Lm Strains Research Articles

FepR as a Central Genetic Target in the Adaptation to Quaternary Ammonium Compounds and Cross-Resistance to Ciprofloxacin in Listeria monocytogenes.

The foodborne pathogen, Listeria monocytogenes, (Lm), frequently undergoes selection pressure associated with the extensive use of disinfectants, such as quaternary ammonium compounds, which are widely used in food processing plants. The repeated exposure to sub-inhibitory biocide concentrations can induce increased tolerance to these compounds, but can also trigger the development of antibiotic resistance, and both increase the risk of food contamination and persistence in food production environments. Although the acquisition of genes can explain biocide tolerance, the genetic mechanisms underlying the adaptive cross-resistance to antibiotics remain unclear. We previously showed that repeated exposure to benzalkonium chloride (BC) and didecyldimethyl ammonium chloride (DDAC) led to reduced susceptibility to ciprofloxacin in Lm strains from diverse sources. Here, we compared the genomes of 16 biocide-adapted and 10 parental strains to identify the molecular mechanisms of fluoroquinolone cross-resistance. A core genome SNP analysis identified various mutations in the transcriptional regulator fepR (lmo2088) for 94% of the adapted strains and mutations in other effectors at a lower frequency. FepR is a local repressor of the MATE fluoroquinolone efflux pump FepA. The impact of the mutations on the structure and function of the protein was assessed by performing in silico prediction and protein homology modeling. Our results show that 75% of the missense mutations observed in fepR are located in the HTH domain of the protein, within the DNA interaction site. These mutations are predicted to reduce the activity of the regulator, leading to the overexpression of the efflux pump responsible for the ciprofloxacin-enhanced resistance.

A European-wide dataset to uncover adaptive traits of Listeria monocytogenes to diverse ecological niches

Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes listeriosis, a serious foodborne illness. In the nature-to-human transmission route, Lm can prosper in various ecological niches. Soil and decaying organic matter are its primary reservoirs. Certain clonal complexes (CCs) are over-represented in food production and represent a challenge to food safety. To gain new understanding of Lm adaptation mechanisms in food, the genetic background of strains found in animals and environment should be investigated in comparison to that of food strains. Twenty-one partners, including food, environment, veterinary and public health laboratories, constructed a dataset of 1484 genomes originating from Lm strains collected in 19 European countries. This dataset encompasses a large number of CCs occurring worldwide, covers many diverse habitats and is balanced between ecological compartments and geographic regions. The dataset presented here will contribute to improve our understanding of Lm ecology and should aid in the surveillance of Lm. This dataset provides a basis for the discovery of the genetic traits underlying Lm adaptation to different ecological niches.

Characterization of Listeria monocytogenes isolated from wildlife in central New York.

Background Listeria monocytogenes (Lm) present in farming soil and food‐processing facilities threatens food safety, but little is known about the carriage of Lm by wildlife.ObjectivesWe estimated the prevalence of faecal Lm shedding among wildlife admitted to a veterinary medical teaching hospital in central New York and characterized a subset of the Lm isolates.MethodsWildlife samples were collected between May 2018 and December 2019. We characterized the Lm isolates by assessing the growth at three temperatures approximating the body temperatures of reptiles (25°C), mammals (37°C), and birds (42°C) and identifying genotypic characteristics related to transmission and virulence.ResultsThe apparent prevalence of faecal Lm shedding was 5.6% [18/324; 95% confidence interval (CI), 3.3%–8.6%]. Among 13 isolates that represented two lineages and 11 clonal complexes, three and five isolates were grouped into the same SNP clusters with human clinical isolates and environmental isolates, respectively. However, specific SNP difference data showed that Lm from wildlife was generally not closely related (>22 SNP differences) to Lm from human clinical sources and the food‐processing environment. While the stress response locus SSI‐2 was absent, SSI‐1 was found in four isolates. Virulence genes prfA, plcA, hly, mpl, actA, plcB, inlA, inlB, inlC, inlE, inlH, inlJ, and inlK were present, without any premature stop codons, in all isolates. Virulence loci Listeria pathogenicity island 3 (LIPI‐3) and LIPI‐4, which have been linked to hypervirulence, and inlG were found in four, three, and seven isolates, respectively.ConclusionsWildlife represents a potential reservoir for genetically diverse and putatively hypervirulent Lm strains. No statistically significant association between growth parameters and hosts was observed. However, compared to lineage I isolates, lineage II isolates showed significantly (p < 0.05) faster growth at 25°C and significantly slower growth at 42°C, suggesting that wildlife Lm isolates that belong to lineages I and II differ in their ability to grow at 25°C and 42°C.

