金黄色葡萄球菌对万古霉素的耐药性及耐药基因检测分析

目的 探讨金黄色葡萄球菌（SA）对万古霉素的耐药性及耐药基因分布情况。方法 收集平顶山市第一人民医院2020年4月至2022年3月临床送检样本分离的288株SA菌株,采用Vitek 2 Compact全自动细菌鉴定和药敏系统以及纸片扩散法进行菌落鉴定与药敏试验;采用聚合酶链反应检测pbp4、mgrA、agr、vraS、vraR、icaA、icaR基因表达。结果 288株SA菌株中筛选出25株万古霉素异质性耐药金黄色葡萄球菌（hVISA）菌株,hVISA阳性率为8.68%,未检测到万古霉素中介耐药金黄色葡萄球菌（VISA）菌株和万古霉素耐药金黄色葡萄球菌（VRSA）菌株;万古霉素敏感金黄色葡萄球菌（VSSA）菌株万古霉素最小抑菌浓度（MIC）主要分布在0.50 mg/L和0.75 mg/L,占比为41.83%和28.90%;hVISA菌株万古霉素MIC主要分布在1.50 mg/L和2.00 mg/L,占比为56.00%和28.00%;VSSA菌株pbp4、agr、icaR的相对表达量均低于hVISA菌株（P<0.05）,而mgrA、vraS、vraR、icaA的相对表达量均高于hV...     

异质性万古霉素中介金黄色葡萄球菌的检测方法和耐药性分析

目的研究比较异质性万古霉素中介金黄色葡萄球菌(hVISA)的几种筛查方法,制定出适合的hVISA的筛查方法。方法收集2018年1月—2019年12月江南大学附属医院临床标本中分离出的耐甲氧西林金黄色葡萄球菌株(MRSA),采用宏量E-test法(MET法)、BHIV3V、BHIV6V及MHA5T法筛查hVISA,最后用菌群曲线分析法(PAP-AUC)确认,比对4种筛查方法的敏感性及特异性。结果538株金黄色葡萄球菌经头孢西丁纸片法筛查出MRSA 266例,MRSA阳性率为49.4%;266株MRSA中经PAP-AUC确认hVISA菌株为17株,检出率为6.4%;MET法筛选出hVISA 21例,其中确认hVISA菌株为14例,敏感性为82.3%,特异性为97.1%。BHIV6V法筛查出hVISA 2株,确认均为hVISA,敏感性为11.8%,特异性100%。BHIV3V法筛查出hVISA 44株,确认为hVISA菌株为11株,敏感性为64.7%,特异性86.7%。MHA5T法筛查出hVISA 56株,确认hVISA为9株,敏感性为52.9%,特异性81.1%。MRSA除对利奈唑胺及万古霉素敏感外,对多种抗菌药物表现为多重耐药。结论本院MRSA检出率为49.4%,高于全国耐药监测网统计数据。hVISA的检出率为6.08%,MET法在敏感性及特异性方面优于其他3种方法,此方法较适合在本院开展hVISA的筛查。检出的MRSA及hVISA除对利奈唑胺及万古霉素敏感外,表现为多种抗菌药物的耐药性,尤其是hVISA具有更高的耐药性,临床治疗时应按照药敏结果选取治疗药物,切勿随意进行经验性用药。     

异质性万古霉素中介金黄色葡萄球菌的分子分型和同源性分析

目的 了解滨州医学院附属医院异质性万古霉素中介金黄色葡萄球菌(hVISA)的分子流行病学现状,为有效预防hVISA的传播提供依据。方法 采用万古霉素琼脂平皿筛选法对263株金黄色葡萄球菌进行初筛,菌群分析-曲线下面积(PAPAUC)法对初筛阳性的hVISA可疑株进行确认。采用多位点序列分型(MLST)、葡萄球菌A蛋白基因(spa)分型和脉冲场凝胶电泳(PFGE)分型方法对hVISA菌株进行分子分型和同源性分析。结果 本院初筛出113株hVISA可疑株,最终确认为hVISA的菌株共22株。MLST分型以ST72型(5株)为主,其次为ST59和ST25型(各4株);spa分型主要为t002、t437以及t2431(各3株)。PFGE聚类分析共得到12种PFGE型别,命名为H1-H12型,相似值处于62.6%～100%之间,其中最主要的是H3型(共6株),其次为H1型(共4株)。结论 hVISA在本院为散发,22株hVISA菌株共检出10种ST型,14种spa型和12种PFGE型。本院hVISA菌株的主要流行克隆为ST72-t2431-H3型,且均来源于儿科,儿科可能存在ST72-t2431...     

