Design of a multi-epitope-based vaccine candidate against Bovine Genital Campylobacteriosis using a reverse vaccinology approach.

BACKGROUND: Bovine Genital Campylobacteriosis (BGC), a worldwide distributed venereal disease caused by Campylobacter fetus subsp. venerealis (Cfv), has a relevant negative economic impact in cattle herds. The control of BGC is hampered by the inexistence of globally available effective vaccines. The present in silico study aimed to develop a multi-epitope vaccine candidate against Cfv through reverse vaccinology. RESULTS: The analysis of Cfv strain NCTC 10354 proteome allowed the identification of 9 proteins suitable for vaccine development. From these, an outer membrane protein, OmpA, and a flagellar protein, FliK, were selected for prediction of B-cell and T-cell epitopes. The top-ranked epitopes conservancy was assessed in 31 Cfv strains. The selected epitopes were integrated to form a multi-epitope fragment of 241 amino acids, which included 2 epitopes from OmpA and 13 epitopes from FliK linked by GPGPG linkers and connected to the cholera toxin subunit B by an EAAAK linker. The vaccine candidate was predicted to be antigenic, non-toxic, non-allergenic, and soluble upon overexpression. The protein structure was predicted and optimized, and the sequence was successfully cloned in silico into a plasmid vector. Additionally, immunological simulations demonstrated the vaccine candidate's ability to stimulate an immune response. CONCLUSIONS: This study developed a novel vaccine candidate suitable for further in vitro and in vivo experimental validation, which may become a useful tool for the control of BGC.

Biochemical and molecular characterization of Campylobacter fetus isolates from bulls subjected to bovine genital campylobacteriosis diagnosis in Spain.

BACKGROUND: Bovine genital campylobacteriosis (BGC) is caused by Campylobacter fetus subsp. venerealis (Cfv) including its biovar intermedius (Cfvi). This sexually transmitted disease induces early reproductive failure causing considerable economic losses in the cattle industry. Using a collection of well-characterized isolates (n = 13), C. fetus field isolates (n = 64) and saprophytic isolates resembling Campylobacter (n = 75) obtained from smegma samples of breeding bulls, this study evaluated the concordance of the most used phenotypic (H2S production in cysteine medium and 1% glycine tolerance) and molecular (PCR) methods for the diagnosis of BGC and assessed possible cross-reactions in the molecular diagnostic methods. RESULTS: Characterization at the subspecies level (fetus vs. venerealis) of C. fetus isolated from bull preputial samples using phenotypic and molecular (PCR targeting nahE and ISCfe1) methods showed moderate concordance (κ = 0.462; CI: 0.256-0.669). No cross-reactions were observed with other saprophytic microaerophilic species or with other Campylobacter species that can be present in preputial samples. Whole genome sequencing (WGS) of discrepant isolates showed 100% agreement with PCR identification. For the differentiation of Cfv biovars, comparison of the H2S test (at 72 h and 5 days of incubation) and a PCR targeting the L-cysteine transporter genes showed higher concordance when H2S production was assessed after 5 days (72 h; κ = 0.553, 0.329-0.778 CI vs. 5 days; κ = 0.881, 0.631-1 CI), evidencing the efficacy of a longer incubation time. CONCLUSIONS: This study confirmed the limitations of biochemical tests to correctly identify C. fetus subspecies and biovars. However, in the case of biovars, when extended incubation times for the H2S test (5 days) were used, phenotypic identification results were significantly improved, although PCR-based methods produced more accurate results. Perfect agreement of WGS with the PCR results and absence of cross-reactions with non-C. fetus saprophytic bacteria from the smegma demonstrated the usefulness of these methods. Nevertheless, the identification of new C. fetus subspecies-specific genes would help to improve BGC diagnosis.

Positive cases of bovine genital campylobacteriosis and bovine trichomonosis in the eradication program of La Pampa, Argentina: 2008-2021.

