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

Abstract

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 28094T, which was higher than that with A. viridans CCUG4311T (94.9%). The consistency of 16S rRNA sequence of strain AV208 was 100% with A. urinaeequi CCUG 28094T and 99.9% with A. viridans CCUG4311T, 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.

Keywords

• Average nucleotide identity
• Whole genome sequencing
• A. viridans
• A. urinaeequi
• Identification