Modulation of Campylobacter jejuni adhesion to biotic model surfaces by fungal lectins and protease inhibitors.

Campylobacter jejuni, a Gram-negative bacterium, is one of the most common causes of foodborne illness worldwide. Its adhesion mechanism is mediated by several bacterial factors, including flagellum, protein adhesins, lipooligosaccharides, proteases, and host factors, such as surface glycans on epithelial cells and mucins. Fungal lectins, specialized carbohydrate-binding proteins, can bind to specific glycans on host and bacterial cells and thus influence pathogenesis. In this study, we investigated the effects of fungal lectins and protease inhibitors on the adhesion of C. jejuni to model biotic surfaces (mucin, fibronectin, and collagen) and Caco-2 cells as well as the invasion of Caco-2 cells. The lectins Marasmius oreades agglutinin (MOA) and Laccaria bicolor tectonin 2 (Tec2) showed remarkable efficacy in all experiments. In addition, different pre-incubations of lectins with C. jejuni or Caco-2 cells significantly inhibited the ability of C. jejuni to adhere to and invade Caco-2 cells, but to varying degrees. Pre-incubation of Caco-2 cells with selected lectins reduced the number of invasive C. jejuni cells the most, while simultaneous incubation showed the greatest reduction in adherent C. jejuni cells. These results suggest that fungal lectins are a promising tool for the prevention and treatment of C. jejuni infections. Furthermore, this study highlights the potential of fungi as a rich reservoir for novel anti-adhesive agents.

Campylobacter jejuni virulence factors: update on emerging issues and trends.

Campylobacter jejuni is a very common cause of gastroenteritis, and is frequently transmitted to humans through contaminated food products or water. Importantly, C. jejuni infections have a range of short- and long-term sequelae such as irritable bowel syndrome and Guillain Barre syndrome. C. jejuni triggers disease by employing a range of molecular strategies which enable it to colonise the gut, invade the epithelium, persist intracellularly and avoid detection by the host immune response. The objective of this review is to explore and summarise recent advances in the understanding of the C. jejuni molecular factors involved in colonisation, invasion of cells, collective quorum sensing-mediated behaviours and persistence. Understanding the mechanisms that underpin the pathogenicity of C. jejuni will enable future development of effective preventative approaches and vaccines against this pathogen.

Modeling strain variability in Campylobacter jejuni thermal inactivation by quantifying the number of strains required.

There is a limited understanding of the survival responses of Campylobacter jejuni during thermal processing, which must be investigated for appropriate risk assessment and processing. Therefore, we aimed to elucidate the survival response of C. jejuni and develop a predictive model considering strain variability and uncertainty, which are important for quantitative microbial risk assessment (QMRA) or risk-based processing control measures. We employed the most probable curve (MPC) method to consider the uncertainty in cell concentrations. Further, the multivariate normal (MVN) distribution served as a model for strain variability in bacterial survival behavior. The prediction curves from the MVN successfully captured the parameter variability of the most probable curves of each strain. More than ten reference strains effectively described the strain variability in parameters using the MVN distribution. The findings indicated that, with sufficient strain data, the MVN could estimate the strain variability, including unknown strains. The multi-level model for strain variability can potentially become a specialized tool for QMRA and risk-based processing controls. The combined approach of MPC and MVN provides valuable insights into strain variability, emphasizing the importance of accounting for variability and uncertainty in predictive models for QMRA and risk-based processing control measures.

Viscosity-dependent determinants of Campylobacter jejuni impacting the velocity of flagellar motility.

IMPORTANCE: Bacteria can adapt flagellar motor output in response to the load that the extracellular milieu imparts on the flagellar filament to enable propulsion. Bacteria can adapt flagellar motor output in response to the load that the extracellular milieu imparts on the flagellar filament to enable propulsion through diverse environments. These changes may involve increasing power and torque in high-viscosity environments or reducing power and flagellar rotation upon contact with a surface. C. jejuni swimming velocity in low-viscosity environments is comparable to other bacterial flagellates and increases significantly as external viscosity increases. In this work, we provide evidence that the mechanics of the C. jejuni flagellar motor has evolved to naturally promote high swimming velocity in high-viscosity environments. We found that C. jejuni produces VidA and VidB as auxiliary proteins to specifically affect flagellar motor activity in low viscosity to reduce swimming velocity. Our findings provide some of the first insights into different mechanisms that exist in bacteria to alter the mechanics of a flagellar motor, depending on the viscosity of extracellular environments.

