Genomic insights into tigecycline non-susceptibility in Clostridioides difficile: the role of the Tet P determinant and efflux mechanisms.

Background: Clostridioides difficile infection (CDI) is a significant cause of nosocomial infections, often arising from antibiotic-induced disruption of the normal gut microbiota, enabling C. difficile proliferation. Tigecycline has proven effective in treating refractory CDI, with resistance to this antibiotic being rare. However, our previous study identified six C. difficile isolates that were non-susceptible to tigecyclin. Methods: To investigate the mechanisms underlying reduced tigecycline susceptibility, the genome of C. difficile strain 16338, which is non-susceptible to tigecycline, was characterized. The tetA(P), tetB(P), and Tet P (including 2,000 bp upstream), were individually cloned into recombinant plasmids and transferred into Escherichia coli to assess their contribution to tigecycline resistance. The role of promoter and terminator sequences was explored by comparing the minimum inhibitory concentrations (MICs) of strains with different combinations. Efflux pump involvement in resistance was evaluated using efflux pump inhibitors. Results: Whole-genome sequencing revealed that C. difficile 16338 lacked plasmids and contained the Tet P determinant on its chromosome. The Tet P determinant contains two coding genes, tetA(P) and tetB(P), and is associated with an insertion sequence containing an 11-bp repeat sequence, which may serve as a recombination site facilitating gene transfer. E. coli strains carrying tetP, tetA(P), or tetB(P) exhibited a 1-fold increase in MIC for tigecycline. Additionally, tetA(P) and tetB(P) primarily mediate resistance to tetracycline (MIC = 32 mg/L) and minocycline (MIC = 32 mg/L), respectively. In addition, strains carrying different P3 promoters and T1 terminator sequences exhibited varying MICs for tigecycline. Efflux pumps were implicated in tigecycline resistance, as inhibitors (CCCP and reserpine) reduced MIC by one-fold. Conclusion: This study identified the Tet P determinant and its related encoding genes tetA(P) and tetB(P) as genetic factors associated with reduced susceptibility to tigecycline in C. difficile. The tetA(P) and tetB(P) genes mediate different resistance to tetracycline and minocycline, showing substrate specificity. Although the efflux mechanism is associated with reduced susceptibility to tigecycline, further studies are needed to determine whether it is only the efflux pump encoded by tetA(P) that is responsible. These findings enhance our understanding of tigecycline resistance in C. difficile and lay the foundation for future studies of potential resistance mechanisms. [ABSTRACT FROM AUTHOR]