Enhancing the targeting performance and prolonging the antibacterial effects of clove essential oil liposomes to Campylobacter jejuni by antibody modification.

The application of plant essential oil liposomes to prevent and control food safety risks caused by Campylobacter jejuni (C. jejuni) still faces challenges such as lack of targeting and low release rate. Here, a bacteria-targeted and protease-activated antibacterial liposome (ACCLPs) was successfully synthesized through encapsulation of clove essential oil (CEO) by film dispersion method, embedding of casein by freeze-thaw method, and conjugation of C. jejuni antibody on the liposome membrane by post-insertion method. The average particle size, the essential oil encapsulation rate, the casein mosaic rate, and the antibody coupling efficiency of ACCLPs were determined as185.87 nm,16.9%,70.1% and 87.5%, respectively. The modification with C. jejuni antibody could significantly improve the targeting of ACCLPs to C. jejuni. Controlled release experiments showed that the exocrine protease from C. jejuni could hydrolyze the embedded casein and perforation on the ACCLPs, thus leading to a bacteria-dependent CEO release and significant prolonging the antibacterial effects of ACCLPs. Application results of ACCLPs on C. jejuni-contaminated foods showed that ACCLPs could effectively inhibit C. jejuni in a variety of meat products, fruits and vegetables and extend their shelf life without significantly affecting food quality. The results above in this work would provide a new view for the development of high efficient liposome-based antibacterial system of plant essential oil.

Combined effect of metabolites produced by a modified Lactobacillus casei and berry phenolic extract on Campylobacter and microbiome in chicken cecum contents.

Campylobacter is one of the most common foodborne bacterial pathogens causing illness, known as campylobacteriosis, in the United States. More than 70% of the campylobacteriosis cases have direct or indirect relation with poultry/poultry products. Currently, both conventional and organic/pasture poultry farmers are searching for sustainable alternative to antibiotics which can reduce colonization and cross-contamination of poultry products with Campylobacter and promote poultry health and growth. Probiotic and their nutritional supplement, known as prebiotic, have become consumers' preferences as alternatives to antibiotics/chemicals. In this study, we evaluated the combined effect of plant-derived prebiotic and probiotic-derived metabolites in reducing growth of Campylobacter in cecum contents, a simulated chicken gut condition. Cecum contents were collected from chickens pre-inoculated with kanamycin-resistant Campylobacter (CJRMKm), were incubated over 48 h time period, while being supplemented with either berry phenolic extract (BPE), cell free cultural supernatant from an engineered probiotic, Lactobacillus casei, or their combination. It was found that combine treatments were able to reduce both inoculated and naturally colonized Campylobacter more effectively. Microbiome analysis using 16S rRNA sequencing also revealed that combine treatments were capable to alter natural microflora positively within chicken cecum contents. Differences were observed in bacterial abundance at both phylum and genus level but did not show significant alteration in alpha diversity due to this treatment. PRACTICAL APPLICATION: The results of this study provide critical information for understanding the potential of synbiotic as an alternative in sustainable poultry farming. The outcomes of this study will lead future direction of using combination of probiotic-derived metabolites and BPE in poultry farming.

In silico prediction and expression analysis of vaccine candidate genes of Campylobacter jejuni.

Campylobacter jejuni (C. jejuni) is the most common food-borne pathogen that causes human gastroenteritis in the United States. Consumption of contaminated poultry products is considered as the major source of human Campylobacter infection. An effective vaccine would be a promising alternative to antibiotic supplements to curb C. jejuni colonization in poultry gastrointestinal (GI) tract. However, the genetic diversity among the C. jejuni isolates makes vaccine production more challenging. Despite many attempts, an effective Campylobacter vaccine is not yet available. This study aimed to identify suitable candidates to develop a subunit vaccine against C. jejuni, which could reduce colonization in the GI tract of the poultry. In the current study, 4 C. jejuni strains were isolated from retail chicken meat and poultry litter samples and their genomes were sequenced utilizing next-generation sequencing technology. The genomic sequences of C. jejuni strains were screened to identify potential antigens utilizing the reverse vaccinology approach. In silico genome analysis predicted 3 conserved potential vaccine candidates (phospholipase A [PldA], TonB dependent vitamin B12 transporter [BtuB], and cytolethal distending toxin subunit B [CdtB]) suitable for the development of a vaccine. Furthermore, the expression of predicted genes during host-pathogen interaction was analyzed by an infection study using an avian macrophage-like immortalized cell line (HD11). The HD11 was infected with C. jejuni strains, and the RT-qPCR assay was performed to determine the expression of the predicted genes. The expression difference was analyzed using ΔΔCt methods. The results indicate that all 3 predicted genes, PldA, BtuB, and CdtB, were upregulated in 4 tested C. jejuni strains irrespective of their sources of isolation. In conclusion, in silico prediction and gene expression analysis during host-pathogen interactions identified 3 potential vaccine candidates for C. jejuni.

