Exploring the occurrence of Listeria in biofilms and deciphering the bacterial community in a frozen vegetable producing environment.

The establishment of Listeria (L.) monocytogenes within food processing environments constitutes a significant public health concern. This versatile bacterium demonstrates an exceptional capacity to endure challenging environmental conditions in the food processing environment, where contamination of food products regularly occurs. The diverse repertoire of stress resistance genes, the potential to colonize biofilms, and the support of a co-existing microbiota have been proposed as root causes for the survival of L. monocytogenes in food processing environments. In this study, 71 sites were sampled after cleaning and disinfection in a European frozen vegetable processing facility, where L. monocytogenes in-house clones persisted for years. L. monocytogenes and L. innocua were detected by a culture-dependent method at 14 sampling sites, primarily on conveyor belts and associated parts. The presence of biofilms, as determined by the quantification of bacterial load and the analysis of extracellular matrix components (carbohydrates, proteins, extracellular DNA) was confirmed at nine sites (12.7%). In two cases, L. innocua was detected in a biofilm. Furthermore, we explored the resident microbial community in the processing environment and on biofilm-positive sites, as well as the co-occurrence of bacterial taxa with Listeria by 16S rRNA gene sequencing. Pseudomonas, Acinetobacter, and Exiguobacterium dominated the microbial community of the processing environment. Using differential abundance analysis, amplicon sequence variants (ASVs) assigned to Enterobacterales (Enterobacter, Serratia, unclassified Enterobacteriaceae) and Carnobacterium were found to be significantly higher abundant in Listeria-positive samples. Several Pseudomonas ASVs were less abundant in Listeria-positive compared to Listeria-negative samples. Acinetobacter, Pseudomonas, Janthinobacterium, Brevundimonas, and Exiguobacterium were key players in the microbial community in biofilms, and Exiguobacterium and Janthinobacterium were more relatively abundant in biofilms. Further, the microbial composition varied between the different areas and the surface materials.

Diverse Listeria monocytogenes in-house clones are present in a dynamic frozen vegetable processing environment.

Listeria (L.) monocytogenes is of global concern for food safety as the listeriosis-causing pathogen is widely distributed in the food processing environments, where it can survive for a long time. Frozen vegetables contaminated with L. monocytogenes were recently identified as the source of two large listeriosis outbreaks in the EU and US. So far, only a few studies have investigated the occurrence and behavior of Listeria in frozen vegetables and the associated processing environment. This study investigates the occurrence of L. monocytogenes and other Listeria spp. in a frozen vegetable processing environment and in frozen vegetable products. Using whole genome sequencing (WGS), the distribution of sequence types (MLST-STs) and core genome sequence types (cgMLST-CT) of L. monocytogenes were assessed, and in-house clones were identified. Comparative genomic analyses and phenotypical characterization of the different MLST-STs and isolates were performed, including growth ability under low temperatures, as well as survival of freeze-thaw cycles. Listeria were widely disseminated in the processing environment and five in-house clones namely ST451-CT4117, ST20-CT3737, ST8-CT1349, ST8-CT6243, ST224-CT5623 were identified among L. monocytogenes isolates present in environmental swab samples. Subsequently, the identified in-house clones were also detected in product samples. Conveyor belts were a major source of contamination in the processing environment. A wide repertoire of stress resistance markers supported the colonization and survival of L. monocytogenes in the frozen vegetable processing facility. The presence of ArgB was significantly associated with in-house clones. Significant differences were also observed in the growth rate between different MLST-STs at low temperatures (4 °C and 10 °C), but not between in-house and non-in-house isolates. All isolates harbored major virulence genes such as full length InlA and InlB and LIPI-1, yet there were differences between MLST-STs in the genomic content. The results of this study demonstrate that WGS is a strong tool for tracing contamination sources and transmission routes, and for identifying in-house clones. Further research targeting the co-occurring microbiota and the presence of biofilms is needed to fully understand the mechanism of colonization and persistence in a food processing environment.

