New Aspects on Listeria monocytogenes ST5-ECVI Predominance in a Heavily Contaminated Cheese Processing Environment

Abstract

The eradication of Listeria monocytogenes from food chains is still a great challenge for the food industry and control authorities since some clonal complexes (CCs) are either better adapted to food processing environments (FPEs) or are globally widespread. In this work, we focus on the in-house evolution of L. monocytogenes genotypes collected from a heavily contaminated FPE whose contamination pattern underwent a massive and yet unexplained change. At the beginning of the sampling in 2010, a high variety of most likely transient L. monocytogenes genotypes was detected belonging to sequence type (ST) 1, ST7, ST21, ST37. After several efforts to intensify the hygiene measures, the variability was reduced to L. monocytogenes ST5 that was dominant in the following years 2011 and 2012. We aimed to elucidate possible genetic mechanisms responsible for the high abundance and persistence of ST5 strains in this FPE. Therefore, we compared the genomes of six L. monocytogenes ST5 strains to the less frequently occurring transient L. monocytogenes ST37 and ST204 from the same FPE as well as the highly abundant ST1 and ST21 isolated in 2010. Whole genome analysis indicated a high degree of conservation among ST5 strains [average nucleotide identity (ANI) 99.93-99.99%; tetranucleotide correlation 0.99998-0.99999]. Slight differences in pulsed field gel electrophoresis (PFGE) patterns of two ST5 isolates could be explained by genetic changes in the tRNA-Arg-TCT prophages. ST5 and ST204 strains harbored virtually identical 91 kbp plasmids related to plasmid group 2 (pLM80 and pLMUCDL175). Interestingly, highly abundant genotypes present in the FPE in 2010 did not harbor any plasmids. The ST5 plasmids harbored an efflux pump system (bcrABC cassette) and heavy metal resistance genes possibly providing a higher tolerance to disinfectants. The pLM80 prototype plasmids most likely provide important genetic determinants for a better survival of L. monocytogenes in the FPE. We reveal short-term evolution of L. monocytogenes strains within the same FPE over a 3 year period and our results suggest that plasmids are important for the persistence of ST5 strains in this FPE.

Keywords: Listeria monocytogenes; food processing environment; multi locus sequence type; persistence; plasmid; whole genome sequencing.

FIGURE 1

Decrease in Listeria monocytogenes genotypic heterogeneity in a cheese processing environment during 2010–2012 based on Rückerl et al. (2014). Different multi-locus sequence types are abbreviated by sequence type (ST). The strain are denoted by 1–15KSM. Further abbreviations: non-food contact surfaces (NFCS) and food-contact surfaces (FCS).

FIGURE 2

Distribution of L. monocytogenes sequence types (STs) in the building compartments I and II isolated from non-food contact surfaces (NFCS), food-contact surfaces (FCS) and raw materials (RM) or products (P) during the study phase (2010–2012).

FIGURE 3

BlastP comparison of L. monocytogenes ST5 genomes from the same FPE compared with L. monocytogenes CFSAN029793. BlastP was performed in Patric (Wattam et al., 2017). The percent sequence identity is indicated in different colors for unidirectional and bidirectional BlastP hits. List of tracks, from outside to inside: L. monocytogenes CFSAN029793, L. monocytogenes 13KSM, L. monocytogenes 11KSM, L. monocytogenes 10KSM, L. monocytogenes 8KSM, L. monocytogenes 6KSM, L. monocytogenes 4KSM.

FIGURE 4

MAUVE alignment of L. monocytogenes 4, 6, 8, 10, 11, 13KSM (ST5), 3KSM (ST204) and reference plasmids plmUCDL175 (ST204, Fox et al., 2016) and pLM80 (ST6; reference strain H7858). Homologous regions are shown in the same color. The height of the similarity profile within each block corresponds to the average level of conservation (Darling et al., 2010). The two contigs of pLM80 and pLMUCDL175 are shown separately.

FIGURE 5

Invasion efficiency of L. monocytogenes strains included in this study in human Caco2 cells. Mean values and standard deviations of the four independent biological replicates are presented. Different letters indicate significant differences (P < 0.05) between the invasion efficiency of the strains.

FIGURE 6

Intracellular growth of L. monocytogenes strains included in this study in human Caco2 cells. Mean values and standard deviations of the four independent biological replicates are presented. Different letters indicate significant differences (P < 0.05) between the invasion efficiency of the strains.