Bovine genital campylobacteriosis, caused by the gram-negative bacteria Campylobacter fetus venerealis, and bovine trichomonosis, caused by the parasite protozoan Tritrichomonas foetus, are venereal diseases that occur with long intercalving periods and abortion. The control of both diseases relies on microbiological testing and culling infected bulls. Vaccination and antibiotic treatment may help in controlling campylobacteriosis but are not recommended for trichomonosis control. Several regions of the world have active control programs for trichomonosis, not campylobacteriosis. In Argentina, the state of La Pampa aims to eradicate trichomonosis and campylobacteriosis by imposing annual diagnostic testing of every bull and slaughtering positive animals. Prior studies indicated a declining trend in the prevalence of campylobacteriosis and trichomonosis in La Pampa. It was also proposed that the prevalence of one disease could be estimated from the prevalence of the other. The purpose of this retrospective analysis of data gathered from 2008 to 2021 was to determine the La Pampa program's efficacy. Descriptive statistics were employed to determine the reason behind the correlation between tricomonosis and campylobacteriosis diagnostic results. The outcomes refute the notion that this program of venereal eradication was a success. Furthermore, an excess of false positives in both diagnoses may have contributed to the correlation between the prevalences of campylobactriosis and trichomonosis. The practice of killing animals without verifying positive results hinders the determination of disease prevalence and results in the death of numerous healthy animals.

Menthol Pretreatment Alleviates Campylobacter jejuni-Induced Enterocolitis in Human Gut Microbiota-Associated IL-10-/- Mice.

Human Campylobacter jejuni infections are of worldwide importance and represent the most commonly reported bacterial enteritis cases in middle- and high-income countries. Since antibiotics are usually not indicated and the severity of campylobacteriosis is directly linked to the risk of developing post-infectious complications, non-toxic antibiotic-independent treatment approaches are highly desirable. Given its health-promoting properties, including anti-microbial and anti-inflammatory activities, we tested the disease-alleviating effects of oral menthol in murine campylobacteriosis. Therefore, human gut microbiota-associated IL-10-/- mice were orally subjected to synthetic menthol starting a week before C. jejuni infection and followed up until day 6 post-infection. Whereas menthol pretreatment did not improve campylobacteriosis symptoms, it resulted in reduced colonic C. jejuni numbers and alleviated both macroscopic and microscopic aspects of C. jejuni infection in pretreated mice vs. controls. Menthol pretreatment dampened the recruitment of macrophages, monocytes, and T lymphocytes to colonic sites of infection, which was accompanied by mitigated intestinal nitric oxide secretion. Furthermore, menthol pretreatment had only marginal effects on the human fecal gut microbiota composition during the C. jejuni infection. In conclusion, the results of this preclinical placebo-controlled intervention study provide evidence that menthol application constitutes a promising way to tackle acute campylobacteriosis, thereby reducing the risk for post-infectious complications.

In-vitro selection of lactic acid bacteria to combat Salmonella enterica and Campylobacter jejuni in broiler chickens.

Campylobacter and Salmonella are the two most prominent foodborne zoonotic pathogens reported in the European Union. As poultry is one of the major sources of these pathogens, it is imperative to mitigate the colonization of these pathogens in poultry. Many strains of lactic acid bacteria (LAB) have demonstrated anti-Salmonella and anti-Campylobacter characteristics to varying degrees and spectrums which are attributed to the production of various metabolites. However, the production of these compounds and consequent antimicrobial properties are highly strain dependent. Therefore, the current study was performed to select a potent LAB and determine its causal attribute in inhibiting Salmonella enterica and Campylobacter jejuni, in-vitro. Six LAB (Lactiplantibacillus plantarum (LP), Lacticaseibacillus casei (LC), Limosilactobacillus reuteri (LR), Lacticaseibacillus rhamnosus (LRh), Leuconostoc mesenteroides (LM) and Pediococcus pentosaceus (PP)) and three serovars of Salmonella enterica (Typhimurium, Enterica and Braenderup) and Campylobacter jejuni were used in the current study. Spot overlays, well diffusion, co-culture and co-aggregation assays against Salmonella and well diffusion assays against Campylobacter jejuni were performed. Organic acid profiling of culture supernatants was performed using HPLC. The results indicated that LRh, LM and PP had the most significant anti-Salmonella effects while LP, LC, LM and PP displayed the most significant anti-Campylobacter effects. Lactic acid and formic acid detected in the culture supernatants seem the most likely source of the anti-Salmonella and anti-Campylobacter effects exhibited by these LAB. In conclusion, Leuconostoc mesenteroides displayed the most significant overall anti-pathogenic effects when compared to the other LAB strains studied, indicating its potential application in-vivo.

