Listeria monocytogenes survives better at lower storage temperatures in regular and low-salt soft and cured cheeses.

The behaviour of Listeria monocytogenes was investigated in soft pasteurized milk cheese elaborated with different salt concentrations (1.17 and 0.30% w/w) and in cured raw sheep milk cheese over storage up to 189 days at different isothermal conditions. Commercial 25-g cheese samples were inoculated with a 4-strain cocktail of L. monocytogenes (serovars 4b, 1/2a, 1/2b and 1/2c) at approximately 104 CFU/g. The inoculated samples were stored at 4 and 22 °C and withdrawn at proper intervals for L. monocytogenes enumeration. The prevalence of the different serovar strains of L. monocytogenes was characterized on soft cheese samples over storage at 4 °C using multiplex PCR. Salt reduction did not affect the survival of L. monocytogenes in soft cheeses and a maximum of 1-log reduction was observed in both regular and low-salt cheeses after 189 days of storage at 4 °C. The pathogen showed greater survival capacity in both soft and cured cheeses during storage at 4 °C compared to the storage at 22 °C, where more than 2.5 log reductions were computed. The fate of L. monocytogenes was described through a Weibull model fitted to survival data. The time required for a first tenfold reduction of the L. monocytogenes population (δ) at 4 °C is around 150 days in soft and 72 days in cured cheeses. At 22 °C, the estimated δ values are at least 60% lower in both cheese types. Among the four L. monocytogenes serovars present in the inoculated cocktail, the serovar 4b strain was the most sensitive to refrigerated storage, while the prevalence of serovar 1/2c strain increased over time in soft cheeses. Overall, the data obtained in this study help to deepen knowledge into factors affecting L. monocytogenes behaviour on cheeses and evidenced the variability between serovars in terms of survival capacity, which may be considered when performing microbial risk assessments.

Associations between Listeria monocytogenes genomic characteristics and adhesion to polystyrene at 8 °C.

Listeria monocytogenes remains a threat to the food system and has led to numerous foodborne outbreaks worldwide. L. monocytogenes can establish itself in food production facilities by adhering to surfaces, resulting in increased resistance to environmental stressors. The aim of this study was to evaluate the adhesion ability of L. monocytogenes at 8 °C and to analyse associations between the observed phenotypes and genetic factors such as internalin A (inlA) genotypes, stress survival islet 1 (SSI-1) genotype, and clonal complex (CC). L. monocytogenes isolates (n = 184) were grown at 8 °C and 100% relative humidity for 15 days. The growth was measured by optical density at 600 nm every 24 h. Adherent cells were stained using crystal violet and quantified spectrophotometrically. Genotyping of inlA and SSI-1, multi-locus sequence typing, and a genome-wide association study (GWAS) were performed to elucidate the phenotype-genotype relationships in L. monocytogenes cold adhesion. Among all inlA genotypes, truncated inlA isolates had the highest mean adhered cells, ABS595nm = 0.30 ± 0.15 (Tukey HSD; P < 0.05), while three-codon deletion inlA isolates had the least mean adhered cells (Tukey HSD; P < 0.05). When SSI-1 was present, more cells adhered; less cells adhered when SSI-1 was absent (Welch's t-test; P < 0.05). Adhesion was associated with clonal complexes which have low clinical frequency, while reduced adhesion was associated with clonal complexes which have high frequency. The results of this study support that premature stop codons in the virulence gene inlA are associated with increased cold adhesion and that an invasion enhancing deletion in inlA is associated with decreased cold adhesion. This study also provides evidence to suggest that there is an evolutionary trade off between virulence and adhesion in L. monocytogenes. These results provide a greater understanding of L. monocytogenes adhesion which will aid in the development of strategies to reduce L. monocytogenes in the food system.

Accurate classification of Listeria species by MALDI-TOF mass spectrometry incorporating denoising autoencoder and machine learning.

