Diametral influence of deoxynivalenol (DON) and deepoxy-deoxynivalenol (DOM-1) on the growth of Campylobacter jejuni with consequences on the bacterial transcriptome.

BACKGROUND: Deoxynivalenol (DON) is a type B trichothecene mycotoxin that is commonly found in cereals and grains worldwide. The presence of this fungal secondary-metabolite raises public-health concerns at both the agriculture and food industry level. Recently, we have shown that DON has a negative impact on gut integrity, a feature also noticed for Campylobacter (C.) jejuni. We further demonstrated that DON increased the load of C. jejuni in the gut and inner organs. In contrast, feeding the less toxic DON metabolite deepoxy-deoxynivalenol (DOM-1) to broilers reduced the Campylobacter load in vivo. Consequently, it can be hypothesized that DON and DOM-1 have a direct effect on the growth profile of C. jejuni. The aim of the present study was to further resolve the nature of this interaction in vitro by co-incubation and RNA-sequencing. RESULTS: The co-incubation of C. jejuni with DON resulted in significantly higher bacterial growth rates from 30 h of incubation onwards. On the contrary, the co-incubation of C. jejuni with DOM-1 reduced the CFU counts, indicating that this DON metabolite might contribute to reduce the burden of C. jejuni in birds, altogether confirming in vivo data. Furthermore, the transcriptomic profile of C. jejuni following incubation with either DON or DOM-1 differed. Co-incubation of C. jejuni with DON significantly increased the expression of multiple genes which are critical for Campylobacter growth, particularly members of the Flagella gene family, frr (ribosome-recycling factor), PBP2 futA-like (Fe3+ periplasmic binding family) and PotA (ATP-binding subunit). Flagella are responsible for motility, biofilm formation and host colonization, which may explain the high Campylobacter load in the gut of DON-fed broiler chickens. On the contrary, DOM-1 downregulated the Flagella gene family and upregulated ribosomal proteins. CONCLUSION: The results highlight the adaptive mechanisms involved in the transcriptional response of C. jejuni to DON and its metabolite DOM-1, based on the following effects: (a) ribosomal proteins; (b) flagellar proteins; (c) engagement of different metabolic pathways. The results provide insight into the response of an important intestinal microbial pathogen against DON and lead to a better understanding of the luminal or environmental acclimation mechanisms in chickens.

Campylobacter jejuni bacteremia in a leukemic child: a nearly missed diagnosis.

Globally, Campylobacter spp. are responsible for most cases of bacterial gastrointestinal infections in humans and although rare, extraintestinal Campylobacter infections have been described. A 2-yearold neutropenic girl with underlying precursor B-cell acute lymphoblastic leukemia presented with a 3-day history of diarrhea. Her stool culture yielded no enteric bacterial pathogens. However, when her blood culture was flagged as positive for bacterial growth, no colonies could be observed on routine bacteriological isolation media. Nonetheless, gram-negative bacilli with seagull and spiral morphologies were seen when the surface of the isolation media used to subculture her blood was Gram-stained. Bacterial colonies were only visible when a subculture was attempted on a Campylobacter blood-free selective agar medium. The organism was identified as Campylobacter jejuni by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Since the organism was erythromycin-resistant and the patient's age precluded the use of tetracycline and ciprofloxacin, an antibiotic regimen consisting of piperacillin-tazobactam and gentamicin was commenced. Her C. jejuni bacteremia resolved following eight days of antibiotic therapy.

Helicobacter pylori and Campylobacter jejuni bacterial holocytochrome c synthase structure-function analysis reveals conservation of heme binding.

