ICU耐亚胺培南铜绿假单胞菌产金属酶检测及其耐药性分析

目的 了解重症监护病房(ICU)耐亚胺培南铜绿假单胞菌(IRPA)的金属β-内酰胺酶(MBLs)的产生情况及其耐药特性,为临床抗感染治疗提供依据.方法 对深圳市观澜人民医院2010年1月至2011年12月ICU分离的587株铜绿假单胞菌(PAE),用双纸片协同试验检测MBLs产生情况,用VITEK 2 compact全自动微生物分析系统检测其对17种抗生素的耐药性.结果 在587株PAE中,IRPA 127株,占21.6％;IRPA对其中7种抗生素为全耐药,对其余10种抗生素的耐药率也在40％以上;IRPA组及亚胺培南敏感铜绿假单胞菌(ISPA)组两组间对常用抗生素的耐药率比较,除AMP、CZO、FTN、SXT、SAM及CTT外,两组其余抗生素的耐药率差异均有统计学意义(P＜0.01);在127株IRPA中,产MBLs 45株,产酶率为35.4％.结论 IRPA的检出率较高,多药耐药现象较严重,产MBLs是PAE对IPM及头孢类抗菌药物耐药的主要原因之一,应参考实验室的药敏结果,合理选用抗菌药物.     

耐亚胺培南铜绿假单胞菌的耐药性和金属酶研究

目的了解广州市某医院烧伤科病房亚胺培南耐药铜绿似单胞菌的耐药谱特点及产生金属酶情况。方法采用Kirby—Bauer法进行药物敏感性试验,采用2-巯基乙醇纸片协同试验筛选产金属酶菌株,对金属酶基因进行聚合酶链反应（PCR）和序列分析。结果48株亚胺培南耐药菌株均为多重耐药菌,对临床常用的多种抗菌药物耐药,敏感率由高到低依次为CIP、TOB、AMK、GEN和FEP。2-巯基乙醇纸片协同试验阳性6株,6株菌金属酶引物VIM-2扩增阳性,经测序证实为VIM-2型金属酶。结论该组亚胺培南耐药铜绿假单胞菌为多重耐药菌,产VIM-2型金属酶。     

大连地区耐亚胺培南铜绿假单胞菌金属β-内酰胺酶检测及耐药性分析

目的了解大连地区分离的耐亚胺培南铜绿假单胞菌的耐药特征,金属β-内酰胺酶携带情况,提供大连地区院内控制铜绿假单胞菌感染的依据。方法选取2013年1月至2014年9月临床分离的400株铜绿假单胞菌,进行菌种鉴定和药物敏感试验;金属酶检测采用E-test试验;PCR法扩增产金属β-内酰胺酶的基因,并对扩增阳性产物进行测序确认。结果 400株铜绿假单胞菌的标本以呼吸道分泌物最多,占81.5%;分布以呼吸内科和ICU病房最高,占总数的19.8%和25.5%;药敏结果发现有89株铜绿假单胞菌对亚胺培南耐药,且多数为多重耐药菌株;400株铜绿假单胞菌经E-test试验进行金属酶表型筛查,有17株金属酶阳性,检出率为19.1%;经PCR扩增金属酶基因阳性的有11株,其中8株为IMP-1,3株为VIM-2,其他几种基因均未检出。结论大连地区耐亚胺培南的89株铜绿假单胞菌多数是多重耐药菌株;产金属β-内酰胺酶在大连地区铜绿假单胞菌对亚胺培南耐药中发挥重要作用,酶的基因型主要为IMP-1和VIM-2。     

多重耐药铜绿假单胞菌的耐药性及耐药基因检测分析

目的探讨呼吸内科病房分离的多重耐药铜绿假单胞菌相关耐药基因。方法采用纸片扩散法进行体外药敏试验,聚合酶链反应（PCR）对其中32株多重耐药铜绿假单胞菌进行相关基因TEM、SHV、CTX-M-9、DHA、VIM、PER、OXA、IMP和OprD2检测,并对耐药基因进行测序。结果 32株多重耐药铜绿假单胞菌对多黏菌素B耐药率是3.1%,对其余抗菌药物耐药率为53%～100%,β-内酰胺酶基因OXA-10、TEM-1、DHA-1、PER-1和IMP-1基因阳性率分别为46.9%、21.9%、15.6%、12.5%和31.3%,未检出SHV基因、CTX-M-9基因和VIM基因;另外OprD2基因缺失率达68.8%。结论呼吸内科病房多重耐药的铜绿假单胞菌携带多种β-内酰胺酶基因,应引起高度重视。     

