Differential gene expression profiling of Listeria monocytogenes in Cacciatore and Felino salami to reveal potential stress resistance biomarkers.

The current study reports a) the in situ transcriptional profiles of Listeria monocytogenes in response to fermented sausage stress and b) an approach in which in situ RT-qPCR data have been combined with advanced statistical techniques to discover potential stress resistance or cell viability biomarkers. Gene expression profiling of the pathogen has been investigated using RT-qPCR to understand how L. monocytogenes responds to the conditions encountered during the fermentation and ripening of sausages. A cocktail of five L. monocytogenes strains was inoculated into the batter of Cacciatore and Felino sausages. The RT-qPCR data showed that the acidic and osmotic stress-related genes were up-regulated. The transcripts of the lmo0669 gene increased during the fermentation and ripening of Cacciatore, whereas gbuA and lmo1421 were up-regulated during the ripening of Felino and Cacciatore, respectively. sigB expression was induced in both sausages throughout the whole process. Finally, the virulence-related gene prfA was down-regulated during the fermentation of Cacciatore. The multivariate gene expression profiling analysis suggested that sigB and lmo1421 or sigB and gbuA could be used as different types of stress resistance biomarkers to track, for example, stress resistance or cell viability in fermented sausages with short (Cacciatore) or long (Felino) maturation times, respectively.

[1]  S. Derzelle,et al.  Tool for Quantification of Staphylococcal Enterotoxin Gene Expression in Cheese , 2010, Applied and Environmental Microbiology.

[2]  D. Sutcliffe.  THE MICROBIOLOGICAL QUALITY OF WATER , 1997 .

[3]  F. Lücke.  Utilization of microbes to process and preserve meat. , 2000, Meat science.

[4]  R. Beumer.  Microbiological hazards and their control: bacteria , 2001 .

[5]  L. Jespersen,et al.  Survival of Listeria monocytogenes in simulated gastrointestinal system and transcriptional profiling of stress- and adhesion-related genes. , 2010, Foodborne pathogens and disease.

[6]  P. Peterkin,et al.  Listeria monocytogenes, a food-borne pathogen , 1991, Microbiological reviews.

[7]  L. Cocolin,et al.  Risk-based control of food-borne pathogens Listeria monocytogenes and Salmonella enterica in the Italian fermented sausages Cacciatore and Felino. , 2015, Meat science.

[8]  T. Abee,et al.  Prediction of Bacillus weihenstephanensis acid resistance: the use of gene expression patterns to select potential biomarkers. , 2013, International journal of food microbiology.

[9]  M. Wiedmann,et al.  Role of σB in Regulating the Compatible Solute Uptake Systems of Listeria monocytogenes: Osmotic Induction of opuC Is σB Dependent , 2003, Applied and Environmental Microbiology.

[10]  D. Fink,et al.  sigmaB-dependent gene induction and expression in Listeria monocytogenes during osmotic and acid stress conditions simulating the intestinal environment. , 2004, Microbiology.

[11]  M. B. Cole,et al.  The effect of pH, salt concentration and temperature on the survival and growth of Listeria monocytogenes. , 1990, The Journal of applied bacteriology.

[12]  T. Abee,et al.  Identification of Sigma Factor σB-Controlled Genes and Their Impact on Acid Stress, High Hydrostatic Pressure, and Freeze Survival in Listeria monocytogenes EGD-e , 2004, Applied and Environmental Microbiology.

[13]  T. Ross,et al.  Responses of Listeria monocytogenes to acid stress and glucose availability monitored by measurements of intracellular pH and viable counts. , 2002, International journal of food microbiology.

[14]  R. Moezelaar,et al.  Phenotypic and Transcriptomic Analyses of Mildly and Severely Salt-Stressed Bacillus cereus ATCC 14579 Cells , 2009, Applied and Environmental Microbiology.

[15]  F. Speleman,et al.  Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes , 2002, Genome Biology.

[16]  Carlo Cantoni,et al.  Analysis of PCR-based methods for characterization of Listeria monocytogenes strains isolated from different sources. , 2005, International journal of food microbiology.

[17]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[18]  R. Karpíšková,et al.  Salt stress‐induced invasiveness of major Listeria monocytogenes serotypes , 2013, Letters in applied microbiology.

[19]  A. Henriksson,et al.  The effect of processed meat and meat starter cultures on gastrointestinal colonization and virulence of Listeria monocytogenes in mice. , 2003, International journal of food microbiology.

[20]  M. Pfaffl,et al.  Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations , 2004, Biotechnology Letters.

[21]  F. Postollec,et al.  mRNA biomarkers selection based on Partial Least Square algorithm in order to further predict Bacillus weihenstephanensis acid resistance. , 2015, Food microbiology.

[22]  R. Moezelaar,et al.  Comparative Transcriptomic and Phenotypic Analysis of the Responses of Bacillus cereus to Various Disinfectant Treatments , 2010, Applied and Environmental Microbiology.

[23]  M. Kubista,et al.  Properties of the reverse transcription reaction in mRNA quantification. , 2004, Clinical chemistry.

[24]  Björn Sjögreen,et al.  The real-time polymerase chain reaction. , 2006, Molecular aspects of medicine.

