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.

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.

Reducing Campylobacter jejuni colonization in broiler chickens by in-feed supplementation with hyperimmune egg yolk antibodies.

Campylobacter infections sourced mainly to poultry products, are the most important bacterial foodborne zoonoses worldwide. No effective measures to control these infections in broiler production exist to date. Here, we used passive immunization with hyperimmune egg yolks to confer broad protection of broilers against Campylobacter infection. Two novel vaccines, a bacterin of thirteen Campylobacter jejuni (C. jejuni) and C. coli strains and a subunit vaccine of six immunodominant Campylobacter antigens, were used for the immunization of layers, resulting in high and prolonged levels of specific immunoglobulin Y (IgY) in the hens' yolks. In the first in vivo trial, yolks (sham, bacterin or subunit vaccine derived) were administered prophylactically in the broiler feed. Both the bacterin- and subunit vaccine-induced IgY significantly reduced the number of Campylobacter-colonized broilers. In the second in vivo trial, the yolks were administered therapeutically during three days before euthanasia. The bacterin IgY resulted in a significant decrease in C. jejuni counts per infected bird. The hyperimmune yolks showed strong reactivity to a broad representation of C. jejuni and C. coli clonal complexes. These results indicate that passive immunization with hyperimmune yolks, especially bacterin derived, offers possibilities to control Campylobacter colonization in poultry.

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.

Antimicrobial, Rheological, and Thermal Properties of Plasticized Polylactide Films Incorporated with Essential Oils to Inhibit Staphylococcus aureus and Campylobacter jejuni.

Polylactide (PLA) is the most mature biobased and biodegradable polymer. Due to its inherent brittleness, the polymer cannot be used as a packaging material without plasticizer. An attempt was made to develop antimicrobial plasticized PLA film by incorporating polyethylene glycol (PEG) and 3 essential oils (EO), namely cinnamon, garlic, and clove by solvent casting method. Physical, thermal, and rheological properties of those films were evaluated for practical applications whereas the antimicrobial properties were tested against Staphylococcus aureus and Campylobacter jejuni-pathogens related to poultry industry. Both PEG and EOs led to the formation of flexible PLA/PEG/EO films with significant drop in the glass transition temperature (Tg ), and mechanical property. Time-temperature superposition (TTS) principle was employed to melt rheology of EO-based films at selected temperature, and rheological moduli superimposed well in an extended frequency range. Among EOs, cinnamon and clove oil-based films (PLA/PEG/CIN and PLA/PEG/CLO) exhibited a complete zone of inhibition against C. jejuni at the maximum concentration (1.6 mL per 2 g PLA/PEG blend) whereas the garlic oil-based film (PLA/PEG/GAR) had the lowest activity.

The antimicrobial effect of spice-based marinades against Campylobacter jejuni on contaminated fresh broiler wings.

The antimicrobial effect of spice-based marinades against Campylobacter jejuni on inoculated fresh broiler wings was investigated. Experiments were carried out with 1 strain of C. jejuni and 6 marinades. Four experimental marinades were composed for the study and contained spices (thyme, rosemary, basil, marjoram, and so on) and different combination of bioactive compounds. Two marinades were commercial and contained spices (black pepper, sweet red pepper, and so on) and chemical additives (monosodium glutamate, sodium diacetate, calcium lactate), 1 commercial marinade was also enriched with bioactive compounds (linalool, cinnamaldehyde, lactic acid). Total aerobic bacterial count was examined to estimate the possible effect of tested marinades on the shelf-life of marinated broiler wings. Study revealed that thyme-based marinade was the most effective against C. jejuni on broiler wings and reduced the numbers of campylobacters by 1.04 log colony forming unit (CFU)/g (P ≤ 0.05) during storage for 168 h at 4 °C temperature. Moreover, it was more effective against C. jejuni than commercial marinade with 0.47 log CFU/g (P ≤ 0.05) reduction effect. Both experimental and commercial marinades had very similar effect on the total aerobic bacterial count. Although experimental and commercial marinades had different effect on pH of broiler wings, this parameter did not show a major impact on the antimicrobial effect of tested marinades (P ≥ 0.05). Our study shows that experimental natural thyme-based marinade can reduce numbers of C. jejuni more effectively than tested commercial marinades.

Reduction of microbiological risk in minced meat by a combination of natural antimicrobials.

