Roles of ramR and tet(A) mutations in conferring tigecycline resistance in carbapenem-resistant Klebsiella pneumoniae clinical isolates [PublishAheadOfPrint]

Tigecycline is regarded as a last-resort treatment for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections, but increasing numbers of tigecycline-resistant K. pneumoniae isolates have been reported. Tigecycline resistance mechanisms are underdetermined in CRKP. This study aimed to elucidate mechanisms underlying tigecycline resistance in 16 tigecycline- and carbapenem-resistant K. pneumoniae (TCRKP) isolates. Mutations in tigecycline resistance determinant genes (ramR, acrR, oqxR, tet(A), tet(L), tet(X), tet(M), rpsJ) were assessed by PCR amplicon sequencing, and mutations in ramR and tet(A) exhibited high prevalences individually (81%) and in combination (63%). Eight functional ramR mutation profiles reducing tigecycline sensitivity were verified by plasmid complementation of wild-type and mutant ramR. Using a site-specific mutant, the most frequent RamR mutation, A19V (60%), had no significant effect on tigecycline susceptibility and upregulation of ramA and acrA. Two tet(A) variants with double frameshift mutations, Type 1 and Type 2, were identified; Type 2 tet(A) is novel. Parent strain transformed with a plasmid carrying Type 1 or Type 2 tet(A) increased tigecycline MIC by 8-fold or 4-fold, respectively. Synergistic effects were observed in strains harboring deficient ramR and mutated tet(A), with an 8-fold increase in tigecycline MIC compared with strains harboring only mutated tet(A). Overall, mutations in the ramR and tet(A) efflux genes constituted the major tigecycline resistance mechanisms among the studied TCRKP isolates. The identification of strains exhibiting the combination of deficient ramR and widespread mutated tet(A) is concerning due to the possible dissemination of increased tigecycline resistance in K. pneumoniae.

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