Study Of The Heterogeneity Of Listeria Monocytogenes Influenced By Major Environmental Factors

L.monocytogenes,which can survive under a wide range of salt concentration,temperature and p H conditions,is a globally important food-borne pathogenic bacterium.It has been shown that pathogenic bacteria isolated from different environments exhibit a wide range of variability in phenotypes such as growth,inactivation,virulence,biofilm formation and resistance,i.e.microbial heterogeneity.as a highly diverse species,L.monocytogenes may differ significantly from strain to strain in terms of virulence potential,environmental adaptation and stress response.Therefore,heterogeneity can affect the accuracy of risk assessment results based on a single standard strain,posing a great challenge to risk assessment efforts.In addition,because the prevalent strains in China differ significantly from those abroad,the parameters and models based on foreign strains cannot scientifically characterize the true risk of L.monocytogenes in China,which has a large uncertainty.Along the food chain,constant exposure to various stressors and resistance to stress support the colonization and persistence of L.monocytogenes in various ecological niches along the food chain.In addition,environmental factors of stress conditions remain under human conditions,such as lower gastric p H,higher bile salt concentrations,etc.Environmental factors with different stress conditions have a significant effect on the growth behavior of different strains.Environmental factors have a significant effect on the virulence gene expression of L.monocytogenes and may enhance the viability of the pathogen and its potential cell invasion ability.The virulence heterogeneity of L.monocytogenes may be related to the differences in the virulence gene expression of different strains by environmental factors.In summary,the main elements of this study address the uncertain effects of environmental factors on the growth,virulence heterogeneity and risk assessment of L.monocytogenes as follows:1)The growth data of 146 strains of L.monocytogenes from different regions of China were measured in pure culture media by high-throughput methods,and growth kinetic parameters were obtained by fitting growth prediction models to analyze the growth differences between different strains and enrich the database of L.monocytogenes growth heterogeneity with"Chinese characteristics".L.monocytogenes growth heterogeneity database with"Chinese characteristics",and provide data support for accurate quantitative risk assessment of L.monocytogenes in China.The results showed that even under optimal conditions,the different strains showed a wide range of growth heterogeneity,but there was no significant correlation with isolation source,lineage and serotype.The variability of the delay period was less than the growth rate;and the clinical strains showed the least growth heterogeneity compared to food and environmental isolates and showed better adaptation to the environment.2)Based on the previous chapter,representative strains were screened and growth data of L.monocytogenes from different sources in China under different stress conditions were determined by high-throughput methods,and growth parameters based onμ_max were fitted to assess the growth ability of L.monocytogenes strains and analyze the effects of major environmental factors on growth between different sources,lineages,virulence and serotypes.The effects of major environmental factors on growth heterogeneity among different sources,lineages,virulence and serotypes were analyzed to provide a basis and direction for the control strategy of L.monocytogenes in the food chain to ensure safe food production.The results showed that L.monocytogenes strains exhibited a wide range of growth heterogeneity under different stress conditions,associated with strain isolation source,serotype,genetic lineage and virulence;clinical strains showed less growth heterogeneity than food-derived strains under different stress conditions;serotype 1/2b strains showed different degrees of growth advantage under different stress conditions;The strongly virulent 4b serotype strains showed significantly better growth ability than other strains under acidic conditions.3)By exploring the effects of major environmental factors on the differential expression of important virulence genes（act A,hly,inl A,prf A,plc A）in different strains,the transcriptional response of L.monocytogenes during food-related stress was assessed in depth,the interaction between environmental factors and virulence was determined,and virulence heterogeneity,environmental factors under stress conditions and virulence genes were elucidated to better control the level and risk of L.monocytogenes in the food chain.The results showed that the changes of virulence gene expression in different strains under stress conditions were not the same with reference to the optimum conditions,showing extensive heterogeneity in virulence gene expression;compared with the optimum conditions at 37℃,high salt stress led to a decrease in virulence gene expression in all strains,and acid stress led to an up-regulation of virulence gene expression in some strains;compared with lineage II,the effect of salt stress on the decrease of virulence gene expression in lineage I strains was relatively small;acid stress caused an up-regulation of virulence gene expression in most lineage I strains.In this paper,we evaluated the effects of major environmental factors on the growth heterogeneity and virulence gene expression heterogeneity of L.monocytogenes strains in China and found that:serotype 1/2b strains showed different degrees of growth advantage under different stress conditions,and their high prevalence in clinical and food samples in China may be related to this.The expression of important virulence genes in different strains under stress conditions existed the expression of important virulence genes in different strains under stress conditions differed,and lineage I strains showed some advantages,especially under acid stress conditions.In conclusion,this paper will provide data and theoretical support for accurate risk assessment of L.monocytogenes in China,and provide arguments for elucidating the intrinsic linkage between virulence heterogeneity,stress phenotypes and virulence genes under stress conditions,and revealing the potential genetic mechanisms leading to phenotypic differences,stress responses and virulence changes.