Deciphering the virulence potential of Listeria monocytogenes in the Norwegian meat and salmon processing industry by combining whole genome sequencing and in vitro data.

Whole genome sequencing (WGS) of foodborne pathogens such as Listeria monocytogenes is globally on the rise in the food industry. It provides an improvement for proactive surveillance and source-tracking and allows in-depth genetic characterization of the pathogen. In the present study, the virulence gene profile including 99 virulence genes of 767 L. monocytogenes isolates from the Norwegian meat and salmon processing industry was characterized. The isolate collection comprised 28 clonal complexes (CCs) that occur globally. We additionally determined the in vitro virulence potential for 13 major CCs in human intestinal epithelial Caco2 cells using cocktails of three to six representative isolates. Our aim was to test whether the virulence potential could be predicted from the virulence gene profiles to estimate the application potential of WGS in risk assessment in the food industry. The virulence gene profiles were highly conserved within the individual CCs and similar among phylogenetically closely related CCs. We observed a CC-associated distribution of accessory virulence genes in addition to different length polymorphisms. Furthermore, we detected different premature stop codons (PMSC) in the inlA gene, which were mainly present in CC9, CC121 and CC5 isolates. Accordingly, CC9 and CC5 were unable to invade Caco2 cells, whereas CC121 showed moderate virulence potential due to the presence of an isolate harboring full-length inlA. The highest invasion was observed for CC403 and CC415, potentially due to the presence of accessory virulence genes. We demonstrated that CC14, which harbored full-length inlA, was unable to invade Caco2 cells due to a low inlA gene expression. Reconstruction of inlA in CC9 and CC121 isolates showed that without the presence of InlA on the cell wall (as detected in the CC9 isolates), invasion into host cells failed. Our study showed that predicting the virulence potential based on genetic virulence profiles provides valuable information for risk assessment in the food industry but also has its limitations. The mere presence of a full-length inlA gene is not sufficient for virulence, but gene expression and the presence of the protein on the cell wall is required for the successful invasion of L. monocytogenes into host cells. Moreover, hypovirulent CCs like CC121 were among the most abundant human clinical isolates in Norway despite harboring a PMSC mutation in the inlA gene. In conclusion, our study highlights that combining genotypic and phenotypic data is of great importance to improve the informative value of applying WGS in the food industry.

Pervasive Listeria monocytogenes Is Common in the Norwegian Food System and Is Associated with Increased Prevalence of Stress Survival and Resistance Determinants.

To investigate the diversity, distribution, persistence, and prevalence of stress survival and resistance genes of Listeria monocytogenes clones dominating in food processing environments in Norway, genome sequences from 769 L. monocytogenes isolates from food industry environments, foods, and raw materials (512 of which were sequenced in the present study) were subjected to whole-genome multilocus sequence typing (wgMLST), single-nucleotide polymorphism (SNP), and comparative genomic analyses. The data set comprised isolates from nine meat and six salmon processing facilities in Norway collected over a period of three decades. The most prevalent clonal complex (CC) was CC121, found in 10 factories, followed by CC7, CC8, and CC9, found in 7 factories each. Overall, 72% of the isolates were classified as persistent, showing 20 or fewer wgMLST allelic differences toward an isolate found in the same factory in a different calendar year. Moreover, over half of the isolates (56%) showed this level of genetic similarity toward an isolate collected from a different food processing facility. These were designated as pervasive strains, defined as clusters with the same level of genetic similarity as persistent strains but isolated from different factories. The prevalence of genetic determinants associated with increased survival in food processing environments, including heavy metal and biocide resistance determinants, stress response genes, and inlA truncation mutations, showed a highly significant increase among pervasive isolates but not among persistent isolates. Furthermore, these genes were significantly more prevalent among the isolates from food processing environments compared to in isolates from natural and rural environments (n = 218) and clinical isolates (n = 111) from Norway. IMPORTANCE Listeria monocytogenes can persist in food processing environments for months to decades and spread through the food system by, e.g., contaminated raw materials. Knowledge of the distribution and diversity of L. monocytogenes is important in outbreak investigations and is essential to effectively track and control this pathogen in the food system. The present study presents a comprehensive overview of the prevalence of persistent clones and of the diversity of L. monocytogenes in Norwegian food processing facilities. The results demonstrate extensive spread of highly similar strains throughout the Norwegian food system, in that 56% of the 769 collected isolates from food processing factories belonged to clusters of L. monocytogenes identified in more than one facility. These strains were associated with an overall increase in the prevalence of plasmids and determinants of heavy metal and biocide resistance, as well as other genetic elements associated with stress survival mechanisms and persistence.