Effects of dietary yeast cell wall supplementation on growth performance, intestinal Campylobacter jejuni colonization, innate immune response, villus height, crypt depth, and slaughter characteristics of broiler chickens inoculated with Campylobacter jejuni at d 21.

A study was conducted to assess the effects of a dietary yeast cell wall (YCW) with and without a Campylobacter jejuni (CJ) challenge. A total of 2,240-day-old Ross 708 males were randomly assigned within 8 treatments with a 4 × 2 factorial design, with 4 diets (negative control, positive control, YCW constant dose (400 g/ton), and YCW step-down dose (800/400/200 g/ton in the starter/grower/finisher diets, respectively) and with and without d 21 CJ oral gavage challenge at 5.2 × 107 CFU/mL. At d 0, 14, 28, and 41 body weights and feed consumption were measured to determine performance. At d 14, 28, and 42, 8 jejunal and ileal histology samples per treatment were collected for villi morphology measurements. At d 22 and 28 (1- and 7-days postinoculation), 24 ileal tissue samples per treatment were collected for relative gene expression analysis. At d 42, 24 cecal content samples per treatment were collected for CJ enumeration. Finally, on d 44, 96 birds per treatment were processed to determine carcass yield and 16 carcass rinses per treatment were collected to determine CJ prevalence after processing. Diet or inoculation did not impact broiler performance (P > 0.05). Limited differences were observed in intestinal morphology, and villus height and crypt depth were different only in the ileum at d 42 (P = 0.0280 and P = 0.0162, respectively). At d 1 postinoculation, differences between treatments inoculated with CJ and PBS were observed in the expression of avian beta defensin 10 (AvBD10), interleukin 1ß (IL-1ß), and interleukin 10 (IL-10) (P < 0.05). At d 7 postinoculation, expression of AvBD10, IL-1ß, and IL-10 was similar among all treatments (P > 0.05). At d 42, all birds, regardless the inoculation, had similar levels of CJ recovered from cecal contents (P > 0.05). After processing, carcass yield and CJ prevalence postchilling was similar in all treatments (P > 0.05). Overall, under the conditions of this study, the addition of YCW during a CJ challenge did not have an impact in growth performance, innate immune response, cecal colonization, carcass yield, or CJ prevalence after processing.

Effect of Probiotic E. coli Nissle 1917 Supplementation on the Growth Performance, Immune Responses, Intestinal Morphology, and Gut Microbes of Campylobacter jejuni Infected Chickens.

Campylobacter jejuni is the most common cause of bacterial foodborne gastroenteritis and holds significant public health importance. The continuing increase of antibiotic-resistant Campylobacter necessitates the development of antibiotic-alternative approaches to control infections in poultry and in humans. Here, we assessed the ability of E. coli Nissle 1917 (EcN; free and chitosan-alginate microencapsulated) to reduce C. jejuni colonization in chickens and measured the effect of EcN on the immune responses, intestinal morphology, and gut microbes of chickens. Our results showed that the supplementation of 3-week-old chickens daily with free EcN in drinking water resulted in a 2.0 log reduction of C. jejuni colonization in the cecum, whereas supplementing EcN orally three times a week, either free or microencapsulated, resulted in 2.0 and 2.5 log reductions of C. jejuni colonization, respectively. Gavaged free and microencapsulated EcN did not have an impact on the evenness or the richness of the cecal microbiota, but it did increase the villous height (VH), crypt depth (CD), and VH:CD ratio in the jejunum and ileum of chickens. Further, the supplementation of EcN (all types) increased C. jejuni-specific and total IgA and IgY antibodies in chicken's serum. Microencapsulated EcN induced the expression of several cytokines and chemokines (1.6 to 4.3-fold), which activate the Th1, Th2, and Th17 pathways. Overall, microencapsulated EcN displayed promising effects as a potential nonantibiotic strategy to control C. jejuni colonization in chickens. Future studies on testing microencapsulated EcN in the feed and water of chickens raised on built-up floor litter would facilitate the development of EcN for industrial applications to control Campylobacter infections in poultry.

