Progress 10/17/16 to 09/30/21
Outputs
Target Audience:Scientists, professionals, and others interested in food safety, particularly Listeria monocytogenes. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?DISSEMINATION DURING THIS PROJECT PERIOD: Three peer-review publications have been published. One oral presentation at a scientific conference (IAFP annual meeting 2021, Phoenix, AZ) was delivered. DISSEMINATION DURING ENITRE PROJECT PERIOD: Eight peer-review publications have been published throughout the duration of this project. Four presentations from graduate students at international scientific conferences were delivered during the duration of this project. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT THIS PROJECT PERIOD: The foodborne pathogen Listeria monocytogenes is responsible for listeriosis, a rare but severe disease in humans, which is acquired primarily through the consumption of contaminated food; particularly "ready-to eat food" is of high risk. Persistence of L. monocytogenes in food production environments is often observed and thus a big challenge for food safety in many areas of food production. A better understanding of these molecular mechanisms is thus urgently needed to increase food safety and to be able to develop better control mechanisms against L. monocytogenes in the long-term. The results of our research this period (described in detail below) provide a better understanding of the contribution and genetics of L. monocytogenes plasmids and plasmid genes to survival in food production environments. We performed the first large-scale survey of plasmids in Listeria monocytogenes. For the first time, we could show that some L. monocytogenes plasmids contribute to UV light stress tolerance. This increased knowledge may provide a better knowledge basis for risk assessment of L. monocytogenes occurrence in food and food processing environments (FPEs) in the future. Objective 2: We have analyzed the contribution of three different L. monocytogenes plasmids to survival under UV-light exposure. For the first time, we could show that some L. monocytogenes plasmids provide increased tolerance to UV light. An increased tolerance to UV light may be advantageous for food-borne pathogens such as L. monocytogenes as UV light is routinely used for disinfection in food production environments. We have performed the first large-scale analysis of plasmid presence, gene content, and conservation in Listeria. We have selected 1921 L. monocytogenes genomes from 33 published studies for our dataset representing 14 highly abundant and common MLST STs. Our results show that overall 54% of all L. monocytogenes strains harbor a putative plasmid. The presence of plasmids was highly different between STs. We show that some highly conserved plasmids are shared between L. monocytogenes strains from different STs, years of isolation, and countries of origin. Thus, some L. monocytogenes plasmids are disseminated globally and are likely important for survival of L. monocytogenes in food and food production environments. To get deeper insight into gene expression patterns of L. monocytogenes plasmids, we have analyzed the gene expression of three different L. monocytogenes strains with three different plasmids subjected to different stress conditions. Differential gene expression (DE) analysis of these plasmids reveals that the number of DE L. monocytogenes plasmid genes ranged from 30 to 45 with log2 fold changes of -2.2 to 6.8 depending on the plasmid. Plasmid-encoded non-coding RNAs were consistently among the highest expressed genes. In addition, we found that the gene expression patterns of the two different L. monocytogenes plasmids were highly divergent despite identical experimental treatments. This suggests plasmid-specific gene expression responses to environmental stimuli and differential plasmid regulation mechanisms between L. monocytogenes strains. Our findings further our understanding of the dynamic expression of L. monocytogenes plasmid-encoded genes in diverse environmental conditions and highlight the need to expand the study of L. monocytogenes plasmid genes' function in stress response. IMPACT OVER ENTIRE PROJECT PERIOD: The results from this project significantly broaden our knowledge about L. monocytogenes plasmids and their role in survival under stress conditions relevant for food and food production environments. Due to the modular nature of Listeria plasmids, our results apply also to other L. monocytogenes strains harboring similar plasmids. As stated above, this increased knowledge may provide a better basis for tiske assessment of L. monocytogenes occurrence in food and FPEs in the future. ACCOMPLISHMENTS OVER ENTIRE PROJECT PERIOD: Objective 1: We have analyzed the genomes of L. monocytogenes strains of different STs (ST1, ST5, ST21, ST37, ST204) from an Austrian food processing plant to determine genetic mechanisms for persistence in this FPE. We show that plasmids are most likely key for the persistence of the ST5 strains in this food processing environment and that plasmids were most likely transferred between different L. monocytogenes STs in this FPE. We have performed transcriptome sequencing of two L. monocytogenes strains under