1. Listeria monocytogenes serovar prevalence and dominance dynamics in meat products: A useful insight into the underrepresentation of serovar 4b strains among food isolates Nikolaos D. Andritsos*, Spiros Paramithiotis, Marios Mataragas, Eleftherios H. Drosinos Laboratory of Food Quality Control and Hygiene, Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, GR-118 55 Athens, Greece * Current working address: Athens Analysis Laboratories S.A., 104 Vas. Sofias Ave., GR-115 27 Athens, Greece; n.andritsos@ergastiria.gr Listeria monocytogenes is the bacterial causative agent of listeriosis, a life-threatening disease in humans and animals. Listeriosis in humans is mainly of a foodborne etiology and despite its low incidence, approximately 20% of diagnosed infections result in death (Mead et al., 1999). The disease is characterized by high hospitalization and case fatality rates among the elderly, the immunocompromized, and pregnant women, rather than among healthy individuals. There are currently 13 known serovars for L. monocytogenes, with serovar 1/2a, 1/2b, 1/2c and 4b strains within this species being responsible for the vast majority (>98%) of the reported human foodborne listeriosis cases (Jacquet et al., 2002). However, human clinical isolates of the pathogen which are usually identified as serovar 4b strains are frequently underrepresented among the food isolates. This may suggest variation in human virulence and/or differences in other ecophysiological attributes for strains of the aforementioned serovars (Kathariou, 2002). Thus, the objective of the present work was to estimate L. monocytogenes serovar prevalence and dominance dynamics in raw and ready-to-eat (RTE) meat products respectively, in order to assess the predominance in meat of serovar strains other than serovar 4b. A four-strain mixture consisting of L. monocytogenes serovars 1/2a, 1/2b, 1/2c, and 4b, isolated from fresh minced pork samples ( n =100) during a field survey using in parallel duplicate plates of PALCAM, ALOA and RAPID’L.mono selective agars (Andritsos et al., 2013), was surface inoculated (ca. 6 log CFU ml ⁻ ¹) on both sides of a sliced RTE meat product (i.e. cooked, cured pork shoulder). The inoculated samples of the product were vacuum packaged and stored at 4° and 10°C. Microbiological analysis of product samples and control (uninoculated) samples was performed according to ISO 11290-2 using PALCAM and incubating plates at 37°C for 48 h. Samples were analyzed during storage at 4°C (days 0, 13, 29) and 10°C (days 0, 2, 7, 15) respectively. The pH was also measured following the analysis of the samples. Serotyping of L. monocytogenes isolates was performed by multiplex PCR (Doumith et al., 2004) (Fig.1), while strains were stored at -20°C in Brain Heart Infusion (BHI) broth supplemented with 20% glycerol. Before use, fresh bacterial culture was obtained by subculturing twice in BHI broth at 37°C for 24 h. The prevalence and dominance ratio(s) of the serotyped L. monocytogenes isolates were estimated for mince and during storage of the artificially contaminated RTE meat product at the two different cold temperature conditions, respectively. The majority of the L. monocytogenes minced pork isolates were identified as serovar 1/2a, while occasional appearance of serovar 1/2b, 1/2c, and 4b strains has been exhibited (Table 1). The serovar dominance dynamics for the RTE meat product showed that even though serovar 4b decreased at both storage temperatures the serovar 4b reduction rate was higher during storage at 10 ⁰ C (Fig.2). On the contrary, serovars 1/2b and 1/2c were largely isolated, except in the case of storage at 4 ⁰ C where the dominance ratio of serovar 1/2b remained relatively constant. Finally, a slight increase (6%) and decrease (8%) in the recoveries of serovar 1/2a were noted compared to all other serovars during storage at low (4 ⁰ C) and high (10 ⁰ C) cold temperature conditions, respectively (Tables 2 & 3). Meat is mainly contaminated with serovar 1/2a, 1/2b and 1/2c strains of L. monocytogenes and this was also the case in the present study; 82.8% of the pathogen’s isolates belonged to these serovars with the majority of them being identified as serovar 1/2a strains. On the other hand, the serovar dominance dynamics for the RTE meat product revealed a substantial decrease in the recovery of serovar 4b at the end of storage, compared to the serogroup 1/2 serovars (i.e. 1/2a, 1/2b and 1/2c), and regardless the cold temperature condition applied (4°C or 10°C). These findings could be used in the interpretation of the underrepresentation of serovar 4b strains among food isolates during the detection of L. monocytogenes in meat and products thereof. Introduction & ObjectivesMaterials & Methods Discussion /ConclusionsFig. 2 (Main Figure) 1)Andritsos et al., Food Microbiol. 2013; 36 :395 2)Doumith et al., J. Clin. Microbiol. 2004; 42 :3819 3)Kathariou, J. Food Protect. 2002; 65 :1811 4)Mead et al., Emerg. Infect. Dis. 1999; 5 :607 5)Jacquet et al., Appl. Environ. Microbiol. 2002; 68 :616 References Table 2: Recoveries of L. monocytogenes serovars during storage of the inoculated RTE meat product at 4°C ¹ number of contaminated samples is given in parenthesis Table 1: Serovar prevalence of L. monocytogenes strains isolated from minced pork samples Table 3: Recoveries of L. monocytogenes serovars during storage of the inoculated RTE meat product at 10°C ¹ dominance ratio (%) of isolated serovar is given in parenthesis 4b1/2c1/2b1/2a Fig.1: Serotyping by Multiplex PCR technique Fig. 2: Dominance dynamics of L. monocytogenes serovars during storage of an artificially contaminated with strains of the pathogen RTE meat product at two different cold temperature conditions (4° and 10°C) 4°C 10°C