Combining a parsimonious mathematical model with infection data from tailor-made experiments to understand environmental transmission.

Although most infections are transmitted through the environment, the processes underlying the environmental stage of transmission are still poorly understood for most systems. Improved understanding of the environmental transmission dynamics is important for effective non-pharmaceutical intervention strategies. To study the mechanisms underlying environmental transmission we formulated a parsimonious modelling framework including hypothesised mechanisms of pathogen dispersion and decay. To calibrate and validate the model, we conducted a series of experiments studying distance-dependent transmission of Campylobacter jejuni in broilers. We obtained informative simultaneous estimates for all three model parameters: the parameter of C. jejuni inactivation, the diffusion coefficient describing pathogen dispersion, and the transmission rate parameter. The time and distance dependence of transmission in the fitted model is quantitatively consistent with marked spatiotemporal patterns in the experimental observations. These results, for C. jejuni in broilers, show that the application of our modelling framework to suitable transmission data can provide mechanistic insight in environmental pathogen transmission.

A secreted sirtuin from Campylobacter jejuni contributes to neutrophil activation and intestinal inflammation during infection.

Histone modifications control numerous processes in eukaryotes, including inflammation. Some bacterial pathogens alter the activity or expression of host-derived factors, including sirtuins, to modify histones and induce responses that promote infection. In this study, we identified a deacetylase encoded by Campylobacter jejuni which has sirtuin activities and contributes to activation of human neutrophils by the pathogen. This sirtuin is secreted from the bacterium into neutrophils, where it associates with and deacetylates host histones to promote neutrophil activation and extracellular trap production. Using the murine model of campylobacteriosis, we found that a mutant of this bacterial sirtuin efficiently colonized the gastrointestinal tract but was unable to induce cytokine production, gastrointestinal inflammation, and tissue pathology. In conclusion, these results suggest that secreted bacterial sirtuins represent a previously unreported class of bacterial effector and that bacterial-mediated modification of host histones is responsible for the inflammation and pathology that occurs during campylobacteriosis.

Butyrate decreases Campylobacter jejuni motility and biofilm partially through influence on LysR expression.

The food pathogen Campylobacter jejuni both colonizes the lower intestines of poultry and infects the lower intestines of humans. The lower intestines of both poultry and humans are also home to a wide range of commensal organisms which compete with an organism like C. jejuni for space and resources. The commensal organisms are believed to protect humans against infection by pathogens of the digestive tract like C. jejuni. The short chain fatty acid (SCFA) butyrate is a metabolite commonly produced by commensal organisms within both the poultry and human digestive tract. We investigated the effect that physiologically relevant concentrations of butyrate have on C. jejuni under in vitro conditions. Butyrate at concentrations of 5 and 20 mM negatively impacted C. jejuni motility and biofilm formation. These two traits are believed important for C. jejuni's ability to infect the lower intestines of humans. Additionally, 20 mM butyrate concentrations were observed to influence the expression of a range of different Campylobacter proteins. Constitutive expression of one of these proteins, LysR, within a C. jejuni strain partially lessened the negative influence butyrate had on the bacteria's motility.

Susceptibility of Campylobacter jejuni to Stressors in Agrifood Systems and Induction of a Viable-but-Nonculturable State.