different stress conditions [lactic acid (1%, pH 3.4), oxidative stress (0.01% hydrogen peroxide)]. Both L. monocytogenes strains used harbor different plasmids; our results also allow first insights into L. monocytogenes plasmid gene expression. Our results reveal massive changes in gene expression in during response to lactic acid stress with more than 2100 differentially expressed (DE) genes. Most strikingly, we found massive upregulation of a gene annotated as a putative Arsenate reductase, which is in line with previous reports indicating massive upregulation of this yet uncharacterized gene in stress conditions. Most notably, the sigmaB-dependent non-coding RNA Rli47 was by far the most highly expressed gene in both strains, suggesting an important role of this non-coding RNA in stress response. We aimed to uncover genes that are differentially expressed during co-cultivation of a L. monocytogenes ST121 strain with other bacteria. L. monocytogenes was co-cultured in broth or on plates with either a gram-negative (Psychrobacter) or a gram-positive (Brevibacterium) cheese rind bacteria. Broth co-cultivations yielded up to 379 and 593 DE genes while plate co-cultivations significantly affected the expression of up to 186 and 490 L. monocytogenes genes, respectively. A cobalamin- dependent gene cluster, responsible for the metabolism of ethanolamine and propylene glycol, significantly increased in expression. This suggests that the cobalamin-dependent gene cluster may be of high importance for survival of L. monocytogenes in food and FPEs. The non-coding RNA rli47 was also significantly upregulated in co-culture on plates accounting for 50-90% of the total reads in these conditions. The upregulation and high expression levels of rli47 under these conditions provides additional evidence for an important role in stress response during mixed-culture conditions. Objective 2: We have generated isogenic pairs of strains from 3 different sequence types (ST5, ST8, ST121) where the wildtype contains the plasmids, and the deletion mutants are devoid of their plasmids. Using these pairs of strains, we could show that plasmids significantly contribute to survival under disinfectants (Benzalkonium chloride), salt stress (15% NaCl), lactic acid (1%), oxidative stress (0.01% hydrogen peroxide), and heat stress (50 or 55 degrees Celsius). We provide first detailed insights into the contribution of L. monocytogenes plasmids to survival under different stress conditions found in food and FPEs. Objective 3: The transcriptome sequencing of L. monocytogenes ST121 strain 6179 during co-cultivation with cheese rind bacteria revealed that the RHS gene (and the other genes in the RHS locus) show a very low expression level and some of them are downregulated under the conditions applied. This suggests that the RHS gene may not be involved in competition with other bacteria - at least under the conditions applied - but this will need further analyses for verification. As a complementary approach, we cloned the C-terminal part of the RHS gene, which we believe represents a toxin domain, into the E. coli expression vector pBAD. Expression of the RHS toxin domain revealed a toxic effect on E. coli growth and survival. We could also show that the toxic effect is most likely due to degradation of tRNAs.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Anast JM, Bobik TA, Schmitz-Esser S. 2020. The cobalamin-dependent gene cluster of Listeria monocytogenes: Implications for virulence, stress response, and food safety. Front Microbiol; 11:601816. doi:10.3389/fmicb.2020.601816
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Schmitz-Esser S, Anast JM, Cortes BW. 2021. A large-scale sequencing-based survey of plasmids in Listeria monocytogenes reveals global dissemination of plasmids. Front Microbiol. 12:653155. doi:10.3389/fmicb.2021.653155
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Anast JM, Schmitz-Esser S. 2021. Certain Listeria monocytogenes plasmids contribute to increased UVC ultraviolet light stress. FEMS Microbiol Lett. 368(17):fnab123. doi: 10.1093/femsle/fnab123.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Cortes B, Seggerman FM, Stroud M, Schmitz-Esser S. 2021. Preliminary Analysis of the Role of the Noncoding RNA Rli47 in the Listeria monocytogenes Response to Lactic Acid Stress. Technical presentation, IAFP (International Association for Food Protection) Annual meeting. July 18-21, 2021, Phoenix, AZ, USA
|
Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Scientists, professionals,and others interested in food safety, particularly Listeria monocytogenes Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Twopeer-review publications have been published. What do you plan to do during the next reporting period to accomplish the goals?Characterize the contribution of the non-coding RNA Rli47 and the putative arsenate reductase to stress survival. using deletion mutants of Rli47 and the putative Arsenate Reductase. Deletion mutants of Rli47 have successfully been generated. Characterization of the contribution of different plasmid genes (clpL and Multicopper oxidase) to stress response. Prepare and submit publication on plasmid (sense and antisense) gene expression.
