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Based on its important role in last-line therapeutics against multidrug-resistant bacteria, tigecycline has been increasingly important in treating infections. However, mounting reports on tigecycline-resistant bacterial strains isolated from different sources are of concern, and molecular mechanisms regarding tigecycline resistance are poorly understood. Riemerella anatipestifer is a Gram-negative, non-motile, non-spore-forming, rod-shaped bacterium, which causes fibrinous pericarditis, perihepatitis, and meningitis in infected ducks. We previously constructed a random transposon mutant library using Riemerella anatipestifer strain CH3, in present study, we described that Riemerella anatipestifer M949_0459 gene is responsible for the bacterial resistance to tigecycline. Using the minimum inhibitory concentration assay, a mutant strain showed significantly increased (about six-fold) tigecycline susceptibility. Subsequently, the knocked-down gene was identified as M949_0459 , a putative flavin adenine dinucleotide-dependent oxidoreductase. To confirm the resistance function, M949_0459 gene was overexpressed in Escherichia coli strain BL21, and the minimum inhibitory concentration analysis showed that the gene product conferred resistance to tigecycline. Additionally, expression of the M949_0459 gene under treatment with tigecycline was measured with quantitative real-time PCR. Results showed that the mRNA expression of M949_0459 gene was elevated under tigecycline treatment with dose range of 1-10 mg/L, and peaked at 4 mg/L. Moreover, two kinds of efflux pump inhibitors, carbonyl cyanide m-chlorophenyl hydrazone and phenylalanine arginyl β -naphthylamide were tested, which showed no function on tigecycline resistance in the strain CH3. Our results may provide insights into molecular mechanisms for chemotherapy in combating Riemerella anatipestifer infections.

Riemerella anatipestifer M949_0459gene is responsible for the bacterial resistance to tigecycline