Listeria develops reduced sanitizer sensitivity but not resistance at recommended sanitizer use levels

Foodborne disease-causing microbes such as Listeria monocytogenes can survive in food processing facilities over decades. By hiding in places that are difficult to clean (“niches”) or in places with high levels of organic material, Listeria may only be exposed to dilute concentrations of sanitizers. Some of these Listeria may survive the dilute sanitizer due to mutations (change in the DNA) or resistance genes and repeated exposure will potentially allow survival of increasing sanitizer concentrations. These resistance mechanisms can be passed on and spread across a population making it more challenging to control Listeria. To better understand the occurrence of resistant bacteria, we will screen Listeria collected from produce processing facilities for sanitizer resistance. We will also determine if continuous exposure to sanitizer can lead to further increased resistance. In addition, we will perform whole genome sequencing and apply bioinformatic tools to identify possible mutations or resistance genes. The data collected in this project will provide tools for more rapid identification of resistant Listeria and will inform industry for which sanitizers to expect resistance as a problem. This information will help in the design of improved sanitation strategies (e.g., sanitizer rotation, use of higher sanitizer concentration that overcome resistance).

Survival and persistence of Listeria spp. and Listeria monocytogenes in packing houses and fresh cut produce processing facilities continues to be a major concern. While “Listeria issues” in these facilities can typically be traced back to survival of these organisms in locations that cannot be (easily) reached by sanitizers (so called niches), there is also a concern about emergence of sanitizer resistant Listeria spp. and L. monocytogenes. The importance of sanitizer resistance in Listeria is also supported by recent reports of Listeria isolates that showed reduced susceptibility to quaternary ammonium compounds (“quats”) and that carry genes (e.g., bcrABC, qacH) that confer reduced sensitivity to quats. In addition to known sanitizer resistance mechanisms, there are concerns that additional unknown mechanisms can lead (i) to resistance to sanitizers other than quats or (ii) to resistance to higher levels of sanitizers, as compared to the current quat resistance mechanisms, which typically only confer reduced sensitivity to quat levels considerably below recommended use levels. Importantly, even reduced susceptibility to sanitizers (to concentrations considerably below recommended use levels) can be of practical concern as it may not be unusual for Listeria to be exposed to reduced sanitizer concentrations in produce operations, for example due to high organic loads, which reduces sanitizer efficacy even if applied at appropriate concentrations. This proposal aims to provide industry with better tools (i) to assess the risk of reduced sanitizer susceptibility in Listeria, (ii) to more rapidly and reliably identify Listeria with reduced sanitizer susceptibility, and (iii) to control Listeria with reduced sanitizer susceptibility. To achieve these goals, we will complete the following three objectives: Obj. 1: Screen > 500 Listeria spp. and L. monocytogenes isolates from packing houses and fresh cut operations for reduced sensitivity to key sanitizers, including quaternary ammonium compounds, sodium hypochlorite and peroxyacetic acid. Obj. 2: Perform whole genome sequencing of Listeria spp. and L. monocytogenes strains identified as showing reduced sanitizer sensitivity to identify mutations and gene acquisitions responsible for reduced sanitizer sensitivity. Obj. 3: Expose selected strains with reduced sanitizer sensitivity to increasing sanitizer concentrations to determine the potential of these strains to become resistant to sanitizer levels close to the recommended use levels (e.g., 200 ppm quat or above).

1. Screen >500 Listeria spp. and L. monocytogenes isolates from packinghouses and fresh-cut operations for reduced sensitivity to key sanitizers, including quaternary ammonium compounds, sodium hypochlorite and peroxyacetic acid. 

2. Perform whole genome sequencing (WGS) of Listeria spp. and L. monocytogenes strains identified as showing reduced sanitizer sensitivity to identify mutations and gene acquisitions responsible for reduced sanitizer sensitivity. 

3. Expose selected strains with reduced sanitizer sensitivity to increasing sanitizer concentrations to determine the potential of these strains to become resistant to sanitizer levels close to the recommended use levels.

Listeria isolates showed a range of responses to sanitizers at use level concentrations, with BC showing a minimum reduction of 2.7 log for all isolates compared to PAA or NaOCl where some isolates reach zero reduction after 30 sec of exposure, indicating application of BC is more likely to achieve target reductions for Listeria. L. monocytogenes is more tolerant than Listeria spp. when exposed to PAA or NaOCl. Differences between L. monocytogenes and Listeria spp. were not observed for BC, suggesting equal likelihood of reduction of all species when BC is used. Prevalence of sanitizer and stress resistance genes, i.e., bcrABC, qacH and SSI-2, are not correlated with sanitizer survival when sanitizer is applied at use level concentrations. Listeria can grow at concentrations of BC up to 18ppm when continuously exposed to sublethal concentrations, supporting the importance of ensuring proper quat concentrations at all locations in a facility. There is a limited risk of sanitizer resistance genes spreading through horizontal gene transfer. Listeria can adapt to sublethal concentrations of BC through point mutations. Sanitizer resistance genes, e.g., bcrABC and qacH, play a limited role in adaptation to sublethal concentrations of BC. Importantly, (i) acquired resistance to low concentrations of BC, or (ii) the presence of sanitizer resistance genes does not increase the survival at use level concentrations. This underlines the importance of well written and followed sanitary standard operation procedures (SSOPs) to ensure sanitizer is applied at target concentrations.