Antiadhesion activity of juniper (Juniperus communis L.) preparations against Campylobacter jejuni evaluated with PCR-based methods.

The food-borne pathogen Campylobacter jejuni can cause bacterial gastrointestinal infections. Biofilm formation amplifies the risk of human infection by improving survival and persistence of C. jejuni in food processing environments and its transmission through the food chain. We aimed to control C. jejuni using an alternative strategy of low doses of Juniperus communis fruit preparations to target bacterial adhesion properties in the first step of biofilm formation. First, we defined the anti-Campylobacter activity of a juniper fruit crude extract and its fractionated biflavonoids, flavone glycosides, and purified amentoflavone, of juniper fruit essential oil and of juniper fruit postdistillation waste material extract. For accurate quantification of adherent C. jejuni, we optimised digital Polymerase Chain Reaction (PCR) and quantitative real-time PCR for construction of standard curves and quantification. We show for the first time that juniper fruit formulations can effectively inhibit adhesion of C. jejuni to polystyrene. Furthermore, ≥94% of the antiadhesion activity of juniper fruit crude extract and juniper fruit essential oil remained under food-related conditions: modified culture medium with glucose, or a stainless steel surface, or mixed co-cultures of C. jejuni and Listeria monocytogenes. This study indicates that addition of juniper fruit formulations can control growth and adhesion of C. jejuni and thus limit food chain transmission of campylobacters.

The Role of luxS in Campylobacter jejuni Beyond Intercellular Signaling.

The full role of the luxS gene in the biological processes, such as essential amino acid synthesis, nitrogen and pyruvate metabolism, and flagellar assembly, of Campylobacter jejuni has not been clearly described to date. Therefore, in this study, we used a comprehensive approach at the cellular and molecular levels, including transcriptomics and proteomics, to investigate the key role of the luxS gene and compared C. jejuni 11168ΔluxS (luxS mutant) and C. jejuni NCTC 11168 (wild type) strains. Transcriptomic analysis of the luxS mutant grown under optimal conditions revealed upregulation of luxS mutant metabolic pathways when normalized to wild type, including oxidative phosphorylation, carbon metabolism, citrate cycle, biosynthesis of secondary metabolites, and biosynthesis of various essential amino acids. Interestingly, induction of these metabolic pathways was also confirmed by proteomic analysis, indicating their important role in energy production and the growth of C. jejuni. In addition, genes important for the stress response of C. jejuni, including nutrient starvation and oxidative stress, were upregulated. This was also evident in the better survival of the luxS mutant under starvation conditions than the wild type. At the molecular level, we confirmed that metabolic pathways were upregulated under optimal conditions in the luxS mutant, including those important for the biosynthesis of several essential amino acids. This also modulated the utilization of various carbon and nitrogen sources, as determined by Biolog phenotype microarray analysis. In summary, transcriptomic and proteomic analysis revealed key biological differences in tricarboxylic acid (TCA) cycle, pyruvate, nitrogen, and thiamine metabolism as well as lipopolysaccharide biosynthesis in the luxS mutant. IMPORTANCE Campylobacter jejuni is the world's leading foodborne bacterial pathogen of gastrointestinal disease in humans. C. jejuni is a fastidious but widespread organism and the most frequently reported zoonotic pathogen in the European Union since 2005. This led us to believe that C. jejuni, which is highly sensitive to stress factors (starvation and oxygen concentration) and has a low growth rate, benefits significantly from the luxS gene. The role of this gene in the life cycle of C. jejuni is well known, and the expression of luxS regulates many phenotypes, including motility, biofilm formation, host colonization, virulence, autoagglutination, cellular adherence and invasion, oxidative stress, and chemotaxis. Surprisingly, this study confirmed for the first time that the deletion of the luxS gene strongly affects the central metabolic pathway of C. jejuni, which improves its survival, showing its role beyond the intercellular signaling system.

Transporters and Efflux Pumps Are the Main Mechanisms Involved in Staphylococcus epidermidis Adaptation and Tolerance to Didecyldimethylammonium Chloride.

Staphylococcus epidermidis cleanroom strains are often exposed to sub-inhibitory concentrations of disinfectants, including didecyldimethylammonium chloride (DDAC). Consequently, they can adapt or even become tolerant to them. RNA-sequencing was used to investigate adaptation and tolerance mechanisms of S. epidermidis cleanroom strains (SE11, SE18), with S. epidermidis SE11Ad adapted and S. epidermidis SE18To tolerant to DDAC. Adaptation to DDAC was identified with up-regulation of genes mainly involved in transport (thioredoxin reductase [pstS], the arsenic efflux pump [gene ID, SE0334], sugar phosphate antiporter [uhpT]), while down-regulation was seen for the Agr system (agrA, arC, agrD, psm, SE1543), for enhanced biofilm formation. Tolerance to DDAC revealed the up-regulation of genes associated with transporters (L-cysteine transport [tcyB]; uracil permease [SE0875]; multidrug transporter [lmrP]; arsenic efflux pump [arsB]); the down-regulation of genes involved in amino-acid biosynthesis (lysine [dapE]; histidine [hisA]; methionine [metC]), and an enzyme involved in peptidoglycan, and therefore cell wall modifications (alanine racemase [SE1079]). We show for the first time the differentially expressed genes in DDAC-adapted and DDAC-tolerant S. epidermidis strains, which highlight the complexity of the responses through the involvement of different mechanisms.

Quantification of Listeria monocytogenes cells with digital PCR and their biofilm cells with real-time PCR.

The purpose of this study was to develop a PCR-based method for quantification of Listeria monocytogenes adhesion in microtitre plates. We optimized isolation of DNA in the microtitre plates using cell lysis, ultrasound treatment, heating, and centrifugation of the lysate. Digital PCR was applied for quantification of L. monocytogenes DNA that was used for construction of the standard curve, and real-time PCR was used for quantification of the attached L. monocytogenes cells. This PCR-based method was applied to quantify different strains of L. monocytogenes at different times of biofilm formation, and to study the anti-adhesive actions of natural bioactive substances (epigallocatechin gallate, (-)-α-pinene). The results show that the PCR-based method developed here can be widely used as a novel approach for adhesion assays and biofilm research.

Novel cost-efficient real-time PCR assays for detection and quantitation of Listeria monocytogenes.

Listeriosis is a serious food-borne infection with mortality rates approaching 30%. Therefore, the rapid, cost-effective, and automated detection of Listeria monocytogenes throughout the food chain continues to be a major concern. Here we describe three novel quantitative real-time PCR assays for L. monocytogenes based on amplification of a target hlyA gene with SYBR Green I chemistry and hydrolysis probe (TaqMan MGB probe). In order to offer sensitive, rapid and robust tool of additional economical value the real-time PCR assays were designed and optimized to only 5 µl-reactions. All assays were evaluated by using different non-reference Listeria strains isolated from various food matrices. Results demonstrated specificity to L. monocytogenes with accurate quantification over a dynamic range of 5-6 log units with R² higher than 0.98 and amplification efficiencies reaching above 92%. The detection and quantification limits were as low as 165 genome equivalents. Comparison of novel assays to commercially available TaqMan® Listeria monocytogenes Detection Kit and previously published studies revealed similar specificity, sensitivity and efficiency, but greater robustness and especially cost-efficiency in the view of smaller reaction volumes and continuous increase in sample throughput.