The aim of the study was to investigate the mode of action of (-)-α-pinene in terms of its modulation of antibiotic resistance in Campylobacter jejuni. Broth microdilution and ethidium bromide accumulation assays were used to evaluate the (-)-α-pinene antimicrobial activity, modulation of antimicrobial resistance, and inhibition of antimicrobial efflux. The target antimicrobial efflux systems were identified using an insertion mutagenesis approach, and C. jejuni adaptation to (-)-α-pinene was evaluated using DNA microarrays. Knock-out mutants of the key up-regulated transcriptional regulators hspR and hrcA were constructed to investigate their roles in C. jejuni adaptation to several stress factors, including osmolytes, and pH, using Biolog phenotypical microarrays. Our data demonstrate that (-)-α-pinene efficiently modulates antibiotic resistance in C. jejuni by decreasing the minimum inhibitory concentrations of ciprofloxacin, erythromycin and triclosan by up to 512-fold. Furthermore, (-)-α-pinene promotes increased expression of cmeABC and another putative antimicrobial efflux gene, Cj1687. The ethidium bromide accumulation was greater in the wild-type strain than in the antimicrobial efflux mutant strains, which indicates that these antimicrobial efflux systems are a target of action of (-)-α-pinene. Additionally, (-)-α-pinene decreases membrane integrity, which suggests that enhanced microbial influx is a secondary mode of action of (-)-α-pinene. Transcriptomic analysis indicated that (-)-α-pinene disrupts multiple metabolic pathways, and particularly those involved in heat-shock responses. Thus, (-)-α-pinene has significant activity in the modulation of antibiotic resistance in C. jejuni, which appears to be mediated by multiple mechanisms that include inhibition of microbial efflux, decreased membrane integrity, and metabolic disruption. These data warrant further studies on (-)-α-pinene to develop its use in the control of antibiotic resistance in Campylobacter.

Campylobacter jejuni is a major foodborne pathogen, and the LuxS-mediated quorum-sensing (QS) system influences its motility, biofilm formation, invasion, host colonization, and virulence. QS therefore represents a target for the control of C. jejuni. The aim of this study was to investigate the correlation of QS inhibition with changes in C. jejuni motility, adhesion to polystyrene surfaces, and adhesion to and invasion of INT407 cells. This was achieved by studying (i) the luxS-deficient mutant and (ii) treatment of C. jejuni with 20 natural extracts as six essential oils, 11 ethanolic extracts, and three pure compounds. Compared to the wild-type, the ΔluxS mutant showed decreased motility, adhesion to polystyrene surfaces, and invasion of INT407 cells. The anti-QS effects of the treatments (n = 15/20) were assayed using Vibrio harveyi BB170 bioluminescence. Moderate positive correlation was shown between C. jejuni QS reduction and reduced motility (τ = 0.492, p = 0.024), adhesion to polystyrene surfaces (τ = 0.419, p = 0.008), and invasion (r = 0.394, p = 0.068). The best overall effect was achieved with a Sedum rosea (roseroot) extract, with 96% QS reduction, a 1.41 log (96%) decrease in adhesion to polystyrene surfaces, and an 82% decrease in invasion. We show that natural extracts can reduce motility, adhesion to polystyrene surfaces, and invasion of INT407 cells by C. jejuni through modulation of the LuxS (QS) system.