Biofilms in Water Hoses of a Meat Processing Environment Harbor Complex Microbial Communities.

Safe and hygienic water distribution is essential for maintaining product quality and safety. It is known that biofilms alter the appearance and microbial quality of water along the distribution chain. Yet, biofilms in water hoses throughout the food processing environment have not been investigated in detail. Here, microbial communities from water hoses and other environmental sites in contact with water, in addition to the source water itself, were studied in the meat processing environment. Biofilms were present in all water hoses as determined by the presence of bacterial DNA and biofilm matrix components (carbohydrates, extracellular DNA, and proteins). The microbial community of the biofilms was dominated by Proteobacteria, represented mainly by Comamonadaceae and Pseudoxanthomonas. Moreover, genera that are associated with an intracellular lifestyle (e.g., Neochlamydia and Legionella) were present. Overall, the microbial community of biofilms was less diverse than the water microbial community, while those from the different sample sites were distinct from each other. Indeed, only a few phyla were shared between the water hose biofilm and the source water or associated environmental samples. This study provides first insights towards understanding the microbiota of water hose biofilms in the food processing environment.

Immunoglobulin G Concentrations in Alpaca Colostrum during the First Four Days after Parturition.

Colostrum provides the newborn with nutrients and immunoglobulins. Immunoglobulins and their intestinal transfer play a major role in the immune system of neonates since they are born agammaglobulinemic. In this study immunoglobulin G (IgG) content was determined in alpaca colostrum and the correlations of the IgG concentration by fat, protein, lactose and minerals were calculated. Colostrum samples were collected daily from 20 multiparous alpaca mares during the first four days after parturition. The IgG concentrations were determined by radial immunodiffusion using a Camelid IgG Test Kit. The IgG concentration decreased significantly from 26,319 mg/dL on day 1 to 3848.8 mg/dL on day 4. There were significant correlations between IgG concentration and the other components of the colostrum. While the correlations between IgG and fat (r = -0.69, p ≤ 0.001) and lactose (r = -0.64, p ≤ 0.001) were negative, the correlations with protein (r = 0.91, p ≤ 0.001), magnesium (r = 0.86, p ≤ 0.001) and cobalt (r = 0.87, p ≤ 0.001) were strongly positive. Due to the strong association, the colostrum protein concentration could be used for a brief estimation of the IgG content.

Luteapyrone, a Novel Æ´-Pyrone Isolated from the Filamentous Fungus Metapochonia lutea.

In the process of screening for new bioactive microbial metabolites we found a novel Æ´-pyrone derivative for which we propose the trivial name luteapyrone, in a recently described microscopic filamentous fungus, Metapochonia lutea BiMM-F96/DF4. The compound was isolated from the culture extract of the fungus grown on modified yeast extract sucrose medium by means of flash chromatography followed by preparative HPLC. The chemical structure was elucidated by NMR and LC-MS. The new compound was found to be non-cytotoxic against three mammalian cell lines (HEK 263, KB-3.1 and Caco-2). Similarly, no antimicrobial activity was observed in tested microorganisms (gram positive and negative bacteria, yeast and fungi).

Virulence Pattern Analysis of Three Listeria monocytogenes Lineage I Epidemic Strains with Distinct Outbreak Histories.

