Numerous vaccination strategies have been evaluated to develop effective vaccines against Campylobacter jejuni colonization in poultry but with limited success. The following experiments were conducted to investigate the effect of biodegradable and biocompatible poly (lactide-co-glycolide) nanoparticle (NP) encapsulated outer membrane proteins (OMP) of C. jejuni. Chickens were vaccinated with different routes [subcutaneous (s/c) or oral] and doses (25, 125, or 250 µg) of candidate nanoparticle vaccine with appropriate control groups. Serum and cloacal fecal samples were taken at regular intervals of time, and the birds were euthanized 7 d postchallenge with C. jejuni. The results were interpreted based on anti-OMP immunoglobulin response in chicken and intestinal colonization of C. jejuni. The C. jejuni colonization in cecal and cloacal contents at 7 d postchallenge was below the detection limit in the s/c vaccinated groups, but the other groups demonstrated varying degrees of colonization. The serum IgA was higher in the group vaccinated s/c with OMP only compared with the rest of the groups. The serum- and fecal-IgY titers were consistently higher in the s/c vaccinated groups (with or without NP) than the rest of the groups. Elevated levels of OMP specific serum antibodies correlated with below the limit of detection levels of Campylobacter colonization in broiler chickens receiving 125 μg of OMP alone and the OMP+NP vaccine s/c. In conclusion, the s/c route of vaccination with or without NP encapsulated OMP of C. jejuni may serve as a candidate vaccine for control of C. jejuni colonization in chickens.

Campylobacter is a leading cause of foodborne bacterial gastroenteritis worldwide and infections can be fatal. The emergence of antibiotic-resistant Campylobacter spp. necessitates the development of new antimicrobials. We identified novel anti-Campylobacter small molecule inhibitors using a high throughput growth inhibition assay. To expedite screening, we made use of a “bioactive” library of 4182 compounds that we have previously shown to be active against diverse microbes. Screening for growth inhibition of Campylobacter jejuni, identified 781 compounds that were either bactericidal or bacteriostatic at a concentration of 200 μM. Seventy nine of the bactericidal compounds were prioritized for secondary screening based on their physico-chemical properties. Based on the minimum inhibitory concentration against a diverse range of C. jejuni and a lack of effect on gut microbes, we selected 12 compounds. No resistance was observed to any of these 12 lead compounds when C. jejuni was cultured with lethal or sub-lethal concentrations suggesting that C. jejuni is less likely to develop resistance to these compounds. Top 12 compounds also possessed low cytotoxicity to human intestinal epithelial cells (Caco-2 cells) and no hemolytic activity against sheep red blood cells. Next, these 12 compounds were evaluated for ability to clear C. jejuni in vitro. A total of 10 compounds had an anti-C. jejuni effect in Caco-2 cells with some effective even at 25 μM concentrations. These novel 12 compounds belong to five established antimicrobial chemical classes; piperazines, aryl amines, piperidines, sulfonamide, and pyridazinone. Exploitation of analogs of these chemical classes may provide Campylobacter specific drugs that can be applied in both human and animal medicine.

Transducer Like Proteins (Tlps), also known as methyl accepting chemotaxis proteins (MCP), enable enteric pathogens to respond to changing nutrient levels in the environment by mediating taxis toward or away from specific chemoeffector molecules. Despite recent advances in the characterization of chemotaxis responses in Campylobacter jejuni, the impact of Tlps on the adaptation of this pathogen to disparate niches and hosts is not fully characterized. The latter is particularly evident in the case of C. jejuni 81-176, a strain that is known to be highly invasive. Furthermore, the cytoplasmic group C Tlps (Tlp5, 6, and 8) were not extensively evaluated. Here, we investigated the role of C. jejuni 81-176 Tlps in chemotaxis toward various substrates, biofilm formation, in vitro interaction with human intestinal cells, and chicken colonization. We found that the Δtlp6 and Δtlp10 mutants exhibited decreased chemotaxis toward aspartate, whereas the Δtlp6 mutant displayed a decreased chemotaxis toward Tri-Carboxylic Acid (TCA) cycle intermediates such as pyruvate, isocitrate, and succinate. Our findings also corroborated that more than one Tlp is involved in mediating chemotaxis toward the same nutrient. The deletion of tlps affected important phenotypes such as motility, biofilm formation, and invasion of human intestinal epithelial cells (INT-407). The Δtlp8 mutant displayed increased motility in soft agar and showed decreased biofilm formation. The Δtlp8 and Δtlp9 mutants were significantly defective in invasion in INT-407 cells. The Δtlp10 mutant was defective in colonization of the chicken proximal and distal gastrointestinal tract, while the Δtlp6 and Δtlp8 mutants showed reduced colonization of the duodenum and jejunum. Our results highlight the importance of Tlps in C. jejuni's adaptation and pathobiology.