Campylobacter jejuni induces differentiation of human neutrophils to the CD16hi /CD62Llo subtype.

The discovery of neutrophil subtypes has expanded what is known about neutrophil functions, yet there is still much to learn about the role of these subtypes during bacterial infection. We investigated whether Campylobacter jejuni induced differentiation of human neutrophils into the hypersegmented, CD16hi /CD62Llo subtype. In addition, we investigated whether C. jejuni-dependent differentiation of this neutrophil subtype induced cancer-promoting activities of human T cells and colonocytes, which were observed in other studies of hypersegmented, CD16hi /CD62Llo neutrophils. We found that C. jejuni causes a significant shift in human neutrophil populations to the hypersegmented, CD16hi /CD62Llo subtype and that those populations exhibit delayed apoptosis, elevated arginase-1 expression, and increased reactive oxygen species production. Furthermore, incubation of C. jejuni-infected neutrophils with human T cells resulted in decreased expression of the ζ-chain of the TCR, which was restored upon supplementation with exogenous l-arginine. In addition, incubation of C. jejuni-infected neutrophils with human colonocytes resulted in increased HIF-1α stabilization and NF-κB activation in those colonocytes, which may result in the up-regulation of protumorigenic genes.

Copper toxicity towards Campylobacter jejuni is enhanced by the nickel chelator dimethylglyoxime.

The nickel (Ni)-chelator dimethylglyoxime (DMG) was found to be bacteriostatic towards Campylobacter jejuni. Supplementation of nickel to DMG-containing media restored bacterial growth, whereas supplementation of cobalt or zinc had no effect on the growth inhibition. Unexpectedly, the combination of millimolar levels of DMG with micromolar levels of copper (Cu) was bactericidal, an effect not seen in select Gram-negative pathogenic bacteria. Both the cytoplasmic Ni-binding chaperone SlyD and the twin arginine translocation (Tat)-dependent periplasmic copper oxidase CueO were found to play a central role in the Cu-DMG hypersensitivity phenotype. Ni-replete SlyD is needed for Tat-dependent CueO translocation to the periplasm, whereas Ni-depleted (DMG-treated) SlyD is unable to interact with the CueO Tat signal peptide, leading to mislocalization of CueO and increased copper sensitivity. In support of this model, C. jejuni ΔslyD and ΔcueO mutants were more sensitive to copper than the wild-type (WT); CueO was less abundant in the periplasmic fraction of ΔslyD or DMG-grown WT cells, compared to WT cells grown on plain medium; SlyD binds the CueO signal sequence peptide, with DMG inhibiting and nickel enhancing the binding, respectively. Injection of Cu-DMG into Galleria mellonella before C. jejuni inoculation significantly increased the insect survival rate compared to the control group. In chickens, oral administration of DMG or Cu-DMG decreased and even abolished C. jejuni colonization in some cases, compared to both water-only and Cu-only control groups. The latter finding is important, since campylobacteriosis is the leading bacterial foodborne infection, and chicken meat constitutes the major foodborne source.

Effect of pepper extracts on the viability kinetics, topography and Quantitative NanoMechanics (QNM) of Campylobacter jejuni evaluated with AFM.