Heme trafficking is essential for cellular function, yet mechanisms of transport and/or heme interaction are not well defined. The System I and System II bacterial cytochrome c biogenesis pathways are developing into model systems for heme trafficking due to their functions in heme transport, heme stereospecific positioning, and mediation of heme attachment to apocytochrome c. Here we focus on the System II pathway, CcsBA, that is proposed to be a bi-functional heme transporter and holocytochrome c synthase. An extensive structure-function analysis of recombinantly expressed Helicobacter pylori and Campylobacter jejuni CcsBAs revealed key residues required for heme interaction and holocytochrome c synthase activity. Homologous residues were previously identified to be required for heme interaction in Helicobacter hepaticus CcsBA. This study provides direct, biochemical evidence that mechanisms of heme interaction are conserved, leading to the proposal that the CcsBA WWD heme-handling domain represents a novel target for therapeutics.

Risk-based critical concentrations of enteric pathogens for recreational water criteria and recommended minimum sample volumes for routine water monitoring.

Concerns are rising about the contamination of recreational waters from human and animal waste, along with associated risks to public health. However, existing guidelines for managing pathogens in these environments have not yet fully integrated risk-based pathogen-specific criteria, which, along with recent advancements in indicators and markers, are essential to improve the protection of public health. This study aimed to establish risk-based critical concentration benchmarks for significant enteric pathogens, i.e., norovirus, rotavirus, adenovirus, Cryptosporidium spp., Giardia lamblia, Campylobacter jejuni, Salmonella spp., and Escherichia coli O157:H7. Applying a 0.036 risk benchmark to both marine and freshwater environments, the study identified the lowest critical concentrations for children, who are the most susceptible group. Norovirus, C. jejuni, and Cryptosporidium presented lowest median critical concentrations for virus, bacteria, and protozoa, respectively: 0.74 GC, 1.73 CFU, and 0.39 viable oocysts per 100 mL in freshwater for children. These values were then used to determine minimum sample volumes corresponding to different recovery rates for culture method, digital polymerase chain reaction and quantitative PCR methods. The results indicate that for children, norovirus required the largest sample volumes of freshwater and marine water (52.08 to 178.57 L, based on the 5th percentile with a 10 % recovery rate), reflecting its low critical concentration and high potential for causing illness. In contrast, adenovirus and rotavirus required significantly smaller volumes (approximately 0.24 to 1.33 L). C. jejuni and Cryptosporidium, which required the highest sampling volumes for bacteria and protozoa, needed 1.72 to 11.09 L and 4.17 to 25.51 L, respectively. Additionally, the presented risk-based framework could provide a model for establishing pathogen thresholds, potentially guiding the creation of extensive risk-based criteria for various pathogens in recreational waters, thus aiding public health authorities in decision-making, strengthening pathogen monitoring, and improving water quality testing accuracy for enhanced health protection.

refMLST: reference-based multilocus sequence typing enables universal bacterial typing.

BACKGROUND: Commonly used approaches for genomic investigation of bacterial outbreaks, including SNP and gene-by-gene approaches, are limited by the requirement for background genomes and curated allele schemes, respectively. As a result, they only work on a select subset of known organisms, and fail on novel or less studied pathogens. We introduce refMLST, a gene-by-gene approach using the reference genome of a bacterium to form a scalable, reproducible and robust method to perform outbreak investigation. RESULTS: When applied to multiple outbreak causing bacteria including 1263 Salmonella enterica, 331 Yersinia enterocolitica and 6526 Campylobacter jejuni genomes, refMLST enabled consistent clustering, improved resolution, and faster processing in comparison to commonly used tools like chewieSnake. CONCLUSIONS: refMLST is a novel multilocus sequence typing approach that is applicable to any bacterial species with a public reference genome, does not require a curated scheme, and automatically accounts for genetic recombination. AVAILABILITY AND IMPLEMENTATION: refMLST is freely available for academic use at https://bugseq.com/academic .

Isolation, characterization, and antimicrobial resistance profiles of Campylobacter jejuni and Campylobacter coli from raw meat of large livestock in Shahrekord, Iran.

