We evaluated the distribution of carbapenem and colistin resistance mechanisms of clinical E. coli and K. pneumoniae isolates from Iran. 165 non-duplicate non-consecutive isolates of K. pneumoniae and E. coli were collected from hospitalized patients admitted to Iran's tertiary care hospitals from September 2016 to August 2018. The isolates were cultured from different clinical specimens, including wound, urine, blood, and tracheal aspirates. Antibiotic susceptibility testing was performed by disc diffusion and microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guideline. The presence of extended spectrum β-lactamases (ESBLs) genes, carbapenemase genes, as well as fosfomycin resistance genes, and colistin resistance genes was also examined by PCR-sequencing. The ability of biofilm formation was assessed with crystal violet staining method. The expression of colistin resistance genes were measured by quantitative reverse transcription-PCR (RT-qPCR) analysis to evaluate the association between gene upregulation and colistin resistance. Genotyping was performed using the multi-locus sequencing typing (MLST). Colistin and tigecycline were the most effective antimicrobial agents with 90.3% and 82.4% susceptibility. Notably, 16 (9.7%) isolates showed resistance to colistin. Overall, 33 (20%), 31 (18.8%), and 95 (57.6%) isolates were categorized as strong, moderate, and weak biofilm-producer, respectively. Additionally, blaTEM, blaSHV, blaCTX-M, blaNDM-1, blaOXA-48-like and blaNDM-6 resistance genes were detected in 98 (59.4%), 54 (32.7%), 77 (46.7%), 3 (1.8%), 17 (10.30%) and 3 (1.8%) isolates, respectively. Inactivation of mgrB gene due to nonsense mutations and insertion of IS elements was observed in 6 colistin resistant isolates. Colistin resistance was found to be linked to upregulation of pmrA-C, pmrK, phoP, and phoQ genes. Three of blaNDM-1 and 3 of blaNDM-6 variants were found to be carried by IncL/M and IncF plasmid, respectively. MLST revealed that blaNDM positive isolates were clonally related and belonged to three distinct clonal complexes, including ST147, ST15 and ST3299. The large-scale surveillance and effective infection control measures are also urgently needed to prevent the outbreak of diverse carbapenem- and colistin-resistant isolates in the future.

中文翻译：

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从伊朗德黑兰住院患者中分离出的耐药大肠杆菌和肺炎克雷伯菌菌株的分子特征

我们评估了来自伊朗的临床大肠杆菌和肺炎克雷伯菌分离株的碳青霉烯类和粘菌素耐药机制的分布。从2016年9月至2018年8月入住伊朗三级医院的住院患者中收集了165株非重复、非连续的肺炎克雷伯菌和大肠杆菌分离株。这些分离株是从不同的临床标本中培养的，包括伤口、尿液、血液、和气管抽吸物。根据临床和实验室标准研究所（CLSI）指南，通过纸片扩散和微量稀释法进行抗生素敏感性测试。还通过 PCR 测序检查了超广谱 β-内酰胺酶 (ESBL) 基因、碳青霉烯酶基因以及磷霉素抗性基因和粘菌素抗性基因的存在。采用结晶紫染色法评估生物膜形成能力。通过定量逆转录 PCR (RT-qPCR) 分析测量粘菌素抗性基因的表达，以评估基因上调与粘菌素抗性之间的关联。使用多位点测序分型（MLST）进行基因分型。粘菌素和替加环素是最有效的抗菌药物，敏感性分别为 90.3% 和 82.4%。值得注意的是，16 个（9.7%）分离株表现出对粘菌素的耐药性。总体而言，33 (20%)、31 (18.8%) 和 95 (57.6%) 分离株分别被归类为强、中和弱生物膜产生者。此外，在 98 例 (59.4%)、54 例 (32.7%)、77 例 (46.7%)、3 例 (1.8%) 中检测到 blaTEM、blaSHV、blaCTX-M、blaNDM-1、blaOXA-48-like 和 blaNDM-6 抗性基因。 ），分别有 17 个（10.30%）和 3 个（1.8%）分离株。在 6 个粘菌素抗性分离株中观察到 mgrB 基因因无义突变和 IS 元件插入而失活。研究发现粘菌素耐药性与 pmrA-C、pmrK、phoP 和 phoQ 基因的上调有关。发现 3 个 blaNDM-1 和 3 个 blaNDM-6 变体分别由 IncL/M 和 IncF 质粒携带。MLST 显示 blaNDM 阳性分离株具有克隆相关性，属于三个不同的克隆复合体，包括 ST147、ST15 和 ST3299。还迫切需要大规模监测和有效的感染控制措施，以防止未来出现多种碳青霉烯类和粘菌素耐药菌株的爆发。