聚合酶链反应检测铜绿假单胞菌的研究现状

聚合酶链反应在病原微生物的检测中应用广泛。近年来已有文献报道其对铜绿假单胞菌的检测，本文就其检测铜绿假单胞菌的引物设计、检测步骤、评价与初步应用情况进行综述。     

708株铜绿假单胞菌的病房分布及药敏分析

目的：了解铜绿假单胞菌的耐药状况。方法：用微量肉汤稀释法鉴定细菌和药敏试验,并对药敏结果进行分析。结果：铜绿假单胞菌是Icu(225／708)、呼吸科(127／708)、胸外科(77／708)等临床科室病员感染的主要病原菌,具多重耐药性,其中对头孢噻肟的耐药率最高,达66．2％,对泰能耐药率为42．9％,对环丙沙星耐药率为37．7％;而对阿米卡星敏感率达83．5％,对哌拉西林／他唑巴坦敏感率达81．1％。结论：铜绿似单胞菌是医院内感染的最常见菌和重要痫原菌,在对其引起的感染进行临床治疗时,第三代头孢菌素和泰能应在连续病原学监测状况下慎用,最好选用含酶抑制剂的药物(如哌拉西林／他唑巴坦,头孢哌酮／舒巴坦)联合氦基糖苷类或氟喹诺酮类药物治疗。这对于抗生素的合理使用及防止耐药菌株的传播都具有重要意义。     

神经外科病房铜绿假单胞菌耐药性监测

目的了解2008年10月至2009年10月神经外科监护病房患者铜绿假单胞菌的药敏及耐药性,为临床合理使用抗生素提供依据。方法菌种经珠海DLMedcal-96细菌测定系统进行鉴定,及该系统肠杆菌属随机体外诊断试剂板进行药敏试验,并作统计分析。结果本院神经外科病房共检出264株病原菌,其中铜绿假单胞菌分离出92株34.85%(92/264),为第一病原检出菌。铜绿假单胞菌对21种抗生素耐药较低的依次为头孢他啶、亚胺培南、美罗培南(均为6.52%)、粘菌素、多粘菌素B(均为8.70%)、左氧氟沙星(17.39%)、头孢吡肟(21.74%)、环丙沙星、氨曲南(均为23.91%)、哌拉西林/他唑巴坦(26.09%)、头孢哌酮/舒巴坦(30.43%)、头孢噻肟(36.96%)。耐药较严重的抗生素为氯霉素(86.96%)、复方新诺明(95.65%)。结论临床应重视药敏试验结果,并及时对耐药结果统计分析,严格控制抗菌药物的使用。     

高度多重耐药绿脓假单胞菌的分子流行病学调查

目的：明确本院烧伤病房不同患者中连续分离到的高度多重耐药绿脓假单胞菌是否由同一起源的菌株传播。方法：用浓度梯度法测定绿脓假单胞菌的最小抑菌浓度,通过重复序列引物聚合酶链反应（rep-PCR),用肠杆菌科基因重复序列和噬菌体重复序两种引物分别对绿脓假单胞菌进行基因分型。结果：抗生素敏感试验显示23株绿脓假单胞菌中13株对12种抗菌药物均耐药,6株仅对哌拉西林／他唑巴坦敏感,对头孢哌酮／舒巴坦中介,而对其他10种抗菌药物高度耐药,另4株为敏感菌（耐药的抗菌药物≤4种）。多重耐药菌株的rep-PCR产物经琼脂糖电泳分析,其基因型完全相同,并与敏感株之间有明显区别。结论：烧伤病房高度多重耐药绿脓假单菌的流行是由同一克隆菌株传播所致。     

铜绿假单胞菌162株耐药性分析

目的了解铜绿假单胞菌对各类抗生素的耐药性，为临床医生选择用药提供依据。方法采用来自各种临床标本分离所得的162株铜绿假单胞菌。用K-B纸片法检测该菌对9种药物的耐药性。结果162株铜绿假单胞菌对9种抗生素的耐药性从高到低依次为：哌啦西林38．9％、左旋氧氟沙星38．3％、哌啦西林／他唑巴坦35．2％、头孢吡肟35．2％、头孢他啶34．6％、奈米卡星34．6％、阿米卡星24．1％、亚胺培南30．9％和头孢哌酮／舒巴坦33．3％。结论铜绿假单胞菌对阿米卡星、亚胺培南和头孢哌酮／他唑巴坦的耐药性相对较低，对哌啦西林的耐药性最高。     