BACKGROUND: Responsibility for food safety must be taken through the entire food-production chain, to avoid consumer cross-contamination. The antimicrobial activities of an Alpinia katsumadai seed extract and epigallocatechin gallate (EGCG), and their combination, were evaluated against individual food-borne pathogenic strains of Listeria monocytogenes, Escherichia coli and Campylobacter jejuni, individually and as a cocktail, in chicken-meat juice and sterile minced meat as food models, and in minced meat with the naturally present microflora, as an actual food sample. RESULTS: The antimicrobial combination of the A. katsumadai extract and EGCG was the most efficient for C. jejuni growth inhibition, followed by inhibition of L. monocytogenes, which was reduced more efficiently in the bacterial cocktail than as an individual strain. The antimicrobial combination added to minced meat at refrigeration temperatures used in the food chain (8 °C) revealed inhibition of these pathogens and inhibition of the naturally present bacteria after 5 days. CONCLUSIONS: The antibacterial efficiencies of the tested combinations are influenced by storage temperature. Food safety can be improved by using the appropriate combination of natural antimicrobials to reduce the microbiological risk of minced meat.

Is allicin able to reduce Campylobacter jejuni colonization in broilers when added to drinking water?

Reducing Campylobacter shedding on the farm could result in a reduction of the number of human campylobacteriosis cases. In this study, we first investigated if allicin, allyl disulfide, and garlic oil extract were able to either prevent C. jejuni growth or kill C. jejuni in vitro. Allyl disulfide and garlic oil extract reduced C. jejuni numbers in vitro below a detectable level at a concentration of 50 mg/kg (no lower concentrations were tested), whereas allicin reduced C. jejuni numbers below a detectable level at a concentration as low as 7.5 mg/kg. In further experiments we screened for the anti-C. jejuni activity of allicin in a fermentation system closely mimicking the broiler cecal environment using cecal microbiota and mucus isolated from C. jejuni-free broilers. During these fermentation experiments, allicin reduced C. jejuni numbers below a detectable level after 24 h at a concentration of 50 mg/kg. In contrast, 25 mg/kg of allicin killed C. jejuni in the first 28 h of incubation, but anti-C. jejuni activity was lost after 48 h of incubation, probably due to the presence of mucin in the growth medium. This had been confirmed in fermentation experiments in the presence of broiler cecal mucus. Based on these results, we performed an in vivo experiment to assess the prevention or reduction of cecal C. jejuni colonization in broiler chickens when allicin was added to drinking water. We demonstrated that allicin in drinking water did not have a statistically significant effect on cecal C. jejuni colonization in broilers. It was assumed, based on in vitro experiments, that the activity of allicin was thwarted by the presence of mucin-containing mucus. Despite promising in vitro results, allicin was not capable of statistically influencing C. jejuni colonization in a broiler flock, although a trend toward lower cecal C. jejuni numbers in allicin-treated broilers was observed.

Antibacterial and cytotoxic activity of isoprenylated coumarin mammea A/AA isolated from Mammea africana.

ETHNOPHARMACOLOGICAL RELEVANCE: The stem bark of Mammea africana is widely distributed in tropical Africa and commonly used in traditional medicine. This study aims to identify the active compound in Mammea africana and to evaluate its antimicrobial and antiproliferative activity. MATERIALS AND METHODS: Methanol extract from the bark of the Mammea africana was separated by liquid-liquid extraction, followed by open column chromatography. A principal antimicrobial compound was purified by high performance liquid chromatography (HPLC) and its structure was elucidated by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). The antibacterial activity of the purified compound was determined using the broth microdilution method against 7 common pathogenic bacteria. The compound was also evaluated for cytotoxicity by cell proliferation assay (MTS) using the mouse embryonic fibroblast cell line NIH 3T3 and the non-small cell lung cancer cell line A549. RESULTS: The purified active compound was determined to be mammea A/AA and was found to be highly active against Campylobacter jejuni (MIC=0.5 µg/ml), Streptococcus pneumoniae (MIC=0.25 µg/ml), and Clostridium difficile (MIC=0.25 µg/ml). The compound exhibited significant antiproliferative activities against both NIH 3T3 and A549 cell lines. CONCLUSION: Mammea A/AA isolated from Mammea africana exerts specific inhibitory activity against Campylobacter jejuni, Streptococcus pneumoniae, and Campylobacter difficile. Mammea A/AA was also found to exhibit significant cytotoxicity against both cancer and normal cell lines.

Bactericidal effect of hydrolysable and condensed tannin extracts on Campylobacter jejuni in vitro.

