The Plasmid-Borne tet (A) Gene Is an Important Factor Causing Tigecycline Resistance in ST11 Carbapenem-Resistant Klebsiella pneumoniae Under Selective Pressure

The emergence and prevalence of tigecycline-resistant have seriously compromised the effectiveness of antimicrobial agents in the treatment of infections. To explore the role of the plasmid-borne (A) gene in tigecycline resistance in carbapenem-resistant (CRKP), a total of 63 CRKP isolates were collected from a tertiary hospital in Hangzhou, China. The minimum inhibitory concentration (MIC) of tigecycline, mutation rate of (A) gene, genetic surroundings of (A)-carrying transmissible plasmid and the contribution of (A) mutation to tigecycline resistance were analyzed using antimicrobial susceptibility test, whole-genome sequencing, tigecycline resistance evolution experiment, and plasmid conjugation experiment. Our results showed that 52.4% (33 isolates) of the test isolates carried the (A) gene; among them, 75.8% (25 isolates) exhibited a tigecycline non-susceptible phenotype (MIC = 4 mg/L). Three clonal groups (cluster I, cluster II, and cluster III) were identified in these (A)-bearing isolates. All 17 isolates belonged to serotype KL21 (cluster I), which differed by only 13 SNPs, suggesting a clonal spread of (A)-positive ST11 with serotype KL21 occurred in the sampling hospital. The induction of tigecycline resistance experiments showed that 71.4% of strains evolved (A) mutations and developed a high-level tigecycline resistance. Eight amino acid substitutions were identified in these mutants. The most common amino acid substitution was A370V, followed by S251A and G300E. Twelve isolates carrying (A) mutants succeeded in the filter mating experiment with a conjugation efficiency of 10 -10 . Tigecycline MICs in EC600 transconjugants with a mutated (A) were 2 to 8-fold higher than those in EC600 transconjugants with a wild-type (A). One ColRNAI/IncFII type and two IncFII type (A)-bearing conjugative plasmids were identified in this study, including a class 1 integron containing multiple antibiotic resistance genes, i.e., (A), , , , , and . Our study revealed the wide-spread situation of plasmid-borne (A) gene in clinical CRKP, and mutation of (A) is a potential driven force that lead to tigecycline resistance.