Controlling the food-borne pathogen Listeria (L.) monocytogenes is of great importance from a food safety perspective, and thus for human health. The consequences of failures in this regard have been ...exemplified by recent large listeriosis outbreaks in the USA and Europe. It is thus particularly notable that tolerance to quaternary ammonium compounds such as benzalkonium chloride (BC) has been observed in many L. monocytogenes strains. However, the molecular determinants and mechanisms of BC tolerance of L. monocytogenes are still largely unknown. Here we describe Tn6188, a novel transposon in L. monocytogenes conferring tolerance to BC. Tn6188 is related to Tn554 from Staphylococcus (S.) aureus and other Tn554-like transposons such as Tn558, Tn559 and Tn5406 found in various Firmicutes. Tn6188 comprises 5117 bp, is integrated chromosomally within the radC gene and consists of three transposase genes (tnpABC) as well as genes encoding a putative transcriptional regulator and QacH, a small multidrug resistance protein family (SMR) transporter putatively associated with export of BC that shows high amino acid identity to Smr/QacC from S. aureus and to EmrE from Escherichia coli. We screened 91 L. monocytogenes strains for the presence of Tn6188 by PCR and found Tn6188 in 10 of the analyzed strains. These isolates were from food and food processing environments and predominantly from serovar 1/2a. L. monocytogenes strains harboring Tn6188 had significantly higher BC minimum inhibitory concentrations (MICs) (28.5 ± 4.7 mg/l) than strains without Tn6188 (14 ± 3.2 mg/l). Using quantitative reverse transcriptase PCR we could show a significant increase in qacH expression in the presence of BC. QacH deletion mutants were generated in two L. monocytogenes strains and growth analysis revealed that ΔqacH strains had lower BC MICs than wildtype strains. In conclusion, our results provide evidence that Tn6188 is responsible for BC tolerance in various L. monocytogenes strains.
The food-borne pathogen Listeria (L.) monocytogenes is able to survive for months and even years in food production environments. Strains belonging to sequence type (ST)121 are particularly found to ...be abundant and to persist in food and food production environments. To elucidate genetic determinants characteristic for L. monocytogenes ST121, we sequenced the genomes of 14 ST121 strains and compared them with currently available L. monocytogenes ST121 genomes. In total, we analyzed 70 ST121 genomes deriving from 16 different countries, different years of isolation, and different origins-including food, animal and human ST121 isolates. All ST121 genomes show a high degree of conservation sharing at least 99.7% average nucleotide identity. The main differences between the strains were found in prophage content and prophage conservation. We also detected distinct highly conserved subtypes of prophages inserted at the same genomic locus. While some of the prophages showed more than 99.9% similarity between strains from different sources and years, other prophages showed a higher level of diversity. 81.4% of the strains harbored virtually identical plasmids. 97.1% of the ST121 strains contain a truncated internalin A (inlA) gene. Only one of the seven human ST121 isolates encodes a full-length inlA gene, illustrating the need of better understanding their survival and virulence mechanisms.
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.
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
, represented mainly by
and
. Moreover, genera that are associated with an intracellular lifestyle (e.g.,
and
) 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.
•Enterobacteriaceae isolates are more often present in severe mastitis cases compared to streptococci.•Staphylococci were the most common udder pathogens.•Low penicillin resistance in S. aureus (14%) ...and streptococci (3%).•Antimicrobial resistance profile is associated with spa types and mastitis pathogen species.
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.
The pathogenicity of the human foodborne pathogen
relies on virulence factors such as internalins. In 2009/2010 two
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
,
and
, and the novel internalin-like protein
in the genomic region of hypervariable genetic hotspot 9 in addition to the standard set of virulence genes. The
prevalence study revealed that these genes rarely occur in
, mainly in minor clonal complexes. To obtain first insights of the role of these genes in the pathogenicity of
, we studied the gene expression under conditions mimicking the ingestion in the host. Expression of
,
,
and
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
or
was deleted. Moreover, the impact of InlP1 and InlP3 in virulence was shown
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
.
Interleukin (IL)-33 is the most recently described member of the IL-1 family of cytokines and it is a ligand of the ST2 receptor. While the effects of IL-33 on the immune system have been extensively ...studied, the properties of this cytokine in the cardiovascular system are much less investigated. Methods/Results- We show here that IL-33 promoted the adhesion of human leukocytes to monolayers of human endothelial cells and robustly increased vascular cell adhesion molecule-1, intercellular adhesion molecule-1, endothelial selectin, and monocyte chemoattractant protein-1 protein production and mRNA expression in human coronary artery and human umbilical vein endothelial cells in vitro as well as in human explanted atherosclerotic plaques ex vivo. ST2-fusion protein, but not IL-1 receptor antagonist, abolished these effects. IL-33 induced translocation of nuclear factor-κB p50 and p65 subunits to the nucleus in human coronary artery endothelial cells and human umbilical vein endothelial cells and overexpression of dominant negative form of IκB kinase 2 or IκBα in human umbilical vein endothelial cells abolished IL-33-induced adhesion molecules and monocyte chemoattractant protein-1 mRNA expression. We detected IL-33 and ST2 on both protein and mRNA level in human carotid atherosclerotic plaques.
