用头孢西丁纸片扩散法、mecA基因PCR法检测MRSA,用琼脂稀释法检测细菌的最低抑菌浓度(MIC),用PCR检测mecI基因,建立错配PCR并用20株mecI基因和mec启动子区分析清楚的菌株对该方法进行评价,用错配PCR分析111株MRSA中mecI基因的突变情况。结果111株mecI阳性的MRSA中,mecI43位突变的5株,mecI202位突变的102株,另有4株MRSA的mecI43位和202位碱基均无突变。结论就其简便易行和准确性而言,错配PCR可用于检测mecI上已知的突变。     

快速检测耐甲氧西林金黄色葡萄球菌mecA基因的PCR方法学评价

摘要：目的：对PCR法直接检测标本中耐甲氧西林金黄色葡萄球菌（MRSA）耐药基因mecA的可靠性进行评估。 方法：用经细菌表型鉴定的70株临床标本分离株进行方法学评估,其中MRSA 22株,非MRSA 48株,检测其最低检测限、重复性、灵敏度和特异性。同时对269份临床标本,包括67份痰液、82份血液、68份尿液以及52份无菌体液进行PCR检测和细菌表型鉴定,分析结果符合率。 结果：PCR法的最低检测限为5×10⁴ CFU/mL;菌数5×10⁶和5×10⁴ CFU/mL标本的批内变异系数（CV）为0.81%、0.94%,批间CV为1.48%、2.03%;检测经表型鉴定的菌株,敏感性和特异性均达到100%。痰液标本PCR检测和细菌表型鉴定符合率为98%,血液、尿液和其他体液标本均为100%。 结论：PCR法检测mecA耐药基因,方便、快速且最低检测下限和重复性等性能指标均比较理想,与表型鉴定符合率较高,适于临床应用。     

耐甲氧西林金黄色葡萄球菌耐药性及耐药基因研究

金黄色葡萄球菌是社区感染和医院感染的主要病原菌之一。其中耐甲氧西林金黄色葡萄球菌（meticillin-resistant staphylococcus aureus,MRSA）具耐多药特征已是临床抗感染化疗的难题之一。我们收集了2003年12月至2004年4月本院分离到的20株MRSA菌，对其进行了耐药性和13内酰胺类耐药相关基因（mecA、TEM）、     

多重聚合酶链反应检测耐甲氧西林葡萄球菌

目的应用一种敏感快速的多重聚合酶链反应（ＰＣＲ）,检测耐甲氧西林的金黄色葡萄球菌（ＭＲＳＡ）和凝固酶阴性葡萄球菌（ＭＲＣＮＳ）。方法临床分离的北京地区金黄色葡萄球菌１２３株,ＭＲＣＮＳ１２２株,用溶壁素和蛋白酶Ｋ制备模板ＤＮＡ,设计葡萄球菌甲氧西林耐药的决定基因,金黄色葡萄球菌独有的一个辅助基因和细菌中均有的１６ＳｒＲＮＡ基因引物,通过多重ＰＣＲ技术对标本进行扩增。结果１２３株金黄色葡萄球菌的ｆｅｍＡ基因１００％（１２３／１２３）阳性,ｍｅｃＡ基因阳性的占１８．７％（２３／１２３）,１２２株ＭＲＣＮＳ的ｆｅｍＡ１００％（１２２／１２２）阴性,ｍｅｃＡ阳性的占２４．６％（３０／１２２）。１６ＳｒＲＮＡ基因片断在多重ＰＣＲ中作为内部参照避免了假阴性结果的出现。结论建立的多重ＰＣＲ技术检测ＭＲＳＡ和ＭＲＣＮＳ具有敏感、快速、特异的特点,是一种可靠的实验诊断手段。     

The distribution of mecA, mecR1 and mecI and sequence analysis of mecI and the mec promoter region in staphylococci expressing resistance to methicillin.

The presence and sequences of genes that regulate the expression of methicillin resistance was investigated in 42 isolates of Staphylococcus aureus and 102 isolates of coagulase-negative staphylococci (CNS). PCR was used to detect mecA and the regulatory genes mecR1 and mecI. In a selected group of isolates, the sequences of mecI and the mec promoter region were also determined and compared with the sequences obtained from pre-MRSA strain N315. The genetic diversity of the collection was assessed by pulsed-field gel electrophoresis (PFGE). mecA was present in 21 S. aureus and 44 CNS. mecR1 was associated with mecA in all S. aureus and in all CNS, except two isolates of Staphylococcus haemolyticus. mecI was present in 48% of mecA-positive S. aureus and 50% of mecA-positive CNS. In six S. aureus isolates, mecI contained a termination codon at nucleotide 202 which would truncate the MecI protein. No mutation was found in the mecI gene of the four other S. aureus and 15 CNS sequenced. Seven isolates of Staphylococcus simulans had a single nucleotide substitution in the mec promoter region. Expression of methicillin resistance could be explained for all mecA-positive staphylococci with mutations within mecI or in the mec promoter region or in which mecI was deleted. However, the 'wild type' sequences observed in four S. aureus and eight CNS suggest that there is another mechanism for overcoming the repression of resistance caused by mecI.     

