Selection for antimicrobial resistance in foodborne pathogens through exposure to ultraviolet light and non-thermal atmospheric plasma decontamination techniques

This study was aimed at assessing whether the repeated exposure of twelve strains of Salmonella spp., Escherichia coli and Listeria monocytogenes to the alternative non-thermal decontamination techniques ultraviolet light (UV-C) and non-thermal atmospheric plasma (NTAP) may cause the emergence of variants showing an increased resistance to clinically relevant antibiotics (ampicillin, cefotaxime, ciprofloxacin, gentamicin, streptomycin, tetracycline, erythromycin, vancomycin and colistin). UV-C and NTAP treatments were applied on the surface of inoculated BHI agar plates. Survivors were recovered and, after 24 hours growth in BHI broth, subjected again to the decontamination treatment and this was repeated for 10 consecutive cycles. A total of 174 strain/decontamination technique/antibiotic combinations were tested and 12 variant strains with an increased resistance to one of the antibiotics studied were identified, with increases in minimum inhibitory concentrations in MH broth ranging from 2 to 256 fold. The variant strains isolated for Salmonella spp. were further characterized through phenotypic screenings and whole genome sequencing (WGS) analyses. Most changes in susceptibility were observed for antibiotics that act at the level of protein synthesis (aminoglycosides, tetracyclines and glycylcyclines) or DNA replication (fluoroquinolones), as well as for polymyxins. No changes in resistance to β-lactams were detected. WGS analyses showed the occurrence of sequence alterations in some antibiotic cellular targets (e.g. gyrA for ciprofloxacin-resistant variants; rpsL for a streptomycin-resistant variant), accompanied by variations in stress response regulators and membrane transporters likely involved in non-selective efflux of antibiotics, which altogether result in an increase of low/medium level in microbial resistance to several antibiotics. IMPORTANCE Emergence and spread of antibiotic resistance along the food chain can be influenced by the different antimicrobial strategies used from farm to fork. This study evidences that two novel, not yet widely used, non-thermal microbial decontamination techniques, ultraviolet light and non-thermal atmospheric plasma, can select variants with increased resistance towards various clinically relevant antibiotics, such as ciprofloxacin, streptomycin, tetracycline and erythromycin. Whole genome analysis of the resistant variants obtained for Salmonella spp. allowed identifying the genetic changes responsible for the observed phenotypes, which suggested that some antimicrobial classes are more susceptible to the cross-resistance phenomena observed. This information is relevant, since these novel decontamination techniques are being proposed as possible alternative green techniques for the decontamination of environments and equipment in food and clinical settings.