Many bacteria can become viable but nonculturable (VBNC) in response to stressors commonly identified in agrifood systems. Campylobacter is able to enter the VBNC state to evade unfavorable environmental conditions, but how food processing can induce Campylobacter jejuni to enter this state and the potential role of foods in inducing the VBNC state in C. jejuni remains largely unknown. In this study, the culturability and viability of C. jejuni cells were investigated under chlorine treatment (25 ppm), aerobic stress (atmospheric condition), and low-temperature (4°C) conditions that mimicked food processing. In addition, the behaviors of C. jejuni cells in ultrahigh-temperature (UHT) and pasteurized milk were also monitored during refrigerated storage. The numbers of viable and culturable C. jejuni cells in both the pure bacterial culture and food matrices were separately determined by propidium monoazide (PMA)-quantitative PCR (qPCR) and plating assay. The C. jejuni cells lost their culturability but partially retained their viability (1% to 10%) once mixed with chlorine. In comparison, ~10% of C. jejuni cells were induced to enter the VBNC state after 24 h and 20 days under aerobic and low-temperature conditions, respectively. The viability of the C. jejuni cells remained stable during the induction process in UHT (>10%) and pasteurized (>10%) milk. The number of culturable C. jejuni cells decreased quickly in pasteurized milk, but culturable cells could still be detected in the end (day 21). In contrast, the number of culturable C. jejuni cells slowly decreased, and they became undetectable after >42 days in UHT milk. The C. jejuni cells responded differently to various stress conditions and survived in high numbers in the VBNC state in agrifood systems. IMPORTANCE The VBNC state of pathogens can pose risks to food safety and public health because the pathogens cannot be detected using conventional microbiological culture-based methods but can resuscitate under favorable conditions to develop virulence. As a leading cause of human gastroenteritis worldwide, C. jejuni can enter the VBNC state to survive in the environment and food-processing chain with high prevalence. In this study, the effect of food-processing conditions and food products on the development of VBNC state in C. jejuni was investigated, providing a better understanding of the interaction between C. jejuni and the agroecosystem. The knowledge elicited from this study can aid in developing novel intervention strategies to reduce the food safety risks associated with this microbe.

Molecular Targets in Campylobacter Infections.

Human campylobacteriosis results from foodborne infections with Campylobacter bacteria such as Campylobacter jejuni and Campylobacter coli, and represents a leading cause of bacterial gastroenteritis worldwide. After consumption of contaminated poultry meat, constituting the major source of pathogenic transfer to humans, infected patients develop abdominal pain and diarrhea. Post-infectious disorders following acute enteritis may occur and affect the nervous system, the joints or the intestines. Immunocompromising comorbidities in infected patients favor bacteremia, leading to vascular inflammation and septicemia. Prevention of human infection is achieved by hygiene measures focusing on the reduction of pathogenic food contamination. Molecular targets for the treatment and prevention of campylobacteriosis include bacterial pathogenicity and virulence factors involved in motility, adhesion, invasion, oxygen detoxification, acid resistance and biofilm formation. This repertoire of intervention measures has recently been completed by drugs dampening the pro-inflammatory immune responses induced by the Campylobacter endotoxin lipo-oligosaccharide. Novel pharmaceutical strategies will combine anti-pathogenic and anti-inflammatory effects to reduce the risk of both anti-microbial resistance and post-infectious sequelae of acute enteritis. Novel strategies and actual trends in the combat of Campylobacter infections are presented in this review, alongside molecular targets applied for prevention and treatment strategies.

A Review of the Advantages, Disadvantages and Limitations of Chemotaxis Assays for Campylobacter spp.

Reproducible qualitative and quantitative assessment of bacterial chemotactic motility, particularly in response to chemorepellent effectors, is experimentally challenging. Here we compare several established chemotaxis assays currently used to investigate Campylobacter jejuni chemotaxis, with the aim of improving the correlation between different studies and establishing the best practices. We compare the methodologies of capillary, agar, and chamber-based assays, and discuss critical technical points, in terms of reproducibility, accuracy, and the advantages and limitations of each.

Research Note: MHCY haplotype impacts Campylobacter jejuni colonization in a backcross [(Line 61 x Line N) x Line N] population.