Listeria monocytogenes belongs to the category of facultative anaerobic bacteria, and is the pathogen of listeriosis, potentially lethal disease for humans. There are many similarities between L. monocytogenes and other non-pathogenic Listeria species, which causes great difficulties for their correct identification. The level of L. monocytogenes contamination in food remains high according to statistics from the Food and Drug Administration. This situation leads to food recall and destruction, which has caused huge economic losses to the food industry. Therefore, the identification of Listeria species is very important for clinical treatment and food safety. This work aims to explore an efficient classification algorithm which could easily and reliably distinguish Listeria species. We attempted to classify Listeria species by incorporating denoising autoencoder (DAE) and machine learning algorithms in matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, convolutional neural networks were used to map the high dimensional original mass spectrometry data to low dimensional core features. By analyzing MALDI-TOF MS data via incorporating DAE and support vector machine (SVM), the identification accuracy of Listeria species was 100%. The proposed classification algorithm is fast (range of seconds), easy to handle, and, more importantly, this method also allows for extending the identification scope of bacteria. The DAE model used in our research is an effective tool for the extraction of MALDI-TOF mass spectrometry features. Despite the fact that the MALDI-TOF MS dataset examined in our research had high dimensionality, the DAE + SVM algorithm was still able to exploit the hidden information embedded in the original MALDI-TOF mass spectra. The experimental results in our work demonstrated that MALDI-TOF mass spectrum combined with DAE + SVM could easily and reliably distinguish Listeria species.

ON-rep-seq as a rapid and cost-effective alternative to whole-genome sequencing for species-level identification and strain-level discrimination of Listeria monocytogenes contamination in a salmon processing plant.

Identification, source tracking, and surveillance of food pathogens are crucial factors for the food-producing industry. Over the last decade, the techniques used for this have moved from conventional enrichment methods, through species-specific detection by PCR to sequencing-based methods, whole-genome sequencing (WGS) being the ultimate method. However, using WGS requires the right infrastructure, high computational power, and bioinformatics expertise. Therefore, there is a need for faster, more cost-effective, and more user-friendly methods. A newly developed method, ON-rep-seq, combines the classical rep-PCR method with nanopore sequencing, resulting in a highly discriminating set of sequences that can be used for species identification and also strain discrimination. This study is essentially a real industry case from a salmon processing plant. Twenty Listeria monocytogenes isolates were analyzed both by ON-rep-seq and WGS to identify and differentiate putative L. monocytogenes from a routine sampling of processing equipment and products, and finally, compare the strain-level discriminatory power of ON-rep-seq to different analyzing levels delivered from the WGS data. The analyses revealed that among the isolates tested there were three different strains. The isolates of the most frequently detected strain (n = 15) were all detected in the problematic area in the processing plant. The strain level discrimination done by ON-rep-seq was in full accordance with the interpretation of WGS data. Our findings also demonstrate that ON-rep-seq may serve as a primary screening method alternative to WGS for identification and strain-level differentiation for surveillance of potential pathogens in a food-producing environment.

Genetic relationships and biofilm formation of Listeria monocytogenes isolated from the smoked salmon industry.

Among pathogens, L. monocytogenes has the capability to persist on Food Processing Environment (FPE), first of all posing safety issues, then economic impact on productivity. The aim of this work was to determine the influence of biofilm forming-ability and molecular features on the persistence of 19 Listeria monocytogenes isolates obtained from FPE, raw and processed products of a cold-smoked salmon processing plant. To verify the phenotypic and genomic correlations among the isolates, different analyses were employed: serotyping, Clonal Complex (CC), core genome Multi-Locus Sequence Typing (cgMLST) and Single Nucleotide Polymorphisms (SNPs) clustering, and evaluation of the presence of virulence- and persistence-associated genes. From our results, the biofilm formation was significantly higher (*P < 0.05) at 37 °C, compared to 30 and 12 °C, suggesting a temperature-dependent behaviour. Moreover, the biofilm-forming ability showed a strain-specific trend, not correlated with CC or with strains persistence. Instead, the presence of internalin (inL), Stress Survival Islet (SSI) and resistance to erythromycin (ermC) genes was correlated with the ability to produce biofilms. Our data demonstrate that the genetic profile influences the adhesion capacity and persistence of L. monocytogenes in food processing plants and could be the result of environmental adaptation in response to the external selective pressure.

Study of the microbial diversity of a panel of Belgian artisanal cheeses associated with challenge studies for Listeria monocytogenes.

