Listeria monocytogenes Sublethal Injury and Viable-but-Nonculturable State Induced by Acidic Conditions and Disinfectants
ABSTRACT The dormancy continuum hypothesis states that in response to stress, cells enter different stages of dormancy ranging from unstressed living cells to cell death, in order to ensure their long-term survival under adverse conditions. Exposure of Listeria monocytogenes cells to sublethal stres...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2021-12-01
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| Series: | Microbiology Spectrum |
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| Online Access: | https://journals.asm.org/doi/10.1128/Spectrum.01377-21 |
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| author | Marianna Arvaniti Panagiotis Tsakanikas Vasiliki Papadopoulou Artemis Giannakopoulou Panagiotis Skandamis |
| author_facet | Marianna Arvaniti Panagiotis Tsakanikas Vasiliki Papadopoulou Artemis Giannakopoulou Panagiotis Skandamis |
| author_sort | Marianna Arvaniti |
| collection | DOAJ |
| description | ABSTRACT The dormancy continuum hypothesis states that in response to stress, cells enter different stages of dormancy ranging from unstressed living cells to cell death, in order to ensure their long-term survival under adverse conditions. Exposure of Listeria monocytogenes cells to sublethal stressors related to food processing may induce sublethal injury and the viable-but-nonculturable (VBNC) state. In this study, exposure to acetic acid (AA), hydrochloric acid (HCl), and two disinfectants, peracetic acid (PAA) and sodium hypochlorite (SH), at 20°C and 4°C was used to evaluate the potential induction of L. monocytogenes strain Scott A into different stages of dormancy. To differentiate the noninjured subpopulation from the total population, tryptic soy agar with 0.6% yeast extract (TSAYE), supplemented or not with 5% NaCl, was used. Sublethally injured and VBNC cells were detected by comparing plate counts obtained with fluorescence microscopy and by using combinations of carboxyfluorescein and propidium iodide (viable/dead cells). Induction of sublethal injury was more intense after PAA treatment. Two subpopulations were detected, with phenotypes of untreated cells and small colony variants (SCVs). SCVs appeared as smaller colonies of various sizes and were first observed after 5 min of exposure to 5 ppm PAA at 20°C. Increasing the stress intensity from 5 to 40 ppm PAA led to earlier detection of SCVs. L. monocytogenes remained culturable after exposure to 20 and 30 ppm PAA for 3 h. At 40 ppm, after 3 h of exposure, the whole population was considered nonculturable, while cells remained metabolically active. These results corroborate the induction of the VBNC state. IMPORTANCE Sublethally injured and VBNC cells may evade detection, resulting in underestimation of a food product’s microbial load. Under favorable conditions, cells may regain their growth capacity and acquire new resistant characteristics, posing a major threat for public health. Induction of the VBNC state is crucial for foodborne pathogens, such as L. monocytogenes, the detection of which relies almost exclusively on the use of culture recovery techniques. In the present study, we confirmed that sublethal injury is an initial stage of dormancy in L. monocytogenes that is followed by the VBNC state. Our results showed that PAA induced SCVs (a phenomenon potentially triggered by external factors) and the VBNC state in L. monocytogenes, indicating that tests of lethality based only on culturability may provide false-positive results regarding the effectiveness of an inactivation treatment. |
| first_indexed | 2024-12-20T15:30:59Z |
| format | Article |
| id | doaj.art-87efd0b3fb31460abc87a58f486f902d |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-12-20T15:30:59Z |
| publishDate | 2021-12-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-87efd0b3fb31460abc87a58f486f902d2022-12-21T19:35:36ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972021-12-019310.1128/Spectrum.01377-21Listeria monocytogenes Sublethal Injury and Viable-but-Nonculturable State Induced by Acidic Conditions and DisinfectantsMarianna Arvaniti0Panagiotis Tsakanikas1Vasiliki Papadopoulou2Artemis Giannakopoulou3Panagiotis Skandamis4Laboratory of Food Quality Control and Hygiene, Department of Food Science and Human Nutrition, Agricultural University of Athens, Athens, GreeceLaboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Human Nutrition, Agricultural University of Athens, Athens, GreeceLaboratory of