万古霉素敏感性减低葡萄球菌筛选及抗生素耐药相关基因检测

目的 了解万古霉素敏感性减低葡萄球菌临床分离株对-内酰胺类、氨基糖苷类、四环素以及万古霉素耐药相关基因存在状况.方法 采用含61tg/mL万古霉素脑心浸液琼脂从临床分离的葡萄球菌中筛选万古霉素中介葡萄球菌和万古霉素耐药葡萄球菌;采用菌谱分析法筛选异质性万古霉素耐药葡萄球菌:E-test法和琼脂稀释法检测其MIC值;PCR技术扩增mecA,aac(6')/aph(2'),aph(3)-Ⅲ,tetM,vanA.vanB和vanC基因,并对阳性扩增产物进行测序.结果 从100株临床分离葡萄球菌中检出7株异质性万古霉素耐药葡萄球菌,并从部分菌株中检出耐药基因,mecA,aac(6')/aph(2'),aph(3')-川基因检出率分别为85.7%,57.1%和85.7%,没有检出tetM,vanA,vanB和vanC.对PCR阳性扩增产物进行测序,BLASTn比对分析,与已登录基因库的相同基因序列具有高度同源性.结论 同甲氧西林耐药葡萄球菌相似,万古霉素敏感性减低葡萄球菌携带多种耐药基因,耐药表型与基因分析支持该菌株多药耐药,该类菌株的检测对于指导临床合理用药具有积极意义.     

耐万古霉素金黄色葡萄球菌的异质性

1997年日本首先报道了对万古霉素中度耐药的金黄色葡萄球菌（VISA），其最低抑菌浓度（MIC）为8mg／L。至2002年6月在美国共确定8株。这些菌株均源于耐甲氧西林的金黄色葡萄球菌（MRSA），且患者接受过较长时间的万古霉素治疗。至今，全球确定的VISA株虽不多，但异质性的VISA（hVISA）却有较多报道。     

呼吸内科金黄色葡萄球菌耐药性与耐药基因检测

目的探讨我院呼吸内科金黄色葡萄球菌的耐药性以及耐药基因的监测情况,旨在为降低医院内金黄色葡萄球菌的感染率提供理论依据。方法收集我院呼吸内科2010年11月至2012年11月感染金黄色葡萄球菌的患者共100例,采用PCR方法对100株菌株的耐药性基因进行检测。结果研究结果表明,青霉素的耐药率高达99%;苯唑青霉素、四环素、庆大霉素和红霉素的耐药率也都达到90%或以上;此外,100株金黄色葡萄球菌中,耐药性基因的检出率高达90%,其中多数为qacA基因。结论医院呼吸内科病房内细菌的耐药性问题非常严重,应该引起足够的重视。为了有效控制耐药菌株的产生,应当合适的使用抗生素并做好隔离措施。     

万古霉素联合利福平对骨髓炎致病菌MRSA的增殖抑制作用及机制

目的:探讨万古霉素联合利福平对骨髓炎致病菌耐甲氧西林金黄色葡萄球菌(MRSA)的毒性作用及作用机制。方法:将万古霉素稀释液与利福平稀释液注入MRSA培养液中,运用Western blot法检测金黄色葡萄球菌蛋白A(Protein A,SpA)的表达,Rt-PCR法检测SpA mRNA的表达。结果:万古霉素对MRSA株蛋白的表达有明显抑制作用,对基因的表达无明显抑制作用,与单一用药的作用比较,万古霉素联合利福平对MRSA的蛋白及基因表达的抑制作用无明显增强。结论:万古霉素对MRSA具有明显的毒性作用,此种作用主要是通过抑制其相关蛋白的表达发挥作用。     