High throughput sequencing could become a powerful tool in food safety. This study was the first to investigate artisanal cheeses from Belgium (31 batches) using metagenetics, in relation to Listeria monocytogenes growth data acquired during a previous project. Five cheese types were considered, namely unripened acid-curd cheeses, smear- and mold-ripened soft cheeses, and Gouda-type and Saint-Paulin-type cheeses. Each batch was analyzed in triplicate the first and the last days of storage at 8 °C. Globally, 2697 OTUs belonging to 277 genera and to 15 phyla were identified. Lactococcus was dominant in all types, but Streptococcus was co-dominant in smear-ripened soft cheeses and Saint-Paulin-type cheeses. The dominant population was not always associated with added starter cultures. Bacterial richness and diversity were significantly higher in both types of soft cheeses than in other categories, including particular genera like Prevotella, Faecalibacterium and Hafnia-Obesumbacterium in mold-ripened cheeses and Brevibacterium, Brachybacterium, Microbacterium, Bacteroides, Corynebacterium, Marinilactibacillus, Fusobacterium, Halomonas and Psychrobacter in smear-ripened soft cheeses. A strong correlation was observed between no growth of L. monocytogenes in a smear-ripened cheese and the presence of an unknown Fusobacterium (relative abundance around 10%). This in silico correlation should be confirmed by further experiments in vitro and in situ.

Mobile Elements Harboring Heavy Metal and Bacitracin Resistance Genes Are Common among Listeria monocytogenes Strains Persisting on Dairy Farms.

Listeria monocytogenes is a foodborne pathogen and a resilient environmental saprophyte. Dairy farms are a reservoir of L. monocytogenes, and strains can persist on farms for years. Here, we sequenced the genomes of 250 L. monocytogenes isolates to investigate the persistence and mobile genetic elements (MGEs) of Listeria strains inhabiting dairy farms. We performed a single-nucleotide polymorphism (SNP)-based phylogenomic analysis to identify 14 monophyletic clades of L. monocytogenes persistent on the farms for ≥6 months. We found that prophages and other mobile genetic elements were, on average, more numerous among isolates in persistent than nonpersistent clades, and we demonstrated that resistance genes against bacitracin, arsenic, and cadmium were significantly more prevalent among isolates in persistent than nonpersistent clades. We identified a diversity of mobile elements among the 250 farm isolates, including three novel plasmids, three novel transposons, and a novel prophage harboring cadmium resistance genes. Several of the mobile elements we identified in Listeria were identical to the mobile elements of enterococci, which is indicative of recent transfer between these genera. Through a genome-wide association study, we discovered that three putative defense systems against invading prophages and plasmids were negatively associated with persistence on farms. Our findings suggest that mobile elements support the persistence of L. monocytogenes on dairy farms and that L. monocytogenes inhabiting the agroecosystem is a potential reservoir of mobile elements that may spread to the food industry. IMPORTANCE Animal-derived raw materials are an important source of L. monocytogenes in the food industry. Knowledge of the factors contributing to the pathogen's transmission and persistence on farms is essential for designing effective strategies against the spread of the pathogen from farm to fork. An increasing body of evidence suggests that mobile genetic elements support the adaptation and persistence of L. monocytogenes in the food industry, as these elements contribute to the dissemination of genes encoding favorable phenotypes, such as resilience against biocides. Understanding of the role of farms as a potential reservoir of these elements is needed for managing the transmission of mobile elements across the food chain. Because L. monocytogenes coinhabits the farm ecosystem with a diversity of other bacterial species, it is important to assess the degree to which genetic elements are exchanged between Listeria and other species, as such exchanges may contribute to the rise of novel resistance phenotypes.

Colonisation dynamics of Listeria monocytogenes strains isolated from food production environments.

Listeria monocytogenes is a ubiquitous bacterium capable of colonising and persisting within food production environments (FPEs) for many years, even decades. This ability to colonise, survive and persist within the FPEs can result in food product cross-contamination, including vulnerable products such as ready to eat food items. Various environmental and genetic elements are purported to be involved, with the ability to form biofilms being an important factor. In this study we examined various mechanisms which can influence colonisation in FPEs. The ability of isolates (n = 52) to attach and grow in biofilm was assessed, distinguishing slower biofilm formers from isolates forming biofilm more rapidly. These isolates were further assessed to determine if growth rate, exopolymeric substance production and/or the agr signalling propeptide influenced these dynamics and could promote persistence in conditions reflective of FPE. Despite no strong association with the above factors to a rapid colonisation phenotype, the global transcriptome suggested transport, energy production and metabolism genes were widely upregulated during the initial colonisation stages under nutrient limited conditions. However, the upregulation of the metabolism systems varied between isolates supporting the idea that L. monocytogenes ability to colonise the FPEs is strain-specific.

Genomic diversity and characterization of Listeria monocytogenes from dry-cured ham processing plants.

