Characterization of integrative and conjugative elements carrying erm(B) and tet(O) resistance determinants in streptococcus uberis isolates from bovine milk in Chiba prefecture, Japan: CompArative GEne cluster analysis toolbox with ICEfinder

Abstract

Objective: We aimed to characterize integrative and conjugative elements (ICEs) in antimicrobial resistant Streptococcs uberis isolates from bovine milk in Chiba, Japan, based on whole-genome sequence (WGS) data.

Results: Of the 101 isolates, we found the 36 isolates harboring erm(B)-tet(O), showing resistance to macrolides-lincosamides-tetracyclines. The 22 isolates were randomly selected and subject to WGS determination. The genomes measured 1.991-2.517 Mbp, with G + C contents of 35.8-36.9%. We used ResFinder-ICEfinder (web-based applications) to search for the antimicrobial resistant genes and ICEs. ResFinder detected combined erm(B)-tet(O)-ant(6)-Ia at the identical contig in each WGS. ICEfinder detected ICEs belonging to the same contigs, which contained erm(B)-tet(O)-ant(6)-Ia complete or partial sequences. Detection of putative ICEs using comparative genomic analysis was performed with identification of other streptococcal ICE resembling S. uberis ICEs. Using comparative genomic analysis (a reference WGS in NZ01 strain), putative ICE base size in UB37 isolate was 77,386-bp that was identical in other 13 isolates. Another similar streptococcal ICE was S. suis ICEnsui78-tet(O)-erm(B) mobile element. For ICE characterization in S. uberis with WGSs, a comparative genomic analysis is required with use of ICEfinder and other annotation tools.

Keywords: Erm(B); Streptococcus uberis; Tet(O); CompArative GEne cluster analysis Toolbox; ICEfinder; Integrative and conjugative element.

Fig. 1

Detection approach of putative Integrative and conjugative elements (ICEs) using comparative genomic analysis. As a reference, we applied a complete genome sequence (nucleotide accession no. CP022435.1) in bovine NZ01 strain from New Zealand in 2014: UB37 isolate was chosen. Similar/identical product comparisons between reference and candidate WGSs are shown in the same colors. Black gradations in interspace among the corresponding genes indicate percent identity. NZ01 and UB37 WGSs possessed identical flanking products of 8-oxo-dGTP diphosphatase and RlmD [23 S rRNA (uracil(1939)-C(5))-methyltransferase]. Putative ICE base size was 77,386-bp. Product organization in ICE was determined. The core products are shown in red letters; the antimicrobial resistance products are shown in blue letters. We performed the re-annotation for ICEUB37 using a Prokaryotic Genomes Annotation Pipeline. To specify ICE components, ICEscreen was applied. To speculate protein function similarities, we used basic local alignment search tool x; the protein functions are shown in brackets

Fig. 2

Putative identification of other streptococcal Integrative and conjugative element (ICE) resembling S. uberis ICEs (UB37/UB68/UB23/UB99), with S. uberis NZ01 WGS. Similar/identical product comparisons between reference and candidate WGSs are shown in the same colors. Black gradations in interspace among the corresponding genes indicate percent identity. Another similar streptococcal ICE was Streptococcus suis strain STC78 ICEnsui78–tet(O)–erm(B) mobile element (nucleotide accession no. ON944185.1), based on the similar product arrangements between UB37/UB68/UB23/UB99 ICEs and ICEnsui78. The core products are shown in red letters; the antimicrobial resistance products are shown in blue letters. We performed the re-annotation for ICEUB37 using a Prokaryotic Genomes Annotation Pipeline. To specify ICE components, ICEscreen was applied. To speculate protein function similarities, we used basic local alignment search tool x; the protein functions are shown in brackets