耐甲氧西林葡萄球菌的耐药性分析

目的 分析临床分离的耐甲氧西林葡萄球菌(MRS)的分布和耐药性,并探讨对耐药菌感染的治疗策略.方法 对临床分离的568株葡萄球菌,药敏试验及耐甲氧西林葡萄球菌的检测均采用Kirby-Bauer琼脂扩散法,操作及结果判断均按美国国家临床实验室标准委员会(NCCLS)颁布的规则(2000年版)标准执行.结果 筛选出金黄色葡萄球菌(SAO)171株,检出率30.1%,凝固酶阴性葡萄球菌(CNS)397株,检出率为69.8%.其中耐甲氧西林金黄色葡萄球菌(MRSA)36.2%,耐甲氧西林凝固酶阴性葡萄球菌(MRSCON)77.5%,MRSA株对喹诺酮类耐药性(21%～48%)明显低于MRSCDN株(84%～89%).MRS除对万古霉素、利复平、呋喃妥因、阿米卡星和少数青霉素类耐药外,MRSA和MRSCON株的耐药性显著高于甲氧西林敏感葡萄球菌(MSSA)和甲氧西林敏感血浆凝固酶阴性葡萄球菌(MSSCON)株(P＜0.05),未发现VRS株.结论 由MRS菌株引起重症感染应首选糖肽类抗生素万古霉素治疗,葡萄球菌是造成医院感染的主要病原菌之一,MRS具有多重耐药性,应长期进行耐药性检测,提高对抗生素耐药性认识.     

金黄色葡萄球菌MgrA蛋白对喹诺酮类药物耐药性影响研究

目的了解金黄色葡萄球菌MgrA蛋白对喹诺酮耐药性的影响,为临床治疗金黄色葡萄球菌感染提供实验室依据。方法以本实验室已经构建的MgrA高表达质粒为研究材料,采用琼脂平板倍比稀释法检测金黄色葡萄球菌耐药株和敏感株、转MgrA组的MIC、凝固酶活性、荧光定量PCR、半定量PCR。结果转MgrA的金黄色葡萄球菌对喹诺酮类药物的耐药性增高,其NorA的表达与MgrA的表达具有线性关系,即MgrA高表达组的NorA表达也增高。结论MgrA蛋白mRNA表达水平与金黄色葡萄球菌氟喹诺酮耐药性相关,其介导耐药的原因可能与NorA基因mRNA表达水平增高有关。     

一株携带VanB基因的异质性耐万古霉素表皮葡萄球菌的发现

目的:利用NCCLS推荐的菌群分析法对南昌地区收集到的耐甲氧西林葡萄球菌(MRS)进行异质性耐万古霉素葡萄球菌的筛查,并对筛查到的菌株进行耐药基因检测和病案回顾性分析。方法:利用NCCLS推荐的菌群分析法进行异质性耐万古霉素菌株的筛查;利用聚合物酶链反应PCR法进行耐万古霉素VanA、VanB、VanC1、VanC2/3检测。结果:从一位患者的褥疮分泌物中分离到的一株异质性耐万古霉素葡萄球菌(代号H40)并检测出了耐万古霉素VanB基因。本研究中所测得的VanB基因序列已登录于NCBI,登录号为:AY960704。结论:异质性耐万古霉素葡萄球菌中检出VanB基因应属首例。     

41株金黄色葡萄球菌耐药性分析及PVL基因检测

目的了解临床分离金葡菌对常用抗生素的耐药性和杀白细胞毒素(PVL)基因携带情况。方法采用头孢西丁纸片扩散法检测耐甲氧西林金黄色葡萄球菌(MRSA),K-B法检测临床分离的41株金葡菌对常用抗生素的敏感性,PCR法检测mecA基因和PVL基因携带情况。结果 41株金葡菌中MRSA占51.2%,MRSA对克林霉素、红霉素、氨基糖苷类和喹诺酮类耐药率明显高于甲氧西林敏感金黄色葡萄球菌(MSSA),MRSA和MSSA对复方新诺明敏感率分别为90.5%和95%,未发现对万古霉素、替考拉林和利奈唑胺耐药株。21株MRSA mecA基因均阳性,41株金葡菌中共检出2株携带PVL基因,MRSA与MSSA各占1株,均分离自骨科病房。结论该院临床分离MRSA耐药率高,应规范临床用药,加强MRSA耐药性监测,加强PVL基因的检测,防止此类菌株的播散。     

