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Background Listeria (L.) monocytogenes strains show a high diversity regarding stress tolerance and virulence potential. Genome studies have mainly focused on specific sequence types (STs) predominantly associated with either food or human listeriosis. This study focused on the prevalent ST155, showing equal distribution among clinical and food isolates. We evaluated the virulence potential of 20 ST155 strains and performed comparative genomic analysis of 130 ST155 strains isolated from food, food processing environments and human listeriosis cases in different countries and years. Results The in vitro virulence assays using human intestinal epithelial Caco2 and hepatocytic HEPG2 cells showed an impaired virulence phenotype for six of the 20 selected ST155 strains. Genome analysis revealed no distinct clustering of strains from the same source category (food, food processing environment, and clinical isolates). All strains harbored an intact inlA and inlB locus, except four strains, which had an internal deletion in the inlA gene. All strains harbored LIPI-1, but prfA was present in a longer variant in six strains, all showing impaired virulence. The longer PrfA variant resulted in lower expression of inlA, inlB, and prfA, and no expression of hly and actA. Regarding stress-related gene content, SSI-1 was present, whereas qacH was absent in all strains. 34.6% of the strains harbored a plasmid. All but one ST155 plasmids showed high conservation and harbored cadA2, bcrABC, and a triphenylmethane reductase. Conclusions This study contributes to an enhanced understanding of L. monocytogenes ST155 strains, being equally distributed among isolates from humans, food, and food processing environments. The conservation of the present genetic traits and the absence of unique inherent genetic features makes these types of STs especially interesting since they are apparently equally adapted to the conditions in food processing environments, as well as in food as to the human host environment. However, a ST155-specific mutation resulting in a longer PrfA variant impaired the virulence potential of several ST155 strains.

Background: The blood-brain barrier (BBB) is altered in several diseases of the central nervous system. For example, the breakdown of the BBB during cerebral ischemia in stroke or traumatic brain injury is a hallmark of the diseases' progression. This functional damage is one key event which is attempted to be mimicked in in vitro models. Recent studies showed the pivotal role of micro-environmental cells such as astrocytes for this barrier damage in mouse stroke in vitro models. The aim of this study was to evaluate the role of micro-environmental cells for the functional, paracellular breakdown in a human BBB cerebral ischemia in vitro model accompanied by a transcriptional analysis. Methods: Transwell models with human brain endothelial cell line hCMEC/D3 in mono-culture or co-culture with human primary astrocytes and pericytes or rat glioma cell line C6 were subjected to oxygen/glucose deprivation (OGD). Changes of transendothelial electrical resistance (TEER) and FITC-dextran 4000 permeability were recorded as measures for paracellular tightness. In addition, qPCR and high-throughput qPCR Barrier chips were applied to investigate the changes of the mRNA expression of 38 relevant, expressed barrier targets (tight junctions, ABC-transporters) by different treatments. Results: In contrast to the mono-culture, the co-cultivation with human primary astrocytes/pericytes or glioma C6 cells resulted in a significantly increased paracellular permeability after 5 h OGD. This indicated the pivotal role of micro-environmental cells for BBB breakdown in the human model. Hierarchical cluster analysis of qPCR data revealed differently, but also commonly regulated clustered targets dependent on medium exchange, serum reduction, hydrocortisone addition and co-cultivations. Conclusions: The co-cultivation with micro-environmental cells is necessary to achieve a functional breakdown of the BBB in the cerebral ischemia model within an in vivo relevant time window. Comprehensive studies by qPCR revealed that distinct expression clusters of barrier markers exist and that these are regulated by different treatments (even by growth medium change) indicating that controls for single cell culture manipulation steps are crucial to understand the observed effects properly.

<p>Biofilms are suggested to be a source of contamination in the food producing environment leading to food spoilage or the transmission of food-borne pathogens. However, to date, research has mainly focused on the presence of (biofilm-forming) bacteria within food processing environments, without analysing the associated biofilm matrix components.</p> <p>The aim of this study was to identify biofilm hotspots in a meat processing environment by analysing the presence of microorganisms (by cultivation and targeted quantitative real-time PCR based on 16S rRNA) and the major matrix components carbohydrates, extracellular DNA and proteins. Sampling included 47 distinct food contact surfaces and 61 distinct non-food contact surfaces from eleven rooms within an Austrian meat processing plant, either during operation or after cleaning and disinfection. Additionally, we isolated and characterized bacteria found in biofilms. The biofilm forming capacity of eleven isolates, was tested, using a static biofilm model. Additionally, two different multi-species settings were tested combining three strains, each. Biofilms were grown on stainless-steel slides for seven days at 10 °C, to mimic conditions found in the food producing environment.</p> <p>Overall, we identified ten biofilm positive sites, among them seven of which were sampled during operation and three after cleaning and disinfection. Five biofilms were detected on food contact surfaces (cutters and associated equipment and a screw conveyor) and five on non-food contact surfaces (drains and water hoses) resulting in 9.3 % of the sites being classified as biofilm positive. From these sites we cultivated bacteria of 29 different genera. The most prevalent bacteria belonged to the genera <em>Brochothrix</em>, <em>Pseudomonas</em> and <em>Psychrobacter</em>. From each biofilm we isolated bacteria from four to 12 different genera, indicating the presence of multi-species biofilms.</p> <p>Culturing of eleven isolates of different species (all detected in the mentioned biofilms, representing typical residential and spoilage bacteria in the meat processing environment) showed that there are differences of individual strains to produce matrix components and biomass on stainless steel slides.  <em>Brochothrix</em>, <em>Carnobacterium</em> and <em>Kocuria</em> produced only detectable amounts of carbohydrates but neither eDNA nor proteins. The <em>Acinetobacter</em> and the <em>Flavobacterium</em> isolates were able to produce two of the measured components and six strains were capable of producing all types of analysed matrix components, among them a <em>Pseudomonas</em> <em>fragi</em> isolate. The minimal mean bacterial load detected was 5.4 log CFU/cm<sup>2</sup> formed by the <em>Psychrobacter</em> strain.</p> <p>Different isolates showed differences in matrix formation ability, possible contributing in different amounts to the matrix production in multi-species biofilms, indicating that multi-species biofilms are a key survival mechanism for microorganisms within the food processing environment.</p> <p>Currently, we are testing two different multi-species biofilms in our model. Hereby we cultivate three species detected in the cutter-associated biofilms and other three species detected in the water hose-associated biofilms together to mimic these biofilms. This work ultimately showed the presence of multi-species biofilms within the meat processing environment, thereby identifying various sources of potential contamination. Data on the presence, formation and composition of biofilms (i.e. chemical and microbiological) will help to prevent and reduce biofilm formation within food processing environments.</p>