Oral curcumin ameliorates acute murine campylobacteriosis.

Introduction: Human infections with the food-borne enteropathogen Campylobacter jejuni are responsible for increasing incidences of acute campylobacteriosis cases worldwide. Since antibiotic treatment is usually not indicated and the severity of the enteritis directly correlates with the risk of developing serious autoimmune disease later-on, novel antibiotics-independent intervention strategies with non-toxic compounds to ameliorate and even prevent campylobacteriosis are utmost wanted. Given its known pleiotropic health-promoting properties, curcumin constitutes such a promising candidate molecule. In our actual preclinical placebo-controlled intervention trial, we tested the anti-microbial and anti-inflammatory effects of oral curcumin pretreatment during acute experimental campylobacteriosis. Methods: Therefore, secondary abiotic IL-10-/- mice were challenged with synthetic curcumin via the drinking water starting a week prior oral C. jejuni infection. To assess anti-pathogenic, clinical, immune-modulatory, and functional effects of curcumin prophylaxis, gastrointestinal C. jejuni bacteria were cultured, clinical signs and colonic histopathological changes quantitated, pro-inflammatory immune cell responses determined by in situ immunohistochemistry and intestinal, extra-intestinal and systemic pro-inflammatory mediator measurements, and finally, intestinal epithelial barrier function tested by electrophysiological resistance analysis of colonic ex vivo biopsies in the Ussing chamber. Results and discussion: Whereas placebo counterparts were suffering from severe enterocolitis characterized by wasting symptoms and bloody diarrhea on day 6 post-infection, curcumin pretreated mice, however, were clinically far less compromised and displayed less severe microscopic inflammatory sequelae such as histopathological changes and epithelial cell apoptosis in the colon. In addition, curcumin pretreatment could mitigate pro-inflammatory innate and adaptive immune responses in the intestinal tract and importantly, rescue colonic epithelial barrier integrity upon C. jejuni infection. Remarkably, the disease-mitigating effects of exogenous curcumin was also observed in organs beyond the infected intestines and strikingly, even systemically given basal hepatic, renal, and serum concentrations of pro-inflammatory mediators measured in curcumin pretreated mice on day 6 post-infection. In conclusion, the anti-Campylobacter and disease-mitigating including anti-inflammatory effects upon oral curcumin application observed here highlight the polyphenolic compound as a promising antibiotics-independent option for the prevention from severe acute campylobacteriosis and its potential post-infectious complications.

Reduction of Campylobacter jejuni contamination by using UVA-LED and sodium hypochlorite on the surface of chicken meat.

Campylobacter jejuni causes gastroenteritis in humans and is a major concern in food safety. Commercially prepared chicken meats are frequently contaminated with C. jejuni, which is closely associated with the diffusion of intestinal contents in poultry processing plants. Sodium hypochlorite (NaClO) is commonly used during chicken processing to prevent food poisoning; however, its antimicrobial activity is not effective in the organic-rich solutions. In this study, we investigated the potential of a new photo-disinfection system, UVA-LED, for the disinfection of C. jejuni-contaminated chicken surfaces. The data indicated that UVA irradiation significantly killed C. jejuni and that its killing ability was significantly facilitated in NaClO-treated chickens. Effective inactivation of C. jejuni was achieved using a combination of UVA and NaClO, even in the organic-rich condition. The results of this study show that synergistic disinfection using a combination of UVA and NaClO has potential beneficial effects in chicken processing systems.

The effect of mesenchymal stem cell conditioned medium incorporated within chitosan nanostructure in clearance of common gastroenteritis bacteria in-vitro and in-vivo.

