Influence of transcriptional activator RamA on expression of multidrug efflux pump AcrAB and tigecycline susceptibility in Klebsiella pneumoniae

Abstract

Tigecycline is an expanded broad-spectrum antibacterial agent that is active against many clinically relevant species of bacterial pathogens, including Klebsiella pneumoniae. The majority of K. pneumoniae isolates are fully susceptible to tigecycline; however, a few strains that have decreased susceptibility have been isolated. One isolate, G340 (for which the tigecycline MIC is 4 microg/ml and which displays a multidrug resistance [MDR] phenotype), was selected for analysis of the mechanism for this decreased susceptibility by use of transposon mutagenesis with IS903phikan. A tigecycline-susceptible mutant of G340, GC7535, was obtained (tigecycline MIC, 0.25 microg/ml). Analysis of the transposon insertion mapped it to ramA, a gene that was previously identified to be involved in MDR in K. pneumoniae. For GC7535, the disruption of ramA led to a 16-fold decrease in the MIC of tigecycline and also a suppression of MDR. Trans-complementation with plasmid-borne ramA restored the original parental phenotype of decreased susceptibility to tigecycline. Northern blot analysis revealed a constitutive overexpression of ramA that correlated with an increased expression of the AcrAB transporter in G340 compared to that in tigecycline-susceptible strains. Laboratory mutants of K. pneumoniae with decreased susceptibility to tigecycline could be selected at a frequency of approximately 4 x 10(-8). These results suggest that ramA is associated with decreased tigecycline susceptibility in K. pneumoniae due to its role in the expression of the AcrAB multidrug efflux pump.

FIG. 1.

Map of the chromosomal region around the IS903φkan insertion site in GC7535. The genetic organization of the ramA locus of K. pneumoniae was described previously (4). Open reading frames are shown by arrows; the unsequenced portions of romA are shown by dashed lines. The site of the IS903φkan insertion in ramA is shown by a vertical triangle.

FIG. 2.

Northern blot analysis of ramA and acrA expression. Total bacterial RNA was isolated from mid-log-phase cultures of G595 (lane 1), G340 (lane 2), and GC7535 (lane 3) and hybridized with ³²P-labeled DNA probes specific to either ramA (A), acrA (B), or rrsE (C). Hybridization with rrsE that encodes 16S rRNA served as an RNA loading control.

FIG. 3.

Western blot analysis of AcrA expression. Total cell lysate proteins of G595 (lane 1), G340 (lane 2), and GC7535 (lane 3) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with polyclonal anti-AcrA antibodies (gift from H. Zgurskaya).