A broad host phage, CP6, for combating multidrug-resistant Campylobacter prevalent in poultry meat.

Campylobacter is a major cause of bacterial foodborne diarrhea worldwide. Consumption of raw or undercooked chicken meat contaminated with Campylobacter is the most common causative agent of human infections. Given the high prevalence of contamination in poultry meat and the recent rise of multi-drug-resistant (MDR) Campylobacter strains, an effective intervention method of reducing bird colonization is needed. In this study, the Campylobacter-specific lytic phage CP6 was isolated from chicken feces. Phage CP6 exhibited a broad host range against different MDR Campylobacter isolates (97.4% of strains were infected). Some biological characteristics were observed, such as a good pH (3-9) stability and moderate temperature tolerance (<50 ℃). The complete genome sequence revealed a linear double-stranded DNA (178,350 bp, group II Campylobacter phage) with 27.51% GC content, including 209 predicted open reading frames, among which only 54 were annotated with known functions. Phylogenetic analysis of the phage major capsid protein demonstrated that phage CP6 was closely related to Campylobacter phage CPt10, CP21, CP20, IBB35, and CP220. CP6 phage exerted good antimicrobial effects on MDR Campylobacter in vitro culture and reduced CFUs of the host cells by up to 1-log compared with the control in artificially contaminated chicken breast meat. Our findings suggested the potential of CP6 phage as a promising antimicrobial agent for combating MDR Campylobacter in food processing.

Novel rpsK / rpsD primer-probe assay improves detection of Campylobacter jejuni and Campylobacter coli in human stool.

Campylobacter causes bacterial enteritis, dysentery, and growth faltering in children in low- and middle-income countries (LMICs). Campylobacter spp. are fastidious organisms, and their detection often relies on culture independent diagnostic technologies, especially in LMICs. Campylobacter jejuni and Campylobacter coli are most often the infectious agents and in high income settings together account for 95% of Campylobacter infections. Several other Campylobacter species have been detected in LMIC children at an increased prevalence relative to high income settings. After doing extensive whole genome sequencing of isolates of C. jejuni and C. coli in Peru, we observed heterogeneity in the binding sites for the main species-specific PCR assay (cadF) and designed an alternative rpsKD-based qPCR assay to detect both C. jejuni and C. coli. The rpsKD-based qPCR assay identified 23% more C.jejuni/ C.coli samples than the cadF assay among 47 Campylobacter genus positive cadF negative samples verified to have C. jejuni and or C. coli with shotgun metagenomics. This assay can be expected to be useful in diagnostic studies of enteric infectious diseases and be useful in revising the attribution estimates of Campylobacter in LMICs.

Comparative pangenomic analysis of Campylobacter fetus isolated from Spanish bulls and other mammalian species.

