[从临床样本中分离的耐碳青霉烯类铜绿假单胞菌分离株抗生素耐药性的分子分析]
[Molecular Analysis of Antibiotic Resistance in CarbapenemResistant Pseudomonas aeruginosa Isolates Isolated from Clinical Samples].
作者信息
Altan Gülşah, Rakici Erva, Özgümüş Osman Birol
机构信息
Kocaeli University Faculty of Medicine, Department of Medical Microbiology, Kocaeli, Türkiye.
Recep Tayyip Erdogan University Faculty of Medicine, Department of Medical Microbiology, Rize, Türkiye.
出版信息
Mikrobiyol Bul. 2024 Apr;58(2):148-170. doi: 10.5578/mb.202498200.
Pseudomonas aeruginosa is an opportunistic pathogen that causes increased morbidity and mortality in risky patient groups. Nowadays, carbapenem resistance has become a threat and resistance genes are spreading among species through mobile genetic elements. The dissemination of carbapenemases among P.aeruginosa is a serious public health concern due to its limited options for the treatment of bacterial infections. In this study, it was aimed to investigate the molecular epidemiology of 47 carbapenem resistant P.aeruginosa (CRPA) isolates derived from various clinical samples from the Central Laboratory Bacteriology Unit of Kocaeli University Research and Training Hospital between October 2021 and March 2023. The rates of resistance to the antibiotics, some carbapenemase and virulence genes, conjugative resistance plasmids, integron gene cassette contents and the clonal similarity of the isolates were investigated and then epidemiologically evaluated. In the study, identification of the bacterial isolates and their susceptibility to some antibiotics (imipenem, meropenem, aztreonam, amikacin, netilmicin, tobramycin, piperacillin, piperacillin/tazobactam, ceftazidime, cefepime, ciprofloxacin and levofloxacin) were determined by the VITEK® 2 Compact automated system. Metallo-beta-lactamase (MBL) production of the isolates was demonstrated by the imipenem/meropenem-EDTA (IMP/MEM-EDTA) combined disc method. Conjugation experiments were performed by the broth mating method. Alkali lysis method was used in plasmid DNA isolations. Co-transferred antibiotic resistances in transconjugants were detected by disc diffusion method. Carbapenemase genes (blaIMP , blaVIM , blaNDM , blaKPC and blaOXA-48 ), integron gene cassettes (class 1 and class 2) and virulence genes (lasR and rhlR) were screened by specific polymerase chain reactions (PCRs). Clonal relationships of the CRPA isolates were investigated by evaluating the DNA f ingerprintings obtained from the ERIC (enterobacterial repetitive intergenic consensus)-PCR assay. The highest resistance rate of the isolates were to levofloxacin, while the lowest resistance rates were observed against tobramycin, gentamicin and amikacin. MBL production was detected in 25 (53.2%) isolates. In conjugation experiments, 12 (25.5%) isolates were detected to harbour conjugative resistance plasmids. In 90% of the CRPA isolates, lasR and rhlR biofilm genes (encoding for the transcriptional activator protein) were detected by PCR. The blaVIM gene was detected in six (12.8%) isolates. The blaNDM gene was detected in five (10.6%) isolates and the blaOXA-48 gene was detected in three (6.4%) isolates. The blaKPC and blaIMP genes were not detected in CRPA isolates. It was determined that two (16.6%) of the isolates that carried the blaVIM gene, one (8.3%) carried the blaNDM gene and one (8.3%) carried the blaOXA-48 gene contained conjugative plasmids.In integron-specific PCRs, intI1 gene was positive in 39 (82.9%) isolates, while class 1 integron gene cassettes were detected in 24 isolates (51%). IntI1 positive six isolates were found to harbour class 1 integron gene cassettes-bearing conjugative plasmids. Class 2 integrons were not found in the CRPA isolates. Dendrogram analysis of ERIC-PCR patterns showed that there was no clonal similarity between the CRPA isolates and the isolates did not spread by cross-contamination. As a result, it has been observed that most of the CRPA isolates which have the potential to form biofilms, are highly resistant to other antibiotic groups other than carbapenems and can co-transfer some resistances (ceftazidime, cefepime, ciprofloxacin, levofloxacin, piperacillin-tazobactam) with conjugative resistance plasmids. It is thought that it would be useful to follow molecular epidemiology in the resistance gene reservoirs of these strains which have the potential to cause epidemics in the clinical arena.