Gastroenteritis infection is a major public health concern worldwide, especially in developing countries due to the high annual mortality rate. The antimicrobial and antibiofilm activity of human mesenchymal stem cell-derived conditioned medium (hMSCsCM) encapsulated in chitosan nanoparticles (ChNPs) was studied in vitro and in vivo against common gastroenteritis bacteria. The synthesized ChNPs were characterized using Zeta potential, scanning electron microscopy (SEM), and dynamic light scattering (DLS) techniques. HMSC-derived conditioned medium incorporated into chitosan NPs (hMSCsCM-ChNPs) composite was fabricated by chitosan nanoparticles loaded with BM-MSCs (positive for CD73 and CD44 markers). The antimicrobial and antibiofilm activity of composite was investigated against four common gastroenteritis bacteria (Campylobacter jejuni ATCC29428, Salmonella enteritidis ATCC13076, Shigella dysenteriae PTCC1188, and E. coli ATCC25922) in-vitro and in-vivo. Majority of ChNPs (96%) had an average particle size of 329 nm with zeta potential 7.08 mV. The SEM images confirmed the synthesis of spherical shape for ChNPs and a near-spherical shape for hMSCsCM-ChNPs. Entrapment efficiency of hMSCsCM-ChNPs was 75%. Kinetic profiling revealed that the release rate of mesenchymal stem cells was reduced following the pH reduction. The antibacterial activity of hMSCsCM-ChNPs was significantly greater than that of hMSCsCM and ChNPs at dilutions of 1:2 to 1:8 (P < 0.05) against four common gastroenteritis bacteria. The number of bacteria present decreased more significantly in the group of mice treated with the hMSCsCM-ChNPs composite than in the groups treated with hMSCsCM and ChNPs. The antibacterial activity of hMSCsCM against common gastroenteritis bacteria in an in vivo assay decreased from > 106 CFU/ml to approximately (102 to 10) after 72 h. Both in vitro and in vivo assays demonstrated the antimicrobial and antibiofilm activities of ChNPs at a concentration of 0.1% and hMSCsCM at a concentration of 1000 µg/ml to be inferior to that of hMSCsCM-ChNPs (1000 µg/ml + 0.1%) composite. These results indicated the existence of a synergistic effect between ChNPs and hMSCsCM. The designed composite exhibited notable antibiofilm and antibacterial activities, demonstrating optimal release in simulated intestinal lumen conditions. The utilization of this composite is proposed as a novel treatment approach to combat gastroenteritis bacteria in the context of more challenging infections.

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

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

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.

Controlled-release casein/cinnamon essential oil nanospheres for the inactivation of Campylobacter jejuni in duck.

Campylobacter jejuni (C. jejuni) is one of the most common foodborne pathogens that cause human sickness mostly through the poultry food chain. Cinnamon essential oil (CEO) has excellent antibacterial ability against C. jejuni growth. This study investigated the antibacterial mechanism of CEO against C. jejuni primarily through metabolism, energy metabolism of essential enzymes (AKPase, ß-galactosidase, and ATPase), and respiration metabolism. Results showed that the hexose monophosphate pathway (HMP) was inhibited, and that the enzyme activity of G6DPH substantially decreased upon treatment with CEO. Analysis of the effect of CEO on the expression of toxic genes was performed by the real-time PCR (RT-PCR). The expression levels of the toxic genes cadF, ciaB, fliA, and racR under CEO treatment were determined. Casein/CEO nanospheres were further prepared for the effective inhibition of C. jejuni and characterized by particle-size distribution, zeta-potential distribution, fluorescence, TEM, and GC-MS methods. Finally, the efficiency of CEO and casein/CEO nanospheres in terms of antibacterial activity against C. jejuni was verified. The casein/CEO nanospheres displayed high antibacterial activity on duck samples. The population of the test group decreased from 4.30 logCFU/g to 0.86 logCFU/g and 4.30 logCFU/g to 2.46 logCFU/g at 4 °C and at 25 °C for C. jejuni, respectively. Sensory evaluation and texture analysis were also conducted on various duck samples.

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.

Refined functional carbohydrates reduce adhesion of Salmonella and Campylobacter to poultry epithelial cells in vitro.

The development of interventions to reduce human foodborne pathogens in the gastrointestinal (GI) tract of chickens will be important for improving the microbial food safety of poultry. Saccharomyces-derived prebiotic refined functional carbohydrates (RFC), composed primarily of ß-glucans, mannanoligosaccharides (MOS), and D-mannose have been demonstrated to reduce GI colonization of Salmonella and Campylobacter when administered to poultry. Although they are presumed to inhibit adhesion of pathogens to the GI epithelium, this functionality of RFC has not been well characterized. In this study, we investigated the effects of RFC and other prebiotics on the adhesion of Salmonella Typhimurium and Campylobacter jejuni to the LMH chicken epithelial cell line in vitro. The reduction of adherent pathogens was observed to be dose-dependent with C. jejuni being more sensitive than Salmonella to inhibition by RFC. Comparison of the primary constituent carbohydrates of RFC found D-mannose to inhibit both pathogens less effectively than ß-glucan and MOS, suggesting that it contributes less to inhibition of pathogen adhesion than the other carbohydrates. Finally, the reduction of adherent pathogens by RFC was compared with that of fructooligosaccharides (FOS), galactooligosaccharides (GOS), and raffinose. All 4 prebiotics inhibited adhesion of both pathogens to chicken epithelial cells. Reduction of adherent Salmonella was greatest with FOS and lowest with GOS, whereas reduction of adherent C. jejuni was greater with RFC and raffinose than with FOS and GOS. These results will inform future research elucidating mechanisms important to adhesion inhibition of pathogens by RFC and other prebiotics.