Campylobacter fetus comprises two closely related mammal-associated subspecies: Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv). The latter causes bovine genital campylobacteriosis, a sexually-transmitted disease endemic in Spain that results in significant economic losses in the cattle industry. Here, 33 C. fetus Spanish isolates were whole-genome sequenced and compared with 62 publicly available C. fetus genomes from other countries. Genome-based taxonomic identification revealed high concordance with in silico PCR, confirming Spanish isolates as Cff (n = 4), Cfv (n = 9) and Cfv biovar intermedius (Cfvi, n = 20). MLST analysis assigned the Spanish isolates to 6 STs, including three novel: ST-76 and ST-77 for Cfv and ST-78 for Cff. Core genome SNP phylogenetic analysis of the 95 genomes identified multiple clusters, revealing associations at subspecies and biovar level between genomes with the same ST and separating the Cfvi genomes from Spain and other countries. A genome-wide association study identified pqqL as a Cfv-specific gene and a potential candidate for more accurate identification methods. Functionality analysis revealed variations in the accessory genome of C. fetus subspecies and biovars that deserve further studies. These results provide valuable information about the regional variants of C. fetus present in Spain and the genetic diversity and predicted functionality of the different subspecies.

Acquisition and clearance dynamics of Campylobacter spp. in children in low- and middle-income countries.

The prevalence of Campylobacter infection is generally high among children in low- and middle-income countries (LMIC), but the dynamics of its acquisition and clearance are understudied. We aim to quantify this process among children under two years old in eight LMIC using a statistical modeling approach, leveraging enzyme-immunoassay-based Campylobacter genus data and quantitative-PCR-based Campylobacter jejuni/coli data from the MAL-ED study. We developed a Markov model to compare the dynamics of acquisition and clearance of Campylobacter across countries and to explore the effect of antibiotic usage on Campylobacter clearance. Clearance rates were generally higher than acquisition rates, but their magnitude and temporal pattern varied across countries. For C. jejuni/coli, clearance was faster than acquisition throughout the two years at all sites. For Campylobacter spp., the acquisition rate either exceeded or stayed very close to the clearance rate after the first half year in Bangladesh, Pakistan and Tanzania, leading to high prevalence. Bangladesh had the shortest (28 and 57 days) while Brazil had the longest (328 and 306 days) mean times from last clearance to acquisition for Campylobacter spp. and C. jejuni/coli, respectively. South Africa had the shortest (10 and 8 days) while Tanzania had the longest (53 and 41 days) mean times to clearance for Campylobacter spp. and C. jejuni/col, respectively. The use of Macrolide accelerated clearance of C. jejuni/coli in Bangladesh and Peru and of Campylobacter spp. in Bangladesh and Pakistan. Fluoroquinolone showed statistically meaningful effects only in Bangladesh but for both Campylobacter groups. Higher prevalence of Campylobacter infection was mainly driven by a high acquisition rate that was close to or surpassing the clearance rate. Acquisition rate usually peaked in 11-17 months of age, indicating the importance of targeting the first year of life for effective interventions to reduce exposures.

Prophylactic Oral Application of Activated Charcoal Mitigates Acute Campylobacteriosis in Human Gut Microbiota-Associated IL-10-/- Mice.

The incidence of human Campylobacter jejuni infections is increasing worldwide. It is highly desirable to prevent campylobacteriosis in individuals at risk for severe disease with antibiotics-independent non-toxic compounds. Activated charcoal (AC) has long been used as an anti-diarrheal remedy. Here, we tested the disease-mitigating effects of oral AC versus placebo in human gut microbiota-associated (hma) IL-10-/- mice starting a week prior to C. jejuni infection. On day 6 post-infection, the gastrointestinal C. jejuni loads were comparable in both infected cohorts, whereas campylobacteriosis symptoms such as wasting and bloody diarrhea were mitigated upon AC prophylaxis. Furthermore, AC application resulted in less pronounced C. jejuni-induced colonic epithelial cell apoptosis and in dampened innate and adaptive immune cell responses in the colon that were accompanied by basal concentrations of pro-inflammatory mediators including IL-6, TNF-α, IFN-γ, and nitric oxide measured in colonic explants from AC treated mice on day 6 post-infection. Furthermore, C. jejuni infection resulted in distinct fecal microbiota shift towards higher enterobacterial numbers and lower loads of obligate anaerobic species in hma mice that were AC-independent. In conclusion, our pre-clinical placebo-controlled intervention study provides evidence that prophylactic oral AC application mitigates acute murine campylobacteriosis.