Strategies are sought to reduce intestinal colonisation of food-producing animals by Campylobacter jejuni, a leading bacterial cause of human foodborne illness worldwide. Presently, we tested the antimicrobial activity of hydrolysable-rich blackberry, cranberry and chestnut tannin extracts and condensed tannin-rich mimosa, quebracho and sorghum tannins (each at 100 mg/mL) against C. jejuni via disc diffusion assay in the presence of supplemental casamino acids. We found that when compared to non-tannin-treated controls, all tested tannins inhibited the growth of C. jejuni and that inhibition by the condensed tannin-rich mimosa and quebracho extracts was mitigated in nutrient-limited medium supplemented with casamino acids. When tested in broth culture, both chestnut and mimosa extracts inhibited growth of C. jejuni and this inhibition was much greater in nutrient-limited than in full-strength medium. Consistent with observations from the disc diffusion assay, the inhibitory activity of the condensed tannin-rich mimosa extracts but not the hydrolysable tannin-rich chestnut extracts was mitigated by casamino acid supplementation to the nutrient-limited medium, likely because the added amino acids saturated the binding potential of the condensed tannins. These results demonstrate the antimicrobial activity of various hydrolysable and condensed tannin-rich extracts against C. jejuni and reveal that condensed tannins may be less efficient than hydrolysable tannins in controlling C. jejuni in gut environments containing high concentrations of amino acids and soluble proteins.

Anti-Campylobacter activities and resistance mechanisms of natural phenolic compounds in Campylobacter.

BACKGROUND: Campylobacter is a major foodborne pathogen and alternative antimicrobials are needed to prevent or decrease Campylobacter contamination in foods or food producing animals. The objectives of this study are to define the anti-Campylobacter activities of natural phenolic compounds of plant origin and to determine the roles of bacterial drug efflux systems in the resistance to these natural phenolics in Campylobacter jejuni. METHODOLOGY/PRINCIPAL FINDINGS: Anti-Campylobacter activities were evaluated by an MIC assay using microdilution coupled with ATP measurement. Mutants of the cmeB and cmeF efflux genes and the cmeR transcriptional repressor gene were compared with the wild-type strain for their susceptibilities to phenolics in the absence and presence of efflux-pump inhibitors (EPIs). The phenolic compounds produced significant, but variable activities against both antibiotic-susceptible and antibiotic resistant Campylobacter. The highest anti-Campylobacter activity was seen with carnosic and rosmarinic acids in their pure forms or in enriched plant extracts. Inactivation of cmeB rendered C. jejuni significantly more susceptible to the phenolic compounds, while mutation of cmeF or cmeR only produced a moderate effect on the MICs. Consistent with the results from the efflux pump mutants, EPIs, especially phenylalanine-arginine ß-naphthylamide and NMP, significantly reduced the MICs of the tested phenolic compounds. Further reduction of MICs by the EPIs was also observed in the cmeB and cmeF mutants, suggesting that other efflux systems are also involved in Campylobacter resistance to phenolic compounds. CONCLUSION/SIGNIFICANCE: Natural phenolic compounds of plant origin have good anti-Campylobacter activities and can be further developed for potential use in controlling Campylobacter. The drug efflux systems in Campylobacter contribute significantly to its resistance to the phenolics and EPIs potentiate the anti-Campylobacter activities of plant phenolic compounds.

Investigating antibacterial effects of garlic (Allium sativum) concentrate and garlic-derived organosulfur compounds on Campylobacter jejuni by using Fourier transform infrared spectroscopy, Raman spectroscopy, and electron microscopy.

Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy were used to study the cell injury and inactivation of Campylobacter jejuni from exposure to antioxidants from garlic. C. jejuni was treated with various concentrations of garlic concentrate and garlic-derived organosulfur compounds in growth media and saline at 4, 22, and 35°C. The antimicrobial activities of the diallyl sulfides increased with the number of sulfur atoms (diallyl sulfide < diallyl disulfide < diallyl trisulfide). FT-IR spectroscopy confirmed that organosulfur compounds are responsible for the substantial antimicrobial activity of garlic, much greater than those of garlic phenolic compounds, as indicated by changes in the spectral features of proteins, lipids, and polysaccharides in the bacterial cell membranes. Confocal Raman microscopy (532-nm-gold-particle substrate) and Raman mapping of a single bacterium confirmed the intracellular uptake of sulfur and phenolic components. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were employed to verify cell damage. Principal-component analysis (PCA), discriminant function analysis (DFA), and soft independent modeling of class analogs (SIMCA) were performed, and results were cross validated to differentiate bacteria based upon the degree of cell injury. Partial least-squares regression (PLSR) was employed to quantify and predict actual numbers of healthy and injured bacterial cells remaining following treatment. PLSR-based loading plots were investigated to further verify the changes in the cell membrane of C. jejuni treated with organosulfur compounds. We demonstrated that bacterial injury and inactivation could be accurately investigated by complementary infrared and Raman spectroscopies using a chemical-based, "whole-organism fingerprint" with the aid of chemometrics and electron microscopy.