Campylobacter jejuni is a pathogen bacterium that causes foodborne gastroenteritis in humans. However, phenolic compounds extracted from natural sources such as capsicum pepper plant (Capsicum annuum L. var. aviculare) could inhibit the growth of C. jejuni. Therefore, different extracts were prepared using ultrasonic extraction (USE), conventional extraction (CE) and thermosonic extraction (TSE). C. jejuni was then exposed to chili extracts to examine the antimicrobial effect and their growth/death bacterial kinetics were studied using different mathematical models. Atomic force microscopy was applied to investigate the microstructural and nanomechanical changes in the bacteria. Extracts obtained by TSE had the highest phenolic content (4.59 ± 0.03 mg/g of chili fresh weight [FW]) in comparison to USE (4.12 ± 0.05 mg/g of chili FW) and CE (4.28 ± 0.07 mg/g of chili FW). The inactivation of C. jejuni was more efficient when thermosonic extract was used. The Gompertz model was the most suitable mathematical model to describe the inactivation kinetics of C. jejuni. Roughness and nanomechanical analysis performed by atomic force microscopy (AFM) provided evidence that the chili extracts had significant effects on morphology, surface, and the reduced Young's modulus of C. jejuni. The novelty of this work was integrating growth/death bacterial kinetics of C. jejuni using different mathematical models and chili extracts, and its relationship with the morphological, topographic and nanomechanical changes estimated by AFM.

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.

Occurrence and multidrug resistance of Campylobacter spp. at duck farms and associated environmental and anthropogenic risk factors in Bangladesh.

BACKGROUND: The alarming rise in multi-drug resistant (MDR) zoonotic pathogens, including Campylobacter spp., has been threatening the health sector globally. In Bangladesh, despite rapid growth in poultry sector little is known about the potential risks of zoonotic pathogens in homestead duck flocks. The aim of this study was to understand the occurrence, species diversity, and multi-drug resistance in Campylobacter spp., and identify the associated risk factors in duck farms in Bangladesh. METHODS: The study involved 20 duck farms at 6 sub-districts of Mymensingh, Bangladesh. Monthly occurrence of Campylobacter spp. in potential sources at the farms during February-September, 2018, was detected by culture and PCR-based methods. Campylobacter isolates were examined for resistance to different antimicrobials. Risk factors, concerning climatic and environmental disposition, farm management, and anthropogenic practices, of Campylobacter infection were estimated by participatory epidemiological tools. RESULTS: Occurrence of Campylobacter spp. was detected in overall 36.90% (155/420) samples, more frequently in drinking water (60%, 30/50), followed by cloacal swab (37.50%, 75/200), egg surface swab (35%, 35/100) and soil of the duck resting places (30%, 15/50) but was not detected in feed samples (n = 20). PCR assays distinguished the majority (61.30%, 95/155) of the isolates as C. coli, while the rest (38.70%, 60/155) were C. jejuni. Notably, 41.7% (25/60) and 31.6% (30/95) strains of C. jejuni and C. coli, respectively, were observed to be MDR. The dynamics of Campylobacter spp., distinctly showing higher abundance during summer and late-monsoon, correlated significantly with temperature, humidity, and rainfall, while sunshine hours had a negative influence. Anthropogenic management-related factors, including, inadequate hygiene practices, use of untreated river water, wet duck shed, flock age (1-6 months), and unscrupulous use of antimicrobials were identified to enhance the risk of MDR Campylobacter infection. CONCLUSION: The present study clearly demonstrates that duck farms contribute to the enhanced occurrence and spread of potentially pathogenic and MDR C. coli and C. jejuni strains and the bacterial dynamics are governed by a combined interaction of environmental and anthropogenic factors. A long-term holistic research at the environment-animal-human interface would be integral to divulge health risk reduction approaches tackling the spread of Campylobacter spp. from duck farms.

Vitamin D Reverses Disruption of Gut Epithelial Barrier Function Caused by Campylobacter jejuni.

Infections by the zoonotic foodborne bacterium Campylobacter jejuni (C. jejuni) are among the most frequent causes of bacterial gastroenteritis worldwide. The aim was to evaluate the relationship between epithelial barrier disruption, mucosal immune activation, and vitamin D (VD) treatment during C. jejuni infection, using intestinal epithelial cells and mouse models focused on the interaction of C. jejuni with the VD signaling pathway and VD treatment to improve C. jejuni-induced barrier dysfunction. Our RNA-Seq data from campylobacteriosis patients demonstrate inhibition of VD receptor (VDR) downstream targets, consistent with suppression of immune function. Barrier-preserving effects of VD addition were identified in C. jejuni-infected epithelial cells and IL-10-/- mice. Furthermore, interference of C. jejuni with the VDR pathway was shown via VDR/retinoid X receptor (RXR) interaction. Paracellular leakiness of infected epithelia correlated with tight junction (TJ) protein redistribution off the TJ domain and apoptosis induction. Supplementation with VD reversed barrier impairment and prevented inhibition of the VDR pathway, as shown by restoration of transepithelial electrical resistance and fluorescein (332 Da) permeability. We conclude that VD treatment restores gut epithelial barrier functionality and decreases bacterial transmigration and might, therefore, be a promising compound for C. jejuni treatment in humans and animals.