Whole-Genome Sequencing Characterization of Virulence Profiles of Listeria monocytogenes Food and Human Isolates and In Vitro Adhesion/Invasion Assessment

Listeria monocytogenes (Lm) is the causative agent of human listeriosis. Lm strains have different virulence potential. For this reason, we preliminarily characterised via Whole-Genome Sequencing (WGS) some Lm strains for their key genomic features and virulence-associated determinants, assigning the clonal complex (CC). Moreover, the ability of the same strains to adhere to and invade human colon carcinoma cell line Caco-2, evaluating the possible correspondence with their genetic virulence profile, was also assessed. The clinical strains typed belonged to clonal complex (CC)1, CC31, and CC101 and showed a very low invasiveness. The Lm strains isolated from food were assigned to CC1, CC7, CC9, and CC121. All CC1 carried the hypervirulence pathogenicity island LIPI-3 in addition to LIPI-1. Premature stop codons in the inlA gene were found only in Lm of food origin belonging to CC9 and CC121. The presence of LIPI2_inlII was observed in all the CCs except CC1. The CC7 strain, belonging to an epidemic cluster, also carried the internalin genes inlG and inlL and showed the highest level of invasion. In contrast, the human CC31 strain lacked the lapB and vip genes and presented the lowest level of invasiveness. In Lm, the genetic determinants of hypo- or hypervirulence are not necessarily predictive of a cell adhesion and/or invasion ability in vitro. Moreover, since listeriosis results from the interplay between host and virulence features of the pathogen, even hypovirulent clones are able to cause infection in immunocompromised people.

Genomic persistence of Listeria monocytogenes strains in dairy and meat processing plants in Italy

Abstract Background Persistence of Listeria monocytogenes (Lm) in food processing plants (FPP) is still considered the major source of food contamination during processing. Correlations among different Lm strains and evaluation of the isolates persistence in FPP environments is an essential tool to improve food safety. The aim of this study was to investigate the genomic characteristics of persistent Lm strains (e.g. Metal and Detergent Resistance, SSIs and genomic island). Methods Totally 221 strains isolated from 10 FPP in the Abruzzo and Molise regions, Italy, were enrolled (4 of dairy products FPP - DP - and 6 of meat products FPP - MP). Sampling was performed in 6 sessions, from March 2017 to May 2018. Strains were sequenced using Illumina platform. The CCs, cgMLST analysis and Metal and Detergent Resistance, SSIs and genomic island (GI) detection was carried using the Pasteur scheme. Results Results returned 13 CCs and 2 singletons. cgMLST clustering showed two persistent clusters DP and 9 clusters in MP. In DP CC3 and CC9 were persistent. While in MP CC5, CC8, CC9, CC37, CC121 and CC155. The presence of SSI-1, was detected in 100% of the persistent strains from DP and 80% from MP, in particular in all strains of CC155, CC5, CC8 and CC9. In CC121 and CC37 it was absent. The presence of loci of Lm GI3, was in 100% of persistent strains of DP and in 52.3% of MP plant, in particular in 100% of CC121, CC8, CC5 and 16.3% of CC9. The 100% of CC121 in DP and MP plant harboured Tn6188 and SSI-2. The inlA PMSC mutations were detected in 100% of CC9 and CC121 strains. Conclusions The persistence of strains inside FPP was confirmed using NGS data, most of the strains were characterised by the presence of genes involved in resistance and adaptation. NGS is confirmed to be a powerful tool to study Lm persistence in FPP, looking also at strains' ecology. Key messages Most of persistent strains showed genes involved in resistance and adaptation in the food processing environment. NGS could help in facing Lm persistence in food producing plants giving new insights on the genomic characteristics of persistent strains.