五地区铜绿假单胞菌PFGE型分析

目的：寻找铜绿假单胞菌流行优势克隆民气候、地域流行病学相关性,观察医院感染的流行趋势与流行特点,指导医院感染的预防与监控。方法：对全国5个地区随机选取的30株铜绿假单胞菌进行脉冲电场凝胶电泳分析,电泳图谱经数据转化后进行聚类分析。结果：仅少数菌株可证实来源的同源性,在相似性85．0％和相似性50．0％水平没有明显的优势克隆存在。在相似性达33．3％时所有菌株可全部被聚为3类。结论：30株菌变异较大,未发现与地区季节相关的流行优势克隆。     

307株铜绿假单胞菌耐药性研究

目的检测本院2007年1月-2009年9月铜绿假单胞菌（PA）耐药性的变化趋势,为临床上耐药PA感染的治疗提供参考。方法采用生物梅里埃公司的API系统进行菌种鉴定,用纸片扩散法（K-B）进行药物敏感性试验,用WHONET5．3进行统计学分析。结果3年间PA对14种抗生素的平均耐药率分别为28．1％、34．0％、35．5％,呈逐年上升的趋势,其中亚胺培南耐药菌株79株。结论及时准确的细菌鉴定及药敏试验,能尽早发现铜绿假单胞菌的院内感染情况,为临床治疗提供重要的实验室依据。     

45株铜绿假单胞菌耐药性分析

目的：分析该院铜绿假单胞菌的临床分布和药敏情况,为临床用药提供参考。方法：按照《全国临床检验操作规程》培养鉴定临床标本中的铜绿假单胞菌,采用Kirby—Bauer纸片扩散法进行抗生素敏感性试验,应用LIS（Laboratory Information System）软件进行药敏情况分析。结果：45株铜绿假单胞菌临床主要分布情况：以呼吸道感染为主;抗生素耐药情况：氨苄青霉素、复方新诺明、头孢呋肟、头孢唑啉的耐药率均达达到100％,庆大霉素、头孢曲松、头孢他啶、氨曲南的耐药率分别为60％、46．7％、46．7％、35．6%,阿米卡星、哌拉西林／他唑巴坦、头孢吡肟、头孢派酮／舒巴坦的耐药率分别为22．2％、22．2％、20％、133％。左氧氟沙星、亚胺培南分别为4．4％和0％。结论：铜绿假单胞菌耐药情况比较严重,耐药率20％以下只有头孢派酮／舒巴坦、左氧氟沙星、亚胺培南。临床用药应根据药敏分析,合理使用抗生素。     

194株铜绿假单胞菌耐药性分析

目的了解铜绿假单胞菌的耐药性,为临床合理选择抗生素提供参考。方法回顾性分析2009年2月至2010年1月益阳市中心医院临床分离的铜绿假单胞菌的分布情况及耐药性。结果临床分离的194株铜绿假单胞菌痰标本分离率最高(90.7%);主要引起神经外科(36.5%)和神经内科(26.3%)以及呼吸内科(20.6%)患者的呼吸道感染;该菌对庆大霉素高度耐药(74.7%),对美洛培南、头孢哌酮/舒巴坦耐药率低,依次为2.1%、6.2%。结论铜绿假单胞菌对碳青霉烯类和β-内酰胺类/β-内酰胺酶抑制剂抗生素耐药率低,可作为对铜绿假单胞菌感染经验性治疗的首选药物。     

重症监护病房66株铜绿假单胞菌耐药性分析

铜绿假单胞菌(PAE)是常见的非发酵革兰阴性杆菌,属条件致病菌,是引起重症监护病房(ICU)医院感染最主要的革兰阴性杆菌。该种细菌极易形成细菌生物被膜,临床上许多难治性感染与此相关,约占医院感染的60%[1]。近年来,     

2011至2013年仁济医院神经外科病房分离的鲍曼不动杆菌的耐药性变迁与同源性分析

目的了解仁济医院神经外科病房分离的鲍曼不动杆菌的耐药性变迁和分子流行特征,分析菌株基因同源性,为临床治疗和医院感染控制提供依据。方法收集2011年7月至2013年6月仁济医院神经外科病房分离的鲍曼不动杆菌共87株,采用琼脂稀释法检测菌株对临床12种抗菌药物的最低抑菌浓度(MIC)。采用多位点序列分型(MLST)技术鉴定基因型别并进一步分析菌株流行特征。结果仁济医院神经外科病房分离的鲍曼不动杆菌对头孢菌素类、喹诺酮类、氨基糖苷类和碳青霉烯类抗菌药物普遍耐药,且耐药率有逐年上升趋势。87株鲍曼不动杆菌分为6个ST型,分别是ST-208(45株)、ST-191(15株)、ST-540(8株)、ST-195(2株)、ST-381(2株)和ST-368(1株),其中ST-208型是主要流行型别。除ST-381外,其余5种型别均属于CC92克隆群,具有基因同源性。结论仁济医院神经外科病房分离的鲍曼不动杆菌耐药率高且有逐年上升趋势,同时存在耐碳青霉烯类鲍曼不动杆菌的播散流行,需加强医院感染控制,防止耐药株传播。     

