Objectives: Antimicrobial agents belonging to the class of β-lactams are used very frequently in human and veterinary medicine. In Enterobacteriaceae, extended-spectrum β-lactamase (ESBL) genes are well studied and are the focus of numerous investigations. In contrast, far less studies focus on other bacterial families. The aim of this study was to identify and analyse the first ESBL gene from Mannheimia haemolytica.
Methods: Susceptibility testing was performed according to CLSI. Plasmids were extracted via alkaline lysis and transferred by electrotransformation into Pasteurellaceae and Escherichia coli recipients. The sequence was determined by whole genome sequencing and confirmed by Sanger sequencing.
Results: The M. haemolytica strain 48 showed high cephalosporin minimal inhibitory concentrations (MICs). A single plasmid, designated pKKM48, with a size of 4323 bp was isolated. Plasmid pKKM48 harboured a novel blaROB gene, tentatively designated blaROB-2, and was transferred to Pasteurella multocida B130 and to Escherichia coli JM107. PCR assays and susceptibility testing confirmed the presence and activity of the blaROB-2 gene in the P. multocida and in the E. coli recipient carrying plasmid pKKM48. The transformants had high MICs for all β-lactam antibiotics. An ESBL phenotype was seen in the E. coli transformant when applying the CLSI double disk confirmatory test for Enterobacteriaceae. The blaROB-2 gene from plasmid pKKM48 differed in three positions from blaROB-1 resulting in two amino acid exchanges and one additional amino acid in the deduced β-lactamase protein. In addition to blaROB-2, pKKM48 harboured mob genes and showed high similarity to other plasmids from Pasteurellaceae.
Conclusions: This study described the first ESBL gene in Pasteurellaceae which may limit the therapeutic options for veterinarians. The transferability to Enterobacteriaceae with the functional activity of the gene in the new host underlines the possibility of the spread of this gene across species or genus boundaries.