Genomic diversity of Listeria monocytogenes isolates from the deboning and slicing areas of three dry-cured ham processing plants was analysed. L. monocytogenes was detected in 58 out of 491 samples from the environment and equipment surfaces, all from the deboning area, with differences in prevalence among facilities. The most frequent PCR-serogroup was IIa (74.1%) followed by IIb and IIc, and only one isolate was serogroup IVb. Twenty different pulsotypes and 11 sequence types (STs) grouped into 10 clonal complexes (CCs) were determined. ST121 (CC121) and ST9 (CC9) were the most abundant. Premature stop codons (PMSC6 and PMSC19) associated with attenuated virulence were found in the inlA sequence in 7 out of 12 selected strains. CC121 strains were strong biofilm formers and some harboured the transposon Tn6188, related with increased tolerance to quaternary ammonium compounds. L. monocytogenes clones considered hypovirulent resulted predominant in the deboning areas. The clonal structure and potential virulence of the isolates could help to establish adequate control measures and cleaning protocols for the comprehensive elimination of the pathogen in dry-cured ham processing environment.

Quantitative risk assessment of Listeria monocytogenes in ready-to-eat (RTE) cooked meat products sliced at retail stores in Greece.

A quantitative microbial risk assessment (QMRA) model predicting the listeriosis risk related to the consumption of Ready- To- Eat (RTE) cooked meat products sliced at retail stores in Greece was developed. The probability of illness per serving assessed for 87 products available in the Greek market was found highly related to the nitrite concentration; products having a lower concentration showed a higher risk per serving. The predicted 95th percentiles of the annual listeriosis cases totaled 33 of which 13 cases were <65 years old and 20 cases ≥65 years old. The highest number of cases was predicted for mortadella, smoked turkey, boiled turkey and parizer, which were the most frequently consumed product categories. Two scenarios for assessing potential interventions to reduce the risk were tested: setting a use-by date of 14 days (these products have no use-by date based on current European Union legislation) and improving the temperature control during domestic storage. The two scenarios resulted in a decrease of the 95th and 99th percentiles of the total annual cases by 97% and 88%, respectively.

Genomic diversity of Listeria monocytogenes isolates from seafood, horticulture and factory environments in New Zealand.

Listeria monocytogenes is a foodborne human pathogen that causes systemic infection, fetal-placental infection in pregnant women causing abortion and stillbirth and meningoencephalitis in elderly and immunocompromised individuals. This study aimed to analyse L. monocytogenes from different sources from New Zealand (NZ) and to compare them with international strains. We used pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and whole-genome single nucleotide polymorphisms (SNP) to study the population structure of the NZ L. monocytogenes isolates and their relationship with the international strains. The NZ isolates formed unique clusters in PFGE, MLST and whole-genome SNP comparisons compared to the international isolates for which data were available. PFGE identified 31 AscI and 29 ApaI PFGE patterns with indistinguishable pulsotypes being present in seafood, horticultural products and environmental samples. Apart from the Asc0002:Apa0002 pulsotype which was distributed across different sources, other pulsotypes were site or factory associated. Whole-genome analysis of 200 randomly selected L. monocytogenes isolates revealed that lineage II dominated the NZ L. monocytogenes populations. MLST comparison of international and NZ isolates with lineage II accounted for 89% (177 of 200) of the total L. monocytogenes population, while the international representation was 45.3% (1674 of 3473). Rarefaction analysis showed that sequence type richness was greater in NZ isolates compared to international trend, however, it should be noted that NZ isolates predominantly came from seafood, horticulture and their respective processing environments or factories, unlike international isolates where there was a good mixture of clinical, food and environmental isolates.

Benzalkonium chloride and heavy metal resistance profiles of Listeria monocytogenes strains isolated from fish, fish products and food-producing factories in Poland.

Phenotypic and genotypic resistance to benzalkonium chloride (BC), cadmium and arsenic was tested (by susceptibility assays and molecular methods) in 287 Listeria monocytogenes strains isolated from fish and fish products, and food-producing factories in Poland. Overall, 40% of the isolates were resistant to BC, 56% to cadmium and 41% to arsenic (57% displayed resistance to more than one of the tested compounds). Among BC-resistant isolates, the most commonly detected resistance determinant was the qacH gene (83%). Three distinct types of cadA gene determining resistance to cadmium were detected, with the cadA1 variant predominant (88%), while most arsenic-resistant isolates (86%) harbored the arsA gene associated with a Tn554-like transposon (one strain harbored two copies of arsA in different arsenic resistance cassettes). 53% of all tested isolates contained plasmids (from 4 kb to > 90 kb in size), which were classified into 11 groups (p1-p11) based on their restriction patterns. Interestingly, 12 isolates harbored the small mobilizable pLMST6-like plasmid pLIS3 encoding multidrug efflux pump EmrC. Clustering analysis of PFGE patterns revealed that these isolates represent several diverse bacterial populations, which strongly suggests mobility of the pLMST6-like plasmids among L. monocytogenes strains and their role in dissemination of BC resistance.

