Antimicrobial Resistance and Virulence-Associated Genes ofPasteurella multocidaandMannheimia haemolyticaIsolated from Polish Dairy Calves with Symptoms of Bovine Respiratory Disease

Abstract

Background: Bovine respiratory disease causes significant economic losses in cattle farming due to mortality, treatment costs, and reduced productivity. It involves viral and bacterial infections, with Pasteurella multocida (P. multocida) and Mannheimia haemolytica (M. haemolytica) as key bacterial pathogens. These bacteria contribute to severe pneumonia and are often found together. A total of 70 bacterial strains were analysed: 48 P. multocida and 22 M. haemolytica, collected from deep nasal swabs or lung and bronchial swabs from affected calves. The bacterial species were confirmed molecularly using PCR, which was also employed to detect antimicrobial resistance and virulence-associated genes. Antimicrobial susceptibility was determined using the broth microdilution method. Results: Antimicrobial resistance varied between the two bacterial species studied. The highest resistance in P. multocida was observed to chlortetracycline 79.2% and oxytetracycline 81.3%, while M. haemolytica showed 63.6% resistance to penicillin and tilmicosin. Multidrug resistance among P. multocida was 27.1%, while among M. haemolytica isolates, it reached 40.9%. The most commonly observed phenotypic resistance patterns were 'chlortetracycline, oxytetracycline' in 37.5% of P. multocida and 'ceftiofur, chlortetracycline, oxytetracycline, penicillin, tilmicosin, tulathromycin' in 18.2% of M. haemolytica. The highest susceptibility was found for fluoroquinolones: P. multocida demonstrated 91.7% susceptibility to enrofloxacin, while 77.3% of M. haemolytica isolates were susceptible to both enrofloxacin and danofloxacin. Multidrug resistance was detected in 31.4% of all tested strains. MIC50 and MIC90 determinations were performed for all tested antimicrobials. All M. haemolytica isolates contained the lkt, gs60, and gcp genes. P. multocida isolates carried the sodA gene, while the hgbB and ompH genes were present in 37.5% and 20.8% of isolates, respectively. The tetH and tetR genes were observed only in P. multocida, at frequencies of 20.8% and 16.7%, respectively. Both species carried the mphE and msrE genes, though at lower frequencies between 6.3% and 14.6%. Conclusions: This study expands the knowledge of the pathogenicity and antimicrobial resistance of P. multocida and M. haemolytica in dairy calves. P. multocida exhibited the highest resistance to tetracyclines, M. haemolytica demonstrated the greatest non-susceptibility to penicillin. Both bacterial species were found to be susceptible to fluoroquinolones. One third of strains showed multidrug resistance.