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 g/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 IS903kan. A tigecycline-susceptible mutant of G340, GC7535, was obtained (tigecycline MIC, 0.25 g/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 ؋ 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.Tigecycline is an expanded broad-spectrum antibiotic representing a new class called the glycylcyclines. The glycylcyclines are semisynthetic derivatives of minocycline and have activity against many bacterial pathogens (2, 14, 15). It has been noted that a few species of gram-negative bacteria, including Pseudomonas aeruginosa, Proteus spp., Providencia spp., and Morganella morganii, are intrinsically less susceptible to tigecycline. Previous studies revealed the involvement of multidrug efflux systems such as MexXY and AcrAB in the decreased tigecycline susceptibility of P. aeruginosa and Proteus mirabilis, respectively (3, 22). These pumps belong to the resistance-nodulation-division (RND) family that combines bacterial transporters with a tripartite architecture and broad substrate specificity (9, 12). Due to the broad substrate specificity of RND pumps, their overexpression usually results in the multidrug resistance (MDR) phenotype.Klebsiella pneumoniae causes infections of wounds, the urinary tract, and the respiratory system. This bacterial species is generally susceptible to tigecycline; however, a few clinical strains with decreased tigecycline susceptibility have been isolated. In this study, one such an isolate, G340, was investigated to determine the mechanism of decreased tigecycline susceptibility in K. pneumoniae.( MATERIALS AND METHODSBacterial strains and growth conditions. The bacterial strains and plasmids used in this study are shown in Table 1. Strains...

A Tripartite Efflux Pump Involved in Gastrointestinal Colonization by Klebsiella pneumoniae Confers a Tolerance Response to Inorganic Acid

“…A few AcrAB multidrug efflux systems have been characterized in Klebsiella spp. (7,20,23,28), but the sequencing data of K. pneumoniae strain MGH78578 show that many others potentially are present, as in closely related E. coli species (3). In this study, we characterized a gene cluster in K. pneumoniae LM21 that exhibits strong homologies with the EefABC RND efflux system-encoding operon, initially described in E. aerogenes (18).…”

Over expression of AdeABC and AcrAB-TolC efflux systems confers tigecycline resistance in clinical isolates of Acinetobacter baumannii and Klebsiella pneumoniae

“…Several studies have shown that tigecycline resistance in K. pneumoniae is associated with the overexpression of the acrAB operon (26) (27) . Additionally, a study by Wang et al found there was a statistically significant linear trend in the expression of acrB as the tigecycline MIC increased (14) .…”

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

“…Thus, mutations in these genes also influence the expression of acrAB (8). In K. pneumoniae and S. enterica, the expression of acrAB is governed by an additional global regulator, RamA (217,267). In Salmonella, field isolates carrying mutations in the regulatory region of ramRA or an insertion in soxR, increasing the expression levels of ramA and soxS, respectively, displayed increased resistance to fluoroquinolones (104).…”