Genotyping of Listeria monocytogenes strains in Italian food-processing environments

Abstract Background In 2019, case fatality from Listeria monocytogenes (Lm) was higher than the previous years confirming Lm as a major public health problem. Food is the main source of Lm infection and cross-contamination from the food-processing environment (FPEs) is the most common route of contamination. The aim of this work was to characterize FPEs Lm strains collected from the National Reference Laboratory (NRL) for Lm in order to evaluate circulating clonal complexes (CCs) and their environmental adaptation. Methods NRL Lm database actually consists of 798 FPEs sequenced strains. In silico MLST was evaluated. Sequences related to prevalent CCs were further investigated using Pasteur's cgMLST scheme and detecting metal and detergent resistance genes and stress survival islet (SSI). Results MLST identified 25 CCs and 4 singleton. Nine isolates gave inexact match with existing alleles and were not classified. CC9 (17.4%) and CC121 (14.2%) were the prevalent clones. CC9 cgMLST analysis revealed a relatedness among strains isolated from the same establishment type (meat or dairy), even if sampled in different regions. Indeed, CC121 cgMLST clustering analysis was strictly associated to the point of sampling. Regarding stress adaptation genes, SSI1 and brcABC genes were detected in 100% and 2.9% of the CC9 strains, respectively. Meanwhile, SSI2 and transposon Tn6188 were detected, respectively, in 100% and 97.3% of CC121 strains. Conclusions Results confirmed, in Italian FPEs, prevalence of hypovirulent Lm strains, as previously observed in Italian food. Tested isolates highlighted adaptation to low pH and high salt concentration (SSI1 genes for CC9), to alkaline and oxidative stress (SSI2 genes for CC121) and benzalkonium chloride tolerance (Tn6188 in CC121) resulting in a best survival under stress conditions. Interestingly, CC9 clustering denoted allelic proximity among strains isolated in different Italian regions paying attention to clones spreading at national level. Key messages Listeria monocytogenes CC9 and CC121 prevalence in Italian establishment confirms environment is the main route of food contamination at production level. Prevalent food-processing environmental Italian Listeria monocytogenes strains denotes adaptation to environmental stress and clones spreading at national level.

Listeria monocytogenes-infected human monocytic derived dendritic cells activate V\u03b39V\u03b42 T cells independently of HMBPP production

Gamma-delta (γδ) T cells express T cell receptors (TCR) that are preconfigured to recognize signs of pathogen infection. In primates, γδ T cells expressing the Vγ9Vδ2 TCR innately recognize (E)-4-hydroxy-3-methyl-but- 2-enyl pyrophosphate (HMBPP), a product of the 2-C-methyl-D-erythritol 4- phosphate (MEP) pathway in bacteria that is presented in infected cells via interaction with members of the B7 family of costimulatory molecules butyrophilin (BTN) 3A1 and BTN2A1. In humans, Listeria monocytogenes (Lm) vaccine platforms have the potential to generate potent Vγ9Vδ2 T cell recognition. To evaluate the activation of Vγ9Vδ2 T cells by Lm-infected human monocyte-derived dendritic cells (Mo-DC) we engineered Lm strains that lack components of the MEP pathway. Direct infection of Mo-DC with these bacteria were unchanged in their ability to activate CD107a expression in Vγ9Vδ2 T cells despite an inability to synthesize HMBPP. Importantly, functional BTN3A1 was essential for this activation. Unexpectedly, we found that cytoplasmic entry of Lm into human dendritic cells resulted in upregulation of cholesterol metabolism in these cells, and the effect of pathway regulatory drugs suggest this occurs via increased synthesis of the alternative endogenous Vγ9Vδ2 ligand isoprenyl pyrophosphate (IPP) and/or its isomer dimethylallyl pyrophosphate (DMAPP). Thus, following direct infection, host pathways regulated by cytoplasmic entry of Lm can trigger Vγ9Vδ2 T cell recognition of infected cells without production of the unique bacterial ligand HMBPP.

