Emergence of a novel tet (X5) variant in Klebsiella spp. isolated from retail meat in Hainan, China

Background Klebsiella has recently been recognized as a potential host of mobile tigecycline-resistant gene tet(X4), posing a serious threat to public health. However, the occurrence of other tet(X) variants in Klebsiella remains uncommon. Methods We identified a novel tet(X5) variant in one Klebsiella pneumoniae and two Klebsiella quasipneumoniae isolates recovered from retail meat in Hainan, China and investigated its phenotypic resistance, genetic context and transferability. Antimicrobial susceptibility was determined by broth microdilution. Whole-genome sequencing, utilizing both Illumina and Nanopore platforms, facilitated the complete characterization of the genome sequence and plasmid structure. Results Resistance gene analysis revealed a novel tet(X5) variant encoding a 385-amino acid protein, designated Tet(X5.5), which exhibited 97.6% identity with the reference Tet(X5) protein. A total of 21 amino acid substitutions were identified, primarily located in the N- and C-terminal regions. Functional assays confirmed that expression of tet(X5.5) increased the tigecycline MIC in E. coli DH5α from 0.125 to 4 mg/L (32-fold). In K. pneumoniae 241CM75B, the tet(X5.5) gene was located on two conjugative plasmids: an ∼170 kb IncFIBK/IncFIIK8 hybrid plasmid and an ∼74 kb IncFIIpCRY plasmid. The other two isolates carried identical IncFIIpCRY plasmids. Conjugation assays confirmed that all tet(X5.5)-carrying plasmids were self-transmissible to E. coli C600, with transfer frequencies ranging from 10−6 to 10−5. Genetic analysis revealed that tet(X5.5) was embedded within an ISCR2-mediated transposon structure that was conserved across distinct plasmid backbones. Conclusions This study reports a novel transferable tet(X5) variant in Klebsiella, underscoring the risk of foodborne transmission and the necessity for integrated surveillance across clinical and agricultural settings.