MRSA中mecA及femB基因的检测与耐药相关性

研究femB、mecA基因在耐甲氧西林金黄色葡萄球菌(MRSA)中的表达与耐药的关系.运用PCR对MRSA的femB、mecA基因进行检测,MRSA耐药检测采用头孢西丁纸片法.40 株金黄色葡萄球菌(下简称金葡菌)通过头孢西丁纸片法,检出 30 株耐头孢西丁的菌株,通过PCR检测这 40 株金葡菌mecA基因,30 株MRSA全部为阳性, femB基因在 30 株MRSA中全部表达,而甲氧西林敏感的金黄色葡萄球菌(MSSA)的未表达.结果可见,PCR能快速准确地鉴定MRSA, mecA基因是MRSA的耐药基因,femB基因是MRSA的耐药相关基因.     

头孢西丁纸片扩散法检测耐甲氧西林溶血葡萄球菌

目的以PCR方法检测溶血葡萄球菌mecA基因为标准,评价头孢西丁纸片扩散法、苯唑西林纸片扩散法和VITEKGPS109卡微量肉汤稀释法检测耐甲氧西林溶血葡萄球菌（methicillin resistant Staphylococcus haemolyticus,MRSH）的敏感度和特异性。方法用PCR扩增临床分离的114株溶血葡萄球菌．检测特异性的mecA基因片段;头孢西丁纸片扩散法、苯唑西林纸片扩散法和VITEK GPS109卡微量肉汤稀释法检测溶血葡萄球菌中耐甲氧西林的溶血葡萄球菌。结果114株溶血葡萄球菌经PCR法检测,MRSH有97株,占85．1％;头孢西丁纸片扩散法、苯唑西林纸片扩散法和VITEKGPS 109卡微量肉汤稀释法检测MRSH分别有92、65、93株．与mecA基因法结果比较,头孢西丁纸片扩散法、苯唑西林纸片扩散法和VITEK GPS109卡微量肉汤稀释法敏感性分别为94．8％（92／97）、67．0％（65／97）、93．8％（91／97）;特异性分别为i00％（17／17）、100％（17／17）、88．2％（15／17）。矿检验,头孢西丁纸片扩散法和mecA基因法差异无显著性。结论头孢西丁纸片扩散法检测MRSH具有很高敏感度和特异性,是筛选和确认MRSH的一种可靠方法。     

2008-2010年舟山某院金黄色葡萄球菌的分布及耐药性变迁

目的 分析舟山医院三年来金黄色葡萄球菌分布及耐药性变迁,并对耐甲氧西林金黄色葡萄球菌(MRSA)与甲氧西林敏感金黄色葡萄球菌(MSSA)的耐药性差异做对比.方法 用ATB Expression半自动微生物分析仪进行菌株鉴定及药敏试验,用K-B法测红霉素、克林霉素、头孢西丁、苯唑西林直径,比较耐甲氧西林金黄色葡萄球菌(MRSA)与甲氧西林敏感金黄色葡萄球菌(MSSA)的耐药性差异.结果 金黄色葡萄球菌对苯唑西林、庆大霉素、红霉素、四环素和克林霉素的耐药率有上升的趋势;MRSA对苯唑西林、庆大霉素、复方新诺明、克林霉素、红霉素、青霉素、喹奴普汀-达福普汀、利福平和四环素的耐药率都明显高于MSSA的耐药率,二者间差异有统计学意义(P＜0.01),D-试验阳性71株,占72.45％.结论 金黄色葡萄球菌的耐药性逐渐升高,特别是对MRSA应引起临床的重视,检测克林霉素诱导型耐药具有重要的临床应用价值.     