Campylobacter spp. genera is one of the most common causes of microbial enteritis worldwide. This study aimed to find out how common Campylobacter organisms were in raw meat from large livestock in Iran, as well as to determine their antibiotic susceptibility profiles. Several 550 fresh, ready-to-eat meat samples were collected from slaughterhouses, butcher shops, and restaurants in the study region. The samples were collected from cattle (n=138), goats (n=102), camels (n=56), and sheep (n=254). Campylobacter spp. were isolated and identified using normal bacteriological methods and polymerase chain reaction (PCR). Genotyping was performed using PCR to identify virulence genes. The disc diffusion technique was used to determine antibiotic susceptibility. The two Campylobacter spp. were found in 84 (15.27%) of the 550 meat samples tested. Cattle and camel samples accounted for the highest (52.38%) and lowest (3.57%) frequencies of Campylobacter spp., respectively. There were significant differences in the prevalence of Campylobacter spp. in cattle (2=43.04 or OR=7.68, CI=3.40-17.30, P<0.01). Campylobacter jejuni and Campylobacter coli accounted for 82.14% (n=69) of Campylobacter spp. isolated from raw meat. While C. jejuni was found in 39.28% of the samples (n=33), C. coli was observed in 42.85% (n=36). Other Campylobacter spp. formed 17.85 % (n=15) of the samples. The most common genotypes observed in C. jejuni bacteria collected from different types of large animal samples were ciaB (100%) and flaA (100%). On the other hand, virbll (7.69%) was the C. jejuni strain found with the lowest incidence in different large animal samples. The most frequent genotypes found in C. coli bacteria were ciaB (100%) and flaA (100%). C. coli isolates dnaJ (0%), wlaN (0%), virbll (0%), and ceuE (0%) were detected with the lowest frequency in several samples from large livestock. Campylobacter spp. isolated from different sample types and sources were 100% sensitive to aphA-3-1 and GM10. The isolates were reported to be resistant to E15 (76.93%), cmeB (69.24%), aadE1 (69.24%), CIP5 (69.24%), and AM10 (69.24%). According to this study, Campylobacter was found in food from factory farming. Consequently, the disease can be transmitted by eating raw or undercooked meat. Therefore, proper handling and preparation of meat meals, as well as hygiene measures from the slaughterhouse to the retailer, are critical in preventing Campylobacter infections.

A Bacteriophage Protein-Based Impedimetric Electrochemical Biosensor for the Detection of Campylobacter jejuni.

Campylobacter jejuni is a common foodborne pathogen found in poultry that can cause severe life-threatening illnesses in humans. It is important to detect this pathogen in food to manage foodborne outbreaks. This study reports a novel impedimetric phage protein-based biosensor to detect C. jejuni NCTC 11168 at 100 CFU/mL concentrations using a genetically engineered receptor-binding phage protein, FlaGrab, as a bioreceptor. The electrochemical impedance spectroscopy (EIS) technique was employed to measure changes in resistance upon interaction with C. jejuni. The sensitivity of the phage protein-immobilized electrode was assessed using the various concentrations of C. jejuni NCTC 11168 ranging from 102-109 colony forming units (CFU)/mL). The change transfer resistance of the biosensor increased with increasing numbers of C. jejuni NCTC 11168 cells. The detection limit was determined to be approximately 103 CFU/mL in the buffer and 102 CFU/mL in the ex vivo samples. Salmonella enterica subsp. enterica serotype Typhimurium-291RH and Listeria monocytogenes Scott A were used as nontarget bacterial cells to assess the specificity of the developed biosensor. Results showed that the developed biosensor was highly specific toward the target C. jejuni NCTC 11168, as no signal was observed for the nontarget bacterial cells.

Genomic diversity of Campylobacter jejuni and Campylobacter coli isolated from the Ethiopian dairy supply chain.