Phenotypic and molecular characterization of erythromycin resistance in Campylobacter jejuni and Campylobacter coli strains isolated from swine and broiler chickens / Caracterização fenotípica e molecular da resistência à eritromicina em cepas de Campylobacter jejuni e Campylobacter coli isoladas de suínos e frangos de corte

Campylobacter spp. is a bacterial agent that causes gastroenteritis in humans and may trigger Guillain-Barré Syndrome (GBS) and is also considered one of the main foodborne diseases in developed countries. Poultry and pigs are considered reservoirs of these microorganisms, as well as raw or undercooked by-products are often incriminated as a source of human infection. Treatment in human cases is with macrolide, such erythromycin, that inhibits the protein synthesis of the microorganism. This study aimed to isolate Campylobacter jejuni and Campylobacter coli from intestinal content samples of broiler chickens (n=20) and swine (n=30) to characterize the erythromycin resistance profile of the strains and to detect molecular mechanisms involved in this resistance. The minimum inhibitory concentration was determined by agar dilution. The Mismatch Amplification Mutation Assay-Polymerase Chain Reaction (MAMA-PCR) was performed to detect mutations at positions 2074 and 2075 of 23S rRNA region, in addition to PCR test to detect the erm(B) gene. From the intestinal content of broiler chickens, 18 strains of C. jejuni and two strains of C. coli were isolated, whereas, from swine samples, no C. jejuni strain and 14 strains of C. coli were isolated. All C. coli strains were resistant, and three C. jejuni strains from broilers chickens were characterized with intermediate resistance to erythromycin. The MIC of the strains ranged from ≤0.5mg/μL to ≥128mg/μL. All resistant strains had the A2075G mutation, and one strain with intermediate resistance had the A2075G mutation. However, the A2074C mutation and the erm(B) gene were not detected. High resistance levels were detected in C. coli strains isolated from swine. The MAMA-PCR is a practical tool for detecting the erythromycin resistance in Campylobacter strains.(AU)

Campylobacter spp. é um agente bacteriano causador de gastroenterite em humanos e associado à síndrome de Guillain-Barré, sendo a campilobacteriose considerada uma das principais enfermidades de origem alimentar. Aves e suínos são importantes reservatórios desses microrganismos e seus produtos derivados crus ou mal cozidos são muitas vezes incriminados como fonte de infecção humana. A primeira escolha para o tratamento em casos humanos são os antimicrobianos da classe dos macrolídeos como à eritromicina. Dentro desse contexto, o objetivo deste estudo foi isolar Campylobacter jejuni e C. coli a partir de 20 amostras de conteúdo intestinal de frangos de corte e de 30 de suínos ao abate e investigar a resistência à eritromicina das estirpes obtidas e os possíveis mecanismos moleculares envolvidos nesta resistência. A concentração inibitória mínima foi determinada pela diluição em ágar e a técnica MAMA-PCR foi utilizada para detecção de mutações nas posições 2074 e 2075 da região 23s rRNA, foi pesquisado também a presença do gene erm(B) pela PCR. A partir do conteúdo intestinal de frangos de corte foram isoladas 18 estirpes de C. jejuni e duas de C. coli, enquanto de suínos foram obtidas 14 estirpes de C. coli e nenhuma estirpe de C. jejuni. Todas as estirpes de C. coli de suínos foram identificadas como resistentes e três estirpes de C. jejuni de frangos foram caracterizadas com resistência intermediária. A CIM das estirpes variou de ≤0,5mg/μL a ≥128mg/μL. Todas as estirpes resistentes tinham a mutação A2075G e uma cepa com resistência intermediária também apresentou a mutação A2075G. Não foi detectada a mutação A2074C ou a presença do gene erm(B) em nenhuma das estirpes obtidas. Os resultados revelam um alto nível de resistência em estirpes de C. coli isoladas de suínos frente a eritromicina. A técnica MAMA PCR utilizada se constitui em uma ferramenta prática para detecção da resistência à eritromicina em estirpes de C. jejuni e C. coli.(AU)

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.