Reduced glycopeptide susceptibility in methicillin-resistant Staphylococcus aureus (MRSA)

Vancomycin and other glycopeptide antibiotics are the current mainstay of therapy for infections caused by methicillin-resistant Staphylococcus aureus (MRSA). However, the high prevalence of MRSA has led to increased use of vancomycin in chronic and seriously ill patients and has resulted in the emergence of MRSA with reduced susceptibility to glycopeptides. Multiple MRSA phenotypes demonstrate reduced susceptibility to glycopeptides. According to the Clinical and Laboratory Standards Institute, vancomycin-intermediate S. aureus (VISA) are now those isolates with minimum inhibitory concentrations (MICs) between 4 microg/mL and 8 microg/mL, whilst heterogeneous VISA (hVISA) strains appear to be susceptible to vancomycin but contain a subpopulation of cells with reduced susceptibility to vancomycin (MICs > or = 4 microg/mL). At this time, MICs for these strains are reported to range between 1 microg/mL and 2 microg/mL. Vancomycin-resistant S. aureus (VRSA) are defined as those having MICs > or = 16 microg/mL. The detection of reduced susceptibility to vancomycin by routine susceptibility testing is unreliable and vancomycin non-susceptibility is most probably being underreported. Reports of reduced clinical efficacy associated with vancomycin MICs between 1 microg/mL and 2 microg/mL have been published. Patients most at risk of infection by hVISA, VISA and VRSA appear to be those with previous exposure to vancomycin. VRSA appears in the elderly and those with chronic leg or decubitus ulcers mainly containing vancomycin-resistant enterococci, which were probably the donor organism of the vanA gene to S. aureus. All MRSA strains recovered from patients whose infections do not respond to vancomycin treatment should be tested accurately for vancomycin susceptibility if these phenotypes are not to be missed. Treatment options for infections due to MRSA with reduced susceptibility to vancomycin are limited. Rapid identification of patients harbouring VRSA, VISA or hVISA as well as prompt isolation and adherence to infection control protocols are paramount in controlling the dissemination of these pathogens.     

金黄色葡萄球菌110株临床分布及耐药性分析

目的 了解临床分离的金黄色葡萄球菌(SA)菌株的临床分布情况及其对常用抗菌药物的耐药性,为临床合理用药提供依据.方法 对临床分离的110株SA菌株采用纸片扩散法(K-B法)检测12种抗菌药物的敏感性,并对药敏结果进行统计分析.结果 110株SA中,甲氧西林敏感金黄色葡萄球菌(MSSA)59株,耐甲氧西林金黄色葡萄球菌(MRSA)51株,MRSA的分离率为46.4％,MRSA对常用抗菌药物的耐药率明显高于MSSA(P＜0.05),未发现耐万古霉素的SA.结论 SA对多种抗菌药物的敏感率均较低,尤其是MRSA耐药株更为严重,临床应根据药敏结果合理选用抗菌药物.     

耐甲氧西林金葡菌的头孢西丁检测法与耐药性分析

目的评价头孢西丁纸片扩散法检测耐甲氧西林金葡菌（MRSA）的效果．了解MRSA的耐药状况。方法用头孢西丁及苯唑西林纸片扩散法、mecA基因聚合酶链反应（PCR）检测MRSA．并以mecA基因PCR法为参考方法与头孢西丁及苯唑西林纸片扩散法检测MRSA进行比较分析。按CLSI（原NCCLS）规定的标准测定MRSA对青霉素等10种抗生素的耐药率（KB法）。结果95株金葡菌（SA）中mecA基因阳性株42株。头孢西丁纸片法筛选出40株MRSA，其中39株mecA基因阳性，方法特异性为97．5％，灵敏度92．9％；苯唑西林纸片法筛选出44株MRSA，其中36株mecA基因阳性．方法特异性为81．8％．灵敏度85．7％。MRSA对青霉素、苯唑西林100％耐药，对万古霉素全部敏感．对红霉素、头孢唑林、克林霉素、氨苄西林／舒巴坦、头孢呋辛、左氧氟沙星、奈替米星呈不同程度耐药。结论头孢西丁纸片扩散法检测MRSA特异性和灵敏度优于苯唑西林纸片扩散法。MRSA具有多重耐药性．须加强其对抗生素的耐药性监测。     

