Linezolid is a critically important antibiotic used to treat human infections caused by MRSA and VRE. While linezolid is not licensed for food-producing animals, linezolid-resistant (LR) isolates have been reported in European countries, including Belgium. To: (i) assess LR occurrence in staphylococci and enterococci isolated from different Belgian food-producing animals in 2019 through selective monitoring; and (ii) investigate the genomes and relatedness of these isolates. Faecal samples (n = 1325) and nasal swab samples (n = 148) were analysed with a protocol designed to select LR bacteria, including a 44–48 h incubation period. The presence of LR chromosomal mutations, transferable LR genes and their genetic organizations and other resistance genes, as well as LR isolate relatedness (from this study and the NCBI database) were assessed through WGS. The LR rate differed widely between animal host species, with the highest rates occurring in nasal samples from pigs and sows (25.7% and 20.5%, respectively) and faecal samples from veal calves (16.4%). WGS results showed that LR determinants are present in a large diversity of isolates circulating in the agricultural sector, with some isolates closely related to human isolates, posing a human health risk. LR dedicated monitoring with WGS analysis could help to better understand the spread of LR. Cross-selection of LR transferable genes through other antibiotic use should be considered in future action plans aimed at combatting antimicrobial resistance and in future objectives for the rational use of antibiotics in a One Health perspective.

Background/Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) represent important antimicrobial resistance threats related to companion animals, which can directly or indirectly lead to adverse health effects in humans and animals living in close contact. Characterizing the phenotypic resistance of MRSA and MRSP to a panel of antimicrobials relevant to both veterinary and human medicine is crucial within a “One Health” framework. Methods: In this study, a total of 79 presumptive MRSA isolates (34 from cats, 45 from dogs) and 110 presumptive MRSP isolates (105 from dogs, 5 from cats) from clinical cases were analysed. Real-time PCR was used to detect the presence of mecA and mecC genes, and susceptibility testing was performed using the Sensititre EUST2 panel. Results: Most of the isolates (88.9%, 168/189) were positive for the mecA gene, while a minority (1.1%, 2/189) were mecC-positive (2 MRSA, 1 dog, 1 cat). MRSP isolates exhibited acquired resistance to a broader range of antibiotics compared to MRSA strains. Furthermore, several isolates demonstrated acquired resistance to antibiotics considered critically important for human medicine. Resistance to vancomycin was found in an MRSP isolate from a dog, and resistance to linezolid in an MRSP isolate from a cat. This study reveals that 83.3% (30/36) of MRSA isolates from dogs and 89.3% (25/28) from cats were multidrug-resistant organisms, while MRSP isolates exhibited multidrug resistance in 99% (101/102) of cases for dogs and 100% (4/4) for cats. Conclusions: The extremely high level of multidrug resistance, with some isolates resistant to critically important antibiotics used in human medicine, highlight the importance of monitoring antimicrobial susceptibility in MRSA and MRSP isolates collected from cats and dogs in a One Health perspective.

Klebsiella pneumoniae of sequence type (ST) 11 is a hyper-epidemic nosocomial clone, which is spreading worldwide among humans and emerging in pets. This is the first report, to the best of our knowledge, of multidrug-resistant (MDR) K. pneumoniae ST11 carrying bla_SCO-1 and bla_DHA-1, isolated from a four-month-old dog in Belgium. Antimicrobial susceptibility testing (AST) of the isolate, performed via broth microdilution following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, revealed resistance to eight different classes of antimicrobials, including carbapenems, in particular ertapenem, third-generation cephalosporins and fluoroquinolones. A hybrid approach, combining long- and short-read sequencing, was employed for in silico plasmid characterization, multi-locus sequence typing (MLST) and the identification and localization of antimicrobial resistance (AMR) and virulence-associated genes. Three plasmids were reconstructed from the whole-genome sequence (WGS) data: the conjugative IncFIB(K), the non-mobilizable IncR and the mobilizable but unconjugative ColRNAI. The IncFIB(K) plasmid carried the bla_SCO-1 gene, whereas IncR carried bla_DHA-1, both alongside several other antimicrobial resistance genes (ARGs). No virulence genes could be detected. Here, we suggest that the resistance to ertapenem associated with susceptibility to imipenem and meropenem in K. pneumoniae could be related to the presence of bla_SCO-1 and bla_DHA-1, combined with permeability defects caused by point mutations in an outer membrane porin (OmpK37). The presence of the bla_SCO-1 gene on a conjugative IncFIB(K) plasmid is worrisome as it can increase the risk of transmission to humans, to animals and to the environment.

The aim of this study was to develop a highly multiplexed bead array to detect genes and/or mutations frequently associated with resistance to antimicrobials of the β-lactam, (fluoro)quinolone, colistin, macrolide and aminoglycoside families in Enterobacteriaceae such as Escherichia coli, Shigella spp. and Salmonella spp. Ligase Chain Reaction and the Luminex® technology were combined in a 53-plex assay designed to target selected genetic markers with 3 internal controls. The AMR-ARRAY consistently detected resistance determinants as compared to phenotypically expressed resistance for 94.7% (856/904) of the assessed resistances. When compared to resistance profiles inferred from whole genome sequencing results, the AMR-ARRAY showed a selectivity and specificity of 99.3% and 100%, respectively. The strong features of the AMR-ARRAY are (i) its competitive cost, currently 18€/sample (ii) its wide analytical scope, currently 50 markers covering 5 antimicrobial families, (iii) its robust and user-friendly design consisting in a single-tube assay conducted in 4 successive steps (iv) its relatively short turnaround time, less than 8 h (v) its ability to detect allelic variability at critical SNPs (vi) its open access and easily upgradable design, with probes sequences, procedure and software source code freely available. The use of the AMR-ARRAY as a screening method in official antimicrobial resistance monitoring could improve the granularity of the collected data and pinpoint remarkable isolates harbouring unusual resistance determinants thereby enabling fit-for-purpose selection of isolates for Whole Genome analysis.