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.

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.

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.

Identification of Novel Bioactive Compound Derived from Rheum officinalis against Campylobacter jejuni NCTC11168.

Gastric diseases are increasing with the infection of Campylobacter jejuni. Late stages of infection lead to peptic ulcer and gastric carcinoma. C. jejuni infects people within different stages of their life, especially childhood, causing severe diarrhea; it infects around two-thirds of the world population. Due to bacterial resistance against standard antibiotic, a new strategy is needed to impede Campylobacter infections. Plants provide highly varied structures with antimicrobial use which are unlikely to be synthesized in laboratories. A special feature of higher plants is their ability to produce a great number of organic chemicals of high structural diversity, the so-called secondary metabolites. Twenty plants were screened to detect their antibacterial activities. Screening results showed that Rheum officinalis was the most efficient against C. jejuni. Fractionation pattern was obtained by column chromatography, while the purity test was done by thin-layer chromatography (TLC). The chemical composition of bioactive compound was characterized using GC-MS, nuclear magnetic resonance, and infrared analysis. Minimal inhibitory concentration (MIC) of the purified compound was 31.25 µg/ml. Cytotoxicity assay on Vero cells was evaluated to be 497 µg/ml. Furthermore, the purified bioactive compound activated human lymphocytes in vitro. The data presented here show that Rheum officinalis could potentially be used in modern applications aimed at the treatment or prevention of foodborne diseases.

Adhesion and invasion of Campylobacter jejuni in chickens with a modified gut microbiota due to antibiotic treatment.

Campylobacter jejuni (C. jejuni) is a predominant cause of foodborne illness in humans, while its colonization in chickens is usually asymptomatic. Antibiotics are not routinely used to treat chickens against C. jejuni, but in the face of other bacterial diseases, C. jejuni may be exposed to antibiotics. In this study, chickens were treated with antibiotics (AT) to modify the gut microbiota composition and compared with untreated chickens (Conv) with respect to changes in C. jejuni-colonization and bacterial-intestine interaction. Groups of AT and Conv chickens were inoculated after an antibiotic-withdrawal time of eight days with one of three different C. jejuni isolates to identify possible strain variations. Significantly higher numbers of colony forming units of C. jejuni were detected in the cecal content of AT birds, with higher colonization rates in the spleen and liver compared to Conv birds independent of the inoculated strain (p < 0.05). Clinical signs and histopathological lesions were only observed in C. jejuni-inoculated AT birds. For the first time we demonstrated C. jejuni invasion of the cecal mucosa in AT chickens and its inter- and intracellular localization by using antigen-straining, and electronic microscopy. This study provides the first circumstantial evidence that antibiotic treatment with lasting modification of the microbiota may provide a suitable environment for C. jejuni invasion also in chickens which may subsequently increase the risk of C. jejuni-introduction into the food chain.

Peracetic acid reduces Campylobacter spp. numbers and total viable counts on broiler breast muscle and drumstick skins during modified atmosphere package storage.

Constant high case numbers of human campylobacteriosis over the last few years show the necessity of efficient strategies to reduce the number of diseases. The aim of this study was to assess the effectiveness of peracetic acid (PAA) as spray application to reduce Campylobacter spp. on chicken meat. For this, the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of 25 Campylobacter jejuni and C. coli isolates were determined. All tested isolates had MICs ranging between 2 to 8 ppm PAA, while MBCs were 1- to 4-fold higher than the MIC. An additional time-kill test, using strain C. jejuni DSM 4688, revealed that after an incubation time of 2 h in medium, supplemented with 1-fold the MIC (4 ppm) of PAA, no surviving C. jejuni cells were detectable. For evaluation of a spraying treatment, C. jejuni DSM 4688 (108 cfu/mL) inoculated chicken drumsticks and native skin-on breast fillets were treated for 30 s with PAA of 1,200 ppm concentration. Samples were packaged in modified atmosphere packages and stored at 4°C until further analysis. On day 1, 6, and 12, the fillets were used for microbial (total viable count), sensory, and physicochemical (color, pH, electrical conductivity) analysis and meat samples for myoglobin redox forms and antioxidant activity were taken. A significant reduction of the total viable counts was seen on day 6 and 12 in comparison to the water control and to the untreated fillets, respectively. Campylobacter jejuni counts on the drumsticks were significantly reduced by PAA application on day 6 and 12 in comparison to the water treatment. Except on day 12, where PAA-treated fillets showed a slightly higher percentage of oxymyoglobin, no significant differences could be found in the sensory and physicochemical measurements as well as in myoglobin and antioxidant activity. Spray application of 1,200 ppm PAA to Campylobacter-contaminated chicken samples led to a significant reduction up to 1.1 log10 of Campylobacter spp. counts without influencing chemical and sensory meat quality parameters.