Campylobacteriosis outbreaks have previously been linked to dairy foods. While the genetic diversity of Campylobacter is well understood in high-income countries, it is largely unknown in low-income countries, such as Ethiopia. This study therefore aimed to conduct the first genomic characterization of Campylobacter isolates from the Ethiopian dairy supply chain to aid in future epidemiological studies. Fourteen C. jejuni and four C. coli isolates were whole genome sequenced using an Illumina platform. Sequences were analyzed using the bioinformatics tools in the GalaxyTrakr platform to identify MLST types, and single nucleotide polymorphisms, and infer phylogenetic relationships among the studied isolates. Assembled genomes were further screened to detect antimicrobial resistance and virulence gene sequences. Among 14 C. jejuni, ST 2084 and ST 51, which belong to the clonal complexes ST-353 and ST-443, respectively, were identified. Among the 4 sequenced C. coli isolates, two isolates belonged to ST 1628 and two to ST 830 from the clonal complex ST-828. The isolates of C. jejuni ST 2084 and ST 51 carried ß-lactam resistance gene blaOXA-605, a fluoroquinolone resistance-associated mutation T86I in the gryA gene, and a macrolide resistance-associated mutation A103V in 50S L22. Only ST 2084 isolates carried the tetracycline resistance gene tetO. Conversely, all four C. coli ST 830 and ST 1628 isolates carried tetO, but only ST 1628 isolates also carried blaOXA-605. Lastly, C. jejuni ST 2084 isolates carried a total of 89 virulence genes, and ST 51 isolates carried up to 88 virulence genes. Among C. coli, ST 830 isolates carried 71 genes involved in virulence, whereas two ST 1628 isolates carried up to 82 genes involved in virulence. Isolates from all identified STs have previously been isolated from human clinical cases, demonstrating a potential food safety concern. This finding warrants further monitoring of Campylobacter in dairy foods in Ethiopia to better understand and manage the risks associated with Campylobacter contamination and transmission.

Burden and epidemiology of Campylobacter species in acute enteritis cases in Burkina Faso.

BACKGROUND: Campylobacter spp. is a significant etiological agent of bacterial gastroenteritis globally. In Burkina Faso (BFA), the actual impact of this pathogen on gastroenteritis is considerably underestimated, primarily due to inadequate surveillance systems. OBJECTIVES: This study aimed to investigate the proportion of Campylobacter species responsible for acute gastroenteritis among patients of all ages in urban and rural areas of BFA, using molecular biology techniques. STUDY DESIGN & METHODS: Between 2018 and 2021, faecal specimens were obtained from 1,295 individuals presenting with acute gastroenteritis. These samples underwent screening for the Campylobacter coli/jejuni/lari complex utilizing real-time polymerase chain reaction (PCR) assays. Subsequently, positive samples were subjected to species-level differentiation through the application of species-specific primers. RESULTS: Campylobacter spp. was detected in 25.0% (324/1,295) of the samples analysed. The majority of positive samples (95%, 308/324) were obtained from children under 5 years of age. Species identification was performed on a subset of 114 isolates, revealing 51 Campylobacter jejuni, 10 Campylobacter coli, and 53 Campylobacter isolates that remained unspeciated. CONCLUSIONS: This study reveals a significant prevalence of Campylobacter species among patients with acute gastroenteritis, with a particularly high incidence observed in children under 5 years of age. Based on these findings, the implementation of routine Campylobacter surveillance in public health laboratories is strongly recommended to better monitor and address this health concern.

Molecular Detection of Virulence-Associated Markers in Campylobacter coli and Campylobacter jejuni Isolates From Water, Cattle, and Chicken Faecal Samples From Kajiado County, Kenya.