Proteomics of Campylobacter jejuni Growth in Deoxycholate Reveals Cj0025c as a Cystine Transport Protein Required for Wild-type Human Infection Phenotypes.

Campylobacter jejuni is a major cause of food-borne gastroenteritis. Proteomics by label-based two-dimensional liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) identified proteins associated with growth in 0.1% sodium deoxycholate (DOC, a component of gut bile salts), and system-wide validation was performed by data-independent acquisition (DIA-SWATH-MS). LC-MS/MS quantified 1326 proteins (∼82% of the predicted C. jejuni proteome), of which 1104 were validated in additional biological replicates by DIA-SWATH-MS. DOC resulted in a profound proteome shift with 512 proteins showing significantly altered abundance. Induced proteins were associated with flagellar motility and antibiotic resistance; and these correlated with increased DOC motility and resistance to polymyxin B and ciprofloxacin. DOC also increased human Caco-2 cell adherence and invasion. Abundances of proteins involved in nutrient transport were altered by DOC and aligned with intracellular changes to their respective carbon sources. DOC increased intracellular levels of sulfur-containing amino acids (cysteine and methionine) and the dipeptide cystine (Cys-Cys), which also correlated with reduced resistance to oxidative stress. A DOC induced transport protein was Cj0025c, which has sequence similarity to bacterial Cys-Cys transporters. Deletion of cj0025c (Δcj0025c) resulted in proteome changes consistent with sulfur starvation, as well as attenuated invasion, reduced motility, atypical morphology, increased antimicrobial susceptibility and poor biofilm formation. Targeted metabolomics showed Δcj0025c could use known C. jejuni amino and organic acid substrates commensurate with wild-type. Medium Cys-Cys levels however, were maintained in Δcj0025c relative to wild-type. A toxic Cys-Cys mimic (selenocystine) inhibited wild-type growth, but not Δcj0025c Provision of an alternate sulfur source (2 mm thiosulfate) restored Δcj0025c motility. Our data confirm that Cj0025c is a Cys-Cys transporter that we have named TcyP consistent with the nomenclature of homologous proteins in other species.

Comparison of relative expressions of genes involved in iron acquisition and regulation in fluoroquinolone-resistant and wild-type Campylobacter jejuni.

Campylobacteriosis caused by C. jejuni is a serious yet common foodborne disease in the U.S. The prevalence of fluoroquinolone-resistant C. jejuni from poultry has continued to increase despite the withdrawal of fluoroquinolone use in the U.S. poultry industry in 2005. To date, no clear selective pressures that explain this effect have been documented. In this study, we investigated limited bioavailability of iron in poultry and enhanced iron uptake and regulation as potential indirect selective pressures conferring fitness advantages in fluoroquinolone-resistant C. jejuni compared to its susceptible wild-type counterpart. Five fluoroquinolone-susceptible C. jejuni isolates were selected from litter collected from commercial broiler farms. Using antibiotic selection, five fluoroquinolone-resistant strains were created. Relative expressions of six genes involved in iron acquisition and regulation were compared between the resistant and susceptible strains using RT-qPCR under normal and iron-limiting conditions. High variability in the relative gene expressions was observed among the strains, with only one resistant strain showing the consistent upregulation of the measured genes compared to the matching susceptible wild-type. Our results suggest that the hypothesis tested in the study may not be an adequate explanation of the molecular mechanism behind the enhanced fitness of fluoroquinolone-resistant C. jejuni compared to susceptible C. jejuni. This study highlights the need for a better understanding of the complex ecology and dynamics of fluoroquinolone resistance in C. jejuni in poultry environment and warrants an examination of fluoroquinolone-resistant C. jejuni strains recovered from the natural broiler chicken environment.

Hepatic Campylobacter jejuni infection in patients with Castleman-Kojima disease (idiopathic multicentric Castleman disease with thrombocytopenia, anasarca, fever, reticulin fibrosis, and organomegaly (TAFRO) syndrome).

