Genetic characterisation of clinical Klebsiella pneumoniae isolates with reduced susceptibility to tigecycline: Role of the global regulator RamA and its local repressor RamR

Laboratory-derived Klebsiella pneumoniae mutants demonstrated that the ramA locus mediated low-level tigecycline resistance. The aim of this study was to elucidate the underlying mechanisms of tigecycline resistance in clinical K. pneumoniae isolates. In total, 106 isolates with tigecycline MICs ranging from 0.125 mg/L to 16 mg/L were collected to determine the correlations between expression of the global regulon ramA, marA, soxS, the acrB pump gene and tigecycline MICs. PCR was used to determine whether mutations in ramR, acrR or the rpsJ gene encoding 30S ribosomal protein S10 were responsible for tigecycline resistance. ramA or ramR inactivation and corresponding trans-complemented strains were used to characterise the contribution of RamA and RamR to tigecycline resistance. Tigecycline MICs were correlated with transcriptional levels of ramA and acrB, but were negatively correlated with marA and soxS. Disrupting ramA strikingly reduced the tigecycline MIC by 16-fold, accompanied by a 0.5-fold downregulation of acrB expression and 3.14- and 3.80-fold upregulation of marA and soxS, respectively. Complementation with plasmid-borne ramA restored the original parental phenotype of decreased tigecycline susceptibility. Of 34 tigecycline-non-susceptible isolates, 21 harbouring diverse mutations in RamR led to ramA overexpression. Disrupting the mutated ramR gene and complementing the mutated ramR gene with a wild-type gene downregulated expression of ramA but maintained the same tigecycline-resistant phenotype as the parental strain; the complemented strain exhibited 4.21- and 27.51-fold increased expression of acrB and marA, respectively. In conclusion, for the majority of tigecycline-resistant K. pneumoniae, ramA, depressed by ramR, was the major factor accounting for tigecycline resistance.