Evaluation of antimicrobial resistance and virulence characteristics in Escherichia coli and Proteus mirabilis isolates from dead Crested Ibis embryos at the artificial breeding base

Bacterial infections of avian embryos can lead to an increase in embryo mortality, and the proliferation of antimicrobial-resistant bacteria aggravates the situation. A low hatching rate also poses a challenge to the population of artificially bred Crested Ibises (Nipponia nippon). This study aims to determine the potential association between bacterial infection and the death of Crested Ibis embryos, and whether there is convergence between antimicrobial resistance and virulence in strain. In this study, 13 Escherichia coli and 12 Proteus mirabilis isolates were recovered from dead Crested Ibis embryos. The pathogenicity examination confirmed the pathogenicity of all isolates, and multiple virulence genes detected by PCR-sequencing demonstrated the presence of irp2 and iucD (100%), fimC and iss (92.31%) in E. coli, and ucaA (58.33%) in P. mirabilis. Antimicrobial susceptibility test demonstrated that isolates were mainly resistant to amoxicillin (E. coli: 76.92%, P. mirabilis: 91.67%), cefazolin (E. coli: 76.92%, P. mirabilis: 91.67%), oxytetracycline (E. coli: 92.31%, P. mirabilis: 75.00%) and sulfamethoxazole-trimethoprim (E. coli: 53.85%, P. mirabilis: 33.33%), and more than 30% of isolates showed multidrug-resistance (MDR). Further analyses detected extended-spectrum β-lactamase (ESBL) genes, of which blaTEM-1 (E. coli: 100%, P. mirabilis: 100%) had the highest frequency, followed by the blaCTX-M-55 (E. coli: 92.31%, P. mirabilis: 50%), blaCTX-M-14 (E. coli: 76.92%, P. mirabilis: 33.33%), blaCTX-M-65 (E. coli: 15.38%, P. mirabilis: 16.67%), and all isolates were negative for blaSHV and blaOXA. Pearson's correlation analysis showed a positive correlation between the presence of β-lactam resistance and ESBL genes, while mainly negative correlations were observed between the presence of ESBL genes and virulence genes. Furthermore, the conjugation experiment and PFGE revealed that the isolates were primarily polyclonal, and there was horizontal transfer of resistance or virulence genes by plasmids. Based on the results, E. coli and P. mirabilis were responsible for embryonic mortality of the ibises in this study. The co-presence and co-transfer of ESBL genes and virulence genes can pose a potential threat to the health of the Crested Ibis, and measures such as prudent use of antimicrobials, and constant surveillance of resistance and pathogenicity, must be implemented at the Crested Ibis breeding base.