Clinical isolate characteristics and demographics of patients with C.jejuni and C.coli infections in Northern Israel, 2015-2021.

C.coli is a significant cause of foodborne gastroenteritis worldwide, with the majority of cases attributed to C.jejuni. Although most clinical laboratories do not typically conduct antimicrobial susceptibility testing for C.coli, the rise in resistant strains has underscored the necessity for such testing and epidemiological surveillance. The current study presents clinical isolate characteristics and demographics of 221 patients with C.coli (coli and jejuni) infections in Northern Israel, between 2015 and 2021. Clinical and demographic data were collected from patient medical records. Susceptibility to erythromycin, tetracycline, ciprofloxacin, and gentamicin was assessed using the standard E-test. No significant correlations were found between bacterial species and patient ethnicity, patient gender, or duration of hospitalization. In contrast, significant differences were found between infecting species and patient age and age subgroup (P < 0.001). Furthermore, erythromycin resistance was observed in only 0.5% of the study population, while resistance to ciprofloxacin, tetracycline, and gentamicin was observed in 95%, 93%, and 2.3% of the population, respectively. The presented study underscores the need for routine surveillance of C.coli antibiotic resistance.

Effect of the polysaccharide capsule and its heptose on the resistance of Campylobacter jejuni to innate immune defenses.

Campylobacter jejuni is a commensal in many animals but causes diarrhea in humans. Its polysaccharide capsule contributes to host colonization and virulence in a strain- and model-specific manner. We investigated if the capsule and its heptose are important for interactions of strain NCTC 11168 with various hosts and their innate immune defenses. We determined that they support bacterial survival in Drosophila melanogaster and enhance virulence in Galleria mellonella. We showed that the capsule had limited antiphagocytic activity in human and chicken macrophages, decreased adherence to chicken macrophages, and decreased intracellular survival in both macrophages. In contrast, the heptose increased uptake by chicken macrophages and supported adherence to human macrophages and survival within them. While the capsule triggered nitric oxide production in chicken macrophages, the heptose mitigated this and protected against nitrosative assault. Finally, the C. jejuni strain NCTC 11168 elicited strong cytokine production in both macrophages but quenched ROS production independently from capsule and heptose, and while the capsule and heptose did not protect against oxidative assault, they favored growth in biofilms under oxidative stress. This study shows that the wild-type capsule with its heptose is optimized to resist innate defenses in strain NCTC 11168 often via antagonistic effects of the capsule and its heptose.

Detection and Distribution of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in Campylobacter jejuni Isolates from Chicken Livers.

Campylobacter jejuni is the leading foodborne bacterial pathogen that causes human gastroenteritis worldwide linked to the consumption of undercooked broiler livers. Application of bacteriophages during poultry production has been used as an alternative approach to reduce contamination of poultry meat by Campylobacter. To make this approach effective, understanding the presence of the bacteriophage sequences in the CRISPR spacers in C. jejuni is critical as they may confer bacterial resistance to bacteriophage treatment. Therefore, in this study, we explored the distribution of the CRISPR arrays from 178 C. jejuni isolated from chicken livers between January and July 2018. Genomic DNA of C. jejuni isolates was extracted, and CRISPR type 1 sequences were amplified by PCR. Amplicons were purified and sequenced by the Sanger dideoxy sequencing method. Direct repeats (DRs) and spacers of CRISPR sequences were identified using the CRISPRFinder program. Further, spacer sequences were submitted to the CRISPRTarget to identify potential homology to bacteriophage types. Even though CRISPR-Cas is reportedly not an active system in Campylobacter, a total of 155 (87%) C. jejuni isolates were found to harbor CRISPR sequences; one type of DR was identified in all 155 isolates. The CRISPR loci lengths ranged from 97 to 431 nucleotides. The numbers of spacers ranged from one to six. A total of 371 spacer sequences were identified in the 155 isolates that could be grouped into 51 distinctive individual sequences. Further comparison of these 51 spacer sequences with those in databases showed that most spacer sequences were homologous to Campylobacter bacteriophage DA10. The results of our study provide important information relative to the development of an effective bacteriophage treatment to mitigate Campylobacter during poultry production.