We hypothesize that IL-33 may contribute to early events in endothelial activation characteristic for the development of atherosclerotic lesions in the vessel wall, by promoting adhesion molecules and proinflammatory cytokine expression in the endothelium.
A large listeriosis outbreak occurred in Austria, Germany and the Czech Republic in 2009 and 2010. The outbreak was traced back to a traditional Austrian curd cheese called "Quargel" which was ...contaminated with two distinct serovar 1/2a Listeria monocytogenes strains (QOC1 and QOC2). In this study we sequenced and analysed the genomes of both outbreak strains in order to investigate the extent of genetic diversity between the two strains belonging to MLST sequence types 398 (QOC2) and 403 (QOC1). Both genomes are highly similar, but also display distinct properties: The QOC1 genome is approximately 74 kbp larger than the QOC2 genome. In addition, the strains harbour 93 (QOC1) and 45 (QOC2) genes encoding strain-specific proteins. A 21 kbp region showing highest similarity to plasmid pLMIV encoding three putative internalins is integrated in the QOC1 genome. In contrast to QOC1, strain QOC2 harbours a vip homologue, which encodes a LPXTG surface protein involved in cell invasion. In accordance, in vitro virulence assays revealed distinct differences in invasion efficiency and intracellular proliferation within different cell types. The higher virulence potential of QOC1 in non-phagocytic cells may be explained by the presence of additional internalins in the pLMIV-like region, whereas the higher invasion capability of QOC2 into phagocytic cells may be due to the presence of a vip homologue. In addition, both strains show differences in stress-related gene content. Strain QOC1 encodes a so-called stress survival islet 1, whereas strain QOC2 harbours a homologue of the uncharacterized LMOf2365_0481 gene. Consistently, QOC1 shows higher resistance to acidic, alkaline and gastric stress. In conclusion, our results show that strain QOC1 and QOC2 are distinct and did not recently evolve from a common ancestor.
In this review we discuss the role of pigment epithelium-derived factor (PEDF) as a possible new target molecule to therapeutically influence cardiovascular disease. PEDF is a multifunctional, ...pleiotropic protein with antiangiogenic, antitumorigenic, antioxidant, anti-inflammatory, antithrombotic, neurotrophic and neuroprotective properties. First identified in retinal pigment epithelium cells, it is expressed in various tissues throughout the body such as the eye, liver and adipose tissue. Recently PEDF has also been characterized in the heart. PEDF has been suggested to have a protective role in atherosclerosis, the main cause of coronary heart disease, myocardial infarction and heart failure due to its anti-inflammatory, antioxidant and antithrombotic effects in the vessel wall and platelets. Additionally PEDF has strong antiangiogenic effects by inducing apoptosis in endothelial cells and by regulating the expression of other angiogenic factors. Therefore blocking of PEDF locally for example in ischemic tissue in the heart might favour angiogenesis, induce neovascularization and lead to increased perfusion of the injured tissue. On the other hand, local overexpression of PEDF restricted to atherosclerotic lesions might block angiogenesis, inflammation and thrombosis at these sites and thus counteract destabilization and rupture of the lesion otherwise caused by inflammatory activation and excessive angiogenesis and inhibit subsequent thrombus formation.
Multiple Listeria monocytogenes strains can be present in the same food sample; moreover, infection with more than one L. monocytogenes strain can also occur. In this study we investigated the impact ...of strain competition on the growth and in vitro virulence potential of L. monocytogenes. We identified two strong competitor strains, whose growth was not (or only slightly) influenced by the presence of other strains and two weak competitor strains, which were outcompeted by other strains. Cell contact was essential for growth inhibition. In vitro virulence assays using human intestinal epithelial Caco2 cells showed a correlation between the invasion efficiency and growth inhibition: the strong growth competitor strains showed high invasiveness. Moreover, invasion efficiency of the highly invasive strain was further increased in certain combinations by the presence of a low invasive strain. In all tested combinations, the less invasive strain was outcompeted by the higher invasive strain. Studying the effect of cell contact on in vitro virulence competition revealed a complex pattern in which the observed effects depended only partially on cell-contact suggesting that competition occurs at two different levels: i) during co-cultivation prior to infection, which might influence the expression of virulence factors, and ii) during infection, when bacterial cells compete for the host cell. In conclusion, we show that growth of L. monocytogenes can be inhibited by strains of the same species leading potentially to biased recovery during enrichment procedures. Furthermore, the presence of more than one L. monocytogenes strain in food can lead to increased infection rates due to synergistic effects on the virulence potential.
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