Genomic characterization of food-borne Listeria monocytogenes isolates from Nanshan district of Shenzhen during 2009-2019

Objective: The study aims to investigate the characteristic baseline information about genetic lineages, drug-resistance genes, virulence genes and evolutionary relationships of food-borne Listeria monocytogenes (Lm) isolates from Nanshan district of Shenzhen. Methods: The whole genomes of 46 Lm isolates during 2009-2019 were extracted and sequenced (Illumina PE-150, 100×). The CLC Genomics Workbench 12.0 was used to assemble and align Lm genomes, analyze their housekeeping genes, drug-resistance genes and virulence genes, and construct a k-mer phylogenetic tree. Results: After assembly, all genomes satisfied analytical conditions (contigs N50>20 kb). The medians of GC content, gene count and gene size were 38.3%, 5 960 and 2 952 608 bp, respectively. Based on the Lm genomic reference database, the local k-mer phylogenetic tree had 14 clusters of which the genetic distance was wide. The 46 Lm isolates were classified as Lineage 1 (21), Lineage 2 (23) and Lineage 3 (2). The most common ST type of Lineage 1 was ST87, followed by ST3, ST59, ST224 and ST429, whereas the major ST types of Lineage 2 included ST8 and ST9, the rest being ST121, ST155, ST199, ST204 and ST321. However, Lineage 3 only had ST299. The part of Lm strains carried five drug-resistance genes, such as fosX (17), tetM (6), dfrG (4), catB3 (1) and mefA (1). Furthermore, all strains possessed nine virulence genes, including flaA, iap, actA, hly, mpl, prfA, plcA, plcB and inlB. Nevertheless, six isolates and three of them respectively carried the mutant inlA and inlJ, and other two isolates lacked inlC. Conclusion: The food-borne Lm isolates from Nanshan district of Shenzhen presented genetic and evolutionary diversity. Noted that the drug-resistant strains, which also owned abundant virulence genes with specific functions, could lead to serious infections, particularly those isolates from raw poultry and Flammulina velutipes. It was implied that the local region was at risk due to Listeriosis by food. This study offered reference for prevention, control and treatment of Lm infection to the Greater Bay Area.

Analysis of Genetic Diversity and Antibiotic Options for Clinical Listeria monocytogenes Infections in China.

BackgroundThe aim of this study was to investigate the mechanism of Listeria monocytogenes (Lm) pathogenicity and resistance. In addition, the effect of existing treatment options against Lm were systematically evaluated.MethodsSix Lm isolates were collected and antimicrobial susceptibility testing of 15 antibiotics were done. Subsequently, whole genome sequencing and bioinformatics analysis were performed. Biofilm formation was evaluated by crystal violet staining. Furthermore, the effect of meropenem, linezolid, penicillin, vancomycin, and trimethoprim/sulfamethoxazole were determined using the time-kill assay.ResultsFour sequence types (STs) were identified (ST1, ST3, ST87, ST451). Multivirulence-locus sequence typing results classified ST87 isolates into cluster. All isolates were resistant to fosfomycin and daptomycin with fosX and mprF. In addition, a total of 80 virulence genes were detected and 72 genes were found in all 6 isolates. Seven genes associated with hemolysin were found in 26530 and 115423. However, due to lack of one genomic island including virulence genes related to flagellar synthesis, isolate 115423 produced less biofilm than 5 other isolates. Although all isolates were susceptible to vancomycin, the in vitro time-kill assay showed that vancomycin monotherapy resulted in less than 2 log10 cerebrospinal fluid (CFU)/mL compared with the initial count. Trimethoprim/sulfamethoxazole at serum or CFU concentrations had bactericidal effect against tested Lm strains at 24 hours.ConclusionsST87 clone was a typical prevalent ST in clinical Lm isolates in China. Trimethoprim/sulfamethoxazole might be greater potential therapeutic option against Lm infections.

Exposure to Quaternary Ammonium Compounds Selects Resistance to Ciprofloxacin in Listeria monocytogenes.

