FepR as a Central Genetic Target in the Adaptation to Quaternary Ammonium Compounds and Cross-Resistance to Ciprofloxacin in Listeria monocytogenes

Abstract

The foodborne pathogen, Listeria monocytogenes, (Lm), frequently undergoes selection pressure associated with the extensive use of disinfectants, such as quaternary ammonium compounds, which are widely used in food processing plants. The repeated exposure to sub-inhibitory biocide concentrations can induce increased tolerance to these compounds, but can also trigger the development of antibiotic resistance, and both increase the risk of food contamination and persistence in food production environments. Although the acquisition of genes can explain biocide tolerance, the genetic mechanisms underlying the adaptive cross-resistance to antibiotics remain unclear. We previously showed that repeated exposure to benzalkonium chloride (BC) and didecyldimethyl ammonium chloride (DDAC) led to reduced susceptibility to ciprofloxacin in Lm strains from diverse sources. Here, we compared the genomes of 16 biocide-adapted and 10 parental strains to identify the molecular mechanisms of fluoroquinolone cross-resistance. A core genome SNP analysis identified various mutations in the transcriptional regulator fepR (lmo2088) for 94% of the adapted strains and mutations in other effectors at a lower frequency. FepR is a local repressor of the MATE fluoroquinolone efflux pump FepA. The impact of the mutations on the structure and function of the protein was assessed by performing in silico prediction and protein homology modeling. Our results show that 75% of the missense mutations observed in fepR are located in the HTH domain of the protein, within the DNA interaction site. These mutations are predicted to reduce the activity of the regulator, leading to the overexpression of the efflux pump responsible for the ciprofloxacin-enhanced resistance.

Adaptation mechanisms ofListeria monocytogenesto quaternary ammonium compounds

“…Strains that carried a mutation in fepR, as well as strains that artificially overexpress the efflux pump FepA exhibited increased tolerance not only towards BAC, but also towards CTAB, ciprofloxacin and gentamycin, indicating that extrusion by the transporter is rather unspecific. This observation further supports the previous hypothesis that de-repression of fepA is the reason for the observed QAC tolerance (Guérin et al 2014;Bland et al 2022;Bolten et al 2022;Douarre et al 2022). We further substantiated this hypothesis by showing that a mutation in the DNA binding domain of FepR resulted in decreased binding to the promoter of the fepRA operon in comparison to the wildtype FepR protein.…”

“…strains (Bolten et al 2022). An independent study of biocide-adapted strains further supported these findings by showing that 94% of the adapted strains possessed mutations in the gene coding for the TetR-like transcriptional regulator FepR that was previously shown to repress its own expression and the expression of the efflux pump encoding gene fepA (Douarre et al 2022). The identified mutations in fepR were thus proposed to increase expression of FepA, resulting in enhanced tolerance towards BAC, as well as norfloxacin and ciprofloxacin (Guérin et al 2014).…”

“…These findings are in accordance with previous studies, which focussed on serial adapted isolated L. monocytogenes and Listeria spp. strains that were exposed to BAC or ciprofloxacin stress and likewise found that the majority of strains acquired mutations in fepR (Bland et al 2022;Bolten et al 2022;Douarre et al 2022). FepR is a TetR-like transcriptional regulator that negatively regulates the fepRA operon, which encodes the regulator itself as well as the MATE family efflux pump FepA (Guérin et al 2014).…”

Quaternary ammonium disinfectants and antiseptics: tolerance, resistance and potential impact on antibiotic resistance

“…Rakic-Martinez et al [ 82 ] found that exposure of Listeria monocytogenes to BAC yielded strains that developed efflux pump-mediated resistance to gentamicin, which is used for treatment of invasive Listeria infections. Another study of L. monocytogenes found that exposure to BAC or DDAC caused increased tolerance to ciprofloxacin MICs, [ 85 ] which was later shown to be caused by mutations in the fepR repressor that affects the fluoroquinolone efflux pump FepA [ 86 ]. It should be noted that the stability of antibiotic resistance demonstrated in the above studies was tested over short time periods in some studies, but none evaluated the persistence of resistance over prolonged time periods.…”

Biofilms as protective cocoons against biocides: from bacterial adaptation to One Health issues

“…Acinetobacter baumannii [129] CepA CHX Col Klebsiella pneumoniae [204] CmeB TCS Amp, Cip, Cm, Ery, Tet, Campylobacter jejuni [205] DinF DDAC Amp, Tet Escherichia coli [203] FepR BAC Cip Listeria monocytogenes [10] MdfA CHP Amp, Cm Escherichia coli [7] TCS Amp, Azi, Cfc, Cfp, Cfz, Cxm, Cip, Ery, Gen, Lin, Lom, Oxa, Tet…”