耐苯唑西林乳酸杆菌mecA基因的分析

目的研究耐苯唑西林乳酸杆菌mecA基因的携带情况,分析其与耐药表型的关系,了解携带该基因质粒的可传递性。方法PCR检测mecA基因,双向测序得到基因序列,质粒电转化试验检测其传递性。结果耐受苯唑西林乳酸杆菌中81.6%的基因组总DNA、质粒DNA、质粒p5.8上均扩增出mecA基因序列保守区域,与GenBank中金黄色葡萄球菌的mecA基因、米酒乳杆菌的类mecA基因相似性分别为100%和59%。质粒p5.8成功电转化入乳酸乳球菌MG1363。结论乳酸杆菌苯唑西林的耐药表型与mecA基因的携带情况基本相符,携带耐药基因的质粒可通过人工转化,在部分细菌间水平传递,乳酸杆菌耐受苯唑西林的原因还需进一步探讨。     

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。     

社区感染金黄色葡萄球菌临床分离株耐药谱研究

目的研究社区获得性金黄色葡萄球菌耐药情况。方法采用常规方法分离金黄色葡萄球菌,用全自动微生物分析仪进行菌种鉴定和药敏试验,采用苯唑西林纸片扩散法检测耐甲氧西林金黄色葡萄球菌(MRSA)。结果社区感染的金黄色葡萄球菌主要分离自精液和尿液,MRSA的分离率为22.0%。MRSA对青霉素和环丙沙星的耐药率均为100%。所有的分离株对呋喃妥因和万古霉素均敏感。MRSA对红霉素、庆大霉素、四环素、复方新诺明、氯霉素和头孢克罗的耐药率分别为92.6%、33.3%、67.7%、92.6%、82.1%和75.1%。MSSA对上述药物的耐药率为75.6%、32.3%、50.5%、7.1%、25.3%和4.5%。结论社区感染的金黄色葡萄球菌主要为甲氧西林敏感的金黄色葡萄球菌,社区感染的MRSA的耐药性较为严重,而MSSA除青霉素和红霉素外耐药率较低。万古霉素对MRSA的体外抗菌活性非常强。     

耐甲氧西林葡萄球菌3种检测方法的实验比较及临床应用

目的 通过对200株临床分离的葡萄球菌耐苯唑西林的检测,比较3种表型检测法的阳性率并对其临床实用性进行评价。方法 采用美国NCCLS 2004年制订的头孢西丁-纸片扩散法、VITEK32微生物鉴定仪检测及苯唑西林盐琼脂法测试mecA介导的葡萄球菌耐药。结果 200株被检测的葡萄球菌中,头孢西丁-纸片扩散法检出耐苯唑西林的阳性率为68％（136／200）;苯唑西林盐琼脂法的阳性率为67．5％（135／200）;VITEK32仪的阳性率为68．5％（137／200）;且耐甲氧西林凝固酶阴性葡萄球菌（MRCNS）的耐药率（86．2％）高于耐甲氧西林金黄色葡萄球菌（26．23％）。3种检测方法结果经χ^2检测差异无显著性。结论 3种方法操作都简便,但各有优缺点,VITEK32仪器检测葡萄球菌耐笨唑西林费用高,一般小型医院不具备条件;盐琼脂筛选法判读直观,但要掌握好孵育时间;NCCLS2004版的纸片扩散法较经济实用,不仅可检测被检菌是否耐笨唑西林,同时还可获得大环内酯-林可霉素-链阳霉素（MLSB）结果,但是当头孢西丁的判断折点在临界时应当复检,建议与盐琼脂筛选法同步检测。     

金黄色葡萄球菌D试验和耐药性分析

目的 了解本地区金黄色葡萄球菌中红霉素对克林霉素诱导耐药表型及其对常用抗菌药物的耐药性.方法 用K-B琼脂扩散法检测耐甲氧西林的金黄色葡萄球菌(MRSA)及双纸片法(D试验)检测红霉素对克林霉素诱导耐药表型,用VITEK 2 Compact进行菌株鉴定和药敏试验.结果 156株金黄色葡萄球菌中MRSA有51株,占32.7％.红霉素对克林霉素诱导耐药共29株,占18.6％,其中MRSA有10株,甲氧西林敏感的金黄色葡萄球菌(MRSS)有19株.金黄色葡萄球菌对多种抗菌药物具有不同的耐药性,对青霉素的耐药性超过95％,对万古霉素、奎努普汀/达福普汀、利奈唑烷和替加环素敏感.结论 临床微生物实验室应加强对金黄色葡萄球菌中克林霉素诱导耐药的检测,临床治疗中也应加强抗菌药物的合理应用以防多耐药菌株的产生.     