Impacts
What was accomplished under these goals?
IMPACT THIS PROJECT PERIOD: The foodborne pathogen Listeria monocytogenes is responsible for listeriosis, a rare but severe disease in humans, which is acquired primarily through the consumption of contaminated food; particularly "ready-to-eat food" is of high risk. Persistence of L. monocytogenes in food production environments is often observed and thus a big challenge for food safety in many areas of food production. A better understanding of these molecular mechanisms is thus urgently needed to increase food safety and to be able to develop better control mechanisms against L. monocytogenes in the long-term. The results of our research this period (described in detail below) provide a better understanding of the contribution and genetics of L. monocytogenes chromosomal and plasmid genes to survival in food production environments. We performed the first large-scale survey of plasmids in Listeria monocytogenes. We show that non-coding RNAs both on the chromosome and the plasmids may be crucial for L. monocytogenes stress survival. This increased knowledge may provide a better knowledge basis for risk assessment of L. monocytogenes occurrence in food and food processing environments in the future. Objective 1: We have performed transcriptome sequencing of two L. monocytogenes strains under different stress conditions [lactic acid (1%, pH 3.4), oxidative stress (0.01% hydrogen peroxide)] and during co-cultivation with commonly found bacteria in cheese rinds and in cheese production environments. Both L. monocytogenes strains used harbor different plasmids; our results also allow first insights into L. monocytogenes plasmid gene expression which will be reported below in Objective 2. Our results reveal massive changes in gene expression in during response to lactic acid stress with more than 2100 differentially expressed (DE) genes. We show upregulation of many genes of the sigmaB regulon, the main stress regulon of L. monocytogenes. Most strikingly, we found massive upregulation of a gene annotated as a putative Arsenate reductase, which is in line with previous reports indicating massive upregulation of this yet uncharacterized gene in stress conditions. We are currently working on the functional characterization of this gene and its role in stress survival by generating a chromosomal deletion mutant. Most notably, the sigmaB-dependent non-coding RNA Rli47 was by far the most highly expressed gene in both strains, suggesting an important role of this non-coding RNA in stress response. A manuscript describing these results has been published (Cortes et al., 2020 Front Microbiol). We aimed to uncover genes that are differentially expressed during co-cultivation of a L. monocytogenes ST121 strain with other bacteria. L. monocytogenes was co-cultured in broth or on plates with either a gram-negative (Psychrobacter) or a gram-positive (Brevibacterium) cheese rind bacteria. Broth co-cultivations with Brevibacterium or Psychrobacter yielded up to 379 and 593 DE genes while plate co-cultivations significantly affected the expression of up to 186 and 490 L. monocytogenes genes, respectively. A cobalamin- dependent gene cluster, responsible for the metabolism of ethanolamine and propylene glycol, significantly increased in expression with 67 of the 68 genes in this cluster upregulated. This suggests that the cobalamin-dependent gene cluster may be of high importance for survival of L. monocytogenes in food and food processing environments. The non-coding RNA rli47 was also significantly upregulated in co-culture on plates incubated for 72 hours with Psychrobacter accounting for 50-90% of the total reads mapped to L. monocytogenes in these conditions. The upregulation and high expression levels of rli47 under these conditions provides additional evidence for an important role in stress response during mixed-culture conditions. A manuscript describing the gene expression results has been published (Anast et al., 2020 PlosOne). Objective 2: We have performed the first large-scale analysis of plasmid presence, gene content, and conservation in Listeria. We have selected 1921 L. monocytogenes genomes from 33 published studies for our dataset representing 14 highly abundant and common MLST STs. Our dataset also contains an equal distribution of clinical, food and environmental strains. Our results show that overall 54% of all L. monocytogenes strains harbor a putative plasmid. The presence of plasmids was highly different between STs and source of the strains with plasmids being significantly more abundant in food and food production strains and in ST121, 5, 3, 8, and 204 strains. The abundance of select stress response genes was also highly different among STs. We show that some highly conserved plasmids are shared (being virtually identical) between L. monocytogenes strains from different STs, years of isolation, and countries of origin. Thus, some highly conserved L. monocytogenes plasmids are disseminated globally and are likely important for survival of L. monocytogenes in food and food production environments. A manuscript describing these results has been submitted for review. To get deeper insight into gene expression patterns of L. monocytogenes plasmids, we are currently analyzing the gene expression of three different L. monocytogenes strains with three different plasmids subjected to different stress conditions. These analyses will significantly increase our understanding of plasmid gene expression focusing on stress response genes and also analyzing antisense gene expression. We identified significant upregulation of yet uncharacterized noncoding RNAs (e.g. rli23, rli28) in response to stress exposure. These analyses are ongoing and a manuscript is expected to be submitted for publication in the first half of 2021. Objective 3: We haven't worked on this objective during this reporting period
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Cortes BW, Naditz AL, Anast JM, Schmitz-Esser S. 2020. Transcriptome sequencing of Listeria monocytogenes reveals major gene expression changes in response to lactic acid stress exposure but a less pronounced response to oxidative stress. Front Microbiol. 10:3110.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Anast JM, Schmitz-Esser S. 2020. The transcriptome of Listeria monocytogenes during co-cultivation with cheese rind bacteria suggests adaptation by induction of ethanolamine and 1,2-propanediol catabolism pathway genes. PLoS One. 15(7):e0233945.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2020
Citation:
Naditz, Annabel, "A comparative analysis of Listeria monocytogenes plasmids: Presence, contribution to stress and conservation" (2020). Graduate Theses and Dissertations. 17859.