Detection of mecA, mecR1 and mecI genes among clinical isolates of methicillin-resistant staphylococci by combined polymerase chain reactions

The distribution of the mec genes mecA, mecR1 and mecI that regulate the expression of methicillin resistance was investigated by PCR in 145 staphylococci of hospital origin. Determination of alterations and deletions in parts of the genes was achieved using 11 sets of primers in combined reactions. Methicillin-resistant Staphylococcus epidermidis strains appeared relatively stable, with 57.9% of isolates containing the whole regulatory region. Alterations within the mecA gene were detected more often in other coagulase-negative staphylococci, which also had a higher percentage with deletions of regulatory genes. Among methicillin-resistant S. aureus, a genetically heterogeneous population was identified, with several alterations and deletions of mec genes.     

Comparison of PCR detection of mecA with methicillin and oxacillin disc susceptibility testing in coagulase-negative staphylococci

The provisional BSAC method for the detection of methicillin sensitivity in coagulase-negative staphylococci (CNS) requires incubation of isolates for 48 h and raises the problem of timely reporting of susceptibility data. The forthcoming withdrawal of methicillin raises another difficulty. We evaluated 42 clinically significant CNS blood culture isolates by PCR, methicillin and oxacillin disc testing and by using methicillin Etests. Our results suggest that, although oxacillin disc susceptibility testing is a reasonable first line step, optimal and timely detection of resistance or susceptibility may require a combination of phenotypic and genotypic methods.     

Detection of methicillin resistance in staphylococci by using a DNA probe.

A DNA probe derived from the PBP 2a gene of the methicillin-resistant Staphylococcus aureus COL was compared with phenotypic microbiologic tests for its ability to identify methicillin-resistant and -susceptible staphylococci. Lysates were applied to nitrocellulose with a dot blot apparatus. Isolates tested were both S. aureus and coagulase-negative staphylococci that had been recovered from a variety of geographic and clinical sources. When compared with a spread plate phenotypic test, the DNA probe gave sensitivity, specificity, and predictive values for both positive and negative tests of 100% for 204 S. aureus isolates (103 positive, 101 negative) and 99, 95, 99, and 95%, respectively, for 249 coagulase-negative staphylococci (210 positive, 39 negative). The probe was more sensitive than broth microdilution and more specific than agar dilution in identifying methicillin-resistant and -susceptible coagulase-negative staphylococci; all tests were equally accurate in identifying the methicillin susceptibility of S. aureus. DNA probe analysis for determining the methicillin susceptibility of staphylococci was rapid, easily interpretable, and equally accurate with radioactive and nonradioactive probes, and it gave results equivalent to the most sensitive microbiologic test for all staphylococcus species studied.     

Simultaneous species identification and detection of methicillin resistance in staphylococci using triplex real-time PCR assay

For rapid identification of methicillin-resistant Staphylococcus aureus, molecular methods are generally targeting mecA and species-specific genes. Sa442 DNA fragment is a popular species-specific target. However, recently, there have been few reports on S. aureus isolates that are negative for Sa442 fragment; therefore, use of single gene or DNA-fragment-specific polymerase chain reaction (PCR) for identification of microbial isolate may result in misidentification. This study includes CoA gene in parallel with Sa442 marker for identification of S. aureus. This further improves the specificity of the assay by checking for 2 determinants simultaneously for the identification of S. aureus and can prevent misidentification of S. aureus isolates lacking Sa442 DNA fragment. In this study, the newly developed triplex real-time PCR assay was compared with a quadruplex conventional gel-based PCR assay using the same primer sets in both assays. The dual-labeled TaqMan probes (ProOligo, France) for these primers were specifically designed and used in a real-time PCR assay. The clinical isolates (n = 152) were subjected to both PCR assays. The results obtained from both assays proved that the primer and probe sets were 100% sensitive and 100% specific for identification of S. aureus and detection of methicillin resistance. This triplex real-time PCR assay represents a rapid and powerful method for S. aureus identification and detection of methicillin resistance.     

