The Analysis of Field Strains Isolated From Food, Animal and Clinical Sources Uncovers Natural Mutations in Listeria monocytogenes Nisin Resistance Genes

Abstract

Nisin is a commonly used bacteriocin for controlling spoilage and pathogenic bacteria in food products. Strains possessing high natural nisin resistance that reduce or increase the potency of this bacteriocin against Listeria monocytogenes have been described. Our study sought to gather more insights into nisin resistance mechanisms in natural L. monocytogenes populations by examining a collection of 356 field strains that were isolated from different foods, food production environments, animals and human infections. A growth curve analysis-based approach was used to access nisin inhibition levels and assign the L. monocytogenes strains into three nisin response phenotypic categories; resistant (66%), intermediate (26%), and sensitive (8%). Using this categorization isolation source, serotype, genetic lineage, clonal complex (CC) and strain-dependent natural variation in nisin phenotypic resistance among L. monocytogenes field strains was revealed. Whole genome sequence analysis and comparison of high nisin resistant and sensitive strains led to the identification of new naturally occurring mutations in nisin response genes associated with increased nisin resistance and sensitivity in this bacterium. Increased nisin resistance was detected in strains harboring RsbU G77S and PBPB3 V240F amino acid substitution mutations, which also showed increased detergent stress resistance as well as increased virulence in a zebra fish infection model. On the other hand, increased natural nisin sensitivity was detected among strains with mutations in sigB , vir , and dlt operons that also showed increased lysozyme sensitivity and lower virulence. Overall, our study identified naturally selected mutations involving pbpB3 ( lm0441 ) as well as sigB , vir , and dlt operon genes that are associated with intrinsic nisin resistance in L. monocytogenes field strains recovered from various food and human associated sources. Finally, we show that combining growth parameter-based phenotypic analysis and genome sequencing is an effective approach that can be useful for the identification of novel nisin response associated genetic variants among L. monocytogenes field strains.

Cold Shock Proteins Promote Nisin Tolerance in Listeria monocytogenes Through Modulation of Cell Envelope Modification Responses

“…Nisin is a widely used bacteriocin for mitigation against foodassociated pathogenic and spoilage bacteria (Bali et al, 2016;Gharsallaoui et al, 2016;Santos et al, 2018;Ibarra-Sánchez et al, 2020;Martinez-Rios et al, 2021). The efficacy of nisin against L. monocytogenes is, however, currently reduced through various intrinsic nisin resistance molecular response systems, most of which we do not yet fully understand (Gravesen et al, 2002;Bergholz et al, 2013;Zhou et al, 2014;Draper et al, 2015;Bucur et al, 2018;Wambui et al, 2020;Pinilla et al, 2021). An improved understanding of these mechanisms will thus enhance our capability to design better strategies for nisin deployment against this pathogen.…”

Potassium Lactate as a Strategy for Sodium Content Reduction without Compromising Salt-Associated Antimicrobial Activity in Salami

“…Using the raw OD 600 data, area under the curve (AUC), was analyzed using GraphPad Prism. AUC has previously been recommended for use in summarizing growth curves [ 58 , 59 , 60 ] and was included in the analysis as it captures the effects of stress and growth conditions on the other three parameters described above. The growth parameters for each strain under normal or stress conditions were further analyzed using the R package “ opm ” as previously described [ 59 , 61 , 62 ].…”

“…AUC has previously been recommended for use in summarizing growth curves [ 58 , 59 , 60 ] and was included in the analysis as it captures the effects of stress and growth conditions on the other three parameters described above. The growth parameters for each strain under normal or stress conditions were further analyzed using the R package “ opm ” as previously described [ 59 , 61 , 62 ]. To facilitate the comparison of the effects of NaCl or NaCl plus KL combination stress on the strains, parameters were normalized relative to their growth under normal conditions vs. growth under stress to correct for inherent variation in growth between strains even in the absence of stress.…”

Table_4.xlsx

“…In response, L. monocytogenes deploys a plethora of response mechanisms such as transporters, efflux pumps, and biofilm formation to facilitate the nutrient acquisition and utilization, stress tolerance, and persistence on food and in the environment (Gandhi and Chikindas, 2007;Ferreira et al, 2014;Buchanan et al, 2017;Stoller et al, 2019;Kragh et al, 2020). Some of these mechanisms have genetic background-based distribution resulting in phenotypic variability among L. monocytogenes strains (Hingston et al, 2017;Muchaamba et al, 2019;Wambui et al, 2020;Gelbicova et al, 2021). For instance, stress survival islet 2 and qacH_Tn6188-encoding benzalkonium chloride (BC) resistance predominantly occur in CC121 (Harter et al, 2017;Gelbicova et al, 2021).…”

“…In contrast, strains carrying an L461T mutation in the pore-forming cytolysin, listeriolysin O (LLO), display significantly reduced virulence (Glomski et al, 2002). Collectively, these differences contribute to genetic background-associated differences in L. monocytogenes virulence and tolerance to food preservatives and cleaning and disinfectant chemicals, including salt, nisin, and quaternary ammonium compounds (Bergholz et al, 2010;Meier et al, 2017;Wambui et al, 2020). Furthermore, these factors and differences in nutrient acquisition and utilization capacities result in variability in the survival and growth of L. monocytogenes strains on food and in food-processing environments (Muchaamba et al, 2019).…”