Clostridium perfringens is an opportunistic pathogen that can cause food poisoning in humans and various enterotoxemia in animal species. Very little is known on the biofilm of C. perfringens and its exposure to subminimal inhibitory concentrations of antimicrobials. This study was undertaken to address these issues. Most of the C. perfringens human and animal isolates tested in this study were able to form biofilm (230/277). Porcine clinical isolates formed significantly more biofilm than the porcine commensal isolates. A subgroup of clinical and commensal C. perfringens isolates was randomly selected for further characterization. Biofilm was found to protect C. perfringens bacterial cells from exposure to high concentrations of tested antimicrobials. Exposure to low doses of some of these antimicrobials tended to lead to a diminution of the biofilm formed. However, a few isolates showed an increase in biofilm formation when exposed to low doses of tylosin, bacitracin, virginiamycin and monensin. Six isolates were randomly selected for biofilm analysis using scanning laser confocal microscopy. Of those, four produced more biofilm in presence of low doses of bacitracin whereas biofilms formed without bacitracin were thinner and less elevated. An increase in the area occupied by bacteria in the biofilm following exposure to low doses of bacitracin was also observed in the majority of isolates. Morphology examination revealed flat biofilms with the exception of one isolate that demonstrated a mushroom-like biofilm. Matrix composition analysis showed the presence of proteins, beta 1-4 linked polysaccharides and extracellular DNA, but no poly-beta-1,6-N-acetyl-D-glucosamine (PNAG). This study brings new information on the biofilm produced by C. perfringens and its exposure to low doses of antimicrobials.

Klebsiella spp. are important opportunistic pathogens commonly defined as environmental clinical mastitis agents. Despite Klebsiella mastitis being clinically impairing in cows and costly to the industry, only a few studies describe Klebsiella isolated from mastitis cases. The aim of this work was to characterize species of Klebsiella involved in clinical mastitis cases in Canada. Klebsiella isolated from clinical mastitis cases (n = 53) were identified to the species level using a biochemical test panel and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The rpoB gene sequence was used as the gold standard method and identified Klebsiella pneumoniae (n = 40), Klebsiella oxytoca (n = 9), Raoultella ornithinolytica (n = 2), and Raoultella planticola (n = 2). Raoultella, a genus closely related to Klebsiella, was also accurately identified using mass spectrometry but not via biochemical testing. Using the disc diffusion technique, 31 (58%) isolates were found to be susceptible to all antimicrobials tested (n = 18). The remaining 22 (42%) isolates were resistant to 1 or more of the following antimicrobials: kanamycin (2%), streptomycin (38%), spectinomycin (13%), sulfisoxazole (13%), and tetracycline (19%). The following antimicrobial resistance genes were identified: tetA, tetB, sul1, strA/strB, and aadA. Random amplified polymorphic DNA revealed the majority of our isolates as unrelated and having different patterns, indicating environmental contamination as the primary source of infection. All isolates were shown to be biofilm producers. In conclusion, although antimicrobial resistance was low for both Klebsiella and Raoultella species, genetically related Klebsiella spp. isolates appeared to be more resistant.