MHCY is a candidate region for influencing immune responses in chickens. MHCY contains multiple specialized, polymorphic MHC class I loci along with loci belonging to 4 additional gene families. In this study, MHCY haplotypes were tested for association with cecal colonization after Campylobacter jejuni infection of a backcross [(Line 61 × Line N) × Line N] population derived from 2 White Leghorn research lines, Line 61 and Line N, that were previously shown to exhibit heritable differences in colonization. Samples were obtained for 51 birds challenged with 108 CFU Campylobacter jejuni at 3 wk of age. Viable C. jejuni in the ceca were enumerated 5 d postinfection and counts were log-transformed for analysis. Birds were assigned to either low or high colonization groups based on the individual count being below or above the mean bacterial count for all birds. The mean bacterial count of the low infection group differed significantly from the high infection group. Sex and MHCB haplotype had similar distributions within the 2 groups. Overall, 7 MHCY haplotypes were found to be segregating. Two were significantly associated with C. jejuni colonization. MHCY Y18 was associated with low colonization (P = 3.00 × 10-5); whereas MHCY Y11a was associated with high colonization (P = 0.008). The MHCY haplotype impacted the mean bacterial count among all birds with MHCY Y18 having the lowest bacterial count compared with MHCY Y11a and all other MHCY (Y5, Y7, Y8, Y11b, and Y11c) haplotypes. These findings support further investigation of the contribution of chicken MHCY in resistance to Campylobacter colonization.

Interplay between DsbA1, DsbA2 and C8J_1298 Periplasmic Oxidoreductases of Campylobacter jejuni and Their Impact on Bacterial Physiology and Pathogenesis.

The bacterial proteins of the Dsb family catalyze the formation of disulfide bridges between cysteine residues that stabilize protein structures and ensure their proper functioning. Here, we report the detailed analysis of the Dsb pathway of Campylobacter jejuni. The oxidizing Dsb system of this pathogen is unique because it consists of two monomeric DsbAs (DsbA1 and DsbA2) and one dimeric bifunctional protein (C8J_1298). Previously, we showed that DsbA1 and C8J_1298 are redundant. Here, we unraveled the interaction between the two monomeric DsbAs by in vitro and in vivo experiments and by solving their structures and found that both monomeric DsbAs are dispensable proteins. Their structures confirmed that they are homologs of EcDsbL. The slight differences seen in the surface charge of the proteins do not affect the interaction with their redox partner. Comparative proteomics showed that several respiratory proteins, as well as periplasmic transport proteins, are targets of the Dsb system. Some of these, both donors and electron acceptors, are essential elements of the C. jejuni respiratory process under oxygen-limiting conditions in the host intestine. The data presented provide detailed information on the function of the C. jejuni Dsb system, identifying it as a potential target for novel antibacterial molecules.

Campylobacter Biofilms: Potential of Natural Compounds to Disrupt Campylobacter jejuni Transmission.

Microbial biofilms occur naturally in many environmental niches and can be a significant reservoir of infectious microbes in zoonotically transmitted diseases such as that caused by Campylobacter jejuni, the leading cause of acute human bacterial gastroenteritis world-wide. The greatest challenge in reducing the disease caused by this organism is reducing transmission of C. jejuni to humans from poultry via the food chain. Biofilms enhance the stress tolerance and antimicrobial resistance of the microorganisms they harbor and are considered to play a crucial role for Campylobacter spp. survival and transmission to humans. Unconventional approaches to control biofilms and to improve the efficacy of currently used antibiotics are urgently needed. This review summarizes the use plant- and microorganism-derived antimicrobial and antibiofilm compounds such as essential oils, antimicrobial peptides (AMPs), polyphenolic extracts, algae extracts, probiotic-derived factors, d-amino acids (DAs) and glycolipid biosurfactants with potential to control biofilms formed by Campylobacter, and the suggested mechanisms of their action. Further investigation and use of such natural compounds could improve preventative and remedial strategies aimed to limit the transmission of campylobacters and other human pathogens via the food chain.

Feeling the Heat: The Campylobacter jejuni HrcA Transcriptional Repressor Is an Intrinsic Protein Thermosensor.

