Search

ABSTRACTOmpK35 and OmpK36 are the major outer membrane porins of Klebsiella pneumoniae. In this study, a virulent clinical isolate was selected to study the role of these two porins in antimicrobial resistance and virulence. The single deletion of ompK36(ΔompK36) resulted in MIC shifts of cefazolin, cephalothin, and cefoxitin from susceptible to resistant, while the single deletion of ompK35(ΔompK35) had no significant effect. A double deletion of ompK35and ompK36(ΔompK35/36) further increased these MICs to high-level resistance and led to 8- and 16-fold increases in the MICs of meropenem and cefepime, respectively. In contrast to the routine testing medium, which is of high osmolarity, susceptibility tests using low-osmolarity medium showed that the ΔompK35mutation resulted in a significant (≥4-fold) increase in the MICs of cefazolin and ceftazidime, whereas a ΔompK36deletion conferred a significantly (4-fold) lower increase in the MIC of cefazolin. In the virulence assays, a significant (P< 0.05) defect in the growth rate was found only in the ΔompK35/36mutant, indicating the effect on metabolic fitness. A significant (P< 0.05) increase in susceptibility to neutrophil phagocytosis was observed in both ΔompK36and ΔompK35/36mutants. In a mouse peritonitis model, the ΔompK35mutant showed no change in virulence, and the ΔompK36mutant exhibited significantly (P< 0.01) lower virulence, whereas the ΔompK35/36mutant presented the highest 50% lethal dose of these strains. In conclusion, porin deficiency in K. pneumoniaecould increase antimicrobial resistance but decrease virulence at the same time.

We report the first clinical Escherichia colistrain EC3000 with concomitant chromosomal colistin and carbapenem resistance. A novel in-frame deletion, Δ6-11 (RPISLR), in pmrBthat contributes to colistin resistance was verified using recombinant DNA techniques. Although being less fit than the wild-type (WT) strain or EC3000 revertant (chromosomal replacement of WT pmrBin EC3000), a portion of serially passaged EC3000 strains preserving colistin resistance without selective pressure raises the concern for further spread.

ABSTRACTTigecycline is regarded as a last-resort treatment for carbapenem-resistant Klebsiella pneumoniae(CRKP) infections, but increasing numbers of tigecycline-resistant K. pneumoniaeisolates have been reported. The tigecycline resistance mechanisms in CRKP are undetermined. This study aimed to elucidate the 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 ramRand tet(A) exhibited high prevalences individually (81%) and in combination (63%). Eight functional ramRmutation 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 or the upregulation of ramAand acrA. Two tet(A) variants with double frameshift mutations, type 1 and type 2, were identified; type 2 tet(A) is novel. A parent strain transformed with a plasmid carrying type 1 or type 2 tet(A) increased the tigecycline MIC by 8-fold or 4-fold, respectively. Synergistic effects were observed in strains harboring no ramRgene and a mutated tet(A), with an 8-fold increase in the tigecycline MIC compared with that in strains harboring only mutated tet(A) being seen. Overall, mutations in the ramRand tet(A) efflux genes constituted the major tigecycline resistance mechanisms among the studied TCRKP isolates. The identification of strains exhibiting the combination of a ramRdeficiency and widespread mutated tet(A) is concerning due to the possible dissemination of increased tigecycline resistance in K. pneumoniae.