Detection of methicillin resistance in coagulase-negative staphylococci and in staphylococci directly from simulated blood cultures using the EVIGENE MRSA Detection Kit

The EVIGENE MRSA Detection Kit was evaluated on coagulase-negative staphylococci (CoNS) from agar plates and on staphylococci directly from positive spiked blood cultures. For the CoNS study, a total of 242 isolates were tested, and of these 237 gave valid test results. For the 237 valid tests, all gave correct mecA classification. For the blood culture procedure, a collection of 51 mecA-positive Staphylococcus aureus, 21 mecA-negative S. aureus, 31 mecA-positive CoNS and 28 mecA-negative CoNS were used for the simulated blood cultures. For the S. aureus strains, all gave valid test results and correct mecA classification. One of the MRSA isolates gave a very faint nuc signal, and another four isolates gave results close to the cut-off of the kit; however, these were still clearly positive when read by the naked eye. For the CoNS isolates, 51 of the 59 strains gave valid results. All of these 51 strains gave correct mecA status. Thus the EVIGENE MRSA Detection Kit can provide fast and accurate determination of methicillin resistance in CoNS. This preliminary study of the blood culture procedure indicates that it is possible to achieve determination of methicillin resistance in staphylococci 8 h after positivity of the blood culture, making same-day detection of methicillin resistance possible.     

Vancomycin-induced deletion of the methicillin resistance gene mecA in Staphylococcus aureus

OBJECTIVE: To elucidate factors that contribute to the development of vancomycin resistance in methicillin-resistant Staphylococcus aureus (MRSA). METHODS: Forty-nine MRSA isolates were subjected to passage selection with vancomycin to isolate mutants with reduced susceptibility to vancomycin. One mutant was chosen for detailed molecular and biochemical characterization. RESULTS: Five vancomycin-resistant mutants (vancomycin MICs, 6-12 mg/L) were obtained in vitro from five MRSA parent isolates. Upon acquisition of vancomycin resistance, all mutants showed a concomitant decrease in oxacillin resistance. In one particular MRSA strain, selection for vancomycin resistance repeatedly produced deletions and rearrangements, including loss of the mecA gene. Pleiotropic phenotypical changes, such as yellow pigment formation, loss of haemolysis, thickened cell wall, increased resistance to lysostaphin and reduced cell wall turnover were observed in this mutant. CONCLUSION: Acquisition of vancomycin resistance in one MRSA strain triggered mecA deletion suggesting that this deletion, coupled to other rearrangements and/or mutations, may be responsible for the increased vancomycin resistance phenotype.     

凝固酶阴性葡萄球菌菌种鉴定与苯唑西林耐药凝固酶阴性葡萄球菌检测准确性

目的 评价凝固酶阴性葡萄球菌(CNS)菌种鉴定与苯唑西林耐药凝固酶阴件葡萄球菌(MRCNS)检测的准确性.方法 139株临床分离CNS,经ID 32 STAPH鉴定到种,用头孢西丁(FOX)、苯唑西林(OXA)纸片扩散法检测MRCNS,以Slidex MRSA detection乳胶凝集法检测青霉素结合蛋白2a(PBP2a)作为参考方法.结果 139株CNS鉴定为8个种,依次为溶血葡萄球菌、表皮葡萄球菌、人葡萄球菌、木糖葡萄球菌、腐生葡萄球菌、耳葡萄球菌、模仿葡萄球菌、沃氏葡萄球菌.FOX纸片法总的敏感度和特异度为99.0%和86.0%;OXA纸片法总的敏感度和特异度为91.7%和74.4%.影响FOX纸片法敏感度的菌种为1株表皮葡萄球菌;影响其特异度的菌种包括木糖葡萄球菌、沃氏葡萄球菌、腐生葡萄球菌;而影响OXA纸片法敏感度的菌种包括溶血葡萄球菌、人葡萄球菌、模仿葡萄球菌、耳葡萄球菌;影响其特异度的菌种包括人葡萄球菌、模仿葡萄球菌、木糖葡萄球菌、耳葡萄球菌、腐生葡萄球菌、沃氏葡萄球菌.结论 FOX纸片法检测MRCNS准确性因菌种不同有所差异,尤其应关注木糖葡萄球菌、沃氏葡萄球菌、腐生葡萄球菌的影响.建议检测MRCNS时,尽可能将CNS鉴定到种,视菌种必要时用PBP2a或mecA基因予以确认.