Unraveling the emergence and population diversity of Listeria monocytogenes in a newly built meat facility through whole genome sequencing.

The food processing environments of a newly opened meat processing facility were sampled in ten visits carried out during its first 1.5 years of activity and analyzed for the presence of Listeria monocytogenes. A total of 18 L. monocytogenes isolates were obtained from 229 samples, and their genomes were sequenced to perform comparative genomic analyses. An increase in the frequency of isolation of L. monocytogenes and in the diversity of sequence types (STs) detected was observed along time. Although the strains isolated belonged to six different STs (ST8, ST9, ST14, ST37, ST121 and ST155), ST9 was the most abundant (8 out of 18 strains). Low (0 and 2) single nucleotide polymorphism (SNP) distances were found between two pairs of ST9 strains isolated in both cases 3 months apart from the same processing room (Lm-1267 and Lm-1705, with a 2 SNPs distance in the core genome; Lm-1265 and Lm-1706, with a 0 SNPs distance), which suggests that these strains may be persistent L. monocytogenes strains in the food processing environment. Most strains showed an in silico attenuated virulence potential either through the truncation of InlA (in 67% of the isolates) or the absence of other virulence factors involved in cell adhesion or invasion. Twelve of the eighteen L. monocytogenes isolates contained a plasmid, which ranged in size from 4 to 87 Kb and harbored stress survival, in addition to heavy metals and biocides resistance determinants. Identical or highly similar plasmids were identified for various sets of L. monocytogenes ST9 isolates, which suggests the clonal expansion and persistence of plasmid-containing ST9 strains in the processing environments of the meat facility. Finally, the analysis of the L. monocytogenes genomes available in the NCBI database, and their associated metadata, evidenced that strains from ST9 are more frequently reported in Europe, linked to foods, particularly to meat and pork products, and less represented among clinical isolates than other L. monocytogenes STs. It also showed that the ST9 strains here isolated were more closely related to the European isolates, which clustered together and separated from ST9 North American isolates.

Analysis of virulence genes and molecular typing of Listeria monocytogenes isolates from human, food, and livestock from 2008 to 2016 in Iran.

The frequency of Listeria monocytogenes isolates collected from a total of 1150 samples including food (n = 300), livestock (n = 50), and human clinical (n = 800) was evaluated during 2008-2016. Antimicrobial resistance patterns, virulence factors, and molecular characteristics of these isolates were analyzed using disk diffusion method, sequencing, serotyping, and pulsed-field gel electrophoresis (PFGE). The analysis of 44 L. monocytogenes isolates showed that 72.7% (32 of 44) of all the isolates belonged to Serotype 1/2c, and 15.9% (7 of 44) belonged to Serotype 3c. All 44 isolates were resistant to one or more antimicrobial agents with the most frequent resistance to penicillin (75%) and tetracycline (47.7%). Of the 44 L. monocytogenes strains, 100, 69.2, and 62.5% of livestock, human, and food strains were resistant to penicillin, respectively. Using pulsed-field gel electrophoresis (PFGE) technique, the isolates' genetic diversity was determined, and 28 PFGE patterns with 8 common (CT) and 20 single types (ST) were identified. This study highlights the high prevalence of Serotype 1/2c in clinical and livestock samples, while different serotypes were observed in food samples. The presence of rare serotypes such as 4c, belonging to the Lineage III, as well as 4e and 1/2c which are infrequent in Iran indicates that paying attention to uncommon serotypes, especially 1/2c, during the listeriosis outbreaks is necessary.

MALDI-ToF MS: A Rapid Methodology for Identifying and Subtyping Listeria monocytogenes.

Listeria monocytogenes is a major food-borne pathogen and causative agent of a fatal disease, listeriosis. Stringent regulatory guidelines and zero tolerance policy toward this bacterium necessitate rapid, accurate, and reliable methods of identification and subtyping. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) has recently become a method of choice for routine identification of pathogens in clinical settings and has largely replaced biochemical assays. Identification relies on well-curated databases such as SARAMIS. Extensive use of SARAMIS to generate consensus mass spectra, in conjunction with statistical analysis, such as partial least square-discriminant analysis and hierarchical cluster analysis, is useful in subtyping bacteria. While MALDI-ToF MS has been extensively used for pathogen detection, its application in bacterial subtyping has been limited. The protocol describes a MALDI-ToF MS workflow as a single tool for simultaneous identification and subtyping of L. monocytogenes directly from solid culture medium.