The heat-shock response, a universal protective mechanism consisting of a transcriptional reprogramming of the cellular transcriptome, results in the accumulation of proteins which counteract the deleterious effects of heat-stress on cellular polypeptides. To quickly respond to thermal stress and trigger the heat-shock response, bacteria rely on different mechanisms to detect temperature variations, which can involve nearly all classes of biological molecules. In Campylobacter jejuni the response to heat-shock is transcriptionally controlled by a regulatory circuit involving two repressors, HspR and HrcA. In the present work we show that the heat-shock repressor HrcA acts as an intrinsic protein thermometer. We report that a temperature upshift up to 42 °C negatively affects HrcA DNA-binding activity to a target promoter, a condition required for de-repression of regulated genes. Furthermore, we show that this impairment of HrcA binding at 42 °C is irreversible in vitro, as DNA-binding was still not restored by reversing the incubation temperature to 37 °C. On the other hand, we demonstrate that the DNA-binding activity of HspR, which controls, in combination with HrcA, the transcription of chaperones' genes, is unaffected by heat-stress up to 45 °C, portraying this master repressor as a rather stable protein. Additionally, we show that HrcA binding activity is enhanced by the chaperonin GroE, upon direct protein-protein interaction. In conclusion, the results presented in this work establish HrcA as a novel example of intrinsic heat-sensing transcriptional regulator, whose DNA-binding activity is positively modulated by the GroE chaperonin.

Reduced Infection Efficiency of Phage NCTC 12673 on Non-Motile Campylobacter jejuni Strains Is Related to Oxidative Stress.

Campylobacter jejuni is a Gram-negative foodborne pathogen that causes diarrheal disease and is associated with severe post-infectious sequelae. Bacteriophages (phages) are a possible means of reducing Campylobacter colonization in poultry to prevent downstream human infections. However, the factors influencing phage-host interactions must be better understood before this strategy can be predictably employed. Most studies have focused on Campylobacter phage binding to the host surface, with all phages classified as either capsule- or flagella-specific. Here we describe the characterization of a C. jejuni phage that requires functional flagellar glycosylation and motor genes for infection, without needing the flagella for adsorption to the cell surface. Through phage infectivity studies of targeted C. jejuni mutants, transcriptomic analysis of phage-resistant mutants, and genotypic and phenotypic analysis of a spontaneous phage variant capable of simultaneously overcoming flagellar gene dependence and sensitivity to oxidative stress, we have uncovered a link between oxidative stress, flagellar motility, and phage infectivity. Taken together, our results underscore the importance of understanding phage-host interactions beyond the cell surface and point to host oxidative stress state as an important and underappreciated consideration for future phage-host interaction studies.

Antibiofilm Potential of Lavandula Preparations against Campylobacter jejuni.

New approaches for the control of Campylobacter jejuni biofilms in the food industry are being studied intensively. Natural products are promising alternative antimicrobial substances to control biofilm production, with particular emphasis on plant extracts. Dried flowers of Lavandula angustifolia were used to produce essential oil (LEO), an ethanol extract (LEF), and an ethanol extract of Lavandula postdistillation waste material (LEW). The chemical compositions determined for these Lavandula preparations included seven major compounds that were selected for further testing. These were tested against C. jejuni for biofilm degradation and removal. Next-generation sequencing was used to study the molecular mechanisms underlying LEO actions against C. jejuni adhesion and motility. Analysis of LEO revealed 1,8-cineol, linalool, and linalyl acetate as the main components. For LEF and LEW, the main components were phenolic acid glycosides, with flavonoids rarely present. The MICs of the Lavandula preparations and pure compounds against C. jejuni ranged from 0.2 mg/ml to 1 mg/ml. LEO showed the strongest biofilm degradation. The reduction of C. jejuni adhesion was ≥1 log10 CFU/ml, which satisfies European Food Safety Authority recommendations. Lavandula preparations reduced C. jejuni motility by almost 50%, which consequently can impact biofilm formation. These data are in line with the transcriptome analysis of C. jejuni, which indicated that LEO downregulated genes important for biofilm formation. LEW also showed good antibacterial and antibiofilm effects, particularly against adhesion and motility mechanisms. This defines an innovative approach using alternative strategies and novel targets to combat bacterial biofilm formation and, hence, the potential to develop new effective agents with biofilm-degrading activities. IMPORTANCE The Lavandula preparations used in this study are found to be effective against C. jejuni, a common foodborne pathogen. They show antibiofilm properties at subinhibitory concentrations in terms of promoting biofilm degradation and inhibiting cell adhesion and motility, which are involved in the initial steps of biofilm formation. These results are confirmed by transcriptome analysis, which highlights the effect of Lavandula essential oil on C. jejuni biofilm properties. We show that the waste material from the hydrodistillation of Lavandula has particular antibiofilm effects, suggesting that it has potential for reuse for industrial purposes. This study highlights the need for efforts directed toward such innovative approaches and alternative strategies against biofilm formation and maintenance by developing new naturally derived agents with antibiofilm activities.