Quinolone and Macrolide-Resistant Campylobacter jejuni in Pediatric Gastroenteritis Patients from Central Iran.

Aims: To determine the prevalence and the antibiotic resistance patterns of Campylobacter jejuni isolated from pediatric diarrhea patients in central Iran. Materials and Methods: Stool specimens (n = 230) were investigated using a modified Gram stain, two specific culture media, and C. jejuni-specific PCR. Antibiotic resistance profiles and relevant resistance genes were determined. Genetic relationships among a selection of the isolates were studied by Fla typing. Results: Out of the 230 diarrhea samples, 48 (20.8%) cases of C. jejuni were identified using modified Gram stain, 45 (19.5%) using the culture media, and 76 (33%) cases were identified using PCR. The highest antibiotic resistance rates were observed in 37 (82.2%) strains against tetracycline, in 32 (71.1%) against ciprofloxacin, and in 31 (68.8%) against erythromycin. Twenty (44.4%) isolates were resistant to ciprofloxacin and erythromycin simultaneously. Genotypic investigations found 36 (97.3%) strains carrying the tet (o) gene, 31 (96.8%) harboring the cmeB gene, 22 (68.7%) strains with the gyrA6 gene, 20 (64.5%) strains containing a 23S rRNA mutation, and 21 (65.6%) strains with the qnrS gene. Fla typing of a random subset of 14 strains revealed 11 different types showing the genomic diversity of the isolates. Strains sharing the same Fla type could be easily distinguished by their resistance gene profile. Conclusions: This is the first study to demonstrate that genetically diverse quinolone-macrolide-resistant C. jejuni is an important cause of gastroenteritis in children from central Iran. Pediatricians should consider these resistance features once the antibiotic prescription is necessary for prevention of possible complications, especially in those under 5 years of age. Of note, most cases of Campylobacter diarrhea are self-limiting and antibiotics should only be prescribed in those cases where severe complications evolve.

Stress Response and Virulence Potential Modulating Effect of Peppermint Essential Oil in Campylobacter jejuni.

Campylobacter jejuni is one of the most common food-borne bacteria that causes gastrointestinal symptoms. In the present study we have investigated the molecular basis of the anti-Campylobacter effect of peppermint essential oil (PEO), one of the oldest EO used to treat gastrointestinal diseases. Transcriptomic, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and proteomic, two-dimensional polyacryl amid gel electrophoresis (2D-PAGE) methods have revealed that, in the presence of a sublethal concentration of PEO, the expression of several virulence-associated genes was decreased (cheY 0.84x; flhB 0.79x; flgE 0.205x; cadF 0.08x; wlaB 0.89x; porA 0.25x; cbf2 4.3x) while impaired motility was revealed with a functional analysis. Scanning electron micrographs of the exposed cells showed that, unlike in the presence of other stresses, the originally curved C. jejuni cells straightened upon PEO exposure. Gaining insight into the molecular background of this stress response, we have revealed that in the presence of PEO C. jejuni dominantly exerts a general stress response that elevates the expression of general stress genes like dnaK, groEL, groES (10.41x, 3.63x, and 4.77x). The most important genes dps, sodB, and katA involved in oxidative stress responses showed however moderate transcriptional elevations (1,58x, 1,55x, and 1,85x).

Pectin or chitosan coating fortified with eugenol reduces Campylobacter jejuni on chicken wingettes and modulates expression of critical survival genes.