耐亚胺培南铜绿假单胞菌金属酶的筛选及基因型研究

目的研究重庆医科大学附属第一医院分离的29株耐亚胺培南铜绿假单胞菌中金属酶（Metallo-β-Lactamase,MBL）的基因型分布情况。方法用亚胺培南-EDTA纸片法筛选29株铜绿假单胞菌中产MBL的铜绿假单胞菌,用PCR扩增法检测29株菌中金属酶VIM和IMP基因。结果29株耐亚胺培南的铜绿假单胞菌中,亚胺培南-EDTA纸片法筛选出MBL阳性菌株5株,阳性率为17％。PCR扩增出IMP基因型有4株,阳性率为14％,均为金属酶IMP-9型,未扩增出VIM基因。以PCR法结果为判定标准,亚胺培南-EDTA纸片法敏感性100％,特异性96％。结论IMP-9型为该院分离的这29株耐亚胺培南的铜绿假单胞菌中主要MBL基因型。本实验中所用的亚胺培南一EDTA纸片法能简单有效的筛选出产MBL的铜绿假单胞菌。     

铜绿假单胞菌对碳青霉烯类药物的耐药机制研究

目的 探讨铜绿假单胞菌对碳青霉烯类药物的耐药机制.方法 收集2008年11月至2009年4月我院临床分离的铜绿假单胞菌31株,根据药敏结果分为碳青霉烯类耐药组(21株)和碳青霉烯类敏感组(10株).另设1株标准株ATCC 27853,用亚胺培南-EDTA(乙二胺四乙酸)抑制试验检测菌株是否产生金屑酶,采用PCR法检测各菌株的外膜孔道蛋白oprD2基因,探讨铜绿假单胞菌对碳青霉烯类抗生素耐药机制.结果 21株耐药株有7株产生金属酶;21株耐药株经oprD2基因扩增,15株阴性,6株阳性,10株敏感株全部阳性.统计学检验结果表明,碳青霉烯类耐药组与敏感组oprD2基因阳性率的差异有极显著性(P＜0.01).结论 oprD2基因缺失和金属酶是本院铜绿假单胞菌对碳青霉烯类抗生素耐药的重要机制.     

多重耐药铜绿假单胞菌金属β-内酰胺酶基因检测及 对多粘菌素B耐药机制探讨

摘要：目的 探讨多重耐药铜绿假单胞菌金属β-内酰胺酶基因检测及对多粘菌素B耐药机制情况。方法 分析2008年2月至2014年2月收集分离的菌株收集200株铜绿假单胞菌,通过琼脂稀释法和E-test法对8种抗生素进行药敏试验,通过PCR对金属β-内酰胺酶基因进行扩增,在对多重耐药铜绿假单胞菌进行体外诱导,获得多粘菌素B的耐药菌株,观察临床分离株和诱导株pmrAB和phoPQ基因扩增和测序情况。结果 200株铜绿假单胞菌对于亚胺培南、美罗培南、环丙沙星、头孢哌酮/舒巴坦耐药率均高于30%,200株铜绿假单胞菌对于阿米卡星耐药率最低仅为10%,敏感率最高达到90%。200株铜绿假单胞菌对于多粘菌素B药敏试验中197株敏感,3株中介,无耐药发生。通过B100、B108、B925三株临床分离株和多粘菌素B诱导耐药株B100d、B108d、B925d的phoP、phoQ和pmrB基因进行扩增。其中B108d号菌株的pmrB基因有两个点突变,在第5365842位的碱基发生了从GGC到AGC的突变,即361个G氨酸变成了S氨酸;第5365864位的碱基发生率从TCG到CCG突变,第368个S氨酸转变为P氨酸。B108d号菌株的1278614位点的碱基从TAC到TTC的突变,第84个Y氨酸变为F氨酸。另外B108d号菌株的phoQ基因中1278416-1278527位的基因发生缺失,进而缺失了152个氨基酸。结论 铜绿假单胞菌耐药率较高,但是没有出现多粘菌素B耐药菌株,诱导出的多粘菌素B耐药的多重铜绿假单胞菌PCR扩增后,出现了pmrB和phoQ基因的碱基突变,同时还有部分DNA片段缺失。     

