Scientific Publications - Work Done by Microbiology Reader Bioscreen C

Food Microbiology, October 1 1997, Vol. 14, No. 5, pp. 403-412

Variability of the response of 66 Listeria monocytogenes and Listeria innocua strains to different growth conditions

C. Begot, I. Lebert and A. Lebert*

ABSTRACT

The growth of 58 strains of Listeria monocytogenes and eight strains of Listeria innocua iso­lated from meat products (68%) and industrial sites (23%), were compared in four con­ditions of temperature, water activity (aw) and pH. Temperatures ranged from 10-37 °C, pH from 5·6-7·0 and aw from 0·96-1. Growths were performed in a meat broth with an auto­mated turbidimeter (Bioscreen C, Labsystem). Growth curves were fitted using the Gom­pertz function, and growth parameters were calculated. The differences between strains in lag phase duration were much greater than in growth rate. The greatest differences occurred at 10 C, pH 7 and aw 0·96 : lag time values ranged from 4 h to 4 days. the Listeria population was separated into five groups, according to the lag time and maximal growth rate values using clustering analysis. The majority of the strains isolated from industrial sites were grouped together and showed faster growth than the others in the four con­ditions studied. The serotype or the nature of the meat from which the strains were isolated did not influence growth. The variability observed among strains raises questions about the consequences in quantitative risk assessment and about the construction of models in pre­dictive modeling.

Introduction

Listeria bacteria are widespread in the environment. Among the different species, Listeria monocytogenes and Listeria innocua are the two most commonly isolated from food processing (Cox et al. 1989). L. monocy­togenes can cause the death of both the very young and immunocompromised individuals. Most cases are traced to the contamination of raw or processed foods with L. monocyto­genes. Listeriosis is therefore a major threat to human health.

Listeria monocytogenes can grow at low temperatures (Walker et al. 1990), low pH (George et al. 1988) and low water activity (aw) (Farber et al. 1992, Nolan et al. 1992); they are therefore able to survive and multi­ply in a wide range of food products.

Work on predictive microbiology has been carried out in an attempt to improve the shelf life and safety of food (Gould 1989). Predic­tive models of microbial growths are set out with respect to the main controlling factors of the environment such as temperature, pH and water activity. Despite variations in growth among strains (Junttila et al. 1988, Walker et al. 1990) numerous models have been constructed using only one or a small pool of strains. In this work, we compared the growth of 66 strains of L. monocytogenes and L. innocua, isolated from meat, meat prod­ucts and industrials sites. Strains were clus­tered according to their growth in a broth medium in four conditions of temperature, pH and aw. Consequences on the construction of models are discussed.

Materials and Methods

Strains

Fifty-eight strains of L. monocytogenes and eight strains of L. innocua were studied: 45 strains were isolated from meat and meat products, 15 from meat and dairy plants (materials, floors, walls) four were involved in outbreaks (Table 1). Five of 13 serotypes of L. monocytogenes were represented: 1/2a, 1/2b, 1/2c, 4b, 4d. Listeria cultures were stored on tryptic soy agar (TSA, Difco, Detroit, MI, USA) slopes at 4 C.

Media

Culture inocula were prepared in a meat medium (MM): meat peptone (Merck) 10 g l 1_, yeast extract (Difco) 5 g l ^1, glucose (Merck) 5 g l ^1. The pH was adjusted to 7·0 with NaOH (Prolabo) 1 mol l ^1. The medium was autoclaved at 120 C for 20 min.

Growth experiments were carried out in a tryptic meat broth (TMB) which was steril­ized by filtration derived from tryptone meat agar (GTV5p, Fournaud et al. 1973): meat extract (Merck) 10 g l ^1, proteose peptone (Merck) 10 g l ^1, tryptone (Difco) 5 g l ^1, glu­cose 5 g l ^1. The medium was buffered with a K2HOP4-KH2PO4 (Merck), 0·1 mol l ¹ solution in proportion 1:1 (v/v) and adjusted to pH 7·0 with NaOH 1 mol l ^1. aw was set by adding NaCl (Merck) in accordance with Chirife and Resnik (1984).

Tryptone sodium chloride medium (TSC) (tryptone 1 g l ^1, NaCl 8·5 g l ^1, pH 7·0) was used for dilutions, and TSA and APT agar (Difco) were used for plate counts and agar slope cultures.

Growth

Growths were measured by optical density in an automated turbidimeter, Bioscreen C (Labsystem, Finland). All strains were inocu­lated in MM and incubated for the same period of time (17 h) at 30 C. All cultures were therefore in stationary phase, thus avo­iding disparities in the subsequent growth kinetics. Nine millilitres of TMB were inocu­lated with the subculture. The inoculum size was confirmed by plate counts. The inocu­lated TMB was dispensed aseptically in 300 µl volumes into honeycomb microplates (10 10) of the Bioscreen C. For each strain, eight successive wells of the same column were filled. The last two wells received the same volume of non-inoculated medium in order to determine the growth medium optical density (OD) and controlled any poss­ible contamination. Ws methodology is simi­lar to that used by Be got et al. (1996). Growth conditions are summarized in Table 2.

Experimental design

A Plackett Burman design (Plackett and Bur­man 1943) was used to test the effects of three factors: temperature, pH and water activity. Each factor was studied twice at two levels: high and low (Table 3). While a fac­torial design would have required eight experiments, four were sufficient with a Plackett Burman design.

Curve fitting

OD data were transferred from the Bioscreen C to Excel software (Microsoft Windows) and transformed for each measurement. Four quantities were calculated at time t: (ODi)_t, the average of the OD of eight replicates; (ODni)t, the average of the OD of the non­inoculated medium

(ΔOD)t=(ODi)t (ODni)t

Log10[(ΔOD)_t/ΔODmin]

where ΔODmin was the lowest ΔOD value above the detection threshold.

In the linear range of the Bioscreen C ( ΔOD<1'2) (Begot et al. 1996), growth curves were fitted using the modified Gompertz equation (Zwietering et al. 1990) (1):