Bi-phasic growth of Listeria monocytogenes in chemically defined medium at low temperatures Nikolaos A. Tyrovouzis a , Apostolos S. Angelidis b , Nikolaos G. Stoforos c, ⁎ a Department of Chemical Engineering, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece b Laboratory of Milk Hygiene and Technology, Department of Food Hygiene and Technology, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece c Department of Food Science and Human Nutrition, Agricultural University of Athens, 11855 Athens, Greece abstract article info Article history: Received 28 November 2013 Received in revised form 16 April 2014 Accepted 22 June 2014 Available online 27 June 2014 Keywords: Modified Welshimer's broth Temperature effects Strain variation Inoculum level effects Refrigeration Modeling The present work reports a novel observation regarding the growth of L. monocytogenes in modified Welshimer's broth (MWB) at low temperatures. Specifically, the direct monitoring of the growth of L. monocytogenes Scott A using plate count data revealed that the pathogen displays a bi-phasic growth pat- tern in MWB at 7 °C. This bi-phasic growth pattern is masked (not observed) when optical density (OD) measurements are used to monitor growth due to the inability of OD readings to detect L. monocytogenes population density increases up to 10 7 CFU/mL. This bi-phasic growth phenomenon was further investigated as a function of growth temperature (4 °C, 7 °C, 10 °C, 14 °C and 18 °C), medium composition (by altering the MWB composition by ten-fold increases in different sets of medium constituents), inoculum level (10 2 , 10 3 , 10 4 , 10 5 , 10 6 , and 10 7 CFU/mL) and L. monocytogenes strain (10 strains). The growth of L. monocytogenes Scott A in MWB at 7 °C, 10 °C and 14 °C was consistently bi-phasic and independent of growth rate; at 18 °C, growth was consistently mono-phasic (single-phase, typical sigmoid growth curves), whereas no growth was observed at 4 °C. The tested modifications in the composition of MWB did not influence the bi-phasic nature of L. monocytogenes Scott A growth at 7 °C, and, overall, we could not point out any strain-, or serotype-specific effects. On the other hand, the initial inoculum level appears to affect the form of the growth curve, as there was a shift towards mono-phasic growth in trials with increasing initial inocula. A mathematical model, based on a stepwise response and described through two sequential sigmoid curves, was used to describe bi-phasic growth and estimate the kinetic parameters of L. monocytogenes growth. An alternative hypothesis, based on the assumption of the existence of two subpopulations, possessing different growth kinetics, materialized under the stress imposed on L. monocytogenes cells due to the combined effect of three factors (defined medium, low temperature and low initial inoculum) was also proposed and formulated. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Listeria monocytogenes is a Gram-positive foodborne pathogen and the causative agent of listeriosis, a disease that can be particularly severe or even fatal for certain high-risk population groups such as neonates, the elderly, and people with a compromised immune system. Contam- ination of food with low levels of Listeria spp. is not an uncommon event (Ryser and Marth, 2007). L. monocytogenes is psychrotrophic and as such, it can proliferate at low temperatures both in laboratory media as well as in certain categories of permissive foods. Therefore, considerable research has been conducted in the last decade in order to elucidate the behavior of L. monocytogenes under chill stress and re- veal the pathogen's underlying physiological adaptive mechanisms (Cacace et al., 2010; Tasara and Stephan, 2006). Defined media are used in (food) microbiology in order to conduct reproducible experiments and avoid confounding by extraneous, often unknown factors originating from the composition of the growth medi- um. Modified Welshimer's broth (MWB) is the chemically defined me- dium that has been most frequently used by researchers working on the physiology and genetics of L. monocytogenes (Premaratne et al., 1991). The composition of MWB is rather simple. MWB was developed by modifying Welshimer's medium (Welshimer, 1963) and it consists of three salts, two of which are phosphate salts added for buffering pur- poses (KH 2 PO 4 , Na 2 HPO 4 ∙7H 2 O, and MgSO 4 ∙7H 2 O), ferric citrate as an iron source, glucose as the sole carbon and energy source, seven amino acids (Leu, Ile, Val, Met, Arg, Cys, and Gln) and four vitamins (riboflavin, thiamine, biotin, and thioctic acid). The present work reports on a novel observation regarding the growth of L. monocytogenes in MWB. Specifically, the direct monitoring of the growth of L. monocytogenes by surface-plating aliquots from cul- tures at selected time intervals on rich, non-selective solid medium International Journal of Food Microbiology 186 (2014) 110–119 ⁎ Corresponding author. Tel.: +30 210 5294706; fax: +30 210 5294682. E-mail address: stoforos@aua.gr (N.G. Stoforos). http://dx.doi.org/10.1016/j.ijfoodmicro.2014.06.021 0168-1605/© 2014 Elsevier B.V. All rights reserved. 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