[25]  A. Holck,et al.  Survival ofEscherichia coliO157:H7,Listeria monocytogenesandSalmonella kentuckyin Norwegian fermented, dry sausage☆ , 1998 .

[26]  L. Leistner,et al.  Basic aspects of food preservation by hurdle technology. , 2000, International journal of food microbiology.

[27]  F. Postollec,et al.  An integrative approach to identify Bacillus weihenstephanensis resistance biomarkers using gene expression quantification throughout acid inactivation. , 2012, Food microbiology.

[28]  M. Mayford,et al.  A GFP-equipped bidirectional expression module well suited for monitoring tetracycline-regulated gene expression in mouse. , 2001, Nucleic acids research.

[29]  Kathryn J. Boor,et al.  Listeria monocytogenes σB Regulates Stress Response and Virulence Functions , 2003 .

[30]  M. Wiedmann,et al.  Contributions of Listeria monocytogenes sigmaB and PrfA to expression of virulence and stress response genes during extra- and intracellular growth. , 2006, Microbiology.

[31]  R. Mitchell,et al.  Fermentation and pathogen control: a risk assessment approach. , 2002, International journal of food microbiology.

[32]  David Sugden,et al.  Quantification of mRNA Using Real Time RT-PCR , 2008 .

[33]  Valentina Alessandria,et al.  EXPRESSION OF VIRULENCE GENES OF LISTERIA MONOCYTOGENES IN FOOD , 2012 .

[34]  L. Jespersen,et al.  Gene transcription and virulence potential of Listeria monocytogenes strains after exposure to acidic and NaCl stress. , 2009, Foodborne pathogens and disease.

[35]  L. Cocolin,et al.  Strain dependent expression of stress response and virulence genes of Listeria monocytogenes in meat juices as determined by microarray. , 2012, International journal of food microbiology.

[36]  L. Cocolin,et al.  Molecular biology: Transcriptomics , 2014 .

[37]  A. Rieu,et al.  Sensitivity to acetic acid, ability to colonize abiotic surfaces and virulence potential of Listeria monocytogenes EGD‐e after incubation on parsley leaves , 2010, Journal of applied microbiology.

[38]  Phillip A. Sharp,et al.  6 – Regulation of Transcription , 1991 .

[39]  M. Wiedmann,et al.  Exposure to Salt and Organic Acids Increases the Ability of Listeria monocytogenes To Invade Caco-2 Cells but Decreases Its Ability To Survive Gastric Stress , 2006, Applied and Environmental Microbiology.

[40]  L. Jespersen,et al.  Relative transcription of Listeria monocytogenes virulence genes in liver pâtés with varying NaCl content. , 2010, International journal of food microbiology.

[41]  M. Wiedmann,et al.  Listeria monocytogenes sigma B regulates stress response and virulence functions. , 2003, Journal of bacteriology.

[42]  U. Purvis,et al.  The microbiological quality of fermented sausage produced under good hygienic practices in Canada , 1987 .

[43]  S. Peterson,et al.  Global gene expression of Listeria monocytogenes to salt stress. , 2012, Journal of food protection.

[44]  S. Loncarevic,et al.  Influence of storage temperature on gene expression and virulence potential of Listeria monocytogenes strains grown in a salmon matrix. , 2010, Food microbiology.

[45]  L. Gorski,et al.  Identification of Genes Induced in Listeria monocytogenes during Growth and Attachment to Cut Cabbage, Using Differential Display , 2005, Applied and Environmental Microbiology.

[46]  Sophia Kathariou,et al.  Listeria monocytogenes virulence and pathogenicity, a food safety perspective. , 2002, Journal of food protection.

[47]  M. Wiedmann,et al.  Sigma(B)-dependent expression patterns of compatible solute transporter genes opuCA and lmo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes. , 2003, Microbiology.

[48]  A. Benson,et al.  Regulation of Transcription of Compatible Solute Transporters by the General Stress Sigma Factor, σB, in Listeria monocytogenes , 2004, Journal of bacteriology.

[49]  Claus Lindbjerg Andersen,et al.  Normalization of Real-Time Quantitative Reverse Transcription-PCR Data: A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data Sets , 2004, Cancer Research.

[50]  F. Grosveld,et al.  Regulation of Transcription , 2007 .

[51]  M. Kubista,et al.  The Prime Technique Real-time PCR Data Analysis , 2007 .

[52]  M. Wiedmann,et al.  Role of sigmaB in regulating the compatible solute uptake systems of Listeria monocytogenes: osmotic induction of opuC is sigmaB dependent. , 2003, Applied and environmental microbiology.

[53]  J. Losos,et al.  Listeria monocytogenes: a foodborne pathogen. , 1988, CMAJ : Canadian Medical Association journal = journal de l'Association medicale canadienne.

[54]  R. Stephan,et al.  Evaluation of housekeeping genes in Listeria monocytogenes as potential internal control references for normalizing mRNA expression levels in stress adaptation models using real-time PCR. , 2007, FEMS microbiology letters.

[55]  R. Moezelaar,et al.  Short- and Long-Term Biomarkers for Bacterial Robustness: A Framework for Quantifying Correlations between Cellular Indicators and Adaptive Behavior , 2010, PloS one.