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.

Nutraceutical intervention protects against bacterial and chemical-induced gastrotoxicity in a non-mammalian model, Galleria mellonella.

Preparations of the fungus Cordyceps sinensis and bovine colostrum are considered nutraceuticals due to their anti-inflammatory, repair and gut alimentation properties in mammalian models. To reduce the reliance on rodents in routine experimentation, we gauged the capacity of nutraceuticals to alleviate gastric damage in an insect surrogate, Galleria mellonella. Larvae were reared on standard or supplemented diets - 10% (w/w) colostrum, 10% (w/w) C. sinensis, or 5% + 5% each - prior to receiving an oral dose of the NSAID indomethacin (30 mg/kg) or challenged with the bacterial pathogen Campylobacter jejuni (1-3 x106) via two inoculation routes. Insects reared on a cordyceps-supplemented diet proved most resistant to indomethacin-induced gut leakiness, and displayed stable health indices after C. jejuni challenge (~77% survival). Insects reared on a colostrum-supplemented diet also showed recalcitrance in the gut, but were more sensitive to C. jejuni when injected directly into the body cavity (50% survival). The nutraceutical blend yielded improved health outcomes when compared to the standard diet, but was not as effective as either nutraceutical alone. Our findings represent clear evidence that insects were more resistant to known chemical and microbial agitators when reared on nutraceutical-supplemented diets - toxicological endpoints that are shared with vertebrate studies.

Presence of Campylobacterjejuni and C. coli in Dogs under Training for Animal-Assisted Therapies.

This study was conducted to evaluate the presence of Campylobacter (C.) jejuni and C. coli in dogs at five dog training centers in Southern Italy. A total of 550 animals were sampled by collecting rectal swabs. The samples were processed to detect thermotolerant Campylobacter spp. by culture and molecular methods. Campylobacter spp. were isolated from 135/550 (24.5-95% confidence interval) dogs. A total of 84 C. jejuni (62.2%) and 51 C. coli (37.8%) isolates were identified using conventional PCR. The dog data (age, sex, breed, and eating habits) were examined by two statistical analyses using the C. jejuni and C. coli status (positive or negative) as dependent variables. Dogs fed home-cooked food showed a higher risk of being positive for C. jejuni than dogs fed dry or canned meat for dogs (50.0%; p < 0.01). Moreover, purebred dogs had a significantly higher risk than crossbred dogs for C. coli positivity (16.4%; p < 0.01). This is the first study on the prevalence of C. jejuni and C. coli in dogs frequenting dog training centers for animal-assisted therapies (AATs). Our findings emphasize the potential zoonotic risk for patients and users involved in AATs settings and highlight the need to carry out ad hoc health checks and to pay attention to the choice of the dog, as well as eating habits, in order to minimize the risk of infection.

Nettle-Leaf Extract Derived ZnO/CuO Nanoparticle-Biopolymer-Based Antioxidant and Antimicrobial Nanocomposite Packaging Films and Their Impact on Extending the Post-Harvest Shelf Life of Guava Fruit.

Green synthesized metal oxide nanoparticles (NPs) have prominent applications in antimicrobial packaging systems. Here we have attempted for the fabrication of chitosan-based nanocomposite film containing Urtica dioica leaf extract derived copper oxide (CuO) and zinc oxide (ZnO) NPs for shelf-life extension of the packaged guava fruits. Electron microscopy and spectroscopy analysis of the CuO and ZnO NPs exhibited nano-scale size, spherical morphologies, and negative ζ-potential values. The NPs possessed appreciable antioxidant and antimicrobial activity (AMA) in order of CuO NPs > ZnO NPs >nettle extract. Therefore, this work establishes for the first time the successful synthesis of CuO NPs and compares its antimicrobial and antioxidant properties with ZnO NPs. On incorporation in chitosan, the polymer nanocomposite films were developed by solvent casting technique. The developed films were transparent, had low antioxidant but substantial AMA. The NP supplementation improved the film characteristics as evident from the decrease in moisture content, water holding capacity, and solubility of the films. The nanocomposite films improved the quality attributes and shelf life of guava fruits by one week on packaging and storage compared to unpackaged control fruits. Therefore, this study demonstrates the higher antimicrobial potential of the nettle leaf extract derived CuO/ZnO NPs for development of antimicrobial nanocomposite films as a promising packaging solution for enhancing the shelf life of various perishable fruits.

