Isolation of a carbapenemase producing Klebisiella in the NUH hospital. OXA-48 Klebsiella pneumoniae

A K. pneuomoniae was isolated from rectal swabs taken to screen for multi-drug resistant Enterobacteriaceae carriage in a Libyan patient transferred to the Queens Medical Centre campus of Nottingham University Hospitals NHS Trust for treatment on severe trauma injuries. Methods: Screening was performed on rectal swabs plated onto MacConkey agar plates with a 10mg ertapenem disc. Antibiotic breakpoints and the modified Hodge test were performed on the suspect colony. Molecular testing was performed using two in-house multiplex PCRs to detect the presence of the most commonly encountered carbapenemase genes. The genome sequence of the isolate was obtained using the Illumina HiSeq platform, assembled and annotated. The antimicrobial resistance gene pool of the organism was determined using the CARD database, and plasmids identified using the PlasmidFinder programme. Results: The K. pneuomonaie isolate was phenotypically confirmed as a carbapenemase producer with resistance to meropenem (>32 µg/ml), piptazobactam (>256 µg/ml), trimethoprim, and co-amoxiclav. The genome sequence identified the isolate as belonging to the globally disseminated ST147 highly-pathogenic, multi-drug resistant lineage. The presence of at least 4 plasmids was determined including pOXA-48. The isolate contained approximately 0.5Mbp of extra-chromosomal DNA and 68 ORFs associated with antimicrobial resistance including Shv-11 and Oxa-30. Conclusion: This is the first report of the characterisation of a K. pneumonia ST147 Oxa-48 producing isolate in the United Kingdom. The study highlights the impressive array of antimicrobial associated genetic elements acquired by this important globally emerging organism, and provides a reference point for comparison of further UK isolates.

本研究从一名因重症创伤转诊至英国诺丁汉大学医院NHS信托基金会女王医疗中心接受治疗的利比亚患者的直肠拭子中，分离得到一株肺炎克雷伯菌（Klebsiella pneumoniae，原文存在笔误，写为K. pneuomoniae），该直肠拭子用于筛查多重耐药肠杆菌科细菌定植情况。

方法：将直肠拭子接种于添加10mg厄他培南药敏纸片的麦康凯琼脂平板进行筛查。对可疑菌落开展抗生素折点检测与改良霍奇试验。采用2种室内自建多重聚合酶链反应（multiplex PCR）检测临床最常见的碳青霉烯酶基因。使用Illumina HiSeq测序平台获取该分离株的全基因组序列，并完成序列组装与注释。通过综合抗生素抗性基因数据库（Comprehensive Antibiotic Resistance Database，CARD）分析该菌株的抗菌药物抗性基因谱，利用PlasmidFinder工具鉴定其携带的质粒。

结果：经表型验证，该分离株为碳青霉烯酶产生菌（原文存在笔误，写为K. pneuomonaie），对美罗培南（>32 μg/ml）、哌拉西林他唑巴坦（原文笔误为piptazobactam，>256 μg/ml）、甲氧苄啶及阿莫西林克拉维酸钾均耐药。全基因组序列分析显示，该分离株属于全球流行的高致病性多重耐药序列型147（Sequence Type 147，ST147）克隆谱系。鉴定出该菌株至少携带4种质粒，其中包含pOXA-48质粒。该分离株携带约0.5 Mbp的染色体外DNA，以及68个与抗菌药物耐药相关的开放阅读框（open reading frames，ORFs），包括Shv-11与Oxa-30基因。

结论：本研究为英国境内首次报道一株携带Oxa-48基因的ST147型肺炎克雷伯菌的特征分析。本研究揭示了这一全球新兴的重要耐药菌所获得的多样抗菌药物耐药相关遗传元件，并为后续英国境内分离株的对比分析提供了参考基准。