In this contribution, the antimicrobial susceptibility toward 11 antibiotics and four biocides of a panel of 205 Listeria monocytogenes (Lm) strains isolated from different ecological niches (i.e., food, animals and natural environment) was evaluated. The impact of exposure to biocides on the antibiotic susceptibilities of Lm was also investigated. Lm strains isolated from food exhibited overall a lower susceptibility (higher minimal inhibitory concentrations, MIC) for ammonium quaternary compounds (QACs) and peracetic acid (PAC) than strains isolated from animals and natural environments. Conversely, the ecological origins of Lm strains did not significantly affect their susceptibilities towards antibiotics. Interestingly, repeated exposure to QACs recurrently led to a decrease in susceptibility toward ciprofloxacin (CIP), a fluoroquinolone antibiotic, largely used in human medicine. Moreover, these lower levels of susceptibility to CIP remained stable in most Lm strains even after subcultures without biocide selection pressure, suggesting an adaptation involving modifications at the genetic level. Results underlined the ability of Lm to adapt to biocides, especially QACs, and the potential link between this adaptation and the selection of resistance toward critical antibiotics such as ciprofloxacin. These data support a potential role of the extensive use of QACs from “farm to fork” in the selection of biocide and antibiotic resistance in pathogenic bacteria such as Lm.

Genomic elements located in the accessory repertoire drive the adaptation to biocides in Listeria monocytogenes strains from different ecological niches

In response to the massive use of biocides for controlling Listeria monocytogenes (hereafter Lm) contaminations along the food chain, strains showing biocide tolerance emerged. Here, accessory genomic elements were associated with biocide tolerance through pangenome-wide associations performed on 197 Lm strains from different lineages, ecological, geographical and temporal origins. Mobile elements, including prophage-related loci, the Tn6188_qacH transposon and pLMST6_emrC plasmid, were widespread across lineage I and II food strains and associated with tolerance to benzalkonium-chloride (BC), a quaternary ammonium compound (QAC) widely used in food processing. The pLMST6_emrC was also associated with tolerance to another QAC, the didecyldimethylammonium-chloride, displaying a pleiotropic effect. While no associations were detected for chemically reactive biocides (alcohols and chlorines), genes encoding for cell-surface proteins were associated with BC or polymeric biguanide tolerance. The latter was restricted to lineage I strains from animal and the environment. In conclusion, different genetic markers, with polygenic nature or not, appear to have driven the Lm adaptation to biocide, especially in food strains but also from animal and the environment. These markers could aid to monitor and predict the spread of biocide tolerant Lm genotypes across different ecological niches, finally reducing the risk of such strains in food industrial settings.

Evaluation of sodium hypochlorite and peroxyacetic acid to prevent transfer of surface‐attached Listeria monocytogenes to produce

AbstractSporadic contamination of produce with Listeria monocytogenes (Lm) is a recurrent problem. Ways for contamination to occur include transfer of the pathogen to produce from processing surfaces or soil particles to which it has become attached. Since it is known that surface‐attached Lm is less susceptible to antimicrobials than Lm grown in liquid culture, the goal of the current study was to determine if Lm grown on surfaces and released into water retained its higher tolerance of antimicrobials. In addition, transfer of Lm from surfaces or soil particles to blueberries, mung beans and spinach leaves in the presence of the antimicrobials, sodium hypochlorite (NaOCl) adjusted to pH 6.8 and peroxyacetic acid (PAA), was studied. The experiments were carried out with a cocktail of six Lm strains and strains obtained from produce processing plants. Results indicated that Lm released from surfaces was as susceptible to the two antimicrobials as Lm grown in liquid culture and was inactivated within seconds by an initial concentration of 100 ppm of NaOCl or PAA. Transfer of Lm grown on stainless steel coupons to blueberries in wash water was not observed at NaOCl concentrations as low as 20 ppm. In contrast, transfer of Lm from washed soil particles (COD = 1.7 ± 0.89 mg/l) in contact with mung beans was observed even at initial NaOCl or PAA concentrations of 250 ppm. Lm released from washed soil particles to which it was attached to spinach leaves could be detected in the presences of 20 ppm of NaOCl and PAA and occasionally even in the presence of 100 and 250 ppm of the antimicrobials.

