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

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.

Bacterial diversity of floor drain biofilms and drain waters in a Listeria monocytogenes contaminated food processing environment.

Sanitation protocols are applied on a daily basis in food processing facilities to prevent the risk of cross-contamination with spoilage organisms. Floor drain water serves along with product-associated samples (slicer dust, brine or cheese smear) as an important hygiene indicator in monitoring Listeria monocytogenes in food processing facilities. Microbial communities of floor drains are representative for each processing area and are influenced to a large degree by food residues, liquid effluents and washing water. The microbial communities of drain water are steadily changing, whereas drain biofilms provide more stable niches. Bacterial communities of four floor drains were characterized using 16S rRNA gene pyrosequencing to better understand the composition and exchange of drain water and drain biofilm communities. Furthermore, the L. monocytogenes contamination status of each floor drain was determined by applying cultivation-independent real-time PCR quantification and cultivation-dependent detection according to ISO11290-1. Pyrosequencing of 16S rRNA genes of drain water and drain biofilm bacterial communities yielded 50,611 reads, which were clustered into 641 operational taxonomic units (OTUs), affiliated to 16 phyla dominated by Proteobacteria, Firmicutes and Bacteroidetes. The most abundant OTUs represented either product- (Lactococcus lactis) or fermentation- and food spoilage-associated phylotypes (Pseudomonas mucidolens, Pseudomonas fragi, Leuconostoc citreum, and Acetobacter tropicalis). The microbial communities in DW and DB samples were distinct in each sample type and throughout the whole processing plant, indicating the presence of indigenous specific microbial communities in each processing compartment. The microbiota of drain biofilms was largely different from the microbiota of the drain water. A sampling approach based on drain water alone may thus only provide reliable information on planktonic bacterial cells but might not allow conclusions on the bacterial composition of the microbiota in biofilms.

Tn6188 - a novel transposon in Listeria monocytogenes responsible for tolerance to benzalkonium chloride.

Controlling the food-borne pathogen Listeria (L.) monocytogenes is of great importance from a food safety perspective, and thus for human health. The consequences of failures in this regard have been exemplified by recent large listeriosis outbreaks in the USA and Europe. It is thus particularly notable that tolerance to quaternary ammonium compounds such as benzalkonium chloride (BC) has been observed in many L. monocytogenes strains. However, the molecular determinants and mechanisms of BC tolerance of L. monocytogenes are still largely unknown. Here we describe Tn6188, a novel transposon in L. monocytogenes conferring tolerance to BC. Tn6188 is related to Tn554 from Staphylococcus (S.) aureus and other Tn554-like transposons such as Tn558, Tn559 and Tn5406 found in various Firmicutes. Tn6188 comprises 5117 bp, is integrated chromosomally within the radC gene and consists of three transposase genes (tnpABC) as well as genes encoding a putative transcriptional regulator and QacH, a small multidrug resistance protein family (SMR) transporter putatively associated with export of BC that shows high amino acid identity to Smr/QacC from S. aureus and to EmrE from Escherichia coli. We screened 91 L. monocytogenes strains for the presence of Tn6188 by PCR and found Tn6188 in 10 of the analyzed strains. These isolates were from food and food processing environments and predominantly from serovar 1/2a. L. monocytogenes strains harboring Tn6188 had significantly higher BC minimum inhibitory concentrations (MICs) (28.5 ± 4.7 mg/l) than strains without Tn6188 (14 ± 3.2 mg/l). Using quantitative reverse transcriptase PCR we could show a significant increase in qacH expression in the presence of BC. QacH deletion mutants were generated in two L. monocytogenes strains and growth analysis revealed that ΔqacH strains had lower BC MICs than wildtype strains. In conclusion, our results provide evidence that Tn6188 is responsible for BC tolerance in various L. monocytogenes strains.