Castleman-Kojima disease, also known as idiopathic multicentric Castleman disease with TAFRO syndrome (iMCD-TAFRO), is a recently recognized systemic inflammatory disorder with a characteristic series of clinical symptoms, including thrombocytopenia (T), anasarca (A), fever (F), reticulin fibrosis (R), and organomegaly (O). Patients with iMCD-TAFRO often develop severe abdominal pain, elevated alkaline phosphatase levels, and systemic inflammation, but the etiological factors are unknown. To investigate the potential role of bacterial infection in the pathogenesis of iMCD-TAFRO, we performed polymerase chain reaction (PCR) for the bacterial 16S rRNA gene with DNA extracted from liver specimens of three patients with iMCD-TAFRO, four patients with amyotrophic lateral sclerosis, and seven patients with inflammatory conditions. Sequencing of the PCR product showed 99% DNA sequence identity with Campylobacter jejuni in all three patients with iMCD-TAFRO and in two patients with inflammatory conditions. Immunohistochemical and electron microscopy analyses could not identify C. jejuni in patients with iMCD-TAFRO. The findings indicated that C. jejuni infection is not the pathological cause of iMCD-TAFRO; however, this ubiquitous bacterium may play a role in uncontrolled systemic hypercytokinemia, possibly through the development of cross-reactive autoantibodies.

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).

Searching for potent and specific antibiotics against pathogenic Helicobacter and Campylobacter strains.

Menaquinone is an obligatory component of the electron-transfer pathway in microorganisms. Its biosynthetic pathway was established by pioneering studies with Escherichia coli and it was revealed to be derived from chorismate by Men enzymes. However, we identified an alternative pathway, the futalosine pathway, operating in some microorganisms including Helicobacter pylori and Campylobacter jejuni, which cause gastric carcinoma and diarrhea, respectively. Because some useful intestinal bacteria, such as lactobacilli, use the canonical pathway, the futalosine pathway is an attractive target for development of chemotherapeutics for the abovementioned pathogens. In this mini-review, we summarize compounds that inhibit Mqn enzymes involved in the futalosine pathway discovered to date.

Campylobacter jejuni bile exposure influences outer membrane vesicles protein content and bacterial interaction with epithelial cells.

Campylobacter jejuni is a prevalent human pathogen and a major cause of bacterial gastroenteritis in the world. In humans, C. jejuni colonizes the intestinal tract and its tolerance to bile is crucial for bacteria to survive and establish infection. C. jejuni produces outer membrane vesicles (OMVs) which have been suggested to be involved in virulence. In this study, the proteome composition of C. jejuni OMVs in response to low concentration of bile was investigated. We showed that exposure of C. jejuni to low concentrations of bile, similar to the concentration in cecum, induced significant changes in the protein profile of OMVs released during growth without affecting the protein profile of the bacteria. This suggests that bile influences a selective packing of the OMVs after bacterial exposure to low bile. A low concentration of bile was found to increase bacterial adhesion to intestinal epithelial cells, likely by an enhanced hydrophobicity of the cell membrane following exposure to bile. The increased bacterial adhesiveness was not associated with increased invasion, instead bile exposure decreased C. jejuni invasion. OMVs released from bacteria upon exposure to low bile showed to increase both adhesion and invasion of non-bile-exposed bacteria into intestinal epithelial cells. These findings suggest that C. jejuni in environments with low concentrations of bile produce OMVs that facilitates colonization of the bacteria, and this could potentially contribute to virulence of C. jejuni in the gut.