46. Tacrolimus Increases Susceptibility to Secondary Infection in a Mouse Model

BackgroundTransplant acceptance requires life-long pharmacological intervention that broadly suppresses recipients’ immunity in order to prevent rejection of foreign graft. In turn, non-specific immune-suppression in these patients is also associated with increased risk of infection from opportunistic pathogens. Currently our knowledge on the effects immune suppressive therapies on adaptive immune components response in patients is limitedMethodsTo investigate this we established a mouse model of post-transplant immune suppression therapy, using tacrolimus. To dissect the effects of tacrolimus on infection susceptibility, tacrolimus-treated mice were infected with a virulent strain of recombinant Listeria monocytogenes (Lm) expressing model antigens. Infection with this transgenic strain of Lm transforms these model antigens into surrogate Lm antigens and allows tracking of pathogen-specific T-cells using MHC tetramer staining.ResultsHere we show, tacrolimus treatment triggered increased susceptibility to secondary, but not primary Lm infection with increased bacterial burden in the liver and spleen tissues. Increased susceptibility during secondary infection paralleled dampened functional activation of Lm-specific CD8+ T cells as indicated by diminished in vivo cytolytic activity. Interestingly, when tacrolimus treatment was initiated only during primary or during secondary infection susceptibility to infection was overturned as both groups of mice had lower bacterial burden in target tissues. This suggests that while tacrolimus treatment does not negatively impact primary immune response, it may dampen the formation of CD8+ T cell memory.ConclusionFurther studies will investigate the long-term durability of blunted pathogen-specific memory and CTL activity triggered by tacrolimus treatment after cessation of therapy. These findings will allow more defined prediction of patient risk of infection allowing for a personalized prophylaxis regimen.Disclosures All Authors: No reported disclosures

A Cross-Protective Vaccine Against 4b and 1/2b Listeria monocytogenes.

Listeria monocytogenes (Lm) is a foodborne zoonotic pathogen that causes listeriosis with a mortality rate of 20–30%. Serovar 4b and 1/2b isolates account for most of listeriosis outbreaks, however, no listeriosis vaccine is available for either prophylactic or therapeutic use. Here, we developed a triple-virulence-genes deletion vaccine strain, and evaluated its safety, immunogenicity, and cross-protective efficiency. The virulence of NTSNΔactA/plcB/orfX was reduced 794-folds compared with the parental strain. Additionally, it was completely eliminated in mice at day 7 post infection and no obvious pathological changes were observed in the organs of mice after prime-boost immunization for 23 days. These results proved that the safety of the Lm vaccine strain remarkably increased. More importantly, the NTSNΔactA/plcB/orfX strain stimulated higher anti-Listeriolysin O (LLO) antibodies, induced significantly higher expression of IFN-γ, TNF-α, IL-17, and IL-6 than the control group, and afforded 100% protection against serovar 4b and 1/2b challenges. Taken together, our research demonstrates that the triple-genes-deletion vaccine has high safety, can elicit strong Th1 type immune response, and affords efficient cross-protection against two serovar Lm strains. It is a promising vaccine for prevention of listeriosis.

Development of a model for the alcoholic fermentation of cocoa beans by a Saccharomyces cerevisiae strain

The aromatic quality of chocolate requires the use of cocoa with high aromatic potential, this being acquired during the fermentation of cocoa beans. Traditional fermentation is still often carried out on a small scale with wild strains of yeasts and acetic bacteria and under poorly controlled conditions leading to cocoa quality ranging from best to worst. This study is the first part of a project aiming to control quality of cocoa to produce high aromatic quality chocolate by using a mixed starter of selected strains of yeast and acetic bacteria and by controlling the conditions of fermentation. To achieve this objective, a mathematical model of the alcoholic fermentation of cocoa beans has been developed. The growth, glucose consumption and ethanol production of Saccharomyces cerevisiae LM strain in synthetic broth were modeled for the most important intrinsic (pH, glucose, ethanol, free nitrogen and oxygen levels) and extrinsic (temperature, oxygen level) fermentation parameters. The model was developed by combining the effects of individual conditions in a multiplicative way using the gamma concept. The model was validated in liquid synthetic medium at two different inoculation levels 104 and 106 CFU/mL with an increase in temperature that recorded during spontaneous fermentations. The model clearly shows that the level of inoculation and the speed of the increase in temperature clearly drive yeast growth, while other factors including pH and ethanol, free nitrogen and oxygen levels have no significant impact on yeast growth.