Treatment with the Probiotic Product Aviguard® Alleviates Inflammatory Responses during Campylobacter jejuni-Induced Acute Enterocolitis in Mice.

Prevalences of Campylobacter (C.) jejuni infections are progressively rising globally. Given that probiotic feed additives, such as the commercial product Aviguard®, have been shown to be effective in reducing enteropathogens, such as Salmonella, in vertebrates, including livestock, we assessed potential anti-pathogenic and immune-modulatory properties of Aviguard® during acute C. jejuni-induced murine enterocolitis. Therefore, microbiota-depleted IL-10-/- mice were infected with C. jejuni strain 81-176 by gavage and orally treated with Aviguard® or placebo from day 2 to 4 post-infection. The applied probiotic bacteria could be rescued from the intestinal tract of treated mice, but with lower obligate anaerobic bacterial counts in C. jejuni-infected as compared to non-infected mice. Whereas comparable gastrointestinal pathogen loads could be detected in both groups until day 6 post-infection, Aviguard® treatment resulted in improved clinical outcome and attenuated apoptotic cell responses in infected large intestines during acute campylobacteriosis. Furthermore, less distinct pro-inflammatory immune responses could be observed not only in the intestinal tract, but also in extra-intestinal compartments on day 6 post-infection. In conclusion, we show here for the first time that Aviguard® exerts potent disease-alleviating effects in acute C. jejuni-induced murine enterocolitis and might be a promising probiotic treatment option for severe campylobacteriosis in humans.

Antimicrobial Resistance Gene Transfer from Campylobacter jejuni in Mono- and Dual-Species Biofilms.

Horizontal gene transfer (HGT) is a driving force for the dissemination of antimicrobial resistance (AMR) genes among Campylobacter jejuni organisms, a leading cause of foodborne gastroenteritis worldwide. Although HGT is well documented for C. jejuni planktonic cells, the role of C. jejuni biofilms in AMR spread that likely occurs in the environment is poorly understood. Here, we developed a cocultivation model to investigate the HGT of chromosomally encoded AMR genes between two C. jejuni F38011 AMR mutants in biofilms. Compared to planktonic cells, C. jejuni biofilms significantly promoted HGT (P < 0.05), resulting in an increase of HGT frequencies by up to 17.5-fold. Dynamic study revealed that HGT in biofilms increased at the early stage (i.e., from 24 h to 48 h) and remained stable during 48 to 72 h. Biofilms continuously released the HGT mutants into supernatant culture, indicating spontaneous dissemination of AMR to broader niches. DNase I treatment confirmed the role of natural transformation in genetic exchange. HGT was not associated with biofilm biomass, cell density, or bacterial metabolic activity, whereas the presence of extracellular DNA was negatively correlated with the altered HGT frequencies. HGT in biofilms also had a strain-to-strain variation. A synergistic HGT effect was observed between C. jejuni with different genomic backgrounds (i.e., C. jejuni NCTC 11168 chloramphenicol-resistant strain and F38011 kanamycin-resistant strain). C. jejuni performed HGT at the frequency of 10-7 in Escherichia coli-C. jejuni biofilms, while HGT was not detectable in Salmonella enterica-C. jejuni biofilms. IMPORTANCE Antimicrobial-resistant C. jejuni has been listed as a high priority of public health concern worldwide. To tackle the rapid evolution of AMR in C. jejuni, it is of great importance to understand the extent and characteristics of HGT in C. jejuni biofilms, which serve as the main survival strategy of this microbe in the farm-to-table continuum. In this study, we demonstrated that biofilms significantly enhanced HGT compared to the planktonic state (P < 0.05). Biofilm cultivation time and extracellular DNA (eDNA) amount were related to varied HGT frequencies. C. jejuni could spread AMR genes in both monospecies and dual-species biofilms, mimicking the survival mode of C. jejuni in food chains. These findings indicated that the risk and extent of AMR transmission among C. jejuni organisms have been underestimated, as previous HGT studies mainly focused on the planktonic state. Future AMR controlling measures can target biofilms and their main component eDNA.