https://lib.dr.iastate.edu/etd/17859
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Dairy scientists and others interested in food safety Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?One journal article has been published. One graduate student delivered an oral presentation on the results of the co-cultivation work at the 2019 International Association for Food Protection (IAFP) meeting in Louisville, KY (July 2019). This graduate student also gave a poster presentation on the co-cultivation work at the International Symposium on Problems in Listeria and Listeriosis (ISOPOL) in Toronto, Canada (Sept 2019). What do you plan to do during the next reporting period to accomplish the goals?Perform detailed analysis of antisense gene expression in L. monocytogenes plasmids. Generate chromosomal deletion mutants of Rli47 and the putative Arsenate Reductase and characterize their contribution to stress survival. Clone plasmid genes (clpL and Multicopper oxidase) into Listeria expression vector pNZ44 to analyze the contribution of these genes to stress response.
Impacts
What was accomplished under these goals?
IMPACT THIS PROJECT PERIOD: The facultative intracellular pathogen Listeria monocytogenes is responsible for listeriosis, a rare but severe disease in humans and animals, which is acquired primarily through the consumption of contaminated food; particularly "ready-to-eat food" is of high risk. Persistence of L. monocytogenes in food production environments is a big challenge for food safety in many areas of food production as persistence of Listeria in food production is widely observed. A better understanding of these molecular mechanisms is thus urgently needed to increase food safety and to be able to develop better control mechanisms against L. monocytogenes in the long-term. The results of our research this period (described below) provide a better understanding of the contribution and genetics of L. monocytogenes chromosomal and plasmid genes to survival in food production environments. We show that non-coding RNAs both on the chromosome and the plasmids may be crucial for L. monocytogenes stress survival. This increased knowledge may provide a better knowledge basis for risk assessment of L. monocytogenes occurrence in food and food processing environments in the future. Objective 1... Genome and transcriptome sequencing of ST121 L. monocytogenes strains. We have performed transcriptome sequencing of one L. monocytogenes ST121 and one ST8 strain under different stress conditions [lactic acid (1%, pH 3.4), oxidative stress (0.01% hydrogen peroxide)] and during co-cultivation with commonly found bacteria in cheese rinds and in cheese production environments. Both L. monocytogenes strains used harbor plasmids of 62 and 88 kbp; our results also allow first insights into L. monocytogenes plasmid gene expression which will be reported below in Objective 2. Our results reveal massive changes in gene expression in during response to lactic acid stress with more than 2100 differentially expressed genes. We show upregulation of many genes of the sigmaB regulon, the main stress regulon of L. monocytogenes. Most strikingly, we found massive upregulation of a gene annotated as a putative Arsenate reducatse, which is in line with previous reports indicating massive upregulation of this yet uncharacterized gene in stress response. Future studies will focus on the functional characterization of this gene and its role in stress survival. Most notably, the sigmaB-dependent non-coding RNA Rli47 was by far the most highly expressed gene in both strains, accounting for an average of 28% and 38% of all mapped reads in the respective chromosomal transcriptomes; suggesting an important role of this non-coding RNA in stress response. A manuscript describing the results is currently under revision. We aimed to uncover genes that are differentially expressed during co-cultivation of a L. monocytogenes ST121 strain with other bacteria. L. monocytogenes was co-cultured in brain heart infusion broth or on plates with either a gram-negative (Psychrobacter) or a gram-positive (Brevibacterium) cheese rind bacteria. Broth co-cultivations with Brevibacterium or Psychrobacter yielded up to 379 and 593 DE genes while plate co-cultivations significantly affected the expression of up to 186 and 490 L. monocytogenes genes, respectively. A cobalamin- dependent gene cluster, responsible for the metabolism of ethanolamine and propylene glycol, significantly increased in expression with 67 of the 68 genes in this cluster upregulated when co-cultivated in broth with Psychrobacter. This suggests that