Decontamination of biofilm and VBNC zoonotic pathogens on the salad leaf phylloplane for enhanced food security and safety

Abstract

Produce-associated outbreaks of foodborne pathogens such as Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica are rising in prominence among outbreaks of foodborne disease. Testing for foodborne pathogens by the agricultural industry relies heavily on culture-based techniques, excluding detection of viable but nonculturable (VBNC) pathogens. Here, a detection method is used that facilitates the use of qPCR on the complex environmental matrices of soil. Targeting the tir gene of E. coli O157, detection of the pathogen in peat-based compost and sand is achieved to a sensitivity of 10 CFU/g. When applied to pristine soil, 310 copies of the gene were detected. Further analysis using PNA-FISH and cell elongation determined the presence of 205 VBNC E. coli O157 cells per gram of soil sample. Resuscitation of the pathogen was achieved through prolonged enrichment in selective media. Decontamination of fresh produce using chlorine washes was simulated using L. monocytogenes and S. enterica serovar Thompson adhered to spinach leaves, resulting in complete VBNC induction of viable cells following two minutes exposure to 50 ppm and 100 ppm chlorine respectively. The infectivity of these chlorine induced VBNC pathogens was assessed in vivo using Caenorhabditis elegans as an animal model. VBNC L. monocytogenes retained its infectivity and caused a significant lifespan reduction (p=0.0064). Together, these data provide evidence of the presence and induction of VBNC foodborne pathogens throughout the food production chain, and determines that VBNC L. monocytogenes presents a threat to food safety.

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

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