两种纸片检测耐甲氧西林葡萄球菌结果比较

目的以苯唑西林琼脂筛选法为标准,比较苯唑西林和头孢西丁2种药敏纸片检测耐甲氧西林葡萄球菌(MRS)结果的准确性和一致性。方法分别用苯唑西林琼脂筛选法,头孢西丁和苯唑西林纸片扩散法检测临床分离的595株葡萄球菌。以琼脂筛选法为标准,计算2种纸片法的敏感性和特异性,比较二者的检测准确率差异是否存在统计学意义。结果金黄色葡萄球菌中,MRSA的检出率为55.97%,两药敏纸片的敏感性和特异性均在97%以上;二者在筛选MRSA的准确率上差异无统计学意义(P>0.05)。凝固酶阴性葡萄球菌中,MRSCN的检出率为69.68%,两纸片的敏感性均为100.00%,特异性分别为92.86%(头孢西丁)和77.38%(苯唑西林)。二者在检测MRSCN的准确率上差异存在统计学意义(P(0.05)。结论常规工作中,头孢西丁可准确检测所有的MRS,但苯唑西林只能用于检测MRSA,不能检测MRSCN。     

乳腺脓肿穿刺液标本病原菌分布及耐药性分析

目的探讨急性化脓性乳腺炎患者脓肿部位感染的病原菌及耐药性状况,指导临床合理使用抗菌药物。方法收集2014年1月至12月期间急性化脓性乳腺炎患者穿刺液标本780份,从中分离病原菌,用法国梅里埃公司生产的API鉴定系统进行细菌菌种鉴定,用K-B纸片扩散法做药敏试验。结果收集的780份标本中共分离出504株病原菌,分离率为64.6%,前五位的病原菌是金黄色葡萄球菌471株(其中MSSA 327株,MRSA 144株),占93.4%,表皮葡萄球菌16株(其中表皮葡萄球菌5株,MRSA11株),占3.2%,α-溶血链球菌11株,占2.2%,β-溶血链球菌4株(其中无乳链球菌3株,化脓性链球菌1株),占0.8%,其他凝固酶阴性葡萄球菌2株,占0.4%。其中327株MSSA对苯唑西林、头孢噻吩、头孢呋新、万古霉素100%敏感,对左氧氟沙星、呋喃妥因、复方磺胺甲唑、四环素、庆大霉素敏感率达90%以上,对青霉素、克林霉素、红霉素的耐药率较高。MRSA对青霉素类和头孢菌素类全部耐药,对克林霉素、红霉素耐药率较高,达80%以上,对左氧氟沙星、呋喃妥因、复方磺胺甲唑、庆大霉素敏感率达90%以上,没有发现对万古霉素耐药的MRSA。MRSA菌株对青霉素、苯唑西林、红霉素、克林霉素、四环素、头孢噻吩、头孢呋新的耐药性明显高于MSSA菌株,差异有统计学意义。结论急性化脓性乳腺炎患者感染的主要病原菌为金黄色葡萄球菌,包括MSSA和MRSA,MRSA对多种抗生素的耐药率明显高于MSSA,MSSA对苯唑西林、第I、II代头孢菌素敏感率较高,因此这些抗菌药物可以作为首选药治疗MSSA引起的急性化脓性乳腺炎,但MRSA对多种抗生素耐药率较高,治疗MRSA引起的急性化脓性乳腺炎,应根据药敏试验结果选用敏感药物。     

Comparison of methods for the detection of methicillin resistance in Staphylococcus aureus isolates from food products