the cobalamin-dependent gene cluster may be of high importance for survival of L. monocytogenes in food and food processing environments. The expression of virulence genes encoding the Listeria adhesion protein LAP and Listeriolysin O were highly upregulated in both plate and broth co-cultivations. The non-coding RNA rli47 was also significantly upregulated in co-culture on plates incubated for 72 hours with Psychrobacter accounting for 50-90% of the total reads mapped to L. monocytogenes in these conditions. The upregulation and high expression levels of rli47 under these conditions provides additional evidence for an important role in stress response during mixed-culture conditions. A manuscript describing these results has been submitted. Objective 2... Determine the contribution of plasmids and prophages and biofilms on ST121 L. monocytogenes persistence. Our results on plasmid contribution to stress response have been published (Naditz et al., 2019). This publication is the first to provide detailed insights into the contribution of L. monocytogenes plasmids to survival under different stress conditions found in food and food production environments. Due to the modular nature of Listeria plasmids, our results apply also to other L. monocytogenes strains harboring similar plasmids. The results described in this publication thus significantly broaden our knowledge about L. monocytogenes plasmids and their role in survival under stress conditions relevant for food and food production environments. Our transcriptome sequencing allowed for the first time in-depth analysis of plasmid gene expression in Listeria. Lactic acid exposure resulted in upregulation of the stress response gene clpL, among others, on the 6179 plasmid. In R479a, a number of uncharacterized plasmid genes were upregulated, indicating a potential role in stress response. Furthermore, an average of 65% of all mapped transcriptome reads for the R479a plasmid following acid stress were mapped to an intergenic region bearing similarity to riboswitches involved in transition metal resistance. This putative plasmid-encoded riboswitch is potentially involved in response to acid exposure and will be characterized functionally in the future. A manuscript describing these results has been submitted. Objective 3... Determine the role of the RHS locus found in ST121 L. monocytogenes in persistence. The transcriptome sequencing of L. monocytogenes ST121 strain 6179 during co-cultivation with cheese rind bacteria revealed that the RHS gene (and the other genes in the RHS locus/operon) show a very low expression level and some of them are downregulated under the conditions applied. This might suggest that the RHS gene may not be involved in competition with other bacteria - at least under the conditions applied - but this will need further and deeper analyses for verification. As a complementary approach, we cloned the C-terminal part of the RHS gene, which we believe represents a toxin domain, into the E. coli expression vector pBAD. Expression of the RHS toxin domain revealed a toxic effect on E. coli growth and survival. Using a bioanalyzer, we could also show that the toxic effect is most likely due to degradation of tRNAs. Objective 4... Determine the role of the RHS locus found in ST121 L. monocytogenes in persistence. We haven't worked on this objective during this reporting period.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Naditz AL, Dzieciol M, Wagner M, Schmitz-Esser S. 2019. Plasmids contribute to food processing environment-associated stress survival in three Listeria monocytogenes ST121, ST8, and ST5 strains. Int J Food Microbiol. 299:39-46. doi: 10.1016/j.ijfoodmicro.2019.03.016
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Dairy scientists and others interested in food safety Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?One journal article published. One graduate student delivered oral presentation on the results of the plasmid work at the 2018 ADSA (American Dairy Science Organization) National Meeting in Knoxville, TN, July 2018. What do you plan to do during the next reporting period to accomplish the goals?The aims for the next reporting period are analyzing the transcriptome data obtained and writing and submission of two manuscripts describing the results of the transcriptome sequencing experiments with one manuscript forstress condition transcriptomicsand one manuscript for the competition conditions transcriptomics. It is planned to express the RHS gene and domains of this genes in E.coli to determine potential toxic effects.
Impacts
What was accomplished under these goals?