Significant contribution of the CmeABC Efflux pump in high-level resistance to ciprofloxacin and tetracycline in Campylobacter jejuni and Campylobacter coli clinical isolates.

BACKGROUND: Campylobacter resistance to antimicrobial agents is regarded as a major concern worldwide. The aim of this study was to investigate the expression of the CmeABC efflux pump and the RAPD-PCR pattern in drug-resistant Campylobacter isolates. METHODS: A total of 283 stool specimens were collected from children under the age of five with diarrhea. The minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin was determined by broth microdilution method and E-test, respectively. Detection of tetracycline and ciprofloxacin determinants was done by amplification of tetO gene and PCR-sequencing of the gyrA gene. The cmeABC transcriptional expression was analyzed by Real-time (RT)-PCR. Clonal correlation of resistant strains was determined by RAPD-PCR genotyping. RESULTS: Out of 283 fecal samples, 20 (7.02%) samples were positive for Campylobacter spp. Analysis of duplex PCR assay of the cadF gene showed that 737 and 461 bp amplicons were corresponding to Campylobacter jejuni and Campylobacter coli, respectively. All of the 17 phenotypically tetracycline-resistant Campylobacter isolates harbored the tetO gene. Also, four phenotypically ciprofloxacin-resistant Campylobacter isolates had a point mutation at codon 257 of the gyrA gene (ACA to ATA; Thr > Ile). High-level expression of the cmeA gene was observed in ciprofloxacin-resistant and high-level tetracycline-resistant Campylobacter isolates, suggesting a positive correlation between the cmeA gene expression level and tetracycline resistance level. Moreover, a statistically significant difference was observed in the cmeA gene expression between ciprofloxacin-resistant and ciprofloxacin-susceptible strains, which signifies the crucial contribution of the efflux pump in conferring multiple drug resistance phenotype among Campylobacter spp. RAPD analysis of Campylobacter isolates exhibited 16 different patterns. Simpsone`s diversity index of RAPD-PCR was calculated as 0.85, showing a high level of homogeneity among the population; however, no clear correlation was detected among tetracycline and/or ciprofloxacin resistant isolates. CONCLUSION: Significant contribution of the CmeABC efflux pump in conferring high-level resistance to tetracycline and ciprofloxacin was observed in C. jejuni and C. coli clinical isolates. The resistant phenotype is suggested to be mediated by CmeABC efflux pumps, the tetO gene, and point mutation of the gyrA gene. Genotyping revealed no clonal correlation among resistant strains, indicating distinct evolution of tetracycline and ciprofloxacin resistant genotypes among the isolates.

Bacillaene Mediates the Inhibitory Effect of Bacillus subtilis on Campylobacter jejuni Biofilms.

Biofilms are the predominant bacterial lifestyle and can protect microorganisms from environmental stresses. Multispecies biofilms can affect the survival of enteric pathogens that contaminate food products, and thus, investigating the underlying mechanisms of multispecies 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 preestablished 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 preestablished C. jejuni biofilms. Furthermore, the coinoculation 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 nonribosomal 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 foodborne infection in humans. This pathogen forms biofilms which enhance survival of C. jejuni in food processing and thus threaten human health. Probiotic bacteria represent a potential alternative in the prevention and control of foodborne 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 coculture and destroys the preestablished 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.