# Characterization of extended-spectrum cephalosporin-resistant Klebsiella recovered from dairy manure in Southern Ontario, Canada

## TL;DR

This study examines antibiotic-resistant Klebsiella bacteria in dairy manure from Ontario, revealing diverse resistance genes and plasmids that could spread between animals and humans.

## Contribution

The study provides the first characterization of ESC-resistant Klebsiella from Canadian livestock, including plasmid and sequence type diversity.

## Key findings

- Eighty-six percent of Klebsiella isolates from dairy manure were multi-drug resistant.
- Variants of CTX-M resistance genes were found on multiple plasmid types and the chromosome.
- Conjugation of resistance plasmids occurred between Klebsiella and E. coli, highlighting potential for gene spread.

## Abstract

Extended-spectrum cephalosporin (ESC)-resistant Klebsiella pneumoniae are a problem in human patients and have been studied extensively. However, there is a paucity of information regarding ESC-resistant K. pneumoniae from livestock in general, and in Canada in particular. This study characterized ESC-resistant K. pneumoniae recovered from dairy manure in Ontario, Canada, and their ESC-resistance plasmids. ESC-resistant K. pneumoniae (n = 73) and K. quasipneumoniae (n = 11) isolates were screened by PCR for blaCTX-M, blaCMY and blaSHV prior to undergoing antimicrobial susceptibility testing using disk diffusion. Isolates from dairy manure carrying blaCTX-M (n = 74), and additional isolates from turkeys (n = 8) and dogs (n = 2), underwent short-read whole genome sequencing (WGS) and a subset of these (n = 35) had additional long-read sequencing and hybrid assembly for confirmation. Isolates were characterized using multi-locus sequence typing (MLST) and antimicrobial resistance (AMR) gene profiles. Thirty known sequence types (STs) and four novel STs were identified for K. pneumoniae, and two STs among K. quasipneumoniae. The isolates were found at various treatment stages of the manure on one farm but were only sporadically found on any of the other farms investigated. The majority of isolates (86%) were multi-drug resistant. Variants of CTX-M were identified in diverse STs and included blaCTX-M-15 (n = 81), blaCTX-M-1 (n = 2) and blaCTX-M-32 (n = 1). The blaCTX-M-15 gene was located on diverse IncF, IncHI1 or IncY replicons as well as on the chromosome, whereas blaCTX-M-1 was harboured on the epidemic IncI1/ST3 plasmid, and blaCTX-M-32 on an IncN plasmid. Plasmids were characterized based on core gene SNPs, replicon types and AMR genes, and compared to plasmids from Escherichia coli from a parallel study. Transfer of blaCTX-M plasmids between bacterial species by conjugation was also assessed. Conjugation of IncI1 and IncFII plasmids occurred from K. pneumoniae to E. coli strains but not vice versa. Notably, IncY replicons were identified as conjugative plasmids and transfer was demonstrated between E. coli strains as well as from K. pneumoniae to E. coli. No strain overlap was observed between dairy manure isolates and those from turkey and dogs, however we did identify similarities between K. pneumoniae MLST and resistance profiles from publicly available human clinical isolates to our isolates found in dairy manure (ST37 and 405), turkeys (ST45) and dogs (ST711).