Characterisation of plasmid-mediated rmtB-1 in Enterobacteriaceae clinical isolates from São Paulo, Brazil

OBJECTIVES The emergence of 16S rRNA methyltranferases (16 RMTAses) has jeopardised the clinical use of aminoglycosides. RmtB is one of the most frequently reported in Gram-negatives worldwide. In this study, we aimed to estimate the frequency of 16S RMTAses encoding genes in Enterobacteriaceae isolated in a three-month period from a tertiary Brazilian hospital. METHODS All Gram-negatives classified as resistant to amikacin, gentamicin, and tobramycin by agar screening were selected for analysis. The presence of 16SRMTases encoding genes was verified by polymerase chain reaction (PCR). Antimicrobial susceptible profile was determined by broth microdilution. The genetic relationship among these isolates was accessed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Selected RmtB-producing isolates were characterised by whole genome sequencing (WGS) analysis. RESULTS Twenty-two of 1,052 (2.1%) Enterobacteriaceae were detected as producers of RmtB-1 [Klebsiella pneumoniae (n = 21) and Proteus mirabilis (n = 1)]. blaKPC-2 was identified among 20 RmtB-1-producing K. pneumoniae isolates that exhibited an identical PFGE and MLST (ST258) patterns. Two K. pneumoniae isolates, the A64216 (not harboring bla KPC-2), A64477 (harboring bla KPC-2) and one P. mirabilis isolate (A64421) were selected for WGS. rmtB-1 and bla KPC-2 genes were carried by distinct plasmids. While a plasmid belonging to the IncFIIk group harbored rmtB-1 in K. pneumoniae, this gene was carried by a non-typable plasmid in P. mirabilis. In the three analysed plasmids, rmtB-1 was inserted on a transposon, downstream a Tn2. CONCLUSION Our findings suggested that the rmtB-1 was harbored by plasmids distinct from those previously reported in Bolivia and China. It suggests that multiple mobilization events might have occurred in South America.

## 研究目的
16S核糖体RNA甲基转移酶（16S rRNA methyltransferases，缩写为16 RMTAses）的出现，严重威胁了氨基糖苷类抗生素的临床应用前景。RmtB是全球范围内革兰氏阴性菌（Gram-negatives）中最常被报道的16S RMTases类型之一。本研究旨在估算巴西某三级医院三个月内分离的肠杆菌科（Enterobacteriaceae）细菌中，携带16S RMTases编码基因的菌株检出率。

## 研究方法
本研究选取所有经琼脂筛选法判定对阿米卡星、庆大霉素及妥布霉素耐药的革兰氏阴性菌进行后续分析。通过聚合酶链反应（polymerase chain reaction，PCR）检测样本中16S RMTases编码基因的存在情况。采用肉汤微量稀释法测定菌株的抗菌药物敏感性谱。通过脉冲场凝胶电泳（pulsed field gel electrophoresis，PFGE）及多位点序列分型（multilocus sequence typing，MLST）分析上述分离株的遗传亲缘关系。选取产RmtB的分离株进行全基因组测序（whole genome sequencing，WGS）分析。

## 研究结果
在1052株肠杆菌科细菌中，共检出22株（2.1%）产RmtB-1的菌株，其中肺炎克雷伯菌（Klebsiella pneumoniae，n=21）21株，奇异变形杆菌（Proteus mirabilis，n=1）1株。在20株产RmtB-1的肺炎克雷伯菌中检出blaKPC-2基因，这些菌株具有完全一致的PFGE及MLST（ST258）型别。选取2株肺炎克雷伯菌（A64216，未携带blaKPC-2；A64477，携带blaKPC-2）及1株奇异变形杆菌（A64421）进行全基因组测序分析。rmtB-1与blaKPC-2基因分别位于不同的质粒上。在肺炎克雷伯菌中，rmtB-1由IncFIIk型质粒携带；而在奇异变形杆菌中，该基因由不可分型质粒承载。在本次分析的3个质粒中，rmtB-1均插入于转座子（transposon）内，位于Tn2转座子的下游区域。

## 研究结论
本研究结果显示，携带rmtB-1的质粒类型与此前玻利维亚及中国报道的质粒存在差异。这提示南美洲可能曾发生过多次rmtB-1的移动传播事件。