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Strain variability of Listeria monocytogenes under NaCl stress elucidated by a high-throughput microbial growth data assembly and analysis protocol


Aalto-Araneda, Mariella; Pöntinen, Anna; Pesonen, Maiju; Corander, Jukka; Markkula, Annukka; Tasara, Taurai; Stephan, Roger; Korkeala, Hannu (2020). Strain variability of Listeria monocytogenes under NaCl stress elucidated by a high-throughput microbial growth data assembly and analysis protocol. Applied and Environmental Microbiology, 86(6):e02378-19.

Abstract

<jats:named-content content-type="genus-species">Listeria monocytogenes</jats:named-content> causes the severe foodborne illness listeriosis and survives in food-associated environments due to its high stress tolerance. A data assembly and analysis protocol for microbial growth experiments was compiled to elucidate the strain variability of <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> stress tolerance. The protocol includes measurement of growth ability under stress (step 1), selection of a suitable method for growth parameter calculation (step 2), comparison of growth patterns between strains (step 3), and biological interpretation of the discovered differences (step 4). In step 1, <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> strains (<jats:italic>n</jats:italic> = 388) of various serovars and origins grown on media with 9.0% NaCl were measured using a Bioscreen C microbiology reader. Technical variability of the growth measurements was assessed and eliminated. In step 2, the growth parameters determined by Gompertz, modified-Gompertz, logistic, and Richards models and model-free splines were compared, illustrating differences in the suitability of these methods to describe the experimental data. In step 3, hierarchical clustering was used to describe the NaCl tolerance of <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> measured by strain-specific variation in growth ability; tolerant strains had higher growth rates and maximum optical densities and shorter lag phases than susceptible strains. The spline parameter area under the curve best classified “poor,” “average,” and “good” growers. In step 4, the tested <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> lineage I strains (serovars 4b and 1/2b) proved to be significantly more tolerant toward 9.0% NaCl than lineage II strains (serovars 1/2a, 1/2c, and 3a). Our protocol provides systematic tools to gain comparable data for investigating strain-specific variation of bacterial growth under stress.
<jats:bold>IMPORTANCE</jats:bold> The pathogen <jats:named-content content-type="genus-species">Listeria monocytogenes</jats:named-content> causes the foodborne disease listeriosis, which can be fatal in immunocompromised individuals. <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> tolerates several environmental stressors and can persist in food-processing environments and grow in foodstuffs despite traditional control measures such as high salt content. Nonetheless, <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> strains differ in their ability to withstand stressors. Elucidating the intraspecies strain variability of <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> stress tolerance is crucial for the identification of particularly tolerant strains. To enhance reliable identification of variability in bacterial stress tolerance phenotypes, we compiled a large-scale protocol for the entire data assembly and analysis of microbial growth experiments, providing a systematic approach and checklist for experiments on strain-specific growth ability. Our study illustrated the diversity and strain-specific variation of <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> stress tolerance with an unprecedented scope and discovered biologically relevant serovar- and lineage-dependent phenotypes of NaCl tolerance.

Abstract

<jats:named-content content-type="genus-species">Listeria monocytogenes</jats:named-content> causes the severe foodborne illness listeriosis and survives in food-associated environments due to its high stress tolerance. A data assembly and analysis protocol for microbial growth experiments was compiled to elucidate the strain variability of <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> stress tolerance. The protocol includes measurement of growth ability under stress (step 1), selection of a suitable method for growth parameter calculation (step 2), comparison of growth patterns between strains (step 3), and biological interpretation of the discovered differences (step 4). In step 1, <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> strains (<jats:italic>n</jats:italic> = 388) of various serovars and origins grown on media with 9.0% NaCl were measured using a Bioscreen C microbiology reader. Technical variability of the growth measurements was assessed and eliminated. In step 2, the growth parameters determined by Gompertz, modified-Gompertz, logistic, and Richards models and model-free splines were compared, illustrating differences in the suitability of these methods to describe the experimental data. In step 3, hierarchical clustering was used to describe the NaCl tolerance of <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> measured by strain-specific variation in growth ability; tolerant strains had higher growth rates and maximum optical densities and shorter lag phases than susceptible strains. The spline parameter area under the curve best classified “poor,” “average,” and “good” growers. In step 4, the tested <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> lineage I strains (serovars 4b and 1/2b) proved to be significantly more tolerant toward 9.0% NaCl than lineage II strains (serovars 1/2a, 1/2c, and 3a). Our protocol provides systematic tools to gain comparable data for investigating strain-specific variation of bacterial growth under stress.
<jats:bold>IMPORTANCE</jats:bold> The pathogen <jats:named-content content-type="genus-species">Listeria monocytogenes</jats:named-content> causes the foodborne disease listeriosis, which can be fatal in immunocompromised individuals. <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> tolerates several environmental stressors and can persist in food-processing environments and grow in foodstuffs despite traditional control measures such as high salt content. Nonetheless, <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> strains differ in their ability to withstand stressors. Elucidating the intraspecies strain variability of <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> stress tolerance is crucial for the identification of particularly tolerant strains. To enhance reliable identification of variability in bacterial stress tolerance phenotypes, we compiled a large-scale protocol for the entire data assembly and analysis of microbial growth experiments, providing a systematic approach and checklist for experiments on strain-specific growth ability. Our study illustrated the diversity and strain-specific variation of <jats:named-content content-type="genus-species">L. monocytogenes</jats:named-content> stress tolerance with an unprecedented scope and discovered biologically relevant serovar- and lineage-dependent phenotypes of NaCl tolerance.

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Item Type:Journal Article, refereed, original work
Communities & Collections:05 Vetsuisse Faculty > Institute of Food Safety and Hygiene
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Biotechnology
Life Sciences > Food Science
Life Sciences > Applied Microbiology and Biotechnology
Physical Sciences > Ecology
Uncontrolled Keywords:Biotechnology, Food Science, Ecology, Applied Microbiology and Biotechnology
Language:English
Date:3 January 2020
Deposited On:15 Feb 2021 16:07
Last Modified:16 Feb 2021 21:01
Publisher:American Society for Microbiology
ISSN:0099-2240
OA Status:Closed
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1128/aem.02378-19

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