Biofilms are the predominant bacterial lifestyle and can protect microorganisms from environmental stresses. Multi-species biofilms can affect the survival of enteric pathogens that contaminate food products, and thus investigating the underlying mechanisms of multi-species biofilms is essential for food safety and human health. In this study, we investigated the ability of the natural isolate Bacillus subtilis PS-216 to restrain Campylobacter jejuni biofilm formation and adhesion to abiotic surfaces as well as to disrupt pre-established C. jejuni biofilms. Using confocal laser scanning microscopy and colony counts, we demonstrate that the presence of B. subtilis PS-216 prevents C. jejuni biofilm formation, decreases growth of the pathogen by 4.2 log10 and disperses 26 h old pre-established C. jejuni biofilms. Furthermore, the co-inoculation of B. subtilis and C. jejuni interferes with the adhesion of C. jejuni to abiotic surfaces reducing it by 2.4 log10. We also show that contact-independent mechanisms contribute to the inhibitory effect of B. subtilis PS-216 on C. jejuni biofilm. Using B. subtilis mutants in genes coding for non-ribosomal peptides and polyketides revealed that bacillaene significantly contributes to the inhibitory effect of B. subtilis PS-216. In summary, we show a strong potential for the use of B. subtilis PS-216 against C. jejuni biofilm formation and adhesion to abiotic surfaces. Our research could bring forward novel applications of B. subtilis in animal production and thus contribute to food safety. IMPORTANCE Campylobacter jejuni is an intestinal commensal in animals (including broiler chickens), but also the most frequent cause of bacterial food-borne infection in humans. This pathogen forms biofilms which mend survival of C. jejuni in food processing and thus threaten human health. Probiotic bacteria represent a potential alternative in the prevention and control of food-borne infections. The beneficial bacterium, Bacillus subtilis has an excellent probiotic potential to reduce C. jejuni in the animal gastrointestinal tract. However, data on the effect of B. subtilis on C. jejuni biofilms are scarce. Our study shows that the B. subtilis natural isolate PS-216 prevents adhesion to the abiotic surfaces and the development of submerged C. jejuni biofilm during co-culture and destroys the pre-established C. jejuni biofilm. These insights are important for development of novel applications of B. subtilis that will reduce the use of antibiotics in human and animal health and increase productivity in animal breeding.

Biofilms provide a protective environment for pathogens such as Campylobacter jejuni, the most prevalent foodborne pathogen, and biofilm formation can enhance bacterial survival in hostile environments. Adhesion of bacteria to the different materials of industrial surfaces is the first step in biofilm formation. Modulation of bacterial adhesion and biofilm formation thus represent important targets in alternative control strategies for reduction of pathogens in food-processing environments. With the high prevalence of C. jejuni and the lack of effective control measures, new control strategies are needed to block adhesion and biofilm formation on food contact surfaces in the food industry, with a focus here on natural antimicrobial phytochemicals. Plants remain a poorly recognized yet vast source of such antimicrobials. Valuable phytochemicals can be obtained directly from plant materials but also from agro-food by-products and waste materials. These materials represent a source of important plant bioactive phytochemicals that are effective for prevention of bacterial adhesion. In this review, we will focus on the anti-adhesion activities of phytochemicals targeted against C. jejuni, on the appropriate methodologies to determine anti-adhesion effects of phytochemicals, on the mechanisms of C. jejuni adhesion, and thus possible targets for reduction and control of this foodborne pathogen in food processing environments.

The culinary herb Satureja montana, known as winter savory, is an ingredient of traditional dishes known in different parts of the world. As an ingredient of foods it has the potential to improve their safety. In this study, the herb’s activity was investigated against Campylobacter jejuni, the leading cause of the most prevalent bacterial gastroenteritis worldwide. The ethanolic extract and essential oil of the herb were chemically characterized and six pure compounds—carvacrol, thymol, thymoquinone, p-cymene, γ-terpinene, and rosmarinic acid—were chosen for further analysis. The antimicrobial activity of the ethanolic extract (MIC 250 mg/L) was 4-fold higher compared to the essential oil. Carvacrol, thymol and thymoquinone had the strongest antimicrobial effect (MIC 31.25 mg/L) and a strong synergistic activity between carvacrol and thymol was determined (FICi 0.2). Strong inhibitory effect on C. jejuni efflux pumps (2-fold inhibition) and disruption of membrane integrity (> 80% disruption) of the herb were determined as modes of action. For resistance against the herb, C. jejuni need efflux pumps, although increased resistance against this herb does not co-occur with increased efflux pump activity, as for antibiotics. This study shows the potential of a common culinary herb for the reduction of the food pathogen C. jejuni without increasing resistance.