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[采用多重PCR法对医院内多重耐药鲍曼不动杆菌分离株中的苯唑西林酶基因进行检测并评估其与重复序列PCR的克隆关系]

[Investigation of oxacillinase genes in nosocomial multidrug-resistant Acinetobacter baumannii isolates by multiplex PCR and evaluation of their clonal relationship with Rep-PCR].

作者信息

Sarı Berrin, Baran Irmak, Alaçam Sema, Mumcuoğlu İpek, Kurşun Şenol, Aksu Neriman

机构信息

Ankara Numune Training and Research Hospital, Medical Microbiology Clinic, Ankara, Turkey.

出版信息

Mikrobiyol Bul. 2015 Apr;49(2):249-58. doi: 10.5578/mb.8884.

Abstract

Acinetobacter baumannii is a major nosocomial pathogen which can cause infections with high morbidity and mortality in hospitalized patients. In recent years A.baumannii has become a serious clinical problem because of the development of resistance to many antibiotics, and especially to carbapenems. The aims of this study were to investigate the oxacillinase genes responsible for carbapenem resistance in multidrug resistant (MDR) A.baumannii strains and to evaluate the clonal relationship between these strains. A total of 62 MDR A.baumannii strains isolated from various clinical specimens (24 tracheal aspirate, 14 wound, 10 blood, 7 urine, 2 abscess, 2 sputum, 2 catheter tip, 1 pleural fluid) of hospitalized patients in intensive care units (n= 42) and other inpatient clinics (n= 20) between February-March 2012, were included in the study. Identification and antibiotic susceptibility of A.baumannii isolates were performed by Vitek-2 automated system (bioMérieux, France), and the identified bacteria were confirmed by Maldi Biotyper (Bruker Daltonics, Germany) system. Imipenem, meropenem, colistin and tigecycline were additionally tested by E-test strips (bioMérieux, France). The presence of carbapenemase-producing OXA genes (blaOXA-23-like, blaOXA-40-like, blaOXA-51-like and blaOXA-58-like) were detected by multiplex PCR (hyplex® CarbOxaID test system, Amplex Diagnostics, Germany) and the clonal relationship between isolates were investigated by rep-PCR method (DiversiLab, bioMérieux, France). In our study, all isolates were found resistant to ampicillin-sulbactam, piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, imipenem, meropenem, ciprofloxacin, levofloxacin and tetracycline, while the resistance rates for amikacin, gentamicin, trimethoprim-sulfamethoxazole, netilmicin and tigecycline were 88.7%, 88.7%, 82.3%, 43.5% and 27.4%, respectively. All A.baumannii isolates were susceptible to colistin. All of the strains were positive for blaOXA-23-like and blaOXA-51-like genes, while blaOXA-40-like and blaOXA-58-like genes were not detected in any of them. Simultaneous cultures from environmental samples collected from inpatient clinics in which MDR A.baumannii strains isolated were negative in terms of A.baumannii growth. In evaluation of clonal relationship between isolates, 48 strains (77.4%) showed greater than 95% similarity and formed a big cluster, named Cluster A. The remaining 14 isolates formed 3 small clusters (each had 2 isolates), named Cluster B, C and D, showing greater than 95% similarity. Majority of isolates (58.3%) in Cluster A were from patients in the surgical intensive care unit, and the first isolate from this cluster was also from a patient in the same unit. In our opinion, isolates from Cluster A may have spread to other clinics from surgical intensive care unit through transferred patients or medical and non-medical devices and equipment. Nosocomial MDR A.baumannii isolates in our hospital are highly resistant to antibiotics and all harboured blaOXA-23-like genes. The rep-PCR analysis of these isolates indicated that a large portion of A.baumannii strains were clonally closely related, and they probably from the same source and common ancestor, and separated shortly from each other. This data emphasizes that the choices of treatment are quite limited for inpatients, and the need for improvement of the infection control measures in our hospital.

摘要

鲍曼不动杆菌是一种主要的医院病原体,可导致住院患者发生感染,发病率和死亡率很高。近年来,由于对多种抗生素尤其是碳青霉烯类抗生素产生耐药性,鲍曼不动杆菌已成为一个严重的临床问题。本研究的目的是调查耐多药(MDR)鲍曼不动杆菌菌株中导致碳青霉烯耐药的氧青霉烷酶基因,并评估这些菌株之间的克隆关系。2012年2月至3月期间,从重症监护病房(n = 42)和其他住院科室(n = 20)的住院患者的各种临床标本(24份气管吸出物、14份伤口标本、10份血液标本、7份尿液标本、2份脓肿标本、2份痰液标本、2份导管尖端标本、1份胸腔积液标本)中分离出62株MDR鲍曼不动杆菌菌株,纳入本研究。鲍曼不动杆菌分离株的鉴定和药敏试验采用Vitek-2自动化系统(法国生物梅里埃公司)进行,鉴定出的细菌通过基质辅助激光解吸电离飞行时间质谱系统(德国布鲁克道尔顿公司)进行确认。亚胺培南、美罗培南、黏菌素和替加环素另外采用E-test试纸条(法国生物梅里埃公司)进行检测。通过多重PCR(hyplex® CarbOxaID检测系统,德国Amplex诊断公司)检测产碳青霉烯酶的OXA基因(blaOXA-23-like、blaOXA-40-like、blaOXA-51-like和blaOXA-58-like)的存在情况,并采用重复PCR方法(DiversiLab,法国生物梅里埃公司)研究分离株之间的克隆关系。在我们的研究中,所有分离株均对氨苄西林-舒巴坦、哌拉西林、哌拉西林-他唑巴坦、头孢他啶、头孢吡肟、亚胺培南、美罗培南、环丙沙星、左氧氟沙星和四环素耐药,而对阿米卡星、庆大霉素、复方磺胺甲恶唑、奈替米星和替加环素的耐药率分别为88.7%、88.7%、82.3%、43.5%和27.4%。所有鲍曼不动杆菌分离株均对黏菌素敏感。所有菌株的blaOXA-23-like和blaOXA-51-like基因均呈阳性,而blaOXA-40-like和blaOXA-58-like基因在所有菌株中均未检测到。从分离出MDR鲍曼不动杆菌菌株的住院科室采集的环境样本的同时培养物中,鲍曼不动杆菌生长呈阴性。在评估分离株之间的克隆关系时,48株(77.4%)显示相似度大于95%,形成一个大的聚类,命名为聚类A。其余14株分离株形成3个小聚类(每个聚类有2株分离株),命名为聚类B、C和D,相似度大于95%。聚类A中的大多数分离株(58.3%)来自外科重症监护病房的患者,该聚类中的第一株分离株也来自同一病房的患者。我们认为,聚类A中的分离株可能通过转诊患者或医疗和非医疗设备从外科重症监护病房传播到其他科室。我院医院获得性MDR鲍曼不动杆菌分离株对抗生素高度耐药,且均携带blaOXA-23-like基因。对这些分离株的重复PCR分析表明,很大一部分鲍曼不动杆菌菌株在克隆上密切相关,它们可能来自同一来源和共同祖先,且彼此分离时间较短。这些数据强调,住院患者的治疗选择非常有限,我院需要改进感染控制措施。

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