Campylobacter is a zoonotic foodborne pathogen that is often linked with gastroenteritis and other extraintestinal infections in humans. This study is aimed at determining the genetic determinants of virulence-encoding genes responsible for flagellin motility protein A (flaA), Campylobacter adhesion to fibronectin F (cadF), Campylobacter invasion antigen B (ciaB) and cytolethal distending toxin (cdt) A (cdtA) in Campylobacter species. A total of 29 Campylobacter coli isolates (16 from cattle, 9 from chicken, and 4 from water samples) and 74 Campylobacter jejuni isolates (38 from cattle, 30 from chicken, and 6 from water samples) described in an earlier study in Kajiado County, Kenya, were examined for the occurrence of virulence-associated genes using polymerase chain reaction (PCR) and amplicon sequencing. The correlations among virulence genes were analyzed using Pearson's correlation coefficient (R) method. Among the 103 Campylobacter strains screened, 89 were found to harbour a single or multiple virulence gene(s), giving an overall prevalence of 86.4%. C. jejuni strains had the highest prevalence of multivirulence at 64.9% (48/74), compared to C. coli (58.6%, 17/29). The ciaB and flaA genes were the most common virulence genes detected in C. jejuni (81.1% [60/74] and 62.2% [46/74], respectively) and in C. coli (each at 62.1%; 18/29). Campylobacter isolates from chicken harboured the most virulence-encoding genes. C. jejuni strains from chicken and cattle harboured the highest proportions of the cdtA and ciaB genes, respectively. All the C. coli strains from water samples harboured the cadF and flaA genes. The results obtained further revealed a significant positive correlation between cadF and flaA (R = 0.733). C. jejuni and C. coli strains from cattle, chicken, and water harbour virulence markers responsible for motility/colonization, invasion, adherence, and toxin production, evoking their important role in campylobacteriosis development among humans and livestock. The identification of cattle, chicken, and water samples as reservoirs of virulent Campylobacter spp. highlights the possible risk to human health. These data on some virulence genes of Campylobacter will assist food safety and public health officials in formulating policy statements.

High Campylobacter diversity in retail chicken: epidemiologically important strains may be missed with current sampling methods.

Campylobacter spp. are leading bacterial gastroenteritis pathogens. Infections are largely underreported, and the burden of outbreaks may be underestimated. Current strategies of testing as few as one isolate per sample can affect attribution of cases to epidemiologically important sources with high Campylobacter diversity, such as chicken meat. Multiple culture method combinations were utilized to recover and sequence Campylobacter from 45 retail chicken samples purchased across Norwich, UK, selecting up to 48 isolates per sample. Simulations based on resampling were used to assess the impact of Campylobacter sequence type (ST) diversity on outbreak detection. Campylobacter was recovered from 39 samples (87%), although only one sample was positive through all broth, temperature, and plate combinations. Three species were identified (Campylobacter jejuni, Campylobacter coli, and Campylobacter lari), and 33% of samples contained two species. Positive samples contained 1-8 STs. Simulation revealed that up to 87 isolates per sample would be required to detect 95% of the observed ST diversity, and 26 isolates would be required for the average probability of detecting a random theoretical outbreak ST to reach 95%. An optimized culture approach and selecting multiple isolates per sample are essential for more complete Campylobacter recovery to support outbreak investigation and source attribution.

CemR atypical response regulator impacts energy conversion in Campylobacteria.

Campylobacter jejuni and Arcobacter butzleri are microaerobic food-borne human gastrointestinal pathogens that mainly cause diarrheal disease. These related species of the Campylobacteria class face variable atmospheric environments during infection and transmission, ranging from nearly anaerobic to aerobic conditions. Consequently, their lifestyles require that both pathogens need to adjust their metabolism and respiration to the changing oxygen concentrations of the colonization sites. Our transcriptomic and proteomic studies revealed that C. jejuni and A. butzleri, lacking a Campylobacteria-specific regulatory protein, C. jejuni Cj1608, or a homolog, A. butzleri Abu0127, are unable to reprogram tricarboxylic acid cycle or respiration pathways, respectively, to produce ATP efficiently and, in consequence, adjust growth to changing oxygen supply. We propose that these Campylobacteria energy and metabolism regulators (CemRs) are long-sought transcription factors controlling the metabolic shift related to oxygen availability, essential for these bacteria's survival and adaptation to the niches they inhabit. Besides their significant universal role in Campylobacteria, CemRs, as pleiotropic regulators, control the transcription of many genes, often specific to the species, under microaerophilic conditions and in response to oxidative stress. IMPORTANCE: C. jejuni and A. butzleri are closely related pathogens that infect the human gastrointestinal tract. In order to infect humans successfully, they need to change their metabolism as nutrient and respiratory conditions change. A regulator called CemR has been identified, which helps them adapt their metabolism to changing conditions, particularly oxygen availability in the gastrointestinal tract so that they can produce enough energy for survival and spread. Without CemR, these bacteria, as well as a related species, Helicobacter pylori, produce less energy, grow more slowly, or, in the case of C. jejuni, do not grow at all. Furthermore, CemR is a global regulator that controls the synthesis of many genes in each species, potentially allowing them to adapt to their ecological niches as well as establish infection. Therefore, the identification of CemR opens new possibilities for studying the pathogenicity of C. jejuni and A. butzleri.