Serotype Assignment by Sero-agglutination, ELISA, and PCR.

For assessing isolates of Listeria monocytogenes, serotype designation is the first subtyping method used. Methodologies used to assign serotype are currently evolving and will eventually be replaced with whole genome sequencing. Traditionally, serotyping has been done with agglutination reactions; however, alternative methods utilizing enzyme linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are common. Described here are the three non-genomic methods and the advantages and disadvantages of each.

Nationwide outbreak of invasive listeriosis associated with consumption of meat products in health care facilities, Germany, 2014-2019.

OBJECTIVES: Invasive listeriosis is a severe foodborne infection caused by Listeria(L.)monocytogenes. The aim of this investigation was to verify and describe a molecular cluster of listeriosis patients and identify factors leading to this outbreak. METHODS: Whole genome sequencing and core genome multilocus sequence typing were used for subtyping L. monocytogenes isolates from listeriosis cases and food samples in Germany. Patient interviews and investigational tracing of foodstuffs offered in health-care facilities (HCF), where some of the cases occurred, were conducted. RESULTS: We identified a German-wide listeriosis outbreak with 39 genetically related cases occurring between 2014 and 2019. Three patients died as a result of listeriosis. After identification of HCF in different regions of Germany for at least 13 cases as places of exposure, investigational tracing of food supplies in six prioritized HCF revealed meat products from one company (X) as a commonality. Subsequently the outbreak strain was analysed in six isolates from ready-to-eat meat products and one isolate from the production environment of company X. No further Sigma1 cases were detected after recall of the meat products from the market and closure of company X (as of August 2020). CONCLUSIONS: Interdisciplinary efforts including whole genome sequencing, epidemiological investigations in patients and investigational tracing of foods were essential to identify the source of infections, and thereby prevent further illnesses and deaths. This outbreak underlines the vulnerability of hospitalized patients for foodborne diseases, such as listeriosis. Food producers and HCF should minimize the risk of microbiological hazards when producing, selecting and preparing food for patients.

Translatability of WGS typing results can simplify data exchange for surveillance and control of Listeria monocytogenes.

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.

Why Are Some Listeria monocytogenes Genotypes More Likely To Cause Invasive (Brain, Placental) Infection?

Although all isolates of the foodborne pathogen Listeria monocytogenes are considered to be pathogenic, epidemiological evidence indicates that certain serovar 4b lineages are more likely to cause severe invasive (neuromeningeal, maternal-fetal) listeriosis. Recently described as L. monocytogenes "hypervirulent" clones, no distinctive bacterial trait has been identified so far that could account for the differential pathogenicity of these strains. Here, we discuss some preliminary observations in experimentally infected mice suggesting that serovar 4b hypervirulent strains may have a hitherto unrecognized capacity for prolonged in vivo survival. We propose the hypothesis that protracted survivability in primary infection foci in liver and spleen-the first target organs after intestinal translocation-may cause L. monocytogenes serovar 4b hypervirulent clones to have a higher probability of secondary dissemination to brain and placenta.

Whole-Genome Sequencing-Based Characterization of Listeria monocytogenes from Fish and Fish Production Environments in Poland.

Listeria monocytogenes, an important foodborne pathogen, may be present in different kinds of food and in food processing environments where it can persist for a long time. In this study, 28 L. monocytogenes isolates from fish and fish manufactures were characterized by whole genome sequencing (WGS). Core genome multilocus sequence typing (cgMLST) analysis was applied to compare the present isolates with publicly available genomes of L. monocytogenes strains recovered worldwide from food and from humans with listeriosis. All but one (96.4%) of the examined isolates belonged to molecular serogroup IIa, and one isolate (3.6%) was classified to serogroup IVb. The isolates of group IIa were mainly of MLST sequence types ST121 (13 strains) and ST8 (four strains) whereas the isolate of serogroup IVb was classified to ST1. Strains of serogroup IIa were further subtyped into eight different sublineages with the most numerous being SL121 (13; 48.1% strains) which belonged to six cgMLST types. The majority of strains, irrespective of the genotypic subtype, had the same antimicrobial resistance profile. The cluster analysis identified several molecular clones typical for L. monocytogenes isolated from similar sources in other countries; however, novel molecular cgMLST types not present in the Listeria database were also identified.