Whole genome sequence-based characterization of Campylobacter isolated from broiler carcasses over a three-year period in a big poultry slaughterhouse reveals high genetic diversity and a recurring genomic lineage of Campylobacter jejuni.

Campylobacter is among the most frequent agents of bacterial gastroenteritis in Europe and is primarily linked to the consumption of contaminated food. The aim of this study was to assess genomic diversity and to identify antimicrobial resistance and virulence genes of 155 Campylobacter isolated from broiler carcasses (neck skin samples) in a large-scale Swiss poultry abattoir over a three-year period. Samples originated from broilers from three different types of farming systems (particularly animal-friendly stabling (PAFS), free-range farms, and organic farms). Campylobacter jejuni (n = 127) and Campylobacter coli (n = 28) were analysed using a whole genome sequencing (WGS) approach (MiniSeq; Illumina). Sequence types (STs) were determined in silico from the WGS data and isolates were assigned into complex types (CTs) using the cgMLST SeqSphere+ scheme. Antimicrobial resistance genes were identified using the Resistance Gene Identifier (RGI), and virulence genes were identified using the virulence factor database (VFDB). A high degree of genetic diversity was observed. Many sequence types (C. jejuni ST19, ST21, ST48, ST50, ST122, ST262 and C. coli ST827) occurred more than once and were distributed throughout the study period, irrespective of the year of isolation and of the broiler farming type. Antimicrobial resistance determinants included blaOXA and tet(O) genes, as well as the T86I substitution within GyrA. Virulence genes known to play a role in human Campylobacter infection were identified such as the wlaN, cstIII, neuA1, neuB1, and neuC1. Subtyping of the Campylobacter isolates identified the occurrence of a highly clonal population of C. jejuni ST21 that was isolated throughout the three-year study period from carcasses from farms with geographically different locations and different farming systems. The high rate of genetic diversity observed among broiler carcass isolates is consistent with previous studies. The identification of a persisting highly clonal C. jejuni ST21 subtype suggests that the slaughterhouse may represent an environment in which C. jejuni ST21 may survive, however, the ecological reservoir potentially maintaining this clone remains unknown.

Molecular characterization of Campylobacter spp. isolates obtained from commercial broilers and native chickens in Southern Thailand using whole genome sequencing.

Chickens are the primary reservoirs of Campylobacter spp., mainly C. jejuni and C. coli, that cause human bacterial gastrointestinal infections. However, genomic characteristics and antimicrobial resistance of Campylobacter spp. in low- to middle-income countries need more comprehensive exploration. This study aimed to characterize 21 C. jejuni and 5 C. coli isolates from commercial broilers and native chickens using whole genome sequencing and compare them to 28 reference Campylobacter sequences. Among the 26 isolates, 13 sequence types (ST) were identified in C. jejuni and 5 ST in C. coli. The prominent ST was ST 2274 (5 isolates, 19.2%), followed by ST 51, 460, 2409, and 6455 (2 isolates in each ST, 7.7%), while all remaining ST (464, 536, 595, 2083, 6736, 6964, 8096, 10437, 828, 872, 900, 8237, and 13540) had 1 isolate per ST (3.8%). Six types of antimicrobial resistance genes (ant(6)-Ia, aph(3')-III, blaOXA, cat, erm(B), and tet(O)) and one point mutations in the gyrA gene (Threonine-86-Isoleucine) and another in the rpsL gene (Lysine-43-Arginine) were detected. The blaOXA resistance gene was present in all isolates, the gyrA mutations was in 95.2% of C. jejuni and 80.0% of C. coli, and the tet(O) resistance gene in 76.2% of C. jejuni and 80.0% of C. coli. Additionally, 203 virulence-associated genes linked to 16 virulence factors were identified. In terms of phenotypic resistance, the C. jejuni isolates were all resistant to ciprofloxacin, enrofloxacin, and nalidixic acid, with lower levels of resistance to tetracycline (76.2%), tylosin (52.3%), erythromycin (23.8%), azithromycin (22.2%), and gentamicin (11.1%). Most C. coli isolates were resistant to all tested antimicrobials, while 1 C. coli was pan-susceptible except for tylosin. Single-nucleotide polymorphisms concordance varied widely, with differences of up to 13,375 single-nucleotide polymorphisms compared to the reference Campylobacter isolates, highlighting genetic divergence among comparative genomes. This study contributes to a deeper understanding of the molecular epidemiology of Campylobacter spp. in Thai chicken production systems.