Campylobacter jejuni and Campylobacter coli infection, determinants and antimicrobial resistance patterns among under-five children with diarrhea in Amhara National Regional State, Northwest Ethiopia.

BACKGROUND: Children with under-five year age disproportionally affected with foodborne illness. Campylobacteriosis is the most common foodborne disease next to Norovirus infection. Macrolides are commonly prescribed as the first line of treatment for Campylobacter gastroenteritis, with fluoroquinolone and tetracycline as secondary options. However, resistance to these alternatives has been reported in various regions worldwide. OBJECTIVE: To determine the prevalence, associated risk-factors and antimicrobial resistance of Campylobacter jejuni and C. coli among under-five children with diarrhea. METHODS: Institution-based cross-sectional study was conducted from November, 2022 to April 2023. The study sites were selected using a random sampling technique, while the study subjects were included using a convenient sampling technique. The data were collected using a structured questionnaire. Stool samples were inoculated onto modified charcoal cefoperazone deoxycholate agar and incubated for 48 hours. The suspected colonies were analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry to confirm the species. Antimicrobial susceptibility testing was performed using a disc diffusion technique. All potential covariates (independent variables) were analyzed one by one using bivariate logistic regression model to identify candidate variables with P value < 0.25. Multivariable logistic analysis was used to identify potential associated factors using the candidate variables. A p value ≤ 0.05 at a 95% confidence interval was statistically significant. RESULT: Among the 428 samples, 7.0% (CI: 4.5-9.3) were confirmed Campylobacter species. The prevalence of C. jejuni and C. coli among under-five children was 5.1% (CI: 3.0-7.0) and 1.9% (CI: 0.7-3.3), respectively. C. jejuni (73.3%) was dominant over C. coli (26.7%). The resident, contact with domestic animals, and parents/guardians education level were significantly associated with campylobacteriosis among under-five children. One-third of the Campylobacter isolates (33.3%, 10/30) were resistant to ciprofloxacin and tetracycline whereas 10.0% (3/30) were resistant to erythromycin. Furthermore, 3.3% (1/30) of the Campylobacter were found to be multidrug-resistant. CONCLUSION: The prevalence of Campylobacter species was 7.0%. The resistance rate of Campylobacter species of ciprofloxacin and tetracycline-resistance strains was 33.3%. Peri-urban residence, contact with domestic animals, and low parental educational statuses were significantly associated factors with increased risk of Campylobacter infection. Continuous surveillance on antimicrobial resistance and health education of personal and environmental hygiene should be implemented in the community.

Active Site Characterization of a Campylobacter jejuni Nitrate Reductase Variant Provides Insight into the Enzyme Mechanism.