Food Quality Control and Hygiene, Department of Food Science and Human Nutrition, Agricultural University of Athens, Athens, GreeceLaboratory of Food Quality Control and Hygiene, Department of Food Science and Human Nutrition, Agricultural University of Athens, Athens, GreeceLaboratory of Food Quality Control and Hygiene, Department of Food Science and Human Nutrition, Agricultural University of Athens, Athens, GreeceABSTRACT The dormancy continuum hypothesis states that in response to stress, cells enter different stages of dormancy ranging from unstressed living cells to cell death, in order to ensure their long-term survival under adverse conditions. Exposure of Listeria monocytogenes cells to sublethal stressors related to food processing may induce sublethal injury and the viable-but-nonculturable (VBNC) state. In this study, exposure to acetic acid (AA), hydrochloric acid (HCl), and two disinfectants, peracetic acid (PAA) and sodium hypochlorite (SH), at 20°C and 4°C was used to evaluate the potential induction of L. monocytogenes strain Scott A into different stages of dormancy. To differentiate the noninjured subpopulation from the total population, tryptic soy agar with 0.6% yeast extract (TSAYE), supplemented or not with 5% NaCl, was used. Sublethally injured and VBNC cells were detected by comparing plate counts obtained with fluorescence microscopy and by using combinations of carboxyfluorescein and propidium iodide (viable/dead cells). Induction of sublethal injury was more intense after PAA treatment. Two subpopulations were detected, with phenotypes of untreated cells and small colony variants (SCVs). SCVs appeared as smaller colonies of various sizes and were first observed after 5 min of exposure to 5 ppm PAA at 20°C. Increasing the stress intensity from 5 to 40 ppm PAA led to earlier detection of SCVs. L. monocytogenes remained culturable after exposure to 20 and 30 ppm PAA for 3 h. At 40 ppm, after 3 h of exposure, the whole population was considered nonculturable, while cells remained metabolically active. These results corroborate the induction of the VBNC state. IMPORTANCE Sublethally injured and VBNC cells may evade detection, resulting in underestimation of a food product’s microbial load. Under favorable conditions, cells may regain their growth capacity and acquire new resistant characteristics, posing a major threat for public health. Induction of the VBNC state is crucial for foodborne pathogens, such as L. monocytogenes, the detection of which relies almost exclusively on the use of culture recovery techniques. In the present study, we confirmed that sublethal injury is an initial stage of dormancy in L. monocytogenes that is followed by the VBNC state. Our results showed that PAA induced SCVs (a phenomenon potentially triggered by external factors) and the VBNC state in L. monocytogenes, indicating that tests of lethality based only on culturability may provide false-positive results regarding the effectiveness of an inactivation treatment.https://journals.asm.org/doi/10.1128/Spectrum.01377-21Listeria monocytogenesVBNC stateacid stressdisinfectantsperacetic acidsublethal injury |
| spellingShingle | Marianna Arvaniti Panagiotis Tsakanikas Vasiliki Papadopoulou Artemis Giannakopoulou Panagiotis Skandamis Listeria monocytogenes Sublethal Injury and Viable-but-Nonculturable State Induced by Acidic Conditions and Disinfectants Microbiology Spectrum Listeria monocytogenes VBNC state acid stress disinfectants peracetic acid sublethal injury |
| title | Listeria monocytogenes Sublethal Injury and Viable-but-Nonculturable State Induced by Acidic Conditions and Disinfectants |
| title_full | Listeria monocytogenes Sublethal Injury and Viable-but-Nonculturable State Induced by Acidic Conditions and Disinfectants |
| title_fullStr | Listeria monocytogenes Sublethal Injury and Viable-but-Nonculturable State Induced by Acidic Conditions and Disinfectants |
| title_full_unstemmed | Listeria monocytogenes Sublethal Injury and Viable-but-Nonculturable State Induced by Acidic Conditions and Disinfectants |
| title_short | Listeria monocytogenes Sublethal Injury and Viable-but-Nonculturable State Induced by Acidic Conditions and Disinfectants |
| title_sort | listeria monocytogenes sublethal injury and viable but nonculturable state induced by acidic conditions and disinfectants |
| topic | Listeria monocytogenes VBNC state acid stress disinfectants peracetic acid sublethal injury |
| url | https://journals.asm.org/doi/10.1128/Spectrum.01377-21 |
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