Downregulation of Klebsiella pneumoniae RND efflux pump genes following indole signal produced by Escherichia coli

Abstract

Background: More than a century has passed since it was discovered that many bacteria produce indole, but research into the actual biological roles of this molecule is just now beginning. The influence of indole on bacterial virulence was extensively investigated in indole-producing bacteria like Escherichia coli. To gain a deeper comprehension of its functional role, this study investigated how indole at concentrations of 0.5-1.0 mM found in the supernatant of Escherichia coli stationary phase culture was able to alter the virulence of non-indole-producing bacteria, such as Pseudomonas aeruginosa, Proteus mirabilis, and Klebsiella pneumoniae, which are naturally exposed to indole in mixed infections with Escherichia coli.

Results: Biofilm formation, antimicrobial susceptibility, and efflux pump activity were the three phenotypic tests that were assessed. Indole was found to influence antibiotic susceptibly of Pseudomonas aeruginosa, Proteus mirabilis and Klebsiella pneumoniae to ciprofloxacin, imipenem, ceftriaxone, ceftazidime, and amikacin through significant reduction in MIC with fold change ranged from 4 to 16. Biofilm production was partially abrogated in both 32/45 Pseudomonas aeruginosa and all eight Proteus mirabilis, while induced biofilm production was observed in 30/40 Klebsiella pneumoniae. Moreover, acrAB and oqxAB, which encode four genes responsible for resistance-nodulation-division multidrug efflux pumps in five isolates of Klebsiella pneumoniae were investigated genotypically using quantitative real-time (qRT)-PCR. This revealed that all four genes exhibited reduced expression indicated by 2^-ΔΔCT < 1 in indole-treated isolates compared to control group.

Conclusion: The outcomes of qRT-PCR investigation of efflux pump expression have established a novel clear correlation of the molecular mechanism that lies beneath the influence of indole on bacterial antibiotic tolerance. This research provides novel perspectives on the various mechanisms and diverse biological functions of indole signaling and how it impacts the pathogenicity of non-indole-producing bacteria.

Keywords: Antibiotic resistance; Biofilm; Efflux pump; Signal molecules.

Fig. 1

Et-Br cartwheel assay showing fluorescence of reference strain (E. coli ATCC 25922) marked with green arrow and eight K. pneumoniae isolates on agar plates containing increasing concentration of Et Br (1-2.5 mg/l). The five isolates with index > 1.5 are marked with numbers (1 to 5)

Fig. 2

Fold changes in the expression of the oqxA, oqxB, acrA, and acrB genes in K. pneumoniae isolates. The bars represent relative expression levels of each indole-treated with corresponding control. Statistically significant differences are indicated by p-values of < 0.05 (*), and < 0.01 (**)

Fig. 3

A representative experiment depicting dissociation curve on the left and amplification curve on the right, displaying the influence of indole on the expression of the RND-type efflux pump gene (acrB) in K. pneumoniae isolates