Strains of the food-borne pathogen Listeria (L.) monocytogenes have diverse virulence potential. This study focused on the virulence of three outbreak strains: the CC1 strain PF49 (serovar 4b) from a cheese-associated outbreak in Switzerland, the clinical CC2 strain F80594 (serovar 4b), and strain G6006 (CC3, serovar 1/2a), responsible for a large gastroenteritis outbreak in the USA due to chocolate milk. We analysed the genomes and characterized the virulence in vitro and in vivo. Whole-genome sequencing revealed a high conservation of the major virulence genes. Minor deviations of the gene contents were found in the autolysins Ami, Auto, and IspC. Moreover, different ActA variants were present. Strain PF49 and F80594 showed prolonged survival in the liver of infected mice. Invasion and intracellular proliferation were similar for all strains, but the CC1 and CC2 strains showed increased spreading in intestinal epithelial Caco2 cells compared to strain G6006. Overall, this study revealed long-term survival of serovar 4b strains F80594 and PF49 in the liver of mice. Future work will be needed to determine the genes and molecular mechanism behind the long-term survival of L. monocytogenes strains in organs.

Bacteria of eleven different species isolated from biofilms in a meat processing environment have diverse biofilm forming abilities.

Biofilms are formed by microorganisms protected by a self-produced matrix, most often attached to a surface. In the food processing environments biofilms endanger the product safety by the transmission of spoilage and pathogenic bacteria. In this study, we characterised the biofilm formation of the following eleven strains isolated from biofilms in a meat-processing environment: Acinetobacter harbinensis BF1, Arthrobacter sp. BF1, Brochothrix thermosphacta BF1, Carnobacterium maltaromaticum BF1, Kocuria salsicia BF1, Lactococcus piscium BF1, Microbacterium sp. BF1, Pseudomonas fragi BF1, Psychrobacter sp. BF1, Rhodococcus erythropolis BF1, Stenotrophomonas sp. BF1. We applied whole- genome sequencing and subsequent genome analysis to elucidate genetic features associated with the biofilm lifestyle. We furthermore determined the motility and studied biofilm formation on stainless steel using a static mono-species biofilm model mimicking the meat processing environment. The biomass and the EPS components carbohydrates, proteins and extracellular DNA (eDNA) of the biofilms were investigated after seven days at 10 °C. Whole-genome analysis of the isolates revealed that all strains except the Kocuria salsicia BF1 isolate, harboured biofilm associated genes, including genes for matrix production and motility. Genes involved in cellulose metabolism (present in 82% of the eleven strains) and twitching motility (present in 45%) were most frequently found. The capacity for twitching was confirmed using plate assays for all strains except Lactococcus piscium BF1, which showed the lowest motility behaviour. Differences in biofilm forming abilities could be demonstrated. The bacterial load ranged from 5.4 log CFU/cm2 (Psychrobacter sp. isolate) to 8.7 log CFU/cm2 (Microbacterium sp. isolate). The amount of the matrix components varied between isolates. In the biofilm of six strains we detected all three matrix components at different levels (carbohydrates, proteins and eDNA), in two only carbohydrates and eDNA, and in three only carbohydrates. Carbohydrates were detected in biofilms of all strains ranging from 0.5 to 4.3 µg glucose equivalents/cm2. Overall, the Microbacterium sp. strain showed the highest biofilm forming ability with high bacterial load (8.7 log CFU/cm2) and high amounts of carbohydrates (2.2 µg glucose equivalents/cm2), proteins (present in all experiments) and eDNA (549 ng/cm2). In contrast, Brochothrix thermosphacta was a weak biofilm former, showing low bacterial load and low levels of carbohydrates in the matrix (6.2 log CFU/cm2 and 0.5 µg glucose equivalents/cm2). This study contributes to our understanding of the biofilm forming ability of bacteria highly abundant in the meat processing environment, which is crucial to develop strategies to prevent and reduce biofilm formation in the food producing environment.

Presence of Microbial Contamination and Biofilms at a Beer Can Filling Production Line.