耐亚胺培南铜绿假单胞菌相关耐药基因的检测

目的对耐亚胺培南（IMP）的铜绿假单胞菌（IRPa）相关耐药基因进行检测。方法 2003年至2009年从临床标本中分离到（P.aeruginosa）共220株,采用三维试验筛选产β-内酰胺酶的铜绿假单胞菌,应用普通PCR和多重PCR分别检测碳青霉烯酶基因和质粒携带的C类头孢菌素酶（AmpC酶）耐药基因,应用荧光定量RT-PCR的方法检测oprD2基因的表达情况。结果共检出43株产β-内酰胺酶的菌株,其中产AmpC酶、超广谱β-内酰胺酶（ESBLs）、金属β-内酰胺酶（MBLs）和未知酶菌株的构成比分别58.14%（25/43）、18.60%（8/43）、4.65%（2/43）和16.28%（7/43）。74株耐亚胺培南的铜绿假单胞菌中,有2株菌携带IMP-9基因,1株菌携带DHA质粒型AmpC酶基因,其他碳青霉烯酶基因检测为阴性。40株菌株oprD2基因表达蛋白量降低,34株oprD2基因表达蛋白量正常。结论 oprD2基因的突变或蛋白表达量降低是IRPa对亚胺培南耐药的主要原因,AmpC酶可水解亚胺培南可能与铜绿假单胞菌对亚胺培南的耐药有一定的关系,而KPC-1酶和MBLs在铜绿假单胞菌对亚胺培南耐药机制中不是主要因素。     

铜绿假单胞菌医院感染调查及耐药分析

目的探讨铜绿假单胞菌(PA)医院感染的临床分布及耐药性,为临床合理使用抗菌药物及控制医院感染提供依据。方法 PA的培养与鉴定严格按照《全国临床检验操作规程》进行。药敏试验采用纸片扩散法(K-B法)。结果收集的359株PA中,分离率居前3位的临床科室依次为ICU、呼吸内科和神经内科,分别占38.1%、17.5%和13.9%;临床标本中以呼吸道标本分离率最高,占62.7%;PA对头孢哌酮/舒巴坦的耐药率最低(9.7%),其次为阿米卡星(13.1%)和头孢他啶(17.8%),另12种抗菌药物的耐药率均较高。结论 PA耐药性已十分严重,临床应根据药敏结果合理使用抗菌药物并加强对细菌耐药性的全面监测。     

ICU患者铜绿假单胞菌获得性肺炎耐药研究

目的监测重症监护病房（ICU）铜绿假单胞菌（Pseudomonas aeruginosa,PA）的感染状况及耐药变迁,指导临床合理使用抗菌药物。方法对2003年至2007年间,我院ICU收治的患者下呼吸道分泌物进行培养及体外耐药试验。结果铜绿假单胞菌是ICU下呼吸道感染的主导致病菌且感染率呈逐年上升的趋势（46．3％-81．0％）,细菌对三代头孢、亚胺培南及氨基糖苷类药物的耐药性也呈逐年上升的趋势,并呈现多重耐药的特性。结论铜绿假单胞菌是ICU院内下呼吸道感染的常见病原菌,其多重耐药性及应引起高度重视。     

铜绿假单胞菌的临床分布和耐药性变迁

目的调查温州医学院附属第一医院铜绿假单胞菌的临床分布和耐药性及其变迁情况,指导临床合理用药和控制耐药菌的产生。方法对该院2003年至2005年437株铜绿假单胞菌进行回顾性分析,用17种抗菌药物进行体外药敏试验。结果铜绿假单胞菌分布以呼吸道感染和创口分泌物感染为主。细菌对第3代头孢菌素耐药率高,对头孢他啶、亚胺培南、头孢哌酮/舒巴坦和氟喹诺酮类药物的耐药率均有逐年增加的趋势。耐药率增加与临床广谱抗菌药物用药量增加有关。结论铜绿假单胞菌的耐药性十分突出,应在药敏指导下合理选用抗菌药物,应该适当控制碳青霉烯类和第3代头孢菌素的使用,减缓耐药菌株的产生。     

The cost of multiple drug resistance in Pseudomonas aeruginosa

The spread of bacterial antibiotic resistance mutations is thought to be constrained by their pleiotropic fitness costs. Here we investigate the fitness costs of resistance in the context of the evolution of multiple drug resistance (MDR), by measuring the cost of acquiring streptomycin resistance mutations (StrepR) in independent strains of the bacterium Pseudomonas aeruginosa carrying different rifampicin resistance (RifR) mutations. In the absence of antibiotics, StrepR mutations are associated with similar fitness costs in different RifR genetic backgrounds. The cost of StrepR mutations is greater in a rifampicin‐sensitive (RifS) background, directly demonstrating antagonistic epistasis between resistance mutations. In the presence of rifampicin, StrepR mutations have contrasting effects in different RifR backgrounds: StrepR mutations have no detectable costs in some RifR backgrounds and massive fitness costs in others. Our results clearly demonstrate the importance of epistasis and genotype‐by‐environment interactions for the evolution of MDR.     

