Novel techniques for the in situ detection of bacteria on salad leaf surfaces

Abstract

Bacteria such as Escherichia coli and Salmonella are capable of causing gastrointestinal illness if ingested with food. There is an urgent need within the food processing industry to improve the efficiency of both the sanitising processes used on fresh produce and the microbiological methods used to ensure its safety. The aims of this study were to develop novel microscopic methods to detect and quantify both indigenous leaf bacteria and viable human pathogens on salad leaves, without the need for recovery and culture, which may reduce the accuracy of quantification. The methods developed could then be used to quantify the number of indigenous and inoculated bacteria in situ on salad leaves as well as to identify factors affecting the quantity and spatial patterning of attachment. In addition, the efficacy of chemical biocides for the reduction of viable Salmonella on salad leaves was considered.

Episcopic Differential Interference Contrast (EDIC) microscopy coupled with epifluorescence was used to rapidly and non-destructively view the natural microflora in situ on spinach leaves. Salmonella enterica serovar Thompson was inoculated onto spinach leaves in order to observe spatial and temporal patterning of colonisation under differing conditions. Salmonella enterica serovar Typhimurium mutants defective in curli fimbriae production were used to assess the role of curli in attachment to abiotic surfaces and leaves. Viability determination of potential viable but non-culturable (VNC), sub-lethally stressed cells was performed to assess the effect of chemical wash treatments on Salmonella attached to fresh spinach.

The results obtained indicate that salad leaves are densely populated with naturally occurring bacteria; these are found predominantly in the margins between leaf epidermal cells as well as around leaf veins and stomata. Cells were present in complex three-dimensional aggregations, suggesting the presence of biofilms. Curli fimbriae were shown to be key in the attachment of Salmonella typhimurium to polystyrene but not to leaf surfaces. When Salmonella thompson was inoculated onto spinach leaves and then subjected to chemical washing in chlorine or Citrox, neither chemical was an effective biocide against the pathogen. Under-reporting of viable cell numbers by plate counting methods indicated that chlorine induced a viable but nonculturable (VNC) state amongst Salmonella.

These findings have important implications for the sanitisation of salads; both stomatal penetration and the formation of biofilms could protect enteric bacteria on leaves from chemical and mechanical disinfection strategies. If human pathogens are able to spread to salad leaves from contaminated soil, irrigation water or directly from animal faeces, and actively penetrate the interior of the leaves, then the microbiological safety of ready-to-eat salads cannot be guaranteed. This study also suggests that current methods for detection of pathogenic bacteria in foods following processing may be under-reporting the threat to consumers due to the induction of the VNC state amongst pathogenic bacteria.

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Identifiers

ORCID for C.W. Keevil: orcid.org/0000-0003-1917-7706

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