Mechanisms of beta-lactam resistance amongst Pseudomonas aeruginosa isolated in an Italian survey

The mechanisms of resistance to beta-lactam antibiotics in 325 isolates of Pseudomonas aeruginosa were examined. These isolates were selected because of their resistance to meropenem and imipenem (breakpoint, >4 mg/L), carbenicillin (>128 mg/L), ceftazidime (>8 mg/L), piperacillin and ticarcillin/clavulanate (>64 mg/L). The most frequent mechanism of resistance was beta-lactamase-independent, so called 'intrinsic resistance', which was found in 183 isolates and was probably due to impermeability and/or efflux mechanisms. beta-Lactamase-mediated resistance was demonstrated in 111 strains (11.1%). Derepression of Ambler Class C chromosomal beta-lactamase was detected in 64 isolates, most of which were resistant to ceftazidime and piperacillin but susceptible to meropenem, whereas secondary plasmid-encoded beta-lactamases were found in 34 isolates, all of them resistant to carboxypenicillins and ureidopenicillins and susceptible to carbapenems. Twelve strains showed more than one plasmid-encoded beta-lactamase plus derepression of chromosomal Class C enzyme. Resistance to carbapenems was independent of resistance to other beta-lactam antibiotics, indicating a different mechanism of resistance, probably due to the loss of the D2 porin. In total, 32 strains were resistant to carbapenems: 24 only to imipenem and eight to both imipenem and meropenem.     

南京地区铜绿假单胞菌的整合子流行性调查

目的调查南京地区铜绿假单胞菌的整合子流行情况,并分析整合子与铜绿假单胞菌耐药的相关性。方法收集98株南京地区临床分离的铜绿假单胞菌,K-B法测定其药敏情况,简并引物PCR法扩增整合子的整合酶基因,对阳性PCR产物采用HinfⅠ内切酶作限制片段长度多态性(RFLP)分析进行整合子分类。结果98株铜绿假单胞菌对哌拉西林等14种抗菌药的耐药率从14.3%到82.7%。40.8%(40/98)的铜绿假单胞菌中检出整合子;PCR-RFLP结果显示均为Ⅰ类整合子,未检出Ⅱ类和Ⅲ类整合子。结论Ⅰ类整合子较广泛地存在于南京地区铜绿假单胞菌中;整合子与铜绿假单胞菌的耐药和多重耐药具有相关性。     

Serotyping of Pseudomonas aeruginosa isolated from clinical specimens.

Serotyping of 168 clinical isolates by the slide agglutination test using TIBS (Toshiba Institute of Biological Science) serotyping sera, which were prepared from Homma's serotype strains at the request of Pseudomonas aeruginosa Serotype Committee in Japan, was performed and the results were compared with serotyping using IMSUT (Institute of Medical Science, University of Tokyo) serotyping sera by the tube agglutination test. When heat-killed antigen from clinical isolates were used, both sets of serotyping sera showed similar results and the slide agglutination test using TIBS serotyping sera could be substituted for the tube agglutination test using IMSUT sera. It was also discovered that serotyping by the slide agglutination test with TIBS serotyping sera could be carried out using live bacteria. However, in serotyping with live bacteria, there is some difficulty in observing the agglutination time. To solve this problem, we propose that positive agglutination should be determined within 60 sec. The authors also performed serotyping of the clinical isolates using Difco serotyping sera prepared by the Difco Company based on Liu's serotyping system, and the results were compared with those obtained with serotyping sera prepared with Homma's serotype strains. The results indicated that in serotyping of clinical isolates using Difco serotyping sera more cross reaction occurred than in serotyping with sera obtained from Homma's serotype strains.     

铜绿假单胞菌临床分离株耐药性监测

目的对临床分离的铜绿假单胞菌的分布情况及耐药特点进行统计分析,为临床合理选用抗菌药物及控制院内感染提供依据。方法采用纸片扩散法(K-B法)进行药敏检测,资料用WHONET5.3软件进行统计分析。结果共分离获得144株铜绿假单胞菌,主要分离自痰液、创面分泌物和脓液标本中;对临床常用抗菌药物敏感性较好的分别是环丙沙星(85.4%)、美洛培南(85.2%)、亚胺培南(83.2%)、头孢他啶(80.6%)、阿米卡星(80%)和哌拉西林/他唑巴坦(80%)。结论本院铜绿假单胞菌是临床呼吸系统及创面分泌物重要感染源,其对临床常用抗菌药物耐药性逐年缓慢上升;对氟喹诺酮类、碳青霉烯类药物及阿米卡星仍维持较高的敏感性。在严重铜绿假单胞菌感染患者的治疗中,β-内酰胺类抗假单胞菌药加氨基糖苷类仍是较好的联合用药组合。应加强对耐药菌株的监控,尤其是耐碳青霉烯铜绿假单胞菌的医院内感染,防止其流行扩散。     