IMPACT THIS PROJECT PERIOD:The facultative intracellular pathogen Listeria (L.) monocytogenes is responsible for listeriosis, a rare but severe disease in humans and animals, which is acquired primarily through the consumption of contaminated food; particularly "ready-to-eat food" is of high risk. Persistence of L. monocytogenes in food production environments is a big challenge for food safety in many areas of food production as persistence of Listeria in food production is widely observed. A better understanding of these molecular mechanisms is thus urgently needed to increase food safety and to be able to develop better control mechanisms against L. monocytogenes in the long-term. The results of our research this period (described below) provide a better understanding of the contribution and genetics of L. monocytogenes plasmids to survival in food production environments. This increased knowledge may provide a better knowledge basis for risk assessment of L. monocytogenes occurrence in food and food processing environments in the future. Objective 1... Genome and transcriptome sequencing of ST121 L. monocytogenes strains We have analyzed the genomes of L. monocytogenes strains of different STs (ST1, ST5, ST21, ST37, ST204) from an Austrian food processing plant to determine genetic mechanisms for persistence in this FPE. One of these strains has also been used for plasmid experiments in Objective 2. We show that plasmids are most likely key for the persistence of the ST5 strains in this food processing environment. Our results also show that plasmids were most likely transferred between different L. monocytogenes STs in this food production plant. The results of this study have been published in a peer-review publication (Muhterem-Uyar et al., 2018, Front Microbiol). We show that plasmids can be important for survival of Listeria monocytogenes in food production environments and that plasmids can be transferred between different L. monocytogenes strains in the same food production environment. We have performed transcriptome sequencing of L. monocytogenes ST121 and ST8 strains under different stress conditions [lactic acid (1%, pH 3.4), oxidative stress (0.01% hydrogen peroxide)] and during co-cultivation with cheese rind bacteria. We have sequenced 48 samples using Illumina NextSeq sequencing technology. We are currently analyzing the data and preparing a publication describing the results of these sequencing approaches. Objective 2... Determine the contribution of plasmids and prophages and biofilms on ST121 L. monocytogenes persistence We have generated isogenic pairs of strains from 3 different sequence types (ST5, ST8, ST121) where the wildtype contains the plasmids, and the deletion mutants are devoid of their plasmids. Using these pairs of strains, we could show that plasmids statistically significantly contribute to survival under disinfectants (Benzalkonium chloride), salt stress (15% NaCl), lactic acid (1%), oxidative stress (0.01% hydrogen peroxide), and heat stress (50 or 55 degrees Celsius). The results have been submitted for publication and the manuscript is currently under revision. This publication will be the first to provide detailed insights into the contribution of L. monocytogenes plasmids to survival under different stress conditions found in food and food production environments. We also provide a detailed sequence analysis of the plasmids of the analyzed strains. Due to the modular nature of Listeria plasmids, our results apply also to other L. monocytogenes strains harboring similar plasmids. The results described in this manuscript will thus significantly broaden our knowledge about L. monocytogenes plasmids and their role in survival under stress conditions relevant for food and food production environments. Objective 3... Determine the role of the RHS locus found in ST121 L. monocytogenes in persistence We have performed transcriptome sequencing of L. monocytogenes ST121 strain 6179 during co-cultivation with cheese rind bacteria (Brevibacterium, Psychrobacter; in broth and on agar plates) to quantitatively analyze the expression of all RHS locus genes and additionally other genes possibly contributing to competition. We are currently analyzing the results of the transcriptome sequencing. Our preliminary results indicate that the RHS gene (and the other genes in the RHS locus/operon) are downregulated under the co-cultivation conditions applied. This might suggest that the RHS gene may not be involved in competition with other bacteria, but will need further and deeper analyses for verification. As a complementary approach, we have started cloning the RHS gene and domains of the RHS gene into E. coli expression vectors (pET16b, pBAD). Objective 4... Analyze the virulence of ST121 L. monocytogenes using transcriptome sequencing and deletion mutants We haven't worked on this objective during this reporting period
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Muhterem-Uyar M, Ciolacu L, Wagner KH, Wagner M, Schmitz-Esser S, Stessl B. New Aspects on Listeria monocytogenes ST5-ECVI Predominance in a Heavily Contaminated Cheese Processing Environment. Front Microbiol. 2018;9:64. doi:
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Naditz, AL, D. Dhar, S. Schmitz-Esser (2018). Elucidating the contribution of Listeria monocytogenes plasmids to survival in dairy foods and production facilities . J. Dairy Sci. Vol. 101, Suppl. 2 Dairy Foods III: Microbiology and Health. Abstract: 397.
|
Progress 10/17/16 to 09/30/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?One journal article. What do you plan to do during the next reporting period to accomplish the goals?The aims for the next reporting period are writing and submission of a manuscript describing the results of the plasmid experiments. It is planned to perform transcriptome sequencing of L. monocytogenes strains under stress and competition conditions and analyze the results of the transcriptome sequencing.