AIMS: To compare several methods for detection of methicillin resistance in Staphylococcus aureus isolates from food. METHODS AND RESULTS: Two hundred S. aureus isolates from food of animal origin were screened for methicillin resistance by a PCR assay specific for the mecA gene, an oxacillin agar screen test and a cefoxitin disk diffusion test. Six out of 200 strains (3%) were found to be methicillin-resistant Staphylococcus aureus (MRSA) by PCR. The oxacillin agar screen test detected only one of the MRSA isolates (sensitivity of 16.7%) and mischaracterized three additional strains as MRSA (specificity of 98.45%). None of the MRSA strains was detected by the cefoxitin test (sensitivity of 0%), while 15 methicillin-susceptible S. aureus (MSSA) strains were misclassified as resistant (specificity of 92.3%). Fifteen MSSA strains displayed a beta-lactamase hyperproducer-like phenotype. The six MRSA (mecA-positive) strains resembled the characteristics of heteroresistant strains. CONCLUSIONS: As MRSA of animal origin may display atypical phenotypes, PCR appears to be more reliable for detection of methicillin resistance in animal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The study stresses the need for implementing the methods of screening S. aureus from food of animal origin for methicillin resistance.     

Quantitative disk diffusion as a convenient method for determining minimum inhibitory concentrations of oxacillin for staphylococci strains

In this study the susceptibility of 58 coagulase-negative staphylococci (CoNS) strains and 58 Staphylococcus aureus strains to oxacillin was evaluated by a novel method called quantitative disk diffusion (DD) method. The results obtained were compared to phenotypic methods as agar dilution (AD) for oxacillin, disk diffusion (DD) for cefoxitin, and related to the presence of the mecA gene detected by PCR. Minimum inhibitory concentrations (MIC) determined by the quantitative DD method were equivalent to MICs determined in the AD method for S. aureus (Student's t test, p=0.99) and CoNS (Student's t test, p=0.97). Incongruent results between PCR mecA gene determinations and the quantitative DD method were obtained in 8 strains (5 S. aureus and 3 CoNS) where the mecA gene expression was blocked. However, oxacillin resistance was detected by the proposed method even in staphylococci strains showing low-level or heterogeneous resistance to the antibiotic while other phenotypic methods failed. The single quantitative DD method is not expensive, it can be performed in any laboratory and permits accurate identification of oxacillin resistant staphylococci.     

Methicillin (Oxacillin)-resistant Staphylococcus aureus strains isolated from major food animals and their potential transmission to humans

From May 2001 to April 2003, various types of specimens from cattle, pigs, and chickens were collected and examined for the presence of methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA). S. aureus was isolated and positively identified by using Gram staining, colony morphology, tests for coagulase and urease activities, and an API Staph Ident system. Among 1,913 specimens collected from the animals, 421 contained S. aureus; of these, 28 contained S. aureus resistant to concentrations of oxacillin higher than 2 micro g/ml. Isolates from 15 of the 28 specimens were positive by PCR for the mecA gene. Of the 15 mecA-positive MRSA isolates, 12 were from dairy cows and 3 were from chickens. Antimicrobial susceptibility tests of mecA-positive MRSA strains were performed by the disk diffusion method. All isolates were resistant to members of the penicillin family, such as ampicillin, oxacillin, and penicillin. All isolates were also susceptible to amikacin, vancomycin, and trimethoprim-sulfamethoxazole. To determine molecular epidemiological relatedness of these 15 animal MRSA isolates to isolates from humans, random amplified polymorphic DNA (RAPD) patterns were generated by arbitrarily primed PCR. The RAPD patterns of six of the isolates from animals were identical to the patterns of certain isolates from humans. The antibiotypes of the six animal isolates revealed types similar to those of the human isolates. These data suggested that the genomes of the six animal MRSA isolates were very closely related to those of some human MRSA isolates and were a possible source of human infections caused by consuming contaminated food products made from these animals.     

Methicillin resistance in staphylococci isolated from subclinical mastitis in sheep

One hundred ovine milk samples were subjected to bacteriological analysis to detect staphylococci. Twenty-four staphylococcal strains isolated were characterised for methicillin resistance with disk diffusion test (DDT) after incubation at 24 and 48 h, oxacillin agar screen test, Minimal Inhibitory Concentration (MIC), nitrocefin test for beta-lactamase production and PCR for the mecA gene. Nine staphylococcal strains resulted resistant in DDT; some differences in the halo diameter at double incubation period were noted; eight of these strains were resistant in MIC test; just one strain was positive to oxacillin agar screen test. All strains were mecA negative by PCR and positive by nitrocefin test. On the basis of these results methicillin-resistant strains can be classified as beta-lactamase hyperproducers.     