Campylobacter jejuni infection in humans is strongly associated with the consumption of contaminated poultry products. With increasing consumer demand for minimally processed and natural product, there is a need for novel intervention strategies for controlling C. jejuni. Antimicrobial coatings are increasingly being used for preventing food contamination due to their efficacy and continuous protection of product. This study investigated the efficacy of pectin and chitosan coating fortified with eugenol to reduce C. jejuni on chicken wingettes. Pectin, chitosan, and eugenol are generally recognized as safe status compounds derived from berries, crustaceans, and cloves respectively. Each wingette was inoculated with a mixture of 4 wild-type strains of C. jejuni (approximately 107 CFU/sample) and randomly assigned to controls, pectin (3%), chitosan (2%), eugenol (0.5, 1, or 2%), or their combinations. Following 1 min of coating, wingettes were air-dried, vacuum sealed, and sampled on 0, 1, 3, 5, and 7 d of refrigerated storage for C. jejuni and aerobic counts (n = 5 wingettes/treatment/d). In addition, the effect of treatments on wingette color and expression of C. jejuni survival/virulence genes was evaluated. All 3 doses of eugenol or chitosan significantly reduced C. jejuni and aerobic bacteria from 0 d through 7 d. Incorporation of 2% eugenol in chitosan improved coating efficiency and reduced C. jejuni counts by approximately 3 Log CFU/sample at the end of 7 d of storage (P < 0.05). Similarly, the antimicrobial efficacy of pectin was improved by 2% eugenol and the coating reduced C. jejuni by approximately 2 Log CFU/sample at 7 d of storage. Chitosan coating with 2% eugenol also showed greater reductions of total aerobic counts as compared to individual treatments of eugenol and chitosan. No significant difference in the color of chicken wingettes was observed between treatments. Exposure of C. jejuni to eugenol, chitosan, or combination significantly modulated select genes encoding for motility, quorum sensing, and stress response. Results demonstrate the potential of pectin or chitosan coating fortified with eugenol as a postharvest intervention against C. jejuni contamination on poultry products.

Dietary supplementation with ferric tyrosine improves zootechnical performance and reduces caecal Campylobacter spp. load in broilers.

1. The objective of this study was to evaluate the effect of ferric tyrosine on the reduction of Campylobacter spp. and zootechnical performance in broilers exposed to Campylobacter spp. using a natural challenge model to simulate commercial conditions. Additionally, the minimum inhibitory concentrations (MICs) of ferric tyrosine against common enteropathogens were evaluated. 2. At the start of the trial, 840 healthy male 1-d-old birds (Ross 308) were randomly allocated to 6 replicate pens of 35 birds each and fed diets containing different concentrations of ferric tyrosine (0, 0.02, 0.05 and 0.2 g/kg) in mash form for 42 d. 3. Broilers fed diets containing ferric tyrosine showed significantly higher body weight at d 42 and weight gain compared to the control group. However, birds fed ferric tyrosine ate significantly more than the control birds so significant improvements in feed conversion rate were not observed. 4. Microbiological analyses of caecal samples collected on d 42 of the study showed, per gram of sample, 2-3 log10 reduction in Campylobacter spp. and 1 log10 reduction in Escherichia coli in the groups fed diets containing ferric tyrosine compared to the control. 5. The MICs of ferric tyrosine was >400 mg/l for C. jejuni and >200 mg/l for E. coli and Salmonella enterica, indicating that ferric tyrosine did not exert antimicrobial activity. 6. The results showed that birds fed ferric tyrosine grew faster and consumed more feed compared to the control group, indicating potential benefits of faster time to reach slaughter weight with no significant reduction on feed efficiency. Moreover, ferric tyrosine significantly reduced caecal Campylobacter spp. and E. coli indicating potential as a non-antibiotic feed additive to lower the risk of infections transmitted through the food chain.

Investigating the Campylobacter jejuni Transcriptional Response to Host Intestinal Extracts Reveals the Involvement of a Widely Conserved Iron Uptake System.

Campylobacter jejuni is a pathogenic bacterium that causes gastroenteritis in humans yet is a widespread commensal in wild and domestic animals, particularly poultry. Using RNA sequencing, we assessed C. jejuni transcriptional responses to medium supplemented with human fecal versus chicken cecal extracts and in extract-supplemented medium versus medium alone. C. jejuni exposed to extracts had altered expression of 40 genes related to iron uptake, metabolism, chemotaxis, energy production, and osmotic stress response. In human fecal versus chicken cecal extracts, C. jejuni displayed higher expression of genes involved in respiration (fdhTU) and in known or putative iron uptake systems (cfbpA, ceuB, chuC, and CJJ81176_1649-1655 [here designated 1649-1655]). The 1649-1655 genes and downstream overlapping gene 1656 were investigated further. Uncharacterized homologues of this system were identified in 33 diverse bacterial species representing 6 different phyla, 21 of which are associated with human disease. The 1649 and 1650 (p19) genes encode an iron transporter and a periplasmic iron binding protein, respectively; however, the role of the downstream 1651-1656 genes was unknown. A Δ1651-1656 deletion strain had an iron-sensitive phenotype, consistent with a previously characterized Δp19 mutant, and showed reduced growth in acidic medium, increased sensitivity to streptomycin, and higher resistance to H2O2 stress. In iron-restricted medium, the 1651-1656 and p19 genes were required for optimal growth when using human fecal extracts as an iron source. Collectively, this implicates a function for the 1649-1656 gene cluster in C. jejuni iron scavenging and stress survival in the human intestinal environment.IMPORTANCE Direct comparative studies of C. jejuni infection of a zoonotic commensal host and a disease-susceptible host are crucial to understanding the causes of infection outcome in humans. These studies are hampered by the lack of a disease-susceptible animal model reliably displaying a similar pathology to human campylobacteriosis. In this work, we compared the phenotypic and transcriptional responses of C. jejuni to intestinal compositions of humans (disease-susceptible host) and chickens (zoonotic host) by using human fecal and chicken cecal extracts. The mammalian gut is a complex and dynamic system containing thousands of metabolites that contribute to host health and modulate pathogen activity. We identified C. jejuni genes more highly expressed during exposure to human fecal extracts in comparison to chicken cecal extracts and differentially expressed in extracts compared with medium alone, and targeted one specific iron uptake system for further molecular, genetic, and phenotypic study.