Antimicrobial edible apple films inactivate antibiotic resistant and susceptible Campylobacter jejuni strains on chicken breast.

UNLABELLED: Campylobacter jejuni is the leading cause of bacterial diarrheal illness worldwide. Many strains are now becoming multidrug resistant. Apple-based edible films containing carvacrol and cinnamaldehyde were evaluated for bactericidal activity against antibiotic resistant and susceptible C. jejuni strains on chicken. Retail chicken breast samples inoculated with D28a and H2a (resistant strains) and A24a (a sensitive strain) were wrapped in apple films containing cinnamaldehyde or carvacrol at 0.5%, 1.5%, and 3% concentrations, and then incubated at 4 or 23 °C for 72 h. Immediately after wrapping and at 72 h, samples were plated for enumeration of viable C. jejuni. The antimicrobial films exhibited dose- and temperature-dependent bactericidal activity against all strains. Films with ≥1.5% cinnamaldehyde reduced populations of all strains to below detection at 23 °C at 72 h. At 4 °C with cinnamaldehyde, reductions were variable for all strains, ranging from 0.2 to 2.5 logs and 1.8 to 6.0 logs at 1.5% and 3.0%, respectively. Films with 3% carvacrol reduced populations of A24a and H2a to below detection, and D28a by 2.4 logs at 23 °C and 72 h. A 0.5-log reduction was observed for both A24a and D28a, and 0.9 logs for H2a at 4 °C at 3% carvacrol. Reductions ranged from 1.1 to 1.9 logs and 0.4 to 1.2 logs with 1.5% and 0.5% carvacrol at 23 °C, respectively. The films with cinnamaldehyde were more effective than carvacrol films. Reductions at 23 °C were greater than those at 4 °C. Our results showed that antimicrobial apple films have the potential to reduce C. jejuni on chicken and therefore, the risk of campylobacteriosis. Possible mechanisms of antimicrobial effects are discussed. PRACTICAL APPLICATION: Apple antimicrobial films could potentially be used in retail food packaging to reduce C. jejuni commonly present on food.

Extracts of edible and medicinal plants in inhibition of growth, adherence, and cytotoxin production of Campylobacter jejuni and Campylobacter coli.

UNLABELLED: Campylobacter spp. is recognized as one of the most common cause of food-borne bacterial gastroenteritis in humans. Campylobacter infection causes campylobacteriosis, which can range from asymptomatic to dysentery-type illnesses with severe complications, such as Guillian-Barre syndrome. Epidemiological studies have revealed that consumption of poultry products is an important risk factor of this disease. Adherence and cytotoxic activity of the bacteria to host mucosal surfaces have been proposed to be critical steps in pathogenesis. Innovative tools for controlling Campylobacter, such as natural products from plants, represent good alternatives for use in foods or as therapeutic agents. In this study, 28 edible or medicinal plants species were analyzed for their bactericidal effects on the growth of Campylobacter jejuni and C. coli. The extracts of Acacia farnesiana, Artemisia ludoviciana, Opuntia ficus-indica, and Cynara scolymus were the most effective against these microorganisms at minimal bactericidal concentrations (MBCs) of 0.3, 0.5, 0.4, and 2.0 mg/mL, respectively. No effect on growth was detected with lower concentrations of extract (25%, 50%, or 75% of the MBC) added to the media. The effect of each extract (75% of the MBC) on adherence and cytotoxicity of C. jejuni and C. coli was evaluated in Vero cells. Adherence of Campylobacter to Vero cells was significantly affected by all the extracts. Cytotoxic activity of bacterial cultures was inhibited by A. farnesiana and A. ludoviciana. These plant extracts are potential candidates to be studied for controlling Campylobacter contamination in foods and the diseases associated with this microorganism. PRACTICAL APPLICATION: Innovative tools for controlling Campylobacter, such as natural products from plants, represent good alternatives for use in foods or as therapeutic agents. The extracts of Acacia farnesiana, Artemisia ludoviciana, Opuntia ficus-indica, and Cynara scolymus were the most effective against these microorganisms. Adherence and cytotoxic activity of the bacteria to host mucosal surfaces which are critical steps in pathogenesis were decreased by these extracts. Our results point to these plants as potential candidates for the control of Campylobacter contamination in foods, the treatment of the diseases associated with this microorganism, and as feed supplements to reduce on-farm prevalence of Campylobacter.