Susceptibility of Pseudomonas aeruginosa strains isolated from hospitalized children

Pseudomonas aeruginosa strains, 1285 in total, were isolated from hospitalized children in the period 1981-1984. Serotypes 011 and 06 and nontypable strains were the most frequently identified. 44.6% of serotype 011 strains and between 12.4 and 37.7% of other serotypes were resistant to gentamicin, or carbenicillin, or both agents. The aminoglycosides with the highest activity against P. aeruginosa isolates were amikacin and netilmicin. Among the seven beta-lactam antibiotics tested the most active were cefsulodin, piperacillin and azlocillin.     

Genotypic analysis of Pseudomonas aeruginosa isolated from ocular infection

Pseudomonas aeruginosa is the causative pathogen of keratitis, conjunctivitis, and dacryocystitis. However little is known about their clinical epidemiology in Japan. In this study we investigated the genotypic characterization and serotype of P. aeruginosa isolates from ocular infections. Thirty-four clinical P. aeruginosa isolates were characterized according to infection type, the type III secretion system (TTSS), serotype, and multilocus sequence typing (MLST). We divided the isolates into four clinical infection types as follows: Contact lens (CL)-related keratitis (CL-keratitis; 15 isolates), non CL-related keratitis (non CL-keratitis; 8 isolates), conjunctivitis (7 isolates), and dacryocystitis (4 isolates). Regarding the TTSS classification and serotyping classification, no significant differences were found among the infection types. Two clusters (I, II) and three subclusters (A, B, C) were classified according to MLST. CL-keratitis isolates with exoU positivity were clustered in II-B, and conjunctivitis was clustered in cluster I. Some linkage was found between the genetic background and CL-keratitis or conjunctivitis.     

铜绿假单胞菌噬菌体的分离鉴定及其生物学特性的分析

目的分离鉴定铜绿假单胞菌噬菌体,并对其生物学特性进行研究。方法从居民生活用水下水道中分离出铜绿假单胞菌噬体,利用噬斑测定法纯化所分离的噬菌体,通过负染法电镜观察噬菌体形态及大小。观察并测定噬菌体一步生产曲线,提取噬菌体核酸并进行酶切电泳。结果利用21株铜绿假单胞菌为宿主菌,从下水道中分离出3株铜绿假单胞菌噬菌体,分别命名为PaP-c01、Pap-c02和Pap-c03。噬菌体Pap-c01能裂解6株铜绿假单胞菌,显示出较宽的噬菌谱,酶切电泳显示其基因组大小为32kb。生长曲线显示其感染宿主菌的潜伏期为30min,暴发时间为55min,裂解量为100。结论分离出的噬菌体具有较广的噬菌谱,潜伏期短,裂解量较大,为噬菌体作为生物制剂用于抗感染治疗打下了基础。     

Genotyping of Pseudomonas aeruginosa isolated from cockroaches and human urine

Molecular-epidemiological analysis of Pseudomonas aeruginosa isolated from cockroaches captured in hospitals and from patient urine was performed, employing randomly amplified polymorphic DNA (RAPD) analysis to investigate the usefulness of RAPD analysis. Four specific bands at positions of 993, 875, 521, and 402 bp were commonly detected using primer 272 in 16 of 45 cockroach-derived strains (35.6%), but not in 21 urine-derived strains. On analysis using primer 208, 4 specific bands at positions of 1,235, 1,138, 1,068, and 303 bp were commonly detected in 15 of the 45 cockroach-derived (33.3%) and 10 of the 21 patient urine-derived (47.6%) strains, in a total of 25 of 66 strains (37.8%). On cluster analysis, 12 (48.5%) and 16 (66.7%) clusters were grouped based on a homology of 89% or greater, using primer 272 and primer 208, respectively, showing that primer 208 was suitable for the confirmation of diversity. Seven patterns were clustered based on 100% homology using either primer, and 6 of these consisted of only cockroach-derived strains. In the individual groups with 100% homology, all strains in the group were isolated at an identical site during the same period. P. aeruginosa isolated from cockroaches showed diverse genotypes suggesting several sources of contamination, indicating the necessity for investigating infection control targeting cockroaches inhabiting hospitals.