Listeria monocytogenes clones in Italian food products: virulence and environmental adaptation

Abstract Background Food is the main source of Listeria monocytogenes (Lm) infection. Lm is a highly heterogeneous species composed of hypervirulent and hypovirulent clones. Understanding the distribution of Lm clonal complexes (CCs) in different food categories has strong implications for risk assessment. The aim of this work was to analyse collection of Lm strains of National Reference Laboratory (NRL Lm) in order to assess link between genetic profile and matrices and the level of pathogenicity of circulating strains based on CCs. Methods NRL Lm database actually consists of 906 sequenced strains isolated in 10 years from 5 food compartments (meat, fish, dairy, vegetables and composite dishes). Epidata were analysed to remove redundant strains based on the same epidemiological description. After that, WGS data from 465 Lm strains were investigated. In silico MLST was defined and Roary 3.12.0 was used to obtain a pan-genome profile. Genes were later uploaded to Pasteur Institute platform for characterization. Results In silico MLST identified 36 CCs and 6 singleton. CC9 (23.0%), CC8 (15.3%) and CC121 (13.3%) were the prevalent CCs. In particular, CC9 was present in 35.2% of meat samples and CC8 in 25.8% of fish samples. Pan genome profile revealed high prevalence (&amp;gt;98%) of genes related to biofilm formation and resistance to environmental stress in CC9 strains and genes involved in tolerance to quaternary ammonium compounds in CC121 strains. Conclusions Results, in particular for meat products, confirmed in Italy, the prevalence of hypovirulent Lm strains previously observed at European Union level. The high presence of stress resistance and disinfectant tolerance genes in these strains could make them able to persist in food-production environment and should be taken into account evaluating the health hazards. In fish product is also relevant the prevalence of CC8 strains which are potentially highly pathogenic and have been responsible of recent European multi country outbreak. Key messages Pangenome of Listeria monocytogenes isolated from Italian food revealed high presence of disinfectant tolerance and stress resistance genes in meat products and virulence genes in fish products. Listeria monocytogenes CC9 and CC121 prevalence in Italian meat product confirms occurrence of hypovirulent strains detected at European Union level.

Biofilm production by Listeria monocytogenes strains: detection with colorimetric analysis

Abstract Background In literature, there are no standardized laboratory methods to detect formed biomass by colorimetric analysis. The purpose of this study was to compare three staining methods and two different wavelengths for determination of biofilm formation of Listeria monocytogenes (Lm) strains. Methods Three strains of Lm isolated from different origin were tested using 96 well polistirene plates at 12 °C and 30 °C, after incubation the wells were subjected to washing, detaching and staining with crystal violet (CV) at 0.2% and 2% (Panreac EU) in 95% ethanol and with Gram's crystal violet solution (Merck KGaA, Germany). The absorbance at 492nm and 540nm wavelengths was read using a spectrophotometer (SIRIO S, Seac, Firenze, Italia). Results The strains incubated at 12 °C displayed production of biofilm when stained with CV 2% and with Gram's crystal violet solution, both at 492 and 540 nm (with better evidence at 540 nm). If CV 0.2% was used to stain and reading at both optical densities there was evidence of weak or no biofilm production. At 30 °C, the biofilm production was displayed at both temperature and with all the stains. For all the strains and for all the conditions tested, the absorbance was greater but not proportional using the Gram's crystal violet solution, versus the CV 0,2% and CV 2%, and absorbance was higher at 540nm versus at 492nm. Conclusions Results confirmed the lack of reproducibility of each of the method used to detect and quantify the biomass produced during a biofilm formation test in vitro and the absence of ratio between the different results obtained using different CV concentration and wavelengths for reading. Key messages Biofilm production at 12 °C could not be adequately detected staining the wells with CV 0,2%. Absorbance could be influenced by the solvent in the stain used (ethanol, methanol or phenol or mixtures). To obtain data for assessment of biomass formation, being the method characterized by poor reproducibility, the laboratory should use at least the same stain and wavelength.

Survival of a serotype 4b strain and a serotype 1/2a strain of Listeria monocytogenes, isolated from a stone fruit outbreak investigation, on whole stone fruit at 4 °C