Characterisation of laboratory-generated vancomycin intermediate resistant Staphylococcus aureus strains

Vancomycin has been the drug of choice for 30 years for the treatment of methicillin-resistant Staphylococcus aureus (MRSA). Emergence of decreased vancomycin susceptibility in MRSA strains presents a significant clinical problem with few therapeutic options. This study was performed to generate and characterise S. aureus strains with reduced susceptibility to vancomycin. Eighteen S. aureus strains were subjected to serial passaging on vancomycin to generate vancomycin intermediate resistant S. aureus (VISA) strains. Minimum inhibitory concentration (MIC) determination was performed for the parent and the passaged cultures with 13 different antibiotics. The strains were tested by the following five methods: simplified population analysis; CDC method; modified vancomycin agar screen; population analysis profile (PAP); and modified population analysis (PAP-area under the curve (AUC) ratio). Phenotypic changes such as doubling time, synergy with beta-lactam antibiotics and effect on norA efflux pumps were also studied for these strains. The result indicated that 8 VISA mutants (vancomycin MICs, 8-16 microg/mL) were generated in vitro from the 18 S. aureus strains. The CDC and modified agar methods proved to be the most sensitive and specific methods for detection of VISA strains. The PAP for all the VISA strains ranged from 12 microg/mL to > 16 microg/mL, with a PAP-AUC ratio of > 1.3. All mutants showed increased doubling time compared with their parent isolate. Synergism of the vancomycin and beta-lactam combinations was observed for all methicillin-resistant mutants. Upon acquisition of vancomycin resistance, a few mutants showed decreased oxacillin resistance. Two VISA strains were chosen for molecular characterisation of the mecA gene and one mutant showed genotypic changes with deletion of mecA. Loss of norA efflux pumps leading to fluoroquinolone sensitivity was also observed in four mutants.     

Comparative in vitro activity of telavancin, vancomycin and linezolid against heterogeneously vancomycin-intermediate Staphylococcus aureus (hVISA)

Selective pressure from glycopeptide use has led to non-susceptible strains of Staphylococcus aureus, including heterogeneously vancomycin-intermediate S. aureus (hVISA). Treatment of hVISA infections with vancomycin has been associated with treatment failure, therefore new treatments are required. The objective of this study was to evaluate the activity of telavancin, vancomycin and linezolid against hVISA clinical strains. Twenty-five hVISA isolates were evaluated for minimum inhibitory concentrations (MICs) by microdilution and for bactericidal activity by time-kill analysis [starting inoculum ca. 10(6)colony-forming units (CFU)/mL and ca. 10(8)CFU/mL] against telavancin, vancomycin and linezolid. MICs for 50% and 90% of the organisms (MIC(50) and MIC(90) values, respectively) were, respectively, 0.5mg/L and 1mg/L for telavancin and 2mg/L and 2mg/L for both vancomycin and linezolid. In time-kill studies, telavancin was bactericidal against all strains at plasma peak and trough concentrations and at low and high inocula. At low inoculum, the time to bactericidal activity (defined as 99.9% kill from initial inoculum) (T(99.9)) for telavancin was 5.6 ± 3.2 h at peak concentration and 12.3 ± 5.2 h at trough concentration. This was superior to vancomycin (P<0.001), which had a T(99.9) of 18.8 ± 2.1 h at peak concentration and 19.1 ± 2.2 h at trough concentration. At high inoculum, telavancin had a T(99.9) of 16.3 ± 3.2 h at peak concentration and 21.4 ± 2.5 h at trough concentration, whilst vancomycin did not consistently achieve bactericidal activity. Linezolid was not bactericidal against any strain at either concentration or inoculum. In conclusion, the killing activity of telavancin against hVISA was found to be superior to vancomycin and linezolid.     