Impacts
What was accomplished under these goals?
IMPACT THIS PROJECT PERIOD: The facultative intracellular pathogen Listeria (L.) monocytogenes is responsible for listeriosis, a rare but severe disease in humans and animals, which is acquired primarily through the consumption of contaminated food; particularly "ready-to-eat food" is of high risk. Persistence of L. monocytogenes in food production environments is a big challenge for food safety in many areas of food production as persistence of Listeria in food production is widely observed. A better understanding of these molecular mechanisms is thus urgently needed to increase food safety and to be able to develop better control mechanisms against L. monocytogenes in the long-term. The results of our research this period (described below) not only provide a better understanding of the genetics of ST121 L. monocytogenes, but may also in the future provide a better knowledge basis for risk assessment of L. monocytogenes occurrence in food and food processing environments. Objective 1... Genome and transcriptome sequencing of ST121 L. monocytogenes strains For this objective, we have determined and analyzed the genomes of 70 ST121 L. monocytogenes strains. The results have been published in a peer-review journal article (Rychli et al., 2017 PloS One). We could show that the main differences between the strains were found in prophage content and prophage conservation. We also detected distinct highly conserved subtypes of prophages inserted at the same genomic locus. While some of the prophages showed more than 99.9% similarity between strains from different sources and years, other prophages showed a higher level of diversity. 81.4% of the strains harbored virtually identical plasmids. 97.1% of the ST121 strains contain a truncated internalin A (inlA) gene. Only one of the seven human ST121 isolates encodes a full-length inlA gene, illustrating the need of better understanding their survival and virulence mechanisms. These results do not only provide a better understanding of the genetics of ST121 L. monocytogenes, but may also in the future provide a better knowledge basis for risk assessment of L. monocytogenes occurrence in food and food processing environments. We have additionally analyzed the genomes of L. monocytogenes ST5 strains from an Austrian food processing plant to determine genetic mechanisms for persistence in this FPE. Some of these strains have also been used for plasmid experiments in Objective 2. A publication describing the results of this study is currently under review. Objective 2... Determine the contribution of plasmids and prophages and biofilms on ST121 L. monocytogenes persistence In this objective, we have generated plasmid deletion mutants of various L. monocytogenes strains: Three ST121 strains, two ST5 and one ST8 strain. We have included other L. monocytogenes STs in our analyses, to provide a broader basis for analyzing the contribution of plasmids on persistence of L. monocytogenes. We have generated isogenic pairs of strains where the wildtype contains the plasmids, and the deletion mutants are devoid of their plasmids. Using these pairs of strains, we could show that plasmids statistically significantly contribute to survival under disinfectants (Benzalkonium chloride), salt stress (15% NaCl), lactic acid (1%), oxidative stress (0.01% hydrogen peroxide), and temperature stress (50 or 55 degrees Celsius). We are currently preparing a publication describing the plasmid results. Objective 3... Determine the role of the RHS locus found in ST121 L. monocytogenes in persistence For this objective, we have started co-cultivation experiments of ST121 strains with cheese bacteria to identify experimental conditions and settings that are suitable for gene expression analysis. Using Reverse Transcriptase PCR we could show that the RHS gene of ST121 strains is expressed under competition conditions. We are also determining experimental protocols and systems for cloning the ST121 RHS gene into E. coli. In a next step, we will perform transcriptome sequencing of ST121 strains under the identified competition conditions to quantitatively analyze the expression of all RHS locus genes and additionally other genes possibly contributing to competition. Objective 4... Analyze the virulence of ST121 L. monocytogenes using transcriptome sequencing and deletion mutants We haven't worked on this objective during this reporting period.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Rychli K, Wagner EM, Ciolacu L, Zaiser A, Tasara T, Wagner M, Schmitz-Esser S. Comparative genomics of human and non-human Listeria monocytogenes sequence type 121 strains. PLoS One. 2017 May 4;12(5):e0176857
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