Mo K-edge X-ray absorption spectroscopy (XAS) is used to probe the structure of wild-type Campylobacter jejuni nitrate reductase NapA and the C176A variant. The results of extended X-ray absorption fine structure (EXAFS) experiments on wt NapA support an oxidized Mo(VI) hexacoordinate active site coordinated by a single terminal oxo donor, four sulfur atoms from two separate pyranopterin dithiolene ligands, and an additional S atom from a conserved cysteine amino acid residue. We found no evidence of a terminal sulfido ligand in wt NapA. EXAFS analysis shows the C176A active site to be a 6-coordinate structure, and this is supported by EPR studies on C176A and small molecule analogs of Mo(V) enzyme forms. The SCys is replaced by a hydroxide or water ligand in C176A, and we find no evidence of a coordinated sulfhydryl (SH) ligand. Kinetic studies show that this variant has completely lost its catalytic activity toward nitrate. Taken together, the results support a critical role for the conserved C176 in catalysis and an oxygen atom transfer mechanism for the catalytic reduction of nitrate to nitrite that does not employ a terminal sulfido ligand in the catalytic cycle.

Five centuries of genome evolution and multi-host adaptation of Campylobacter jejuni in Brazil.

Consumption of raw, undercooked or contaminated animal food products is a frequent cause of Campylobacter jejuni infection. Brazil is the world's third largest producer and a major exporter of chicken meat, yet population-level genomic investigations of C. jejuni in the country remain scarce. Analysis of 221 C. jejuni genomes from Brazil shows that the overall core and accessory genomic features of C. jejuni are influenced by the identity of the human or animal source. Of the 60 sequence types detected, ST353 is the most prevalent and consists of samples from chicken and human sources. Notably, we identified the presence of diverse bla genes from the OXA-61 and OXA-184 families that confer beta-lactam resistance as well as the operon cmeABCR related to multidrug efflux pump, which contributes to resistance against tetracyclines, macrolides and quinolones. Based on limited data, we estimated the most recent common ancestor of ST353 to the late 1500s, coinciding with the time the Portuguese first arrived in Brazil and introduced domesticated chickens into the country. We identified at least two instances of ancestral chicken-to-human infections in ST353. The evolution of C. jejuni in Brazil was driven by the confluence of clinically relevant genetic elements, multi-host adaptation and clonal population growth that coincided with major socio-economic changes in poultry farming.

Dual bacteriocin and extracellular vesicle-mediated inhibition of Campylobacter jejuni by the potential probiotic candidate Ligilactobacillus salivarius UO.C249.

Campylobacter jejuni (C. jejuni) is one of the most common causes of foodborne infections worldwide and a major contributor to diarrheal diseases. This study aimed to explore the ability of commensal gut bacteria to control C. jejuni infection. Bacterial strains from the intestinal mucosa of broilers were screened in vitro against C. jejuni ATCC BAA1153. The cell-free supernatant (CFS) of Ligilactobacillus salivarius UO.C249 showed potent dose-dependent antimicrobial activity against the pathogen, likely due to the presence of bacteriocin-like moieties, as confirmed by protease treatment. Genome and exoproteome analyses revealed the presence of known bacteriocins, including Abp118. The genome of Lg. salivarius UO.C249 harbors a 1.8-Mb chromosome and a 203-kb megaplasmid. The strain was susceptible to several antibiotics and had a high survival rate in the simulated chicken gastrointestinal tract (GIT). Post-protease treatment revealed residual inhibitory activity, suggesting alternative antimicrobial mechanisms. Short-chain fatty acid (SCFA) quantification confirmed non-inhibitory levels of acetic (24.4 ± 1.2 mM), isovaleric (34 ± 1.0 µM), and butyric (32 ± 2.5 µM) acids. Interestingly, extracellular vesicles (EVs) isolated from the CFS of Lg. salivarius UO.C249 were found to inhibit C. jejuni ATCC BAA-1153. Proteome profiling of these EVs revealed the presence of unique proteins distinct from bacteriocins identified in CFS. The majority of the identified proteins in EVs are located in the membrane and play roles in transmembrane transport and peptidoglycan degradation, peptidase, proteolysis, and hydrolysis. These findings suggest that although bacteriocins are a primary antimicrobial mechanism, EV production also contributes to the inhibitory activity of Lg. salivarius UO.C249 against C. jejuni. IMPORTANCE: Campylobacter jejuni (C. jejuni) is a major cause of gastroenteritis and a global public health concern. The increasing antibiotic resistance and lack of effective alternatives in livestock production pose serious challenges for controlling C. jejuni infections. Therefore, alternative strategies are needed to control this pathogen, especially in the poultry industry where it is prevalent and can be transmitted to humans through contaminated food products. In this study, Ligilactobacillus salivarius UO.C249 isolated from broiler intestinal mucosa inhibited C. jejuni and exhibited important probiotic features. Beyond bacteriocins, Lg. salivarius UO.C249 secretes antimicrobial extracellular vesicles (EVs) with a unique protein set distinct from bacteriocins that are involved in transmembrane transport and peptidoglycan degradation. Our findings suggest that beyond bacteriocins, EV production is also a distinct inhibitory signaling mechanism used by Lg. salivarius UO.C249 to control C. jejuni. These findings hold promise for the application of probiotic EVs for pathogen control.