Prevention of intestinal Campylobacter jejuni colonization in broilers by combinations of in-feed organic acids.

AIM: We have tested the effect of various combinations of formic acid and sorbate on Campylobacter jejuni colonization in broiler chickens to reduce the colonization of this zoonotic pathogen in broiler chicken flocks. METHODS AND RESULTS: Chickens were offered feed supplemented with different concentrations and combinations of formic acid and/or potassium sorbate. We found little or no effect on the Camp. jejuni colonization levels in chickens that were given feed supplemented with formic acid alone. A combination of 1.5% formic acid and 0.1% sorbate reduced the colonization of Camp. jejuni significantly, while a concentration of 2.0% formic acid in combination with 0.1% sorbate prevented Camp. jejuni colonization in chickens. This inhibition was replicated in two independent trials with a combination of three different Camp. jejuni strains. CONCLUSIONS: Our results show a novel and promising intervention strategy to reduce the incidence of Camp. jejuni in poultry products and to obtain safer food. SIGNIFICANCE AND IMPACT OF THE STUDY: To ensure food safety, a reduction of the carcass contamination with Camp. jejuni through reduced colonization of this pathogen in broiler chicken flocks is important. A range of organic acids as additives in feed and drinking water have already been evaluated for this purpose. However, no studies have yet shown a complete inhibition of Camp. jejuni colonization in broiler chickens.

Selecting for development of fluoroquinolone resistance in a Campylobacter jejuni strain 81116 in chickens using various enrofloxacin treatment protocols.

AIMS: To determine the effect of various enrofloxacin dose regimes on the colonization and selection of resistance in Campylobacter jejuni strain 81116P in experimentally colonized chickens. METHODS AND RESULTS: Two experiments were undertaken, in which 14-day-old chickens were colonized with 1 × 10(7) -1 × 10(9 ) CFU g(-1) Camp. jejuni strain 81116P and then treated with enrofloxacin at 12-500 ppm in drinking water for various times. Caecal colonization levels were determined at various time-points after start-of-treatment, and the susceptibility of recovered isolates to ciprofloxacin was monitored. Resistance was indicated by growth on agar containing 4 µg ml(-1) ciprofloxacin, MICs of 16 µg ml(-1) and the Thr86Ile mutation in gyrA. Enrofloxacin at doses of 12-250 ppm reduced Camp. jejuni colonization over the first 48-72 h after start-of-treatment. The degree of reduction in colonization was dose, but not treatment time, dependent. In all cases, maximal colonization was re-established within 4-6 days. Fluoroquinolone-resistant organisms were recoverable within 48 h of start-of-treatment; after a further 24 h all recovered isolates were resistant. In contrast, a dose of 500 ppm enrofloxacin reduced colonization to undetectable levels within 48 h, and the treated birds remained Campylobacter negative throughout the remaining experimental period. By high pressure liquid chromatography, for all doses, the maximum concentrations of enrofloxacin and ciprofloxacin in the caecal contents were detected at the point of treatment completion. Thereafter, levels declined to undetectable by 7 days post-treatment withdrawal. CONCLUSIONS: In a model using chickens maximally colonized with Camp. jejuni 81116P, treatment with enrofloxacin, at doses of 12-250 ppm in drinking water, enables the selection, and clonal expansion, of fluoroquinolone-resistant organisms. However, this is preventable by treatment with 500 ppm of enrofloxacin. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of chickens with enrofloxacin selects for resistance in Camp. jejuni in highly pre-colonized birds. However, a dose of 500 ppm enrofloxacin prevented the selection of resistant campylobacters.

Reduction of Campylobacter jejuni and Campylobacter coli in poultry skin by fruit extracts.