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Clavibacter michiganensis subsp. michiganensis (Cmm) is a Gram-positive seed-transmitted bacterial phytopathogen responsible for substantial economic losses by adversely affecting tomato production worldwide. A high-throughput, cell-based screen was adapted to identify novel small molecule growth inhibitors to serve as leads for future bactericide development. A library of 4,182 compounds known to be bioactive against Saccharomyces cerevisiae was selected for primary screening against Cmm wild-type strain C290 for whole-cell growth inhibition. Four hundred sixty-eight molecules (11.2% hit rate) were identified as bacteriocidal or bacteriostatic against Cmm at 200 μM. Seventy-seven candidates were selected based on Golden Triangle analyses for secondary screening. Secondary screens showed that several of these candidates were strain-selective. Several compounds were inhibitory to multiple Cmm strains as well as Bacillus subtilis, but not to Pseudomonas fluorescens, Mitsuaria sp., Lysobacter enzymogenes, Lactobacillus rhamnosus, Bifidobacterium animalis, or Escherichia coli. Most of the compounds were not phytotoxic and did not show overt host toxicity. Using a novel 96-well bioluminescent Cmm seedling infection assay, we assessed effects of selected compounds on pathogen infection. The 12 most potent novel molecules were identified by compiling the scores from all secondary screens combined with the reduction of pathogen infection in planta. When tested for ability to develop resistance to the top-12 compounds, no resistant Cmm were recovered, suggesting that the discovered compounds are unlikely to induce resistance. In conclusion, here we report top-12 compounds that provide chemical scaffolds for future Cmm-specific bactericide development.

Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Polyphosphate kinases 1 and 2 (PPK1 and PPK2) regulate several cellular processes, including the biosynthesis of the bacterial cell wall. Despite their importance, whether PPK1 and PPK2 modulate the composition of C. jejuni outer membrane constituents (OMCs) and consequently impact its interaction with host cells remains unknown. Our comparative analysis between C. jejuni wild type, Δppk1, and Δppk2 strains showed qualitative and quantitative differences in the total OMC composition among these strains. Importantly, these OMC variations observed on the C. jejuni polyphosphate kinase mutants are directly related to their capacity to invade, survive, and alter the immune response of intestinal epithelial cells in vitro. Specifically, sub-fractionation of the C. jejuni OMC indicated that OMC proteins are uniquely associated with bacterial invasion, whereas C. jejuni OMC proteins, lipids, and lipoglycans are all associated with C. jejuni intracellular survival. This study provides new insights regarding the function of polyphosphate kinases and their role in C. jejuni infection.

Cryptosporidium hominis and Cryptosporidium parvum are the major Cryptosporidium species that infect humans. Earlier studies in gnotobiotic piglets, model susceptible to both, showed that piglets recovered from infection with C. hominis were fully protected against challenge with same species but incompletely protected against C. parvum challenge. In the present study, piglets were infected with C. parvum first, and after recovery were re-challenged with C. parvum or C. hominis. Again, full protection was only observed when piglets were challenged with the homologous parasite strain. Although the two species are genetically/antigenically almost identical, they do not confer complete protection against each other.