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.

Campylobacter occurrence and antimicrobial resistance profile in under five-year-old diarrheal children, backyard farm animals, and companion pets.

Campylobacteriosis disproportionately affects children under five in low-income countries. However, epidemiological and antimicrobial resistance (AMR) information at the children-animal interface is lacking. We hypothesized that Campylobacter is a major cause of enteritis in children in Ethiopia, and contact with animals is a potential source of transmission. The objective of the study was to determine Campylobacter occurrence and its AMR in children under five with diarrhea, backyard farm animals, and companion pets. Stool from 303 children and feces from 711 animals were sampled. Campylobacter was isolated through membrane filtration on modified charcoal cefoperazone deoxycholate agar plates under microaerobic incubation, and the technique showed to be feasible for use in regions lacking organized laboratories. Typical isolates were characterized with MALDI-TOF MS and multiplex PCR. Of 303 children, 20% (n = 59) were infected, with a higher proportion in the 6 to 11-month age group. Campylobacter occurred in 64% (n = 14) of dogs and 44% (n = 112) of poultry. Campylobacter jejuni was present in both a child and animal species in 15% (n = 23) of 149 households positive for Campylobacter. MICs using the gradient strip diffusion test of 128 isolates displayed resistance rates of 20% to ciprofloxacin and 11% to doxycycline. MICs of ciprofloxacin and doxycycline varied between C. coli and C. jejuni, with higher resistance in C. coli and poultry isolates. Campylobacter infection in children and its prevalent excretion from backyard poultry and dogs is a understudied concern. The co-occurrence of C. jejuni in animals and children suggest household-level transmission As resistance to ciprofloxacin and doxycycline was observed, therapy of severe campylobacteriosis should consider susceptibility testing. Findings from this study can support evidence-based diagnosis, antimicrobial treatment, and further investigations on the spread of AMR mechanisms for informed One Health intervention.

Interplay of two small RNAs fine-tunes hierarchical flagella gene expression in Campylobacter jejuni.

Like for many bacteria, flagella are crucial for Campylobacter jejuni motility and virulence. Biogenesis of the flagellar machinery requires hierarchical transcription of early, middle (RpoN-dependent), and late (FliA-dependent) genes. However, little is known about post-transcriptional regulation of flagellar biogenesis by small RNAs (sRNAs). Here, we characterized two sRNAs with opposing effects on C. jejuni filament assembly and motility. We demonstrate that CJnc230 sRNA (FlmE), encoded downstream of the flagellar hook protein, is processed from the RpoN-dependent flgE mRNA by RNase III, RNase Y, and PNPase. We identify mRNAs encoding a flagella-interaction regulator and the anti-sigma factor FlgM as direct targets of CJnc230 repression. CJnc230 overexpression upregulates late genes, including the flagellin flaA, culminating in longer flagella and increased motility. In contrast, overexpression of the FliA-dependent sRNA CJnc170 (FlmR) reduces flagellar length and motility. Overall, our study demonstrates how the interplay of two sRNAs post-transcriptionally fine-tunes flagellar biogenesis through balancing of the hierarchically-expressed components.

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.