ABSTRACT: Contamination of beer arises in 50% of all events at the late stages of production, in the filling area. This is where biofilms, a consortia of microorganisms embedded in a matrix composed of extracellular polymeric substances, play a critical role. To date, most studies have focused on the presence of (biofilm-forming) microorganisms in the filling environment. Our aim was to characterize the microbial status as well as the presence of possible biofilms at a can filling line for beer by determining the presence of microorganisms and their associated matrix components (carbohydrates, proteins and extracellular DNA [eDNA]). For 23 sampling sites, targeted quantitative PCR confirmed the presence of microorganisms at 10 sites during operation and at 3 sites after cleaning. The evaluation of carbohydrates, eDNA, and proteins showed that 16 sites were positive for at least one component during operation and 4 after cleaning. We identified one potential biofilm hotspot, namely the struts below the filler, harboring high loads of bacteria and yeast, eDNA, carbohydrates, and proteins. The protein pattern was different from that of beer. This work deepens our understanding of biofilms and microorganisms found at the filling line of beer beverages at sites critical for production.

Generation of Nonpolar Deletion Mutants in Listeria monocytogenes Using the "SOEing" Method.

The ability to manipulate chromosomally encoded genes is a fundamental biological tool for the analysis of gene function. Here, we provide in greater depth a protocol for the creation of nonpolar unlabelled gene deletions in Listeria monocytogenes that are facilitated by the splicing overlap extension PCR technique. For mutagenesis in L. monocytogenes, we describe the pKSV7 plasmid-based approach, which facilitates the introduction of a spliced amplicon in place of the corresponding segment of chromosomal DNA.

Virulence characterization and comparative genomics of Listeria monocytogenes sequence type 155 strains.

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.

Identification of biofilm hotspots in a meat processing environment: Detection of spoilage bacteria in multi-species biofilms.

Biofilms are comprised of microorganisms embedded in a self-produced matrix that normally adhere to a surface. In the food processing environment they are suggested to be a source of contamination leading to food spoilage or the transmission of food-borne pathogens. To date, research has mainly focused on the presence of (biofilm-forming) bacteria within food processing environments, without measuring the associated biofilm matrix components. Here, we assessed the presence of biofilms within a meat processing environment, processing pork, poultry and beef, by the detection of microorganisms and at least two biofilm matrix components. Sampling included 47 food contact surfaces and 61 non-food contact surfaces from eleven rooms within an Austrian meat processing plant, either during operation or after cleaning and disinfection. The 108 samples were analysed for the presence of microorganisms by cultivation and targeted quantitative real-time PCR based on 16S rRNA. Furthermore, the presence of the major matrix components carbohydrates, extracellular DNA and proteins was evaluated. Overall, we identified ten biofilm hotspots, 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 biofilm positive samples, we cultivated bacteria of 29 different genera. The most prevalent bacteria belonged to the genera Brochothrix (present in 80 % of biofilms), Pseudomonas and Psychrobacter (isolated from 70 % biofilms). From each biofilm we isolated bacteria from four to twelve different genera, indicating the presence of multi-species biofilms. This work ultimately determined the presence of multi-species biofilms within the meat processing environment, thereby identifying various sources of potential contamination. Especially the identification of biofilms in water hoses and associated parts highlights the need of a frequent monitoring at these sites. The knowledge gained about the presence and composition of biofilms (i.e. chemical and microbiological) will help to prevent and reduce biofilm formation within food processing environments.

The Novel Internalins InlP1 and InlP4 and the Internalin-Like Protein InlP3 Enhance the Pathogenicity of Listeria monocytogenes.

