Purpose: The study aims to correlate the changes in expression of stress- and virulence-associated genes of L. monocytogenes following habituation under suboptimal pH and NaCl, with the survival under extreme acid stress.
Methods: Tryptic Soy Broth, supplemented with 0.6% Yeast Extract (TSBYE) with various combinations of pH (4.6-6.4) and NaCl (2-10% w/v) was prepared in triplicate, inoculated with two L. monocytogenes strains (C5, 6179) separately and stored at 7°C for thirteen days. Growth followed by survival (log reductions, DpH:2.0-values) against severe acid stress (TSBYE, pH 2.0 adjusted with HCl) were assessed on day 2, 4, 6, 8, 10 and 13. Relative transcription of gad2, sigB and prfA, compared to control (pH 7.2, day 0) were estimated with quantitative RT-PCR.
Results: Inter-strain variation in log changes, regarding growth, was evident at pH<5.5 and NaCl>8%w/v, manifested by C5 exhibiting higher survival (2-3 log reduction) than 6179, which reduced by 4-6 log CFU/ml by the end of storage. During storage, growth/no growth interface for C5 was slightly shifted to higher NaCl and lower pH values than the respective interface of 6179, while for both strains, survival/death interfaces post acid challenge were similar (P > 0.05) throughout storage. Principal Component Analysis pointed out that high relative transcription levels of gad2 (Fold Changes>30) and sigB upregulation (FC>1) correlate with increased D-values (biphasic inactivation curve, D1:0.8-5min) and acid resistance (2-4 log reductions). Noteworthy, induction of prfA prior or post acid challenge reveals the underlying potential risk resulting from exposure to suboptimal or lethal conditions.
Significance: Correlating transcriptional response of L. monocytogenes with growth/no growth or survival/death interface, results in stochastic description of stress resistance and virulence. Such a description is of high value for designing safe intervention strategies. It also assists in better understanding the impact of processing on bacterial physiology and integrates –omics in quantitative microbial risk assessment. This work has been supported by the project “Efficacy of NOVEL analytical techniques to prEdict the quality and safetY of newly developed pErishable food products 11SYN_2_1528” co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the O.P. “Competitiveness and Entrepreneurship (OPC II)” ROP Macedonia – Thrace, ROP Crete and Aegean Islands, ROP Thessaly – Mainland Greece – Epirus, ROP Attica, Framework NSRF 2007-2013, COOPERATION 2011.