Influence of a specific amino acid pattern in the diet on the course of an experimental Campylobacter jejuni infection in broilers.

Campylobacter jejuni (C. jejuni) is one of the most important zoonotic pathogens worldwide. In Europe, the majority of the cases are caused by consuming contaminated poultry meat. The objective of the present study was to investigate potential effects of different crude protein levels in complete diets for broilers on infection dynamics of C. jejuni after experimental infection. In total, 300 commercial broilers line Ross 308 were divided into 4 different groups, including 5 replications of 15 chickens each. The chickens were fed a conventional diet (212 g CP/kg DM) and a protein-reduced test diet (190 g CP/kg DM) supplemented with essential amino acids. This resulted simultaneously in lower amino-acid concentrations preferentially utilized by C. jejuni, such as aspartate, glutamate, proline, and serine. One group of each feeding concept was infected artificially with C. jejuni at day 21 by applying an oral C. jejuni inoculum containing 4.17 ± 0.09 log10 cfu of C. jejuni to 3 of 15 chickens, called "seeders." Feeding the test diet resulted in a significant reduction (P < 0.001) in CP intake (31.5 ± 1.20 g CP/broiler/day and 27.7 ± 0.71 g CP/broiler/day, respectively), a significant decrease (P < 0.05) in crude mucin in excreta (55.7 ± 8.23 g/kg DM and 51.9 ± 7.62 g/kg DM, respectively), and in goblet cell number in cecal crypts (P < 0.05; 15.1 ± 5.71 vs. 13.6 ± 5.91 goblet cells/crypt). In groups receiving the test diet, the excretion of C. jejuni was significantly reduced in seeders by 1.9 log10 cfu/g excreta at day 23 (3.38a ± 2.55 vs. 1.47b ± 2.20; P = 0.033). At day 25, prevalence of C. jejuni in cloacal swabs amounted to 53.3% in the group fed the test diet and 75.7% in the control group, respectively (P < 0.05). In summary, a definite amino acid pattern in the broiler diets could contribute to a development of an effective feeding strategy to reduce the prevalence of C. jejuni infection in chickens (Patent No 17187659.2-1106).

Therapeutic administration of enrofloxacin in mice does not select for fluoroquinolone resistance in Campylobacter jejuni.

Enrofloxacin is registered for therapeutic use in beef cattle to treat bovine respiratory disease in Canada. A murine model was used to experimentally examine the impact of therapeutic administration of enrofloxacin on fluoroquinolone resistance development in Campylobacter jejuni. Administration of enrofloxacin to mice via subcutaneous injection or per os routes resulted in equivalent levels of bioactive enrofloxacin within the intestine, but bioactivity was short-lived (<48 h after cessation). Enrofloxacin administration did not affect densities of total bacteria, Firmicutes, or Bacteroidetes in digesta and had modest impacts on densities of Enterobacteriaceae. All mice inoculated with C. jejuni NCTC 11168 became persistently colonized by the bacterium. Enrofloxacin reduced C. jejuni cell densities within the cecal and colonic digesta for all treatments, and densities shed in feces as a function of antibiotic duration. None of the C. jejuni isolates recovered from mice after administration of enrofloxacin (n = 260) developed resistance to ciprofloxacin regardless of method or duration of administration. Furthermore, only modest shifts in the minimum inhibitory concentration of the isolates by treatment were noted. The study findings indicate that the risk posed by short-term subcutaneous administration of enrofloxacin for the development of fluoroquinolone resistance in mammals is low.