Effects of a dietary direct-fed microbial and Ferulago angulata extract on growth performance, intestinal microflora, and immune function of broiler chickens infected with Campylobacter jejuni.

Colonization of the gastrointestinal tract by potentially pathogenic bacteria and their shedding in animal feces is a fundamental factor for both animal health and human food safety. This study was conducted to evaluate the efficacy of salinomycin (Sal), direct-fed microbial (DFM), and Ferulago angulata hydroalcoholic extract (FAE) against Campylobacter jejuni in broiler chickens in a 6-week pilot-scale study. A total of six hundred and seventy two 1-day-old broiler chickens were equally divided into 6 groups (each consisting of 8 replicates of 14 birds): negative control (NC; untreated and uninfected); positive control (PC; untreated, infected with C. jejuni); PC + Sal; PC + DFM; PC + 200 mg/kg of FAE (FAE200); or PC + 400 mg/kg of FAE (FAE400). All these groups (except NC) were challenged with C. jejuni on day 15. The results showed that all experimental treatments improved (P < 0.05) average daily gain compared with the PC group, and the best value was observed in the NC and FAE400 groups throughout the entire experimental period (day 1-42). The overall feed conversion ratio and mortality rate, as well as the population of C. jejuni (day 24 and 42) and Coliforms (day 42) in the ileum and cecum, were higher (P < 0.05) in broiler chickens fed with the PC diet than for chickens in the other groups, except those in the FAE200 group. Immune responses revealed that among challenged birds, those that were fed diets DFM and FAE400 had significantly higher IgG (day 24 and 42), IgA (day 24), IL-6 (day 24), and gamma interferon (day 24 and 42) concentrations than the PC group. In conclusion, dietary FAE, especially at a high level of inclusion in broiler diet (400 mg/kg), could beneficially influence the immune status, as well as improve growth performance and intestinal microflora under Campylobacter challenge, which was comparable to those of Sal and DFM supplements.

Possible reservoirs of thermotolerant Campylobacter at the farm between rearing periods and after the use of enrofloxacin as a therapeutic treatment.

Campylobacteriosis is a zoonosis and the most frequent cause of food-borne bacterial enteritis in humans. C. jejuni and C. coli are the most common species implicated in campylobacteriosis. Broilers and their products are considered the most important food sources of human infections. The aim of the present study was to evaluate the presence of thermotolerant Campylobacter in different reservoirs at the farm, and the permanence of this pathogen during four consecutive rearing periods. The samples were taken from the same house farm in the downtime period and during the last week of broiler rearing, prior to their slaughter during four consecutive cycles. Different reservoirs as potential sources of Campylobacter were analysed. The prevalence of Campylobacter in vectors was 23% in A. diaperinus larvae, 20% in wild birds, 13% in A. diaperinus adults, and 9% in flies; as regards fomites, the prevalence was 50% in workers' boots, 27% in litter, and 21% in feed, while in broilers it was 80%. Campylobacter jejuni was the most detected species (51%) in the samples analysed. In addition, some Campylobacter genotypes persisted in the house farm throughout consecutive rearing periods, indicating that those strains remain during downtime periods. However, our study could not identify the Campylobacter sources in the downtime periods because all the samples were negative for Campylobacter isolation. In addition, a remarkable finding was the effect of the use of enrofloxacin (as a necessary clinical intervention for flock health) in cycle 3 on the Campylobacter population. No Campylobacter could be isolated after that clinic treatment. Afterwards, we found a greater proportion of C. coli isolates, and the genotypes of those isolates were different from the genotypes found in the previous rearing periods. In conclusion, the effect of the use of enrofloxacin during the rearing period changed the Campylobacter species proportion, and this finding is particularly interesting for further evaluation. Furthermore, more studies should be conducted with the aim of detecting the Campylobacter sources between rearing periods.

