Abstract The microbiological safety of fresh produce is monitored almost exclusively by detection methods. However, bacterial foodborne pathogens are known to enter a viable but nonculturable (VBNC) state in response to environmental stresses such as chlorine, commonly used for fresh produce decontamination. Here, complete VBNC induction of green fluorescent protein (GFP) tagged Listeria monocytogenes and Salmonella enterica Thompson was achieved by exposure to 12 ppm and 3 ppm chlorine respectively. Pathogens were subjected to chlorine washes following incubation on spinach leaves. Culture data revealed that total viable L. monocytogenes and Salmonella Thompson populations became VBNC by 50 and 100 ppm chlorine, respectively, while enumeration by direct viable count found chlorine caused a reduction in viability of less than 1 log. The pathogenicity of chlorine-induced VBNC L. monocytogenes and Salmonella Thompson was assessed using Caenorhabditis elegans. Ingestion of VBNC pathogens by C. elegans resulted in a significant reduction of lifespan (p=0.0064 and p<0.0001), and no significant lifespan reduction was observed between VBNC and culturable L. monocytogenes treatments. L. monocytogenes was visualised beyond the nematode intestinal lumen, indicating resuscitation and cell invasion. These data emphasise the risk that VBNC foodborne pathogens could pose to public health should they continue to go undetected.
Abstract The microbiological safety of fresh produce is monitored almost exclusively by detection methods. However, bacterial foodborne pathogens are known to enter a viable but nonculturable (VBNC) state in response to environmental stresses such as chlorine, commonly used for fresh produce decontamination. Here, complete VBNC induction of green fluorescent protein (GFP) tagged Listeria monocytogenes and Salmonella enterica Thompson was achieved by exposure to 12 ppm and 3 ppm chlorine respectively. Pathogens were subjected to chlorine washes following incubation on spinach leaves. Culture data revealed that total viable L. monocytogenes and Salmonella Thompson populations became VBNC by 50 and 100 ppm chlorine, respectively, while enumeration by direct viable count found chlorine caused a reduction in viability of less than 1 log. The pathogenicity of chlorine-induced VBNC L. monocytogenes and Salmonella Thompson was assessed using Caenorhabditis elegans. Ingestion of VBNC pathogens by C. elegans resulted in a significant reduction of lifespan (p=0.0064 and p<0.0001), and no significant lifespan reduction was observed between VBNC and culturable L. monocytogenes treatments. L. monocytogenes was visualised beyond the nematode intestinal lumen, indicating resuscitation and cell invasion. These data emphasise the risk that VBNC foodborne pathogens could pose to public health should they continue to go undetected.
VBNC_phylloplane_paper_non_GFP_Salmonella_mBio_v3_CLEAN.docx