Pharmacokinetic and antimicrobial activity of a new carvacrol-based product against a human pathogen, Campylobacter jejuni.

AIM: In vitro and in vivo studies were conducted to test a new carvacrol-based product designed to delay the carvacrol release so that it could reach the caeca of broiler chickens in order to control Campylobacter jejuni. METHODS AND RESULTS: Antimicrobial activity of carvacrol, a constituent of oregano and thyme essential oil, has been demonstrated against C. jejuni in vitro, and this compound was found beneficial for broiler growth. Here, we tested a new liquid formulation that did not change the antibacterial efficacy of carvacrol against C. jejuni in vitro, as assessed by broth microdilution. The mode of action of carvacrol also remained unchanged as illustrated by electronic microscopy. A pharmacokinetic assay monitored carvacrol of the solid galenic formulation in the avian digestive tract and this showed that this compound was mainly found in the last part (caeca, large intestine) and in the droppings. Extremely low concentrations of free carvacrol were present in blood plasma, with larger amounts of carvacrol metabolites: carvacrol glucuronide and sulphate. A qPCR analysis showed that the solid galenic form of carvacrol added at 5 kg per tonne of food (i.e. 9·5 mg of carvacrol per kg of bodyweight per day) significantly decreased the C. jejuni caecal load by 1·5 log. CONCLUSIONS: The new liquid formulation was as effective as unformulated carvacrol in vitro. In vivo the solid galenic form seems to delay the carvacrol release into the caeca and presented interesting results on C. jejuni load after 35 days. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggested that this product could be promising to control Campylobacter contamination of broilers.

TYPLEX® Chelate, a novel feed additive, inhibits Campylobacter jejuni biofilm formation and cecal colonization in broiler chickens.

Reducing Campylobacter spp. carriage in poultry is challenging, but essential to control this major cause of human bacterial gastroenteritis worldwide. Although much is known about the mechanisms and route of Campylobacter spp. colonization in poultry, the literature is scarce on antibiotic-free solutions to combat Campylobacter spp. colonization in poultry. In vitro and in vivo studies were conducted to investigate the role of TYPLEX® Chelate (ferric tyrosine), a novel feed additive, in inhibiting Campylobacter jejuni (C. jejuni) biofilm formation and reducing C. jejuni and Escherichia coli (E. coli) colonization in broiler chickens at market age. In an in vitro study, the inhibitory effect on C. jejuni biofilm formation using a plastic bead assay was investigated. The results demonstrated that TYPLEX® Chelate significantly reduces biofilm formation. In an in vivo study, 800 broilers (one d old) were randomly allocated to 4 dietary treatments in a randomized block design, each having 10 replicate pens with 20 birds per pen. At d 21, all birds were challenged with C. jejuni via seeded litter. At d 42, cecal samples were collected and tested for volatile fatty acid (VFA) concentrations and C. jejuni and E. coli counts. The results showed that TYPLEX® Chelate reduced the carriage of C. jejuni and E. coli in poultry by 2 and 1 log10 per gram cecal sample, respectively, and increased cecal VFA concentrations. These findings support TYPLEX® Chelate as a novel non-antibiotic feed additive that may help produce poultry with a lower public health risk of Campylobacteriosis.

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.

Microbiota-Derived Short-Chain Fatty Acids Modulate Expression of Campylobacter jejuni Determinants Required for Commensalism and Virulence.

Campylobacter jejuni promotes commensalism in the intestinal tracts of avian hosts and diarrheal disease in humans, yet components of intestinal environments recognized as spatial cues specific for different intestinal regions by the bacterium to initiate interactions in either host are mostly unknown. By analyzing a C. jejuni acetogenesis mutant defective in converting acetyl coenzyme A (Ac-CoA) to acetate and commensal colonization of young chicks, we discovered evidence for in vivo microbiota-derived short-chain fatty acids (SCFAs) and organic acids as cues recognized by C. jejuni that modulate expression of determinants required for commensalism. We identified a set of C. jejuni genes encoding catabolic enzymes and transport systems for amino acids required for in vivo growth whose expression was modulated by SCFAs. Transcription of these genes was reduced in the acetogenesis mutant but was restored upon supplementation with physiological concentrations of the SCFAs acetate and butyrate present in the lower intestinal tracts of avian and human hosts. Conversely, the organic acid lactate, which is abundant in the upper intestinal tract where C. jejuni colonizes less efficiently, reduced expression of these genes. We propose that microbiota-generated SCFAs and lactate are cues for C. jejuni to discriminate between different intestinal regions. Spatial gradients of these metabolites likely allow C. jejuni to locate preferred niches in the lower intestinal tract and induce expression of factors required for intestinal growth and commensal colonization. Our findings provide insights into the types of cues C. jejuni monitors in the avian host for commensalism and likely in humans to promote diarrheal disease.IMPORTANCECampylobacter jejuni is a commensal of the intestinal tracts of avian species and other animals and a leading cause of diarrheal disease in humans. The types of cues sensed by C. jejuni to influence responses to promote commensalism or infection are largely lacking. By analyzing a C. jejuni acetogenesis mutant, we discovered a set of genes whose expression is modulated by lactate and short-chain fatty acids produced by the microbiota in the intestinal tract. These genes include those encoding catabolic enzymes and transport systems for amino acids that are required by C. jejuni for in vivo growth and intestinal colonization. We propose that gradients of these microbiota-generated metabolites are cues for spatial discrimination between areas of the intestines so that the bacterium can locate niches in the lower intestinal tract for optimal growth for commensalism in avian species and possibly infection of human hosts leading to diarrheal disease.

