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Viable-but-nonculturable Listeria monocytogenes and Salmonella enterica serovar Thompson induced by chlorine stress remain infectious

Viable-but-nonculturable Listeria monocytogenes and Salmonella enterica serovar Thompson induced by chlorine stress remain infectious
Viable-but-nonculturable Listeria monocytogenes and Salmonella enterica serovar Thompson induced by chlorine stress remain infectious

The microbiological safety of fresh produce is monitored almost exclusively by culture-based detection methods. However, bacterial food-borne pathogens are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses such as chlorine, which is commonly used for fresh produce decontamination. Here, complete VBNC induction of green fluorescent protein-tagged Listeria monocytogenes and Salmonella enterica serovar Thompson was achieved by exposure to 12 and 3 ppm chlorine, respectively. The pathogens were subjected to chlorine washing 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 counting found that chlorine caused a <1-log reduction in viability. The pathogenicity of chlorine-induced VBNC L. monocytogenes and Salmonella Thompson was assessed by using Caenorhabditis elegans. Ingestion of VBNC pathogens by C. elegans resulted in a significant life span reduction (P = 0.0064 and P < 0.0001), and no significant difference between the life span reductions caused by the VBNC and culturable L. monocytogenes treatments was observed. L. monocytogenes was visualized beyond the nematode intestinal lumen, indicating resuscitation and cell invasion. These data emphasize the risk that VBNC food-borne pathogens could pose to public health should they continue to go undetected. IMPORTANCE Many bacteria are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses. VBNC cells cannot be detected by standard laboratory culture techniques, presenting a problem for the food industry, which uses these techniques to detect pathogen contaminants. This study found that chlorine, a sanitizer commonly used for fresh produce, induces a VBNC state in the food-borne pathogens Listeria monocytogenes and Salmonella enterica. It was also found that chlorine is ineffective at killing total populations of the pathogens. A life span reduction was observed in Caenorhabditis elegans that ingested these VBNC pathogens, with VBNC L. monocytogenes as infectious as its culturable counterpart. These data show that VBNC food-borne pathogens can both be generated and avoid detection by industrial practices while potentially retaining the ability to cause disease.

Animals, Anti-Infective Agents/metabolism, Caenorhabditis elegans, Chlorine/metabolism, Disease Models, Animal, Listeria monocytogenes/drug effects, Listeriosis/microbiology, Microbial Viability/drug effects, Salmonella Infections/microbiology, Salmonella enterica/drug effects, Spinacia oleracea/microbiology, Survival Analysis, Virulence
2150-7511
Highmore, Callum
35d9d9e5-4f25-4dcf-876f-753f7a1a11d2
Warner, Jennifer C
4158c840-4073-4e4f-82f3-a9137535f07b
Rothwell, Steve D.
d28296e0-a91f-4293-9724-470f7986d566
Wilks, Sandra
86c1f41a-12b3-451c-9245-b1a21775e993
Keevil, Charles
cb7de0a7-ce33-4cfa-af52-07f99e5650eb
Highmore, Callum
35d9d9e5-4f25-4dcf-876f-753f7a1a11d2
Warner, Jennifer C
4158c840-4073-4e4f-82f3-a9137535f07b
Rothwell, Steve D.
d28296e0-a91f-4293-9724-470f7986d566
Wilks, Sandra
86c1f41a-12b3-451c-9245-b1a21775e993
Keevil, Charles
cb7de0a7-ce33-4cfa-af52-07f99e5650eb

Highmore, Callum, Warner, Jennifer C, Rothwell, Steve D., Wilks, Sandra and Keevil, Charles (2018) Viable-but-nonculturable Listeria monocytogenes and Salmonella enterica serovar Thompson induced by chlorine stress remain infectious. mBio, 9 (2), [e00540-18]. (doi:10.1128/mBio.00540-18).

Record type: Article

Abstract

The microbiological safety of fresh produce is monitored almost exclusively by culture-based detection methods. However, bacterial food-borne pathogens are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses such as chlorine, which is commonly used for fresh produce decontamination. Here, complete VBNC induction of green fluorescent protein-tagged Listeria monocytogenes and Salmonella enterica serovar Thompson was achieved by exposure to 12 and 3 ppm chlorine, respectively. The pathogens were subjected to chlorine washing 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 counting found that chlorine caused a <1-log reduction in viability. The pathogenicity of chlorine-induced VBNC L. monocytogenes and Salmonella Thompson was assessed by using Caenorhabditis elegans. Ingestion of VBNC pathogens by C. elegans resulted in a significant life span reduction (P = 0.0064 and P < 0.0001), and no significant difference between the life span reductions caused by the VBNC and culturable L. monocytogenes treatments was observed. L. monocytogenes was visualized beyond the nematode intestinal lumen, indicating resuscitation and cell invasion. These data emphasize the risk that VBNC food-borne pathogens could pose to public health should they continue to go undetected. IMPORTANCE Many bacteria are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses. VBNC cells cannot be detected by standard laboratory culture techniques, presenting a problem for the food industry, which uses these techniques to detect pathogen contaminants. This study found that chlorine, a sanitizer commonly used for fresh produce, induces a VBNC state in the food-borne pathogens Listeria monocytogenes and Salmonella enterica. It was also found that chlorine is ineffective at killing total populations of the pathogens. A life span reduction was observed in Caenorhabditis elegans that ingested these VBNC pathogens, with VBNC L. monocytogenes as infectious as its culturable counterpart. These data show that VBNC food-borne pathogens can both be generated and avoid detection by industrial practices while potentially retaining the ability to cause disease.

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Infectious chlorine-induced VBNC foodborne pathogens - Accepted Manuscript
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Accepted/In Press date: 19 March 2018
e-pub ahead of print date: 17 April 2018
Published date: 17 April 2018
Keywords: Animals, Anti-Infective Agents/metabolism, Caenorhabditis elegans, Chlorine/metabolism, Disease Models, Animal, Listeria monocytogenes/drug effects, Listeriosis/microbiology, Microbial Viability/drug effects, Salmonella Infections/microbiology, Salmonella enterica/drug effects, Spinacia oleracea/microbiology, Survival Analysis, Virulence

Identifiers

Local EPrints ID: 418842
URI: http://eprints.soton.ac.uk/id/eprint/418842
ISSN: 2150-7511
PURE UUID: 86df001d-1ccd-4671-88cd-7395a38906cb
ORCID for Sandra Wilks: ORCID iD orcid.org/0000-0002-4134-9415
ORCID for Charles Keevil: ORCID iD orcid.org/0000-0003-1917-7706

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Date deposited: 23 Mar 2018 17:30
Last modified: 18 Feb 2021 16:58

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Contributors

Author: Callum Highmore
Author: Jennifer C Warner
Author: Steve D. Rothwell
Author: Sandra Wilks ORCID iD
Author: Charles Keevil ORCID iD

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