Indole-Induced Activities of β-Lactamase and Efflux Pump Confer Ampicillin Resistance in Pseudomonas putida KT2440.

Jisun Kim,Chulwoo Park,Bora Shin,Woojun Park

doi:10.3389/fmicb.2017.00433

Abstract

Indole, which is widespread in microbial communities, has received attention because of its effects on bacterial physiology. Pseudomonas putida and Pseudomonas aeruginosa can acquire ampicillin (Amp) resistance during growth on indole-Amp agar. Transcriptome, mutant, and inhibitor studies have suggested that Amp resistance induced by indole can be attributed to increased gene expression of ttgAB encoding two genes of RND-type multidrug efflux operons and an ampC encoding β-lactamase. Expression, enzyme activities, and mutational analyses indicated that AmpC β-lactamase is important for acquiring Amp resistance of P. putida in the presence of indole. Here, we show, for the first time, that volatile indole increased Amp-resistant cells. Consistent with results of the volatile indole assay, a low concentration of indole in liquid culture promoted growth initially, but led to mutagenesis after indole was depleted, which could not be observed at high indole concentrations. Interestingly, ttgAB and ampC gene expression levels correlate with the concentration of indole, which might explain the low number of Amp-mutated cells in high indole concentrations. The expression levels of genes involved in mutagenesis, namely rpoS, recA, and mutS, were also modulated by indole. Our data indicates that indole reduces Amp-induced heterogeneity by promoting expression of TtgABC or MexAB-OprM efflux pumps and the indole-induced β-lactamase in P. putida and P. aeruginosa.

Highlights

IntroductionIndole has received a great deal of attention because of its broad range of effects on bacterial physiology, including biofilm formation (Lee et al, 2007; Kim and Park, 2013; Kim et al, 2015), quorum sensing (Chu et al, 2012; Sabag-Daigle et al, 2012; Kim and Park, 2013), virulence (Lee et al, 2009; Chu et al, 2012; Nikaido et al, 2012), spore formation (Stamm et al, 2005), plasmid stabilization (Field and Summers, 2012), and antimicrobial resistance (hereafter, AMR) (Hirakawa et al, 2005; Lee H.H. et al, 2010; Nikaido et al, 2012; Vega et al, 2012, 2013)
Among the genes involved in β-lactam resistance (Nakae et al, 1999; Quale et al, 2006; Kong et al, 2010), genes encoding resistance nodulation cell division (RND) efflux pumps/transporters and bacterial secretion systems, were increased by indole plus Amp or only indole (Supplementary Tables S5–S7), suggesting that they might contribute primarily to the acquisition of indole-induced Amp resistance
189, 193, and 162 genes were upregulated by indole, Amp, and indole plus Amp, respectively, indicating that both chemicals can cause a broad range of transcriptional responses during long-term exposure of P. putida

Summary

Introduction

Indole has received a great deal of attention because of its broad range of effects on bacterial physiology, including biofilm formation (Lee et al, 2007; Kim and Park, 2013; Kim et al, 2015), quorum sensing (Chu et al, 2012; Sabag-Daigle et al, 2012; Kim and Park, 2013), virulence (Lee et al, 2009; Chu et al, 2012; Nikaido et al, 2012), spore formation (Stamm et al, 2005), plasmid stabilization (Field and Summers, 2012), and antimicrobial resistance (hereafter, AMR) (Hirakawa et al, 2005; Lee H.H. et al, 2010; Nikaido et al, 2012; Vega et al, 2012, 2013). Indole is transported or diffused outside of cells, where indole concentrations commonly reach 1–2 mM, Effect of Indole on Ampicillin Resistance and sometimes up to 5 mM, in the stationary growth phase (Li and Young, 2013). Research on how exogenous indole is transported into different bacterial cells, the effects of indole on various cellular processes, and molecular mechanisms for sensing indole remain to be established. When indole is imported across a cellular membrane, the electrochemical potential and adenosine triphosphate (ATP) concentration inside of the cell can decrease and the NADH/NAD+ ratio is modulated (Piñero-Fernandez et al, 2011; Chimerel et al, 2012; Kim et al, 2013)

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