Chemical mutagenesis ofListeria monocytogenesfor increased tolerance to benzalkonium chloride shows independent genetic underpinnings and off-target antibiotic resistance

Abstract

AbstractListeria monocytogenes, a potentially fatal foodborne pathogen commonly found in food processing facilities, creates a significant economic burden that totals more than $2 billion annually in the United States due to outbreaks. Quaternary ammonium compounds (QACs), including benzalkonium chloride (BAC), are among the most widely used sanitizers to inhibit the growth and spread ofL. monocytogenesfrom food processing facilities. However, resistance to QACs has been increasing inL. monocytogenesand different genetic mechanisms conferring resistance have been discovered. Here, we used ethyl methanesulfonate (EMS) to chemically mutagenize the BAC-susceptible strain,L. monocytogenesFSL-N1-304. We isolated two mutants with increased tolerance to BAC compared to the parental strain. Next, we assessed the off-target effect of increased tolerance to BAC by measuring the minimum inhibitory concentrations (MICs) of a diverse set of antibiotics, revealing thatmut-1andmut-2displayed significantly increased resistance to fluoroquinolone antibiotics compared to the parental strain. We then sequenced the genomes of the parental strain and both mutants to identify mutations that may be involved in the increased resistance to BAC. We identified 3 and 29 mutations inmut-1andmut-2, respectively.mut-1contained nonsynonymous mutations indagK(a diacylglycerol kinase), lmo2768 (a permease-encoding gene), andlmo0186(resuscitation promoting factor).mut-2contained a nonsense mutation in the nucleotide excision repair enzyme UvrABC system protein B encoding gene,uvrB, which likely accounts for the higher number of mutations observed. Transcriptome analysis in the presence of BAC revealed that genes related to the phosphotransferase system and internalins were upregulated in both mutants, suggesting their significance in the BAC stress response. These two mutants provide insights into alternative mechanisms for increased BAC tolerance and could further our understanding of howL. monocytogenespersists in the food processing environment.