Kahraman Elmas Pınar, Toptan Hande, Otlu Barış, Köroğlu Mehmet, Altındiş Mustafa
Sakarya University Faculty of Medicine, Department of Medical Microbiology, Sakarya, Turkey.
Inonu University Faculty of Medicine, Department of Medical Microbiology, Malatya, Turkey.
Mikrobiyol Bul. 2019 Apr;53(2):134-143. doi: 10.5578/mb.67914.
The emergence and spread of multi-drug-resistant (MDR), extended-spectrum beta-lactamase (ESBL) producing carbapenem-resistant members of Enterobacteriaceae family has become a worldwide health problem. Carbapenem resistance caused by blaKPC, blaNDM gene regions are sporadic and blaOXA-48 gene region is endemic in our country. The aim of this study was to determine the presence of blaOXA-232, blaOXA-181, blaOXA-162, blaOXA-204, blaOXA-244, blaOXA-163, blaOXA-245 genes in OXA-48 like carbapenemase producing Klebsiella pneumoniae isolates. The isolates used in this study were provided from the Medical Microbiology Laboratory collection of Sakarya University Sakarya Training and Research Hospital. Identification and antibiotic susceptibility tests were determined by the VITEK 2® automated system (biomerieux, France) and the carbapenemase production of isolates was determined by the modified Hodge test. Minimal inhibitor concentration (MIC) values were determined with broth microdilution method. The isolates containing the blaOXA-48-like gene region were identified by real-time polymerase chain reaction (Rt-PCR) method using consensus primers. In "High Resolution Melting Analysis (HRMA)" method carried out by using "Type-it HRM PCR" (Qiagen, Hilden, Germany) kit, isolates which showed a deviation in melting temperatures (Tm) were selected with the suspicion of OXA-48 variant. The sequence analysis (ABI 3500, Applied Biosystems, USA) was carried out to determine which variants were present in these isolates. Compatibility of MIC values was determined between VITEK 2® and the microdilution method with the rate of 82% for imipenem, 77% for meropenem and 90% for ertapenem in carbapenemase-producing K.pneumoniae isolates. In 45 of 100 K.pneumoniae isolates, the blaOXA-48-like gene region was found to be positive by the Rt-PCR method. For the determination of OXA-48 variants, these 45 isolates were evaluated by HRMA method. The sequence analysis revealed that 41 (91.2%) isolates contained blaOXA-48/blaOXA-245 gene regions, while 2 (4.4%) isolates were found to contain blaOXA-181 gene regions and 2 (4.4%) isolates were found to contain blaOXA-244 gene regions. This is the first study to determine OXA-48 and OXA-244 positivity in blaOXA-48-like gene regions in Turkey. As a result of this study, the OXA-48-like gene region was found to be 45%, of which 4.4% had blaOXA-181 and 4.4% had blaOXA-244 gene regions. The detection of blaOXA-48-like gene regions will guide for the selection of antibiotics in critical patient groups.
肠杆菌科中产生超广谱β-内酰胺酶（ESBL）的耐多药（MDR）碳青霉烯类耐药菌的出现和传播已成为一个全球性的健康问题。由blaKPC、blaNDM基因区域引起的碳青霉烯类耐药较为零散，而blaOXA - 48基因区域在我国呈地方性流行。本研究的目的是确定产OXA - 48型碳青霉烯酶的肺炎克雷伯菌分离株中blaOXA - 232、blaOXA - 181、blaOXA - 162、blaOXA - 204、blaOXA - 244、blaOXA - 163、blaOXA - 245基因的存在情况。本研究中使用的分离株来自萨卡里亚大学萨卡里亚培训和研究医院的医学微生物实验室收藏。通过VITEK 2®自动化系统（法国生物梅里埃公司）进行鉴定和抗生素敏感性试验，通过改良 Hodge试验确定分离株的碳青霉烯酶产生情况。采用肉汤微量稀释法测定最低抑菌浓度（MIC）值。使用通用引物通过实时聚合酶链反应（Rt - PCR）方法鉴定含有blaOXA - 48样基因区域的分离株。在使用“Type - it HRM PCR”（德国希尔德市Qiagen公司）试剂盒进行的“高分辨率熔解分析（HRMA）”方法中，选择熔解温度（Tm）出现偏差的分离株怀疑为OXA - 48变体。进行序列分析（美国应用生物系统公司ABI 3500）以确定这些分离株中存在哪些变体。在产碳青霉烯酶的肺炎克雷伯菌分离株中，VITEK 2®和微量稀释法之间的MIC值兼容性确定为：亚胺培南为82%，美罗培南为77%，厄他培南为90%。在100株肺炎克雷伯菌分离株中，有45株通过Rt - PCR方法检测到blaOXA - 48样基因区域呈阳性。为了确定OXA - 48变体，采用HRMA方法对这45株分离株进行评估。序列分析显示，41株（91.2%）分离株含有blaOXA - 48/blaOXA - 245基因区域，2株（4.4%）分离株含有blaOXA - 181基因区域，2株（4.4%）分离株含有blaOXA - 244基因区域。这是土耳其首次确定blaOXA - 48样基因区域中OXA - 48和OXA - 244阳性的研究。本研究结果显示，blaOXA - 48样基因区域为45%，其中4.4%含有blaOXA - 181基因区域，4.4%含有blaOXA - 244基因区域。blaOXA - 48样基因区域的检测将为重症患者群体的抗生素选择提供指导。