Antimicrobial susceptibility of Gram-positive bacterial isolates from the Asia-Pacific region and an in vitro evaluation of the bactericidal activity of daptomycin, vancomycin, and teicoplanin: a SENTRY Program Report (2003-2004)

Medical centres in eight countries in the Asia-Pacific region provided 2391 isolates for the SENTRY Antimicrobial Surveillance Program during 2003-2004 to determine their susceptibility to several antimicrobial classes, including daptomycin. Daptomycin, vancomycin and teicoplanin minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for 120 isolates of Staphylococcus aureus, which included wild-type (WT) methicillin-resistant S. aureus (MRSA) and strains with decreased susceptibility to vancomycin (hetero-vancomycin-intermediate S. aureus (hVISA)). Oxacillin-resistant staphylococcal isolates were much less susceptible to the other tested agents compared with oxacillin-susceptible strains. Vancomycin resistance was higher among Enterococcus faecium (10.3%) than Enterococcus faecalis (0.4%), and macrolide resistance was high both for beta-haemolytic (17.7%) and viridans group (48.7%) streptococci. Daptomycin (MIC for 90% of the organisms (MIC(90))=0.5-1mg/L) was two-fold more potent than vancomycin, with >99% susceptibility when tested against staphylococci. All tested isolates of E. faecalis (MIC(90)=2mg/L) and beta-haemolytic streptococci (MIC(90)=0.5mg/L) were susceptible to daptomycin. Daptomycin MIC and MBC values were slightly higher for the hVISA isolates compared with WT-MRSA, with MBC/MIC ratios of only 1-2 for both groups. The MBC/MIC ratio for vancomycin was often greater when tested against these strains, particularly hVISA. In contrast, teicoplanin MBC/MIC ratios were significantly higher, with many of the strains showing values consistent with tolerance (>or=32). Daptomycin was demonstrated to have excellent in vitro activity when tested against Gram-positive isolates collected from Asia-Pacific countries, including hVISA strains.     

Antimicrobial resistance of Staphylococcus aureus isolated from skin infections

The antimicrobial susceptibility of 229 strains of Staphylococcus aureus isolated from various skin infections was determined against 22 antimicrobial agents by the agar dilution method. The clinical isolates were most sensitive to vancomycin, teicoplanin, mupirocin and fusidic acid. No strains were resistant to vancomycin or teicoplanin. Three strains were highly resistant (MIC > or =100 mg/l) to mupirocin and eight strains to fusidic acid. The MIC(50) of all antimicrobials, except for gentamicin, were below 3.13 mg/l. The incidence of resistance to penicillin, cephalosporins and clindamycin ranged from 20 to 30%. The occurrence of gentamicin, erythromycin and roxithromycin resistance was high at 55.2, 39.6 and 39.1%, respectively. Methicillin resistance occurred in 21.0% of strains. The incidence of organisms with MIC > or =3.13 mg/l to oxacillin was 24.3%. These results were comparable to the average rate of MRSA in Japanese dermatological specimens.     

山西临汾地区耐甲氧西林金黄色葡萄球菌临床分布及耐药性分析

目的了解山西临汾地区临床标本分离出的耐甲氧西林金黄色葡萄球菌(MRSA)临床分布及对常用抗菌药物的耐药性,为临床合理应用抗生素提供参考依据。方法 API系统鉴定细菌,用MIC法ATBSTAPH5板条进行药物敏感分析,MRSA用头孢西丁纸片扩散法(K-B法)筛选,用WHONET 5.4软件进行统计学分析。结果共检出金黄色葡萄球菌137株,其中MRSA 59株,MRSA的平均检出率为30.1%。MRSA主要分布在重症监护病房(35.6%)和神经外科(18.6%)。感染标本以痰(59.3%)、伤口分泌物(32.2%)为主。MRSA对β-内酰胺类、大环内酯类、喹诺酮类、氨基糖苷类、林可霉素、四环素的耐药率均>80%,但对复方磺胺甲(口恶)唑敏感率高(84.8%)。未发现对万古霉素、替考拉宁、达福普汀和呋喃妥因耐药的菌株。结论临汾地区金黄色葡萄球菌中MRSA分离率低,MRSA耐药严重且呈多重耐药。