This study was conducted to characterize the antimicrobial resistance determinants and investigate plasmid colocalization of tetracycline and macrolide genes in Enterococcus faecalis and Enterococcus faecium from broiler chicken and turkey flocks in Canada. A total of 387 E. faecalis and E. faecium isolates were recovered from poultry cecal contents from five processing plants. The percentages of resistant E. faecalis and E. faecium isolates, respectively, were 88.1 and 94% to bacitracin, 0 and 0.9% to chloramphenicol, 0.7 and 14.5% to ciprofloxacin, 72.6 and 80.3% to erythromycin, 3.7 and 41% to flavomycin, 9.6 and 4.3% (high-level resistance) to gentamicin, 25.2 and 17.1% (high-level resistance) to kanamycin, 100 and 94% to lincomycin, 0 and 0% to linezolid, 2.6 and 20.5% to nitrofurantoin, 3 and 27.4% to penicillin, 98.5 and 89.7% to quinupristin-dalfopristin, 7 and 12.8% to salinomycin, 46.7 and 38.5% (high-level resistance) to streptomycin, 95.6 and 89.7% to tetracycline, 73 and 75.2% to tylosin, and 0 and 0% to vancomycin. One predominant multidrug-resistant phenotypic pattern was identified in both E. faecalis and E. faecium (bacitracin, erythromycin, lincomycin, quinupristin-dalfopristin, tetracycline, and tylosin). These isolates were further examined by PCR and sequencing for the genes encoding their antimicrobial resistance. Various combinations of vatD, vatE, bcrR, bcrA, bcrB, bcrD, ermB, msrC, linB, tetM, and tetO genes were detected, and ermB, tetM, and bcrB were the most common antimicrobial resistance genes identified. For the first time, plasmid extraction and hybridization revealed colocalization of tetO and ermB genes on a ca. 11-kb plasmid in E. faecalis isolates, and filter mating experiments demonstrated its transferability. Results indicate that the intestinal enterococci of healthy poultry, which can contaminate poultry meat at slaughter, could be a reservoir for quinupristin-dalfopristin, bacitracin, tetracycline, and macrolide resistance genes.

Antimicrobial resistance (AMR) represents a global threat to both human and animal health and has received increasing attention over the years from different stakeholders. Certain AMR bacteria circulate between humans, animals, and the environment, while AMR genes can be found in all ecosystems. The aim of the present review was to provide an overview of antimicrobial use in food-producing animals and to document the current status of the role of farm animals in the spread of AMR to humans. The available body of scientific evidence supported the notion that restricted use of antimicrobials in farm animals was effective in reducing AMR in livestock and, in some cases, in humans. However, most recent studies have reported that livestock have little contribution to the acquisition of AMR bacteria and/or AMR genes by humans. Overall, strategies applied on farms that target the reduction of all antimicrobials are recommended, as these are apparently associated with notable reduction in AMR (avoiding co-resistance between antimicrobials). The interconnection between human and animal health as well as the environment requires the acceleration of the implementation of the ‘One Health’ approach to effectively fight AMR while preserving the effectiveness of antimicrobials.

Phenotypic bacitracin resistance has been reported in Clostridium perfringens. However, the genes responsible for the resistance have not yet been characterized. Ninety-nine C. perfringens isolates recovered from broilers and turkeys were tested for phenotypic bacitracin resistance. Bacitracin MIC₉₀ (>256 µg/ml) was identical for both turkey and chicken isolates; whereas MIC₅₀ was higher in turkey isolates (6 µg/ml) than in chicken isolates (3 µg/ml). Twenty-four of the 99 isolates showed high-level bacitracin resistance (MIC breakpoint >256 µg/ml) and the genes encoding for this resistance were characterized in C. perfringens c1261_A strain using primer walking. Sequence analysis and percentages of amino acid identity revealed putative genes encoding for both an ABC transporter and an overproduced undecaprenol kinase in C. perfringens c1261_A strain. These two mechanisms were shown to be both encoded by the putative bcrABD operon under the control of a regulatory gene, bcrR. Efflux pump inhibitor thioridazine was shown to increase significantly the susceptibility of strain c1261_A to bacitracin. Upstream and downstream from the bcr cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Pulsed-field gel electrophoresis with prior double digestion with I-CeuI/MluI enzymes followed by hybridization analyses revealed that the bacitracin resistance genes bcrABDR were located on the chromosome. Semi-quantitative RT-PCR demonstrated that this gene cluster is expressed under bacitracin stress. Microarray analysis revealed the presence of these genes in all bacitracin resistant strains. This study reports the discovery of genes encoding for a putative ABC transporter and an overproduced undecaprenol kinase associated with high-level bacitracin resistance in C. perfringens isolates from turkeys and broiler chickens.