Molecular characteristics of CTX-M β-lactamase-producing and quinolone-resistant Escherichia coli among deer in a popular tourist spot in Japan

Abstract

Introduction. Antimicrobial resistance (AMR) is a growing global concern. Clonal lineages of CTX-M β-lactamase-producing Escherichia coli (CTXE) and quinolone-resistant E. coli (QREC) were disseminated among the deer population in a famous tourist destination (Nara Park; NP) in Japan. Hypothesis/gap statement. The molecular characteristics of CTXE or QREC isolates, which could pose a threat to public health, have not been elucidated. Aim. This study aimed to characterize the genetic traits of CTXE and QREC isolates derived from NP deer and compare them with lineages prevalent worldwide. Methodology. Sixteen CTXE and three QREC isolates recovered from NP deer faeces between 2018 and 2020 were analysed using whole-genome sequencing (WGS). For endemic lineages, phylogenetic trees were constructed against the isolates registered in the EnteroBase database using the core genome SNP scheme. Results. The most prevalent lineage in NP deer was ST3580. Several pandemic lineages, such as sequence type (ST) 38, ST58 and ST117, were included. The QREC lineages prevalent among deer were designated as extra-intestinal pathogenic E. coli or uropathogenic E. coli (UPEC). Thirteen of the 24 antimicrobial resistance genes (ARGs) were considered high-risk ARG families. Chromosomal integration of bla _CTX-M-15 was observed in all plasmid-negative isolates. Phylogenetic analysis suggested relationships between NP isolates and isolates sourced from the environment or poultry. Conclusion. ST3580 has a high potential for clonal dissemination. Furthermore, multiple clinically relevant lineages of CTXE and QREC are endemic in NP deer; however, they could be less virulent than isolates belonging to the same lineages, which could cause severe infectious diseases. Further studies are required to investigate the relationship between chromosomal integration of plasmid-encoded genes and the stable propagation of AMR bacteria in wildlife and the environment.

Keywords: CTX-M β-lactamase; Escherichia coli; WGS; chromosomal insertion; quinolone-resistant; sika deer.

Fig. 1.. ARGs, VGs and plasmid replicon of CTXE and QREC isolates derived from deer in NP between July 2018 and July 2020. Black, red and blue cells represent the presence of each gene or plasmid replicon. Underlined and bolded ARGs are ranked as first and second high-risk ARGs, respectively [26].

Fig. 2.. Chromosomal insertion of bla_CTX-M-15 mediated by IS1380. (a) I52, I197 and E129 and (b) C165. In each isolate, the nucleotide sequence around bla_CTX-M-15 was homologous with Klebsiella pneumoniae strain F16KP0096 plasmid (GenBank accession No. CP052151) ((a) I52 contig 7, I197 contig 9 and E129 contig10, between 167 085 and 178 237 bp; (b) C165 contig33, between 21 557 and 23950; C165 coting 64, between 106 and 12,105). Downstream and upstream of insertion exhibited high similarity with (a) E. coli strain LH09-a chromosome (GenBank accession No. CP100544) and (b) E. coli strain EC0880B chromosome (GenBank accession No. OX460315). Yellow, red, green and blue arrows represent genes encoding IS or transposase, β-lactamase, antimicrobial resistance other than β-lactamase and other functions including hypothetical protein, respectively. The numerical numbers in pink-shaded areas indicate the identity score (%) between two sequences.

Fig. 3.. Maximum-likelihood tree based on cgSNP scheme compared with metadata from the EnteroBase database. (a) ST3580 O8:H12 and (b) ST117 O119:H4. GrapeTree clusters containing NP deer isolates were submitted for cgSNP analysis to construct a maximum likelihood tree based on RaxML (Figs S1 and S2, red circle). Each leaf was coloured according to source type and annotated using the EnteroBase sample barcode, which was highlighted in yellow in case of NP deer isolate.