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通过全基因组测序和平均核苷酸同一性分析鉴定尿微球菌分离株。

Identification of an Aerococcus urinaeequi isolate by whole genome sequencing and average nucleotide identity analysis.

机构信息

Department of Laboratory Medicine, Nanjing Drum Tower Hospital, Affiliated Hospital of Nanjing University Medical School, Nanjing, Jiangsu Province, PR China.

Department of Laboratory Medicine, Nanjing Drum Tower Hospital, Affiliated Hospital of Nanjing University Medical School, Nanjing, Jiangsu Province, PR China.

出版信息

J Glob Antimicrob Resist. 2022 Jun;29:353-359. doi: 10.1016/j.jgar.2022.04.013. Epub 2022 Apr 25.

DOI:10.1016/j.jgar.2022.04.013
PMID:35477007
Abstract

OBJECTIVES

Identification and classification of microorganisms is one of the most important but difficult and challenging issues in microbiology. Whole genome sequencing (WGS), which can give a thorough understanding for the genome of bacteria strain, has been universally used for studying bacterial classification, evolution, and drug-related resistant genes. We in this study aimed to identify a Gram-positive, microaerophilic, catalase-negative cocci strain named AV208, which has shown resistance to vancomycin, by whole genome's average nucleotide identity (ANI) and high-throughput sequencing technology.

METHODS

The AV208 strain was identified by following commercially available identification systems, including API 20 Strep system and Vitek 2 Compact Gram-positive identification system for biochemical phenotypic test. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and 16S rRNA gene sequencing were used for confirmation identification. The whole genome of AV208 was sequenced by using high throughput sequencing technology and ANI between AV208, and its phylogenetic neighbours were analysed by the Orthologous Average Nucleotide Identity Tool (OAT) software. Polymerase chain reaction (PCR) and DNA sequencing were used to investigate the potential molecular mechanism for vancomycin resistance.

RESULTS

The AV208 strain was isolated from an ascites sample from a patient with chronic kidney disease who showed extensive resistance to the drugs detected, such as vancomycin with MIC >256 μg/mL. With combination of biochemical phenotypic test, MALDI-TOF-MS and 16S rRNA gene sequencing, the AV208 strain was tentatively identified as an Aercoccus viridans. By using complete genome sequence, we found a 96.24% ANI between strain AV208 and Aerococcus urinaeequi CCUG 28094, which was higher than that with A. viridans CCUG4311 (94.9%). The consistency of 16S rRNA sequence of strain AV208 was 100% with A. urinaeequi CCUG 28094 and 99.9% with A. viridans CCUG4311, with only one base difference between them. PCR and sequencing for van genes revealed that AV208 was positive for the vanA gene. A Tn1546 transposon-like structure with vanA gene was found in the genome, which was predicted locating in plasmid, causing vancomycin resistance phenotypes.

CONCLUSION

Average nucleotide identity analysis based on whole genome sequence is an accurate and effective method for identification of bacteria, especially for strains that are not discernible by existing methods such as Aerococcus.

摘要

目的

微生物的鉴定和分类是微生物学中最重要但也是最困难和具有挑战性的问题之一。全基因组测序(WGS)可以深入了解细菌株的基因组,已被广泛用于研究细菌分类、进化和与药物相关的耐药基因。我们在这项研究中,旨在通过全基因组平均核苷酸同一性(ANI)和高通量测序技术,鉴定一株对万古霉素耐药的革兰阳性、微需氧、过氧化氢酶阴性球菌菌株 AV208。

方法

采用市售鉴定系统,包括 API 20 Strep 系统和 Vitek 2 Compact 革兰阳性鉴定系统进行生化表型试验,对 AV208 菌株进行鉴定。基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)和 16S rRNA 基因测序用于确认鉴定。采用高通量测序技术对 AV208 全基因组进行测序,并利用 Orthologous Average Nucleotide Identity Tool(OAT)软件分析 AV208 及其系统发育上的近缘种的 ANI。聚合酶链反应(PCR)和 DNA 测序用于研究万古霉素耐药的潜在分子机制。

结果

AV208 株从一名患有慢性肾病的患者的腹水样本中分离得到,该患者对检测到的多种药物表现出广泛耐药,例如万古霉素的 MIC 值>256μg/ml。结合生化表型试验、MALDI-TOF-MS 和 16S rRNA 基因测序,AV208 株被初步鉴定为鸟肠球菌。通过使用全基因组序列,我们发现菌株 AV208 与解鸟氨酸阿克曼菌 CCUG 28094 之间的 ANI 为 96.24%,高于与鸟肠球菌 CCUG4311 的 ANI(94.9%)。菌株 AV208 的 16S rRNA 序列与解鸟氨酸阿克曼菌 CCUG 28094 的一致性为 100%,与鸟肠球菌 CCUG4311 的一致性为 99.9%,两者仅相差一个碱基。对 van 基因进行 PCR 和测序显示,AV208 对 vanA 基因呈阳性。在基因组中发现了一个携带 vanA 基因的 Tn1546 转座子样结构,这导致了万古霉素耐药表型。

结论

基于全基因组序列的平均核苷酸同一性分析是一种准确有效的细菌鉴定方法,特别是对于那些无法通过现有方法(如阿克曼菌)识别的菌株。

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