铜绿假单胞菌的分布特点耐药性分析

目的分析3年来我院患者铜绿假单胞菌的耐药特点,为临床合理用药提供依据,并有助于医院感染预防与控制。方法对2010年至2012年我院各类感染患者标本中分离获得的铜绿假单胞菌,采用纸片扩散法（K-B法）检测抗菌药物的敏感性,并用WHONET5．3软件对药敏结果进行统计分析。结果3年分离的铜绿假单胞菌共计369株,其对多黏菌素B无耐药,对亚胺培南、美罗培南、头孢哌酮／舒巴坦、哌拉西彬他唑巴坦和阿米卡星的耐药率较低（6．81％～22．73％）,对其他抗菌药物的耐药率较高（32．47％～73．38％）。结论铜绿假单胞菌对多种抗菌药物均具有较高的耐药性,临床上治疗该菌感染时应根据药敏检测结果选择抗菌药物。     

Antibiotic resistance study of clinical strains of Pseudomonas aeruginosa]

The study of 40 clinical strains of Ps. aeruginosa isolated from the wound surfaces of the patients showed that all the isolates were resistant to one or several antibiotics. The number of the strains resistant to 5, 4, 3, 2 or 1 drug was 5, 22.5, 25. 30 or 17.5 per cent respectively. Fifteen strains carried resistance plasmids capable of conjugative transfer. Eleven out of 21 plasmids controlled resistance to chloramphenicol, 7 plasmids controlled resistance to streptomycin and sulfanylamides, 1 plasmid controlled resistance to streptomycin and chloramphenicol. The presence of two types of the plasmids controlling resistance to chloramphenicol and streptomycin + sulfanylamides respectively was found. All the plasmids proved to be capable of conjugative transfer between the strains of Ps. aeruginosa ML (PAO). The frequency of the plasmid conjugative transfer in such crosses ranged from 10(-6) to 10(-3). Most of the plasmids belonged to the incompatibility groups P-2 and P-7. One plasmid belonged to the incompatibility group P-5. It should be noted that about a half of the plasmids (11 out of 21) belonged to the incompatibility group P-7 which up to the present time was conditional, since was represented by a single plasmid Rms 148.     

Antimicrobial resistance of Pseudomonas aeruginosa biofilms

Resistance to antimicrobial agents is the most important feature of biofilm infections. As a result, infections caused by bacterial biofilms are persistent and very difficult to eradicate. Although several mechanisms have been postulated to explain reduced susceptibility to antimicrobials in bacterial biofilms, it is becoming evident that biofilm resistance is multifactorial. The contribution of each of the different mechanisms involved in biofilm resistance is now beginning to emerge.     

Plasmid-determined resistance to chromate in Pseudomonas aeruginosa

Abstract Resistance to chromate in five independent Pseudomonas aeruginosa clinical isolates was transferred by conjugation to P. aeruginosa strain PU21. All chromate-resistant transconjugants contained large plasmids that also conferred resistance to inorganic mercury. One of these plasmids, pUM505, increased the resistance to CrO₄²⁻ and decreased the accumulation of intracellular ⁵¹CrO₄²⁻ by the host cells as compared to the plasmidless strain PU21.     

Adaptive resistance to polymyxin in Pseudomonas aeruginosa due to an outer membrane impermeability mechanism

The isolated outer membrane from cells of a Pseudomonas aeruginosa strain exhibiting adaptive resistance to polymyxin was not affected by polymyxin treatment, as monitored by electron microscopy of negatively stained preparations. This was in sharp contrast with extensive disruption by polymyxin of the outer membranes of the parent polymyxin-sensitive strain and the resistant strain following reversion to greater polymyxin sensitivity. The isolated cytoplasmic membrane of the polymyxin-resistant strain, on the other hand, remained sensitive to the disruptive effects of polymyxin treatment. The permeability characteristics of the resistant strains appear to be altered, as indicated by differences in minimal inhibitory concentrations for a variety of antibiotics between the polymyxin-sensitive and polymyxin-resistant strains. No evidence was found for a polymyxin-inactivating enzyme in osmotic shock fluid from the polymyxin-resistant strain. No evidence for a cytoplasmic membrane repair mechanism was found in the polymyxin-resistant strain. These observations suggest that the mechanism of adaptive polymyxin resistance in this model system is the alteration of the outer membrane so that it excludes polymyxin from reaching the still sensitive cytoplasmic membrane.