# Antimicrobial Resistance, Virulence Factors and Plasmid Replicon Patterns of Klebsiella pneumoniae and Klebsiella grimontii Isolates from Bovine Mastitic Milk in the Northwest of Portugal: Pilot Genomic Characterization

## TL;DR

This study characterizes the antimicrobial resistance and virulence of Klebsiella isolates from bovine mastitis in Portugal, revealing new resistance patterns and plasmid features.

## Contribution

The study reports novel resistance profiles and plasmid replicon patterns in bovine mastitis-associated Klebsiella isolates.

## Key findings

- K. pneumoniae-1DH1 showed resistance to multiple antibiotics including colistin and nitrofurantoin.
- K. grimontii-2DH2 was found to carry a plasmid replicon with both blaCTX-M and astA genes.
- Phylogenomic analysis showed K. pneumoniae-1DH1 clustered with human isolates, while K. grimontii-2DH2 grouped with environmental isolates.

## Abstract

Background: Bovine mastitis (BM) remains an economically significant disease in the global dairy industry. Multidrug resistance (MDR) in Klebsiella pneumoniae and Klebsiella grimontii has increased in recent years, representing an area of concern related to BM. Methods: Bovine mastitis 1-DH1 and 2-DH2 isolates (n = 2) were investigated. Antimicrobial susceptibility testing was performed using the Neg-Urine-Combo98 panel. Antimicrobial resistance genes (ARGs), virulence factor (VF) genes and plasmid replicons were identified by whole-genome sequencing (WGS). Phylogenomic analyses were performed for a visual comparison of the genomes. Results: Phenotypically, isolates 1-DH1 and 2-DH2 were identified as K. pneumoniae-1DH1 and Klebsiella oxytoca, respectively; the latter was subsequently confirmed as K. grimontii-2DH2 by WGS. K. pneumoniae-1DH1 (20.0%, 5/25) exhibited phenotypic resistance to amoxicillin–clavulanic acid, ampicillin (AM), cefuroxime, colistin (Cl), and nitrofurantoin (Fd), whereas K. grimontii-2DH2 (8.0%, 2/25) was resistant to AM and nalidixic acid (NA). In total, 31 and 32 ARGs and 10 and 15 VF genes were identified in K. pneumoniae-1DH1 and K. grimontii-2DH2, respectively. Two plasmid replicons were detected in K. pneumoniae-1DH1 (one harboring the blaCTX-M ARG) compared to one in K. grimontii-2DH2 (encoding both the blaCTX-M ARG and the astA VF gene) isolates, respectively. Phylogenomic analysis showed that K. pneumoniae-1DH1 clustered most closely with human-derived K. pneumoniae isolates, whereas K. grimontii-2DH2 grouped with environmental K. grimontii isolates. Conclusions: Novel phenotypic resistance profiles were observed in the BM-associated K. pneumoniae-1DH1 (MDR, Cl and Fd) and K. grimontii-2DH2 (AM and NA) isolates. Notably, K. grimontii-2DH2 harbored a plasmid replicon carrying both the blaCTX-M ARG and the astA VF gene.