Evaluation of methicillin-resistance in Staphylococcus aureus by the agar disk diffusion method and PCR

Staphylococcus aureus is a widespread human pathogen. One the most striking characteritics of this bacterium is resistance to methicillin and all beta-lactam antibiotics. The agar disk diffusion method is the most widely used in vitro susceptibility test, but recently molecular methods, e.g. Polymerase Chain Reaction, have been also introduced. We compared the detection of methicillin resistant coagulase positive Staphylococcus aureus isolated from clinical materials in Silesian microbiological laboratories by diffusion method and PCR through the detection of nuc and mec A genes. Our results show that PCR used for the detection of mec A gene increases the detection of methicillin-resistant Staphylococcus aureus strains by 10% as compared to the agar disk diffusion method. Among Staphylococcus aureus strains, detected as methicillin-resistant, 17% of organisms showed no presence of mec A gene.     

金黄色葡萄球菌的耐药性分析及基因分型研究

目的通过分析上海地区院内分离金黄色葡萄球菌的药敏谱型及对耐甲氧西林的金黄色葡萄球菌（MRSA）进行基因谱型的研究,了解金黄色葡萄球菌的院内流行状况。方法对临床分离出的43株金黄色葡萄球菌进行药敏试验和SCCmec基因盒的多重PCR检测,并将结果整合后用MEGA3．1软件分析其进化相关关系。结果药敏结果显示43株金葡菌对青霉素和甲氧西林的耐药率最高。甲氧西林的耐药率达到62．8％。MecA阳性菌株SCCmec的分型显示均为Ⅱ型或Ⅲ型,且所占比例相近,未见Ⅰ型和Ⅳ型。进化树分析发现了在同一医院中亲缘关系相近的菌株,为院内感染流行株。结论MecA基因介导的MRSA在分离菌株中所占比例高,存在院内感染爆发性流行。     

Analysis of borderline-resistant strains of methicillin-resistant Staphylococcus aureus using polymerase chain reaction.

Identification of methicillin-resistant Staphylococcus aureus by drug-susceptibility tests alone poses a serious problem, because a considerable number of clinical S. aureus isolates are borderline resistant to methicillin. To circumvent this problem, we have developed a quick and sensitive method of PCR amplification for the detection of mecA gene, which, coding for PBP2', is the specific genetic element of methicillin-resistant Staphylococcus aureus. This method made it possible to identify MRSA strains in a short time using as few as 30 cells as a starting material for template DNA. Using this method, we found that the strains of borderline methicillin-resistance could be accurately identified. We also found one S. aureus clinical strain, T3, which lacked mecA gene in spite of its resistance to methicillin.     

Characteristics of defective strains of methicillin resistance Staphylococcus aureus that do not produce coagulase or CF

The aim of the study was to estimate frequency of coagulase-negative and CF-negative strains among methicillin-resistant Staphylococcus aureus (MRSA) and to assess their homogeneicity in respect of genotype, phagotype and drug resistance pattern. A total of 186 MRSA strains collected from different hospitals in Gdańsk region were studied. Gens: nuc, mecA, and coa were identified by PCR method. The coagulase tube test for staphylocoagulase and the slide test for clumping factor were used. Coagulase-negative and CF-negative MRSA strains were confirmed by PCR-RFLP method of coa gene; phage typing and drug resistance pattern were evaluated by disc diffusion test. The results of the study showed low frequency of both coagulase-negative and CF-negative MRSA strains (7.25% and 3.76% respectively). Among MRSA population tested the simultaneous occurrence of the strains lacking coagulase and clumping factor was not observed. All coagulase negative MRSA had coagulase gene (coa) and differed from CF-negative strains in respect of coa gene.