The pathogenicity of the human foodborne pathogen Listeria monocytogenes relies on virulence factors such as internalins. In 2009/2010 two L. monocytogenes strains were responsible for a serious listeriosis outbreak in Austria, Germany, and the Czech Republic. One of these clones, QOC1, which caused 14 cases including five fatalities, encodes the novel internalins inlP1, inlPq and inlP4, and the novel internalin-like protein inlP3 in the genomic region of hypervariable genetic hotspot 9 in addition to the standard set of virulence genes. The in silico prevalence study revealed that these genes rarely occur in L. monocytogenes, mainly in minor clonal complexes. To obtain first insights of the role of these genes in the pathogenicity of L. monocytogenes, we studied the gene expression under conditions mimicking the ingestion in the host. Expression of inlP1, inlP3, inlPq and inlP4 was increased under gastric stress and in intracellular bacteria grown in intestinal epithelial cells. Furthermore, colonization of the liver and the spleen was slightly, but significantly reduced 72 h post infection in an oral mouse infection model when inlP1 or inlP4 was deleted. Moreover, the impact of InlP1 and InlP3 in virulence was shown in vitro in human intestinal epithelial cells. In this study we conclusively demonstrate a potential contribution of uncommon novel internalins and an internalin-like protein to the pathogenicity of L. monocytogenes.

The relationship between clinical signs and microbiological species, spa type, and antimicrobial resistance in bovine mastitis cases in Austria.

Bovine mastitis, an inflammation of the udder usually caused by bacteria, is the most common disease in dairy cattle worldwide with a negative economic impact on the dairy industry. In this study 3020 quarter milk samples from 647 dairy cows on 166 Austrian farms were collected and microbial species, spa type for Staphylococcus (S.) aureus and antimicrobial susceptibility were analysed. A multinomial logistic regression model was applied to investigate the effect of possible categorical influencing covariates on the microbiological findings. Additionally, a generalized linear model was used to analyse the effects of genotype and pathogen species on the occurrence of antimicrobial resistance. Staphylococci were the most common (17% of samples) udder pathogens including 32 different S. aureus spa types. The occurrence of pathogen groups was significantly associated with the clinical mastitis score. Enterobacteriaceae isolates had a significantly higher probability of being present in severe mastitis cases compared to streptococci. Benzylpenicillin and tetracycline were the most common resistance in S. aureus including 14% and 11% resistant isolates. Whereas 16% and 13% of coagulase-negative staphylococci (CNS) isolates were resistant to tetracycline and clindamycin. Overall the proportion of Enterobacteriaceae isolates resistant to at least one antibiotic agents was high (55% of isolates), whereas only 3% benzylpenicillin resistant streptococci were detected. Associations were detected between antimicrobial resistance and particular species of Enterobacteriaceae, CNS and specific S. aureus spa types. In conclusion we present in this study data on causative udder pathogen species and their antimicrobial resistance, which are of great importance for mastitis management and prevention.

Listeria monocytogenes Isolated from Illegally Imported Food Products into the European Union Harbor Different Virulence Factor Variants.

Unregulated international flow of foods poses a danger to human health, as it may be contaminated with pathogens. Recent studies have investigated neglected routes of pathogen transmission and reported the occurrence of Listeria monocytogenes in food illegally imported into the European Union (EU), either confiscated at four international airports or sold illegally on the Romanian black market. In this study we investigated the genotype diversity and the amino acid sequence variability of three main virulence factors of 57 L. monocytogenes isolates. These isolates were derived from 1474 food samples illegally imported into the EU and originated from 17 different countries. Multilocus sequence typing revealed 16 different sequence types (STs) indicating moderate genotype diversity. The most prevalent STs were ST2, ST9, and ST121. The pulsed-field gel electrophoresis (PFGE) analysis resulted in 34 unique pulsotypes. PFGE types assigned to the most prevalent STs (ST2, ST9, and ST121) were highly related in their genetic fingerprint. Internalin A (InlA) was present in 20 variants, including six truncated InlA variants, all harbored by isolates of ST9 and ST121. We detected eight ST-specific listeriolysin O (LLO) variants, and among them, one truncated form. The actin-assembly-inducing protein ActA was present in 15 different ST-specific variants, including four ActA variants with an internal truncation. In conclusion, this study shows that L. monocytogenes, isolated from illegally imported food, have moderate genotype diversity, but diverse virulence factors variants, mainly of InlA.

