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Microbial genomics 7(1), 000491 (2021). doi:10.1099/mgen.0.000491, Published by Microbiology Society, London

Where classical epidemiology has proven to be inadequate for surveillance and control of foodborne pathogens, molecular epidemiology, using genomic typing methods, can add value. However, the analysis of whole genome sequencing (WGS) data varies widely and is not yet fully harmonised. We used genomic data on 494 Listeria monocytogenes isolates from ready-to-eat food products and food processing environments deposited in the strain collection of the German National Reference Laboratory to compare various procedures for WGS data analysis and to evaluate compatibility of results. Two different core genome multilocus sequence typing (cgMLST) schemes, different reference genomes in single nucleotide polymorphism (SNP) analysis and commercial as well as open-source software were compared. Correlation of allele distances from the different cgMLST approaches was high, ranging from 0.97 to 1, and unified thresholds yielded higher clustering concordance than scheme-specific thresholds. The number of detected SNP differences could be increased up to a factor of 3.9 using a specific reference genome compared with a general one. Additionally, specific reference genomes improved comparability of SNP analysis results obtained using different software tools. The use of a closed or a draft specific reference genome did not make a difference. The harmonisation of WGS data analysis will finally guarantee seamless data exchange, but, in the meantime, knowledge on threshold values that lead to comparable clustering of isolates by different methods may improve communication between laboratories. We therefore established a translation code between commonly applied cgMLST and SNP methods based on optimised clustering concordances. This code can work as a first filter to identify WGS-based typing matches resulting from different methods, which opens up a new perspective for data exchange and thereby accelerates time-critical analyses, such as in outbreak investigations.

We compared the consistency, accuracy and reproducibility of next-generation short read sequencing between ten laboratories involved in food safety (research institutes, state laboratories, universities and companies) from Germany and Austria. Participants were asked to sequence six DNA samples of three bacterial species (Campylobacter jejuni, Listeria monocytogenes and Salmonella enterica) in duplicate, according to their routine in-house sequencing protocol. Four different types of Illumina sequencing platforms (MiSeq, NextSeq, iSeq, NovaSeq) and one Ion Torrent sequencing instrument (S5) were involved in the study. Sequence quality parameters were determined for all data sets and centrally compared between laboratories. SNP and cgMLST calling were performed to assess the reproducibility of sequence data collected for individual samples. Overall, we found Illumina short read data to be more accurate (higher base calling accuracy, fewer miss-assemblies) and consistent (little variability between independent sequencing runs within a laboratory) than Ion Torrent sequence data, with little variation between the different Illumina instruments. Two laboratories with Illumina instruments submitted sequence data with lower quality, probably due to the use of a library preparation kit, which shows difficulty in sequencing low GC genome regions. Differences in data quality were more evident after assembling short reads into genome assemblies, with Ion Torrent assemblies featuring a great number of allele differences to Illumina assemblies. Clonality of samples was confirmed through SNP calling, which proved to be a more suitable method for an integrated data analysis of Illumina and Ion Torrent data sets in this study.

We report here the draft genome sequence of a Listeria monocytogenes strain, core genome multilocus sequence typing (cgMLST) complex type 2521 (CT2521), isolated from ready-to-eat meat sausage related to a protracted and supraregional listeriosis outbreak (Sigma1) in Germany from 2014 to 2019.

Background Various molecular typing methods are used for the surveillance of foodborne pathogens and outbreak investigations, differing widely in information content and discriminatory power. Presently, not least because of the rapid technological development, the focus is shifting to whole genome sequencing (WGS) as an analytical tool. As a result of globalisation of food trade, a comprehensive understanding of the association between the occurrence of human infections and causative pathogens has to be established to monitor and to prevent their spread. In this respect, the accuracy of WGS clearly supersedes that of previous tools. Scope and approach Our review describes the status quo of WGS in surveillance and outbreak investigations of foodborne pathogens through the example of Listeria monocytogenes. It highlights the value of WGS in trace-back of infections to food sources and provides an overview of methods used for data generation (wet lab) and analysis (dry lab). Altogether, progress but also challenges in the worldwide practical application of WGS for bacterial typing are described. Key findings and conclusions The current status of WGS differs widely between countries and even laboratory sites. A consensus has to be found concerning methods, quality measures, thresholds for data generation and analysis as well as rules for data sharing. International harmonisation is going to be indispensable on the way to data exchangeability which will finally support global control of foodborne pathogens.

Emerging infectious diseases 23(10), 1680-1683 (2017). doi:10.3201/eid2310.161623, Published by CDC, Atlanta, Ga.

Foodborne pathogens such as L. monocytogenes can persist in food production environments for a long time, causing perennial outbreaks. Hence, bacterial pathogens are able to survive cleaning and disinfection procedures. Accordingly, they may be repeatedly exposed to sublethal concentrations of disinfectants, which might result in bacterial adaptation to these biocides. Furthermore, antibiotic coresistance and cross-resistance are known to evolve under biocide selection pressure in vitro. Hence, antimicrobial tolerance seems to play a crucial role in the resilience and persistence of foodborne pathogens in the food chain and might reduce therapeutic options in infectious diseases., Contamination of food during processing is recognized as a main transmission route of Listeria monocytogenes. To prevent microbial contamination, biocides are widely applied as disinfectants in food processing plants. However, there are concerns about the development of antimicrobial resistance in foodborne pathogens due to widespread biocide usage. In our study, 93 L. monocytogenes isolates from German food production facilities were (i) tested for biocide and antibiotic susceptibility using broth microdilution assays, (ii) analyzed for links between reduced biocide susceptibility and antibiotic resistance, and (iii) characterized by whole-genome sequencing, including the detection of genes coding for biocide tolerance, antibiotic resistance, and other virulence factors. Fifteen L. monocytogenes isolates were tolerant to benzalkonium chloride (BAC), and genes conferring BAC tolerance were found in 13 of them. Antibiotic resistance was not associated with biocide tolerance. BAC-tolerant isolates were assigned to 6 multilocus sequence type (MLST) clonal complexes, and most of them harbored internalin A pseudogenes with premature stop codons or deletions (n = 9). Our study demonstrated a high genetic diversity among the investigated isolates including genotypes that are frequently involved in human infections. Although in vitro adaptation studies to biocides have raised concerns about increasing cross-resistance to antibiotics, our results do not provide evidence for this phenomenon in field isolates. IMPORTANCE Foodborne pathogens such as L. monocytogenes can persist in food production environments for a long time, causing perennial outbreaks. Hence, bacterial pathogens are able to survive cleaning and disinfection procedures. Accordingly, they may be repeatedly exposed to sublethal concentrations of disinfectants, which might result in bacterial adaptation to these biocides. Furthermore, antibiotic coresistance and cross-resistance are known to evolve under biocide selection pressure in vitro. Hence, antimicrobial tolerance seems to play a crucial role in the resilience and persistence of foodborne pathogens in the food chain and might reduce therapeutic options in infectious diseases.