Role of nitric oxide in the control of biofilm and zoonotic pathogen colonisation on the salad leaf phylloplane

Fresh produce such as salad crops are generally grown outdoors in soil and irrigated by surface water abstractions. As a consequence crops are exposed to the risk of contamination by a range of pathogenic bacteria. Indeed, numerous outbreaks of gastrointestinal disease have been identified as being caused by ingestion of fresh produce so contaminated. Leading UK salad producers such as Vitacress go to considerable lengths to ensure pathogen-free soil. For example, no animal grazing or manures for 5 years pre cropping; no compost with animal manure for 2 years pre-cropping unless audited and tested batch by batch to prove free of Salmonella and low counts of E. coli etc. Consequently, there are many codes in place to ensure pathogen-free soil. However, it is recognised that outdoor crops will get contaminated, for example by animal intruders, birds, and of course poor quality irrigation water that has been contaminated up stream of abstraction. These vectors may potentially carry zoonotic pathogens such as Salmonella, E. coli O157 and Campylobacter. The risk of infection is much greater than for the majority of other food types because these are cooked or preserved by pickling, fermentation etc. At present, the most important means of sanitising fresh produce is by washing with chlorine post-harvest. However, there are safety and consumer preference concerns connected to the use of chlorine. Alternative technologies are beginning to be advocated, driven by the desire of large multiple retailers to get away from reliance on chlorine. These include the use of ozonation augmented with UV irradiation, hydrogen peroxide and citrox. There is now an urgent need to utilise our knowledge of microbial physiology and genomics to develop new procedures to assess the ability of these newer sanitisation technologies to decontaminate important foodborne pathogens on the fresh produce surface. Indeed, do motile pathogens migrate into stomata and below the leaf outer surface, making them more inaccessible to the currently used sanitisers? Do they interact, structurally or physiologically, with the microflora that is already present on the leaf in ways that might interfere with their removal or sanitation? An Industrial case studentship nearing completion has shown that for motile Salmonella, both of these mechanisms occur, and that these attached pathogens are highly resistant to disinfection and some become sub-lethally stressed by the treatments, making them difficult to recover using conventional culture techniques due to their viable but nonculturable (VNC) state. This VNC state may help explain why agents causing major outbreaks of foodborne disease go undetected at source. New microscopy and resuscitation techniques have been developed to assess the microbial quality of harvested salads, indicating the limitations of the current codes and growing practices in excluding natural biofilms and pathogens from the production chain, as well confirming that existing and novel sanitisation technologies are ineffective in removing any potential pathogen risk. Recent work has indicated that nitric oxide producing systems may have the potential to release biofilms and zoonotic pathogens from the salad leaf phylloplane, probably through interplay with their di-cyclic GMP regulatory pathways for attachment/detachment, and also now render them susceptible to conventional disinfection strategies. These observations will be confirmed for the agents of major salad borne outbreaks of disease, E. coli O157 and Salmonella enterica, and the mechanisms of the nitric oxide response at the phylloplane elucidated. This knowledge will be transferred into the salad processing factory environment to ensure the safe supply of salads to the public. The combination of microbiology, molecular biology and engineering expertise proposed in the project should lead to reduced incidence of disease transmission due to consumption of contaminated fresh produce.