Gentisaldehyde and Its Derivative 2,3-Dihydroxybenzaldehyde Show Antimicrobial Activities Against Bovine Mastitis Staphylococcus aureus.

Bovine mastitis is a worldwide disease of dairy cattle associated with significant economic losses for the dairy industry. One of the most common pathogens responsible for mastitis is Staphylococcus (S.) aureus. Due to the development and spreading of antibiotic resistance, the search for novel antimicrobial substances against S. aureus is of great importance. The aim of this study was to evaluate two dihydroxybenzaldehydes for the prevention of bovine mastitis. Therefore we determined the minimal inhibitory concentration (MICs) of gentisaldehyde (2,5-dihydroxybenzaldehyde) and 2,3-dihydroxybenzaldehyde of a diverse set of 172 bovine mastitis S. aureus isolates using an automated robot-based microdilution method. To characterize the bovine isolates we determined the genotype by spa-typing, the antimicrobial resistance to eight antibiotic classes using the disk diffusion method and the MICs of three commonly used antiseptics (benzalkonium chloride, chlorhexidine, and iodine). Further we investigated the cytotoxicity of gentisaldehyde and 2,3-dihydroxybenzaldehyde in bovine mammary epithelial MAC-T cells using the XTT assay. The S. aureus strains showed a high genetic diversity with 52 different spa-types, including five novel types. Antibiotic susceptibility testing revealed that 24% of isolates were resistant to one antimicrobial agent and 3% of isolates were multi-resistant. The occurrence of antibiotic resistance strongly correlated with the spa-type. Both dihydroxybenzaldehydes showed antimicrobial activities with a MIC50 of 500 mg/L. The MIC of gentisaldehyde significantly correlated with that of 2,3-dihydroxybenzaldehyde, whereas no correlation was observed with the MIC of the three antiseptics. Cytotoxicity testing using bovine mammary epithelial MAC-T cells revealed that gentisaldehyde and 2,3-dihydroxybenzaldehyde show low toxicity at MIC50 and MIC90 concentrations. In conclusion, gentisaldehyde and 2,3-dihydroxybenzaldehyde exhibited antimicrobial activities against a diverse range of bovine mastitis S. aureus strains at low-cytotoxic concentrations. Therefore, both compounds are potential candidates as antiseptics to prevent bovine mastitis and to reduce the use of antibiotics in dairy cows.

The impact of shelf life on exposure as revealed from quality control data associated with the quargel outbreak.

A cluster of 34 human cases of listeriosis was traced to consumption of contaminated quargel cheese, a sour milk specialty sold in Austria, Germany and Czech Republic. Here, we try to assess how many portions were consumed by the Austrian population at a certain contamination level (CL). In total, 1623 cheese lots were produced during the outbreak period resulting in >3 million portions of cheese delivered to the market. From 650 sets of quality control data provided by the food business operator, we reconstructed the contamination scenario over time and identified 84 lots that were found to be positive. With regard to another sixteen lots, a CL was found ranging from one to 3,84 log10 CFU L. monocytogenes/g, measured in product stored between one to 23 days after production. However the number of storage days at home before consumption is unknown. To resolve this issue, we modelled the theoretical CL of the product if consumed either 20, 30, 40 or 50 days post production. We found that 10 lots (approx. 27,350 portions) would have been contaminated at CLs higher than 3 log10 CFU L. monocytogenes/g if all cheese had been consumed after 20 days of storage. This number shifts to 20 lots (approx. 54,700 portions) after 30 days of storage. If all cheese had been consumed at the end of shelf life (50 days of storage), theoretically 242,5 lots would have exceeded a CL of 6 log10 CFU L. monocytogenes/g. We concluded that the extended shelf life given to the product was a driver of the outbreak scenario. It is stunning to note that so few cases were reported in spite of consumers' massive exposure to L. monocytogenes. We hypothesized that a low pathogenicity of both quargel outbreak clones (QOC1 and QOC2) could have contributed to this discrepancy. Our hypothesis was falsified since both strains QOC1 and QOC2 are fully virulent in an oral infection mouse model, showing even higher pathogenicity than the reference strain EGDe.