Campylobacter sp.: Pathogenicity factors and prevention methods-new molecular targets for innovative antivirulence drugs?

Infections caused by bacterial species from the genus Campylobacter are one of the four main causes of strong diarrheal enteritis worldwide. Campylobacteriosis, a typical food-borne disease, can range from mild symptoms to fatal illness. About 550 million people worldwide suffer from campylobacteriosis and lethality is about 33 million p.a. This review summarizes the state of the current knowledge on Campylobacter with focus on its specific virulence factors. Using this knowledge, multifactorial prevention strategies can be implemented to reduce the prevalence of Campylobacter in the food chain. In particular, antiadhesive strategies with specific adhesion inhibitors seem to be a promising concept for reducing Campylobacter bacterial load in poultry production. Antivirulence compounds against bacterial adhesion to and/or invasion into the host cells can open new fields for innovative antibacterial agents. Influencing chemotaxis, biofilm formation, quorum sensing, secretion systems, or toxins by specific inhibitors can help to reduce virulence of the bacterium. In addition, the unusual glycosylation of the bacterium, being a prerequisite for effective phase variation and adaption to different hosts, is yet an unexplored target for combating Campylobacter sp. Plant extracts are widely used remedies in developing countries to combat infections with Campylobacter. Therefore, the present review summarizes the use of natural products against the bacterium in an attempt to stimulate innovative research concepts on the manifold still open questions behind Campylobacter towards improved treatment and sanitation of animal vectors, treatment of infected patients, and new strategies for prevention. KEY POINTS: • Campylobacter sp. is a main cause of strong enteritis worldwide. • Main virulence factors: cytolethal distending toxin, adhesion proteins, invasion machinery. • Strong need for development of antivirulence compounds.

Antibacterial Mode of Action of the Daucus carota Essential Oil Active Compounds against Campylobacter jejuni and Efflux-Mediated Drug Resistance in Gram-Negative Bacteria.

Today, an alarming rise of bacterial gastroenteritis in humans resulting from consuming Campylobacter-tainted foods is being observed. One of the solutions for mitigating this issue may be the antibacterial activity of essential oils. In the present research, we propose to study the antibacterial activity against Campylobacter and other Gram-negative bacteria of Daucus carota essential oil and its active molecules. In addition, a few chemically synthesized molecules such as (E)-methylisoeugenol, Elemicin, and eugenol were also studied. The results showed that the essential oil itself and its most active component, (E)-methylisoeugenol, exhibited bactericidal effects. Similar effects were detected using purified and chemically synthesized molecules. Also, it was observed that the Daucus carota essential oil and its active molecules affected intracellular potassium and intracellular ATP contents in Campylobacter cells. Inhibition of the membrane bound FOF1-ATPase was also observed. Eventually, for the first time, the efflux mechanism of active molecules of Daucus carota essential oil was also identified in gamma proteobacteria and its specific antibacterial activity against Campylobacter jejuni was associated with the lack of this efflux mechanism in this species.

Validation of the T86I mutation in the gyrA gene as a highly reliable real time PCR target to detect Fluoroquinolone-resistant Campylobacter jejuni.

BACKGROUND: Campylobacter jejuni is a leading cause of bacterial diarrhea worldwide, and increasing rates of fluoroquinolone (FQ) resistance in C. jejuni are a major public health concern. The rapid detection and tracking of FQ resistance are critical needs in developing countries, as these antimicrobials are widely used against C. jejuni infections. Detection of point mutations at T86I in the gyrA gene by real-time polymerase chain reaction (RT-PCR) is a rapid detection tool that may improve FQ resistance tracking. METHODS: C. jejuni isolates obtained from children with diarrhea in Peru were tested by RT-PCR to detect point mutations at T86I in gyrA. Further confirmation was performed by sequencing of the gyrA gene. RESULTS: We detected point mutations at T86I in the gyrA gene in 100% (141/141) of C. jejuni clinical isolates that were previously confirmed as ciprofloxacin-resistant by E-test. No mutations were detected at T86I in gyrA in any ciprofloxacin-sensitive isolates. CONCLUSIONS: Detection of T86I mutations in C. jejuni is a rapid, sensitive, and specific method to identify fluoroquinolone resistance in Peru. This detection approach could be broadly employed in epidemiologic surveillance, therefore reducing time and cost in regions with limited resources.