In the summer of 2014, a multistate outbreak of listeriosis associated with contaminated stone fruit (peach and nectarine) was reported. A serotype 4b variant Listeria monocytogenes (Lm) strain of singleton Sequence Type (ST) 382 was isolated from clinical samples and stone fruit associated with the outbreak. A serotype 1/2b Lm strain of ST5, Clonal Complex 5 was isolated only from outbreak-associated stone fruit, not from clinical samples. Here we investigated the fate of the serotype 4b and 1/2b strains, at two inoculation levels (high level at 3.7 logCFU/fruit and low level at 2.7 logCFU/fruit), on the surfaces of white peach, yellow peach and yellow nectarine stored at 4 °C for 26 days. After rinsing the fruits, we determined the Lm levels in the rinsates and on the peels. We enumerated Lm using a direct plating method and compared two chromogenic agars. The Lm populations rapidly declined in the first 3 days and then declined more slowly until Day 19/21. The maximum decline was 1.6 logCFU/fruit on yellow peach inoculated with serotype 4b at high level. For fruits inoculated with high-level Lm, the lowest level of Lm (1.7 logCFU/fruit) was observed on for white peach inoculated with serotype 1/2b, and the highest level of Lm (2.6 logCFU/fruit) on Day 19/21 was observed on yellow peach inoculated with the serotype 1/2b strain. For fruits inoculated with low-level Lm, the lowest level of Lm (1.3 logCFU/fruit) was observed on yellow nectarine inoculated with either the serotype 4b or 1/2b strain, and the highest level of Lm (1.7 logCFU/fruit) on Day 19/21 was observed on yellow peach inoculated with ST382. The D-values ranged from 15 days to 28 days. Lm remained viable until the end of storage (Day 26), but the levels were not significantly different from those on Day 19/21. The types of stone fruit and Lm strain did not significantly affect the survival of Lm. These results demonstrate that contaminated stone fruit can carry a potential risk for causing listeriosis in susceptible populations. Comparison of direct plating results using two chromogenic agars showed that RAPID' L. mono and Agar Listeria Ottavani & Agosti performed equivalently for enumerating Lm on stone fruit. The fruit rinsing recovered 80% to 84% of Lm from fruit surfaces.

Active Lysogeny in Listeria Monocytogenes Is a Bacteria-Phage Adaptive Response in the Mammalian Environment

SummarySome Listeria monocytogenes (Lm) strains harbor a prophage within the comK gene, which renders it inactive. During Lm infection of macrophage cells, the prophage turns into a molecular switch, promoting comK gene expression and therefore Lm intracellular growth. During this process, the prophage does not produce infective phages or cause bacterial lysis, suggesting it has acquired an adaptive behavior suited to the pathogenic lifestyle of its host. In this study, we demonstrate that this non-classical phage behavior, named active lysogeny, relies on a transcriptional response that is specific to the intracellular niche. While the prophage undergoes lytic induction, the process is arrested midway, preventing the transcription of the late genes. Further, we demonstrate key phage factors, such as LlgA transcription regulator and a DNA replicase, that support the phage adaptive behavior. This study provides molecular insights into the adaptation of phages to their pathogenic hosts, uncovering unusual cooperative interactions.

Susceptibility of aging mice to listeriosis: Role of anti-inflammatory responses with enhanced Treg-cell expression of CD39/CD73 and Th-17 cells

Foodborne Listeria monocytogenes (Lm) causes serious illness and death in immunosuppressed hosts, including the elderly population. We investigated Lm susceptibility and inflammatory cytokines in geriatric mice. Young-adult and old mice were gavaged with a Lm strain Lmo-InlAm. Tissues were assayed for Lm burden and splenocytes were analyzed for Th1/Th2/Th17/Treg responses and expression of CD39 and CD73. Old Lm-infected mice lost body-weight dose-dependently, had higher Lm colonization, and showed higher inflammatory responses than Lm-infected young-adult mice. After infection, IL-17 levels increased significantly in old mice whereas IFN-γ levels were unchanged. Levels of IL-10 and Treg cells were increased in infected old mice as compared to infected young-adult mice. Age-dependent enhanced expression of CD39/CD73 was observed in purified Treg prior to infection, suggesting increased baseline adenosine production in old mice. Lm lysate-treated splenocytes from older mice produced significantly higher levels of IL-10, IL17, and IL-1β, produced less IFN-γ and IL-2, and proliferated less than splenocytes from young-adult mice. Data suggests that older mice maybe more susceptible to Lm infection due to an imbalance of Th cell responses with disproportionate and persistent anti-inflammatory responses. Lm infection enhanced differentiation of proinflammatory Th17 cells, which may also exacerbate pathological responses during listeriosis.