A robust high-throughput fungal biosensor assay for the detection of estrogen activity.

Estrogenic active compounds are present in a variety of sources and may alter biological functions in vertebrates. Therefore, it is crucial to develop innovative analytical systems that allow us to screen a broad spectrum of matrices and deliver fast and reliable results. We present the adaptation and validation of a fungal biosensor for the detection of estrogen activity in cow derived samples and tested the clinical applicability for pregnancy diagnosis in 140 mares and 120 cows. As biosensor we used a previously engineered genetically modified strain of the filamentous fungus Aspergillus nidulans, which contains the human estrogen receptor alpha and a reporter construct, in which ß-galactosidase gene expression is controlled by an estrogen-responsive-element. The estrogen response of the fungal biosensor was validated with blood, urine, feces, milk and saliva. All matrices were screened for estrogenic activity prior to and after chemical extraction and the results were compared to an enzyme immunoassay (EIA). The biosensor showed consistent results in milk, urine and feces, which were comparable to those of the EIA. In contrast to the EIA, no sample pre-treatment by chemical extraction was needed. For 17ß-estradiol, the biosensor showed a limit of detection of 1ng/L. The validation of the biosensor for pregnancy diagnosis revealed a specificity of 100% and a sensitivity of more than 97%. In conclusion, we developed and validated a highly robust fungal biosensor for detection of estrogen activity, which is highly sensitive and economic as it allows analyzing in high-throughput formats without the necessity for organic solvents.

Stress Survival Islet 2, Predominantly Present in Listeria monocytogenes Strains of Sequence Type 121, Is Involved in the Alkaline and Oxidative Stress Responses.

The foodborne pathogen Listeria monocytogenes is able to survive a variety of stress conditions leading to the colonization of different niches like the food processing environment. This study focuses on the hypervariable genetic hot spot lmo0443 to lmo0449 haboring three inserts: the stress survival islet 1 (SSI-1), the single-gene insert LMOf2365_0481, and two homologous genes of the nonpathogenic species Listeria innocua: lin0464, coding for a putative transcriptional regulator, and lin0465, encoding an intracellular PfpI protease. Our prevalence study revealed a different distribution of the inserts between human and food-associated isolates. The lin0464-lin0465 insert was predominantly found in food-associated strains of sequence type 121 (ST121). Functional characterization of this insert showed that the putative PfpI protease Lin0465 is involved in alkaline and oxidative stress responses but not in acidic, gastric, heat, cold, osmotic, and antibiotic stresses. In parallel, deletion of lin0464 decreased survival under alkaline and oxidative stresses. The expression of both genes increased significantly under oxidative stress conditions independently of the alternative sigma factor σB Furthermore, we showed that the expression of the protease gene lin0465 is regulated by the transcription factor lin0464 under stress conditions, suggesting that lin0464 and lin0465 form a functional unit. In conclusion, we identified a novel stress survival islet 2 (SSI-2), predominantly present in L. monocytogenes ST121 strains, beneficial for survival under alkaline and oxidative stresses, potentially supporting adaptation and persistence of L. monocytogenes in food processing environments.IMPORTANCEListeria monocytogenes strains of ST121 are known to persist for months and even years in food processing environments, thereby increasing the risk of food contamination and listeriosis. However, the molecular mechanism underlying this remarkable niche-specific adaptation is still unknown. Here, we demonstrate that the genomic islet SSI-2, predominantly present in L. monocytogenes ST121 strains, is beneficial for survival under alkaline and oxidative stress conditions, which are routinely encountered in food processing environments. Our findings suggest that SSI-2 is part of a diverse set of molecular determinants contributing to niche-specific adaptation and persistence of L. monocytogenes ST121 strains in food processing environments.