Campylobacter spp. are a major cause of foodborne bacterial gastroenteritis in humans, and current methods to control Campylobacter contamination in foods are not completely successful. Plants are a promising source of antimicrobial agents, particularly given the growing interest in "all natural" foods. In this study, the antimicrobial activity of extracts from 28 edible plants against Campylobacter jejuni and Campylobacter coli was evaluated in vitro and in a poultry skin model. Nine of 28 extracts exhibited antimicrobial activity in a diffusion assay, and MBCs were determined for the three most active extracts, i.e., lime, plum, and sour orange peel (MBCs of 2 to 3 mg/ml). Mixtures of the lime, plum, and sour orange peel extracts were applied to chicken skin inoculated with 10(5) CFU of Campylobacter to test for synergistic or antagonist effects. After incubation (48 h at 4 degrees C) with any extract mixture, no Campylobacter CFUs were detectable. A panel of tasters determined that the mixture of lime and plum gave the best flavor to chicken wings. These active extracts from edible fruits are simple to prepare and are alternatives to reduce or eliminate Campylobacter contamination of chicken products.

Potential of coriander (Coriandrum sativum) oil as a natural antimicrobial compound in controlling Campylobacter jejuni in raw meat.

Twelve essential oils were tested in vitro for antimicrobial activities against several strains of Campylobacter jejuni, a pathogen causing food-borne diseases worldwide. Using disk diffusion and minimal inhibitory concentration determination assays, we noted that coriander oil exhibited the strongest antimicrobial activity against all tested strains. The oil had a bactericidal effect on the target bacteria. In evaluating the antimicrobial potency of coriander oil against C. jejuni on beef and chicken meat at 4 degrees C and 32 degrees C, it was found that the oil reduced the bacterial cell load in a dose-dependent manner. The type of meat and temperature did not influence the antimicrobial activity of the oil. This study indicates the potential of coriander oil to serve as a natural antimicrobial compound against C. jejuni in food.

Investigations into the antibacterial activities of phytotherapeutics against Helicobacter pylori and Campylobacter jejuni.

The prevalence of gastric diseases is increasing with H. pylori, the causative agent of acute and chronic gastritis, being a major predisposing factor for peptic ulcer disease and gastric carcinoma. C. jejuni is the most common cause of enteric infections, particularly among children, resulting in severe diarrhoea. Increasing drug resistance of these bacteria against standard antibiotics, and the more widespread use of herbal medicines, favours investigations into additional anti-Helicobacter and anti-Campylobacter effects of phytotherapeutics that are already used for their beneficial effects on bowel and digestive functions. Twenty-one hydroethanol herbal extracts and four essential oils were screened for antibacterial activity using a modification of a previously described micro-dilution assay and compared with the inhibitory effects of antibiotics. The herbal extracts showing the highest growth inhibition of C. jejuni were Calendula officinalis, Matricaria recutita, Zingiber officinale, Salvia officinalis, Foeniculum vulgare and Silybum marianum. Agrimonia eupatoria, Hydrastis canadensis, Filipendula ulmaria and Salvia officinalis were the most active herbal extracts in inhibiting the growth of H. pylori. This study provides evidence for additional beneficial effects of phytotherapeutics marketed for their gastrointestinal effects and identifies new beneficial antibacterial effects for some herbal medicines not currently recommended for gastrointestinal problems.

Antimicrobial resistance in selected bacteria from poultry.

This paper reviews the present state of antimicrobial resistance (AMR) in the zoonotic bacteria Salmonella, Campylobacter jejuni and Campylobacter coli, and in Escherichia coli from chickens and turkeys. For Salmonella, the frequencies and patterns of AMR vary depending on time, region, serovar, the particular farm, layers versus broilers, and the antimicrobial agent. There is usually a higher frequency of AMR in Salmonella from turkeys compared with Salmonella from chickens. Clonal and horizontal transmission of AMR occur and there is concern about the spread of transmissible plasmids that encode extended spectrum cephalosporinases. Resistance to fluoroquinolones is generally low. For Campylobacter, resistance to tetracycline is usually at moderate to high frequency, resistance to quinolones/fluoroquinolones varies from low to high, and resistance to macrolides is usually low. There are high levels of fluoroquinolone resistance in some countries. Avian pathogenic E. coli are often highly resistant, especially to tetracycline, streptomycin, and sulfonamides. Plasmid-mediated resistance is common. High levels of resistance to ciprofloxacin have been reported from China. Commensal E. coli from poultry have similar patterns of resistance but at lower frequencies. Integron associated resistance occurs commonly in Salmonella and E. coli but has not been detected in Campylobacter.