E. coli Nissle microencapsulation in alginate-chitosan nanoparticles and its effect on Campylobacter jejuni in vitro.

Microencapsulation enhances the oral delivery of probiotic bacteria. In this study, the probiotic Escherichia coli Nissle 1917 (EcN) was microencapsulated using alginate and chitosan nanoparticles. The result showed 90% encapsulation yield of EcN, and the encapsulated EcN displayed significantly (P < 0.05) increased survival in low pH (1.5), high bile salt concentration (4%), and high temperature (70 °C). The most effective cryopreservatives of EcN during freezing and thawing was skim milk and sucrose. Exposure to microencapsulated EcN significantly (P < 0.05) reduced the Campylobacter jejuni growth by 2 log CFU. The rate of EcN release from microcapsule was 9.2 × 105 cell min-1, and the appropriate model to describe its release kinetics was zero order. Importantly, the entrapment of EcN inside the microcapsule did not eliminate the exterior diffusion of EcN produced antioxidant compounds. In addition, the EcN microcapsule efficiently adhered to intestinal HT-29 cells and the pre-treatment of HT-29 cells with EcN-microcapsule for 4 h significantly (P < 0.05) reduced the invasion (1.9 log) of C. jejuni; whereas, completely abolished the intracellular survival. Furthermore, HT-29 cells pre-treated with encapsulated EcN in PCR array showed decreased expression (> 1.5-fold) of genes encoding chemokines, toll-like receptors, interleukins, and tumor necrosis factors. In conclusion, the alginate-chitosan microcapsule can provide effectual platform to deliver probiotic EcN and thereby can reduce the Campylobacter infection in chickens and humans.

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.

Active Chicken Meat Packaging Based on Polylactide Films and Bimetallic Ag-Cu Nanoparticles and Essential Oil.

Plasticized polylactide (PLA) composite films with multifunctional properties were created by loading bimetallic silver-copper (Ag-Cu) nanoparticles (NPs) and cinnamon essential oil (CEO) into polymer matrix via compression molding technique. Rheological, structural, thermal, barrier, and antimicrobial properties of the produced films, and its utilization in the packaging of chicken meat were investigated. PLA/PEG/Ag-Cu/CEO composites showed a very complex rheological system where both plasticizing and antiplasticizing effects were evident. Thermal properties of plasticized PLA film with polyethylene glycol (PEG) enhanced considerably with the reinforcement of NPs whereas loading of CEO decreased glass transition, melting, and crystallization temperature. The barrier properties of the composite films were reduced with the increase of CEO loading (P < 0.05). Their optical properties were also modified by the addition of both CEO and Ag-Cu NPs. The changes in the molecular organization of PLA composite films were visualized by FTIR spectra. Rough and porous surfaces of the films were evident by scanning electron microscopy. The effectiveness of composite films was tested against Salmonella Typhimurium, Campylobacter jejuni and Listeria monocytogenes inoculated in chicken samples, and it was found that the films loaded with Ag-Cu NPs and 50% CEO showed maximum antibacterial action during 21 days at the refrigerated condition. The produced PLA/Ag-Cu/CEO composite films can be applied to active food packaging. PRACTICAL APPLICATION: The nanoparticles and essential oil loaded PLA composite films are capable of exhibiting antimicrobial effects against Gram (+) and (-) bacteria, and extend the shelf-life of chicken meat. The bionanocomposite films showed the potential to be manufactured commercially because of the thermal stability of the active components during the hot-press compression molding process. The developed bionanocomposites could have practical importance and open a new direction for the active food packaging to control the spoilage and the pathogenic bacteria associated with the fresh chicken meat.

The Host Antimicrobial Protein Calgranulin C Participates in the Control of Campylobacter jejuni Growth via Zinc Sequestration.

Campylobacter jejuni is a leading cause of bacterially derived gastroenteritis worldwide. Campylobacter is most commonly acquired through the consumption of undercooked poultry meat or through drinking contaminated water. Following ingestion, Campylobacter adheres to the intestinal epithelium and mucus layer, causing toxin-mediated inflammation and inhibition of fluid reabsorption. Currently, the human response to infection is relatively unknown, and animal hosts that model these responses are rare. As such, we examined patient fecal samples for the accumulation of the neutrophil protein calgranulin C during infection with Campylobacter jejuni In response to infection, calgranulin C was significantly increased in the feces of humans. To determine whether calgranulin C accumulation occurs in an animal model, we examined disease in ferrets. Ferrets were effectively infected by C. jejuni, with peak fecal loads observed at day 3 postinfection and full resolution by day 12. Serum levels of interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-α) significantly increased in response to infection, which resulted in leukocyte trafficking to the colon. As a result, calgranulin C increased in the feces of ferrets at the time when C. jejuni loads decreased. Further, the addition of purified calgranulin C to C. jejuni cultures was found to inhibit growth in a zinc-dependent manner. These results suggest that upon infection with C. jejuni, leukocytes trafficked to the intestine release calgranulin C as a mechanism for inhibiting C. jejuni growth.