The In Vitro and In Vivo Effect of Carvacrol in Preventing Campylobacter Infection, Colonization and in Improving Productivity of Chicken Broilers.

The current trend in reducing the antibiotic usage in animal production imposes urgency in the identification of novel biocides. The essential oil carvacrol, for example, changes the morphology of the cell and acts against a variety of targets within the bacterial membranes and cytoplasm, and our in vitro results show that it reduces adhesion and invasion of chicken intestinal primary cells and also biofilm formation. A trial was conducted to evaluate the effects of dietary supplementation of carvacrol at four concentrations (0, 120, 200, and 300 mg/kg of diet) on the performance of Lactobacillus spp., Escherichia coli, Campylobacter spp., and broilers. Each of the four diets was fed to three replicates/trial of 50 chicks each from day 0 to 35. Our results show that carvacrol linearly decreased feed intake, feed conversion rates and increased body weight at all levels of supplementation. Plate count analysis showed that Campylobacter spp. was only detected at 35 days in the treatment groups compared with the control group where the colonization occurred at 21 days. The absence of Campylobacter spp. at 21 days in the treatment groups was associated with a significant increase in the relative abundance of Lactobacillus spp. Also, carvacrol was demonstrated to have a significant effect on E. coli numbers in the cecum of the treatment groups, at all supplementation levels. In conclusion, this study shows for the first time that at different concentrations, carvacrol can delay Campylobacter spp., colonization of chicken broilers, by inducing changes in gut microflora, and it demonstrates promise as an alternative to the use of antibiotics.

Antimicrobial Use for and Resistance of Zoonotic Bacteria Recovered from Nonhuman Primates.

As a growing threat to human and animal health, antimicrobial resistance (AMR) has become a central public-health topic. Largescale surveillance systems, such as the National Antimicrobial Resistance Monitoring System (NARMS), are now established to monitor and provide guidance regarding AMR, but comprehensive literature on AMR among NHP is sparse. This study provides data regarding current antimicrobial use strategies and the prevalence of AMR in zoonotic bacteria recovered from NHP within biomedical research institutions. We focused on 4 enteric bacteria: Shigella flexneri, Yersinia enterocolitica, Y. pseudotuberculosis, and Campylobacter jejuni. Fifteen veterinarians, 7 biomedical research institutions, and 4 diagnostic laboratories participated, providing susceptibility test results from January 2012 through April 2015. Veterinarians primarily treated cases caused by S. flexneri, Y. enterocolitica, and Y. pseudotuberculosis with enrofloxacin but treated C. jejuni cases with azithromycin and tylosin. All isolates were susceptible to the associated primary antimicrobial but often showed resistance to others. Specifically, S. flexneri isolates frequently were resistant to erythromycin (87.5%), doxycycline (73.7%), and tetracycline (38.3%); Y. enterocolitica isolates to ampicillin (100%) and cefazolin (93.6%); and C. jejuni isolates to methicillin (99.5%) and cephalothin (97.5%). None of the 58 Y. pseudotuber-culosis isolates was resistant to any tested antimicrobial. Notably, resistance patterns were not shared between this study's NHP isolates and human isolates presented by NARMS. Our findings indicate that zoonotic bacteria from NHP diagnostic samples are broadly susceptible to the antimicrobials used to treat the clinical infections. These results can help veterinarians ensure effective antimicrobial therapy and protect staff by minimizing occupational risk.