铜绿假单胞菌是一种机会致病菌,会增加高危患者群体的发病率和死亡率。如今,碳青霉烯耐药性已成为一种威胁,耐药基因正通过移动遗传元件在物种间传播。碳青霉烯酶在铜绿假单胞菌中的传播是一个严重的公共卫生问题,因为治疗细菌感染的选择有限。在本研究中,旨在调查2021年10月至2023年3月间从科贾埃利大学研究与培训医院中央实验室细菌学单元的各种临床样本中分离出的47株耐碳青霉烯铜绿假单胞菌(CRPA)的分子流行病学。研究了这些分离株对多种抗生素的耐药率、一些碳青霉烯酶和毒力基因、接合耐药质粒、整合子基因盒内容物以及分离株的克隆相似性,然后进行了流行病学评估。在该研究中,通过VITEK® 2 Compact自动化系统确定细菌分离株的鉴定及其对一些抗生素(亚胺培南、美罗培南、氨曲南、阿米卡星、奈替米星、妥布霉素、哌拉西林、哌拉西林/他唑巴坦、头孢他啶、头孢吡肟、环丙沙星和左氧氟沙星)的敏感性。通过亚胺培南/美罗培南-EDTA(IMP/MEM-EDTA)联合纸片法证明分离株产生金属β-内酰胺酶(MBL)。通过肉汤交配法进行接合实验。采用碱裂解法进行质粒DNA分离。通过纸片扩散法检测转接合子中共同转移的抗生素耐药性。通过特异性聚合酶链反应(PCR)筛选碳青霉烯酶基因(blaIMP、blaVIM、blaNDM、blaKPC和blaOXA-48)、整合子基因盒(1类和2类)和毒力基因(lasR和rhlR)。通过评估从ERIC(肠杆菌重复基因间共有序列)-PCR分析获得的DNA指纹图谱来研究CRPA分离株的克隆关系。分离株的最高耐药率是对左氧氟沙星,而对妥布霉素、庆大霉素和阿米卡星的耐药率最低。在25株(53.2%)分离株中检测到MBL产生。在接合实验中,检测到12株(25.5%)分离株携带接合耐药质粒。在90%的CRPA分离株中,通过PCR检测到lasR和rhlR生物膜基因(编码转录激活蛋白)。在6株(12.8%)分离株中检测到blaVIM基因。在5株(10.6%)分离株中检测到blaNDM基因,在3株(6.4%)分离株中检测到blaOXA-48基因。在CRPA分离株中未检测到blaKPC和blaIMP基因。确定携带blaVIM基因的分离株中有2株(16.6%)、携带blaNDM基因的1株(8.3%)和携带blaOXA-48基因的1株(8.3%)含有接合质粒。在整合子特异性PCR中,intI1基因在39株(82.9%)分离株中呈阳性,而在24株(51%)分离株中检测到1类整合子基因盒。发现6株intI1阳性分离株携带带有1类整合子基因盒的接合质粒。在CRPA分离株中未发现2类整合子。ERIC-PCR图谱的聚类分析表明,CRPA分离株之间没有克隆相似性,且分离株不是通过交叉污染传播的。结果发现,大多数有形成生物膜潜力的CRPA分离株对碳青霉烯类以外的其他抗生素组具有高度耐药性,并且可以通过接合耐药质粒共同转移一些耐药性(头孢他啶、头孢吡肟、环丙沙星、左氧氟沙星、哌拉西林-他唑巴坦)。认为在这些有可能在临床领域引起流行的菌株的耐药基因库中跟踪分子流行病学是有用的。