Monitoring antimicrobial resistance in Campylobacter isolates of chickens and turkeys at the slaughter establishment level across the United States, 2013-2021.

Foodborne infections with antimicrobial-resistant Campylobacter spp. remain an important public health concern. Publicly available data collected by the National Antimicrobial Resistance Monitoring System for Enteric Bacteria related to antimicrobial resistance (AMR) in Campylobacter spp. isolated from broiler chickens and turkeys at the slaughterhouse level across the United States between 2013 and 2021 were analysed. A total of 1,899 chicken-origin (1,031 Campylobacter coli (C. coli) and 868 Campylobacter jejuni (C. jejuni)) and 798 turkey-origin (673 C. coli and 123 C. jejuni) isolates were assessed. Chicken isolates exhibited high resistance to tetracycline (43.65%), moderate resistance to ciprofloxacin (19.5%), and low resistance to clindamycin (4.32%) and azithromycin (3.84%). Turkey isolates exhibited very high resistance to tetracycline (69%) and high resistance to ciprofloxacin (39%). The probability of resistance to all tested antimicrobials, except for tetracycline, significantly decreased during the latter part of the study period. Turkey-origin Campylobacter isolates had higher odds of resistance to all antimicrobials than isolates from chickens. Compared to C. jejuni isolates, C. coli isolates had higher odds of resistance to all antimicrobials, except for ciprofloxacin. The study findings emphasize the need for poultry-type-specific strategies to address differences in AMR among Campylobacter isolates.

Identification of knowledge gaps in whole-genome sequence analysis of multi-resistant thermotolerant Campylobacter spp.

BACKGROUND: Campylobacter spp. is the most frequent cause of bacterial food-borne gastroenteritis and a high priority antibiotic resistant bacterium according to the World Health Organization (WHO). European monitoring of thermotolerant Campylobacter spp. does not reflect the global burden of resistances already circulating within the bacterial population worldwide. METHODS: We systematically compared whole genome sequencing with comprehensive phenotypic antimicrobial susceptibility, analyzing 494 thermotolerant Campylobacter poultry isolates from Vietnam and Germany. Any discrepancy was checked by repeating the wet lab and improving the dry lab part. Selected isolates were additionally analyzed via long-read Oxford Nanopore technology, leading to closed chromosomes and plasmids. RESULTS: Overall, 22 different resistance genes and gene variants (e. g. erm(B), aph(3')-IIIa, aph(2'')-If, catA, lnu(C), blaOXA, sat4) and point mutations in three distinct genes (gyrA, 23S rRNA, rpsL) associated with AMR were present in the Campylobacter isolates. Two AMR genes were missing in the database and one falsely associated with resistance. Bioinformatic analysis based on short-read data partly failed to identify tet(O) and aadE, when the genes were present as duplicate or homologous gene variants. Intriguingly, isolates also contained different determinants, redundantly conferring resistance to chloramphenicol, gentamicin, kanamycin, lincomycin and streptomycin. We found a novel tet(W) in tetracycline sensitive strains, harboring point mutations. Furthermore, analysis based on assemblies from short-read data was impaired to identify full length phase variable aad9, due to variations of the poly-C tract within the gene. The genetic determinant responsible for gentamicin resistance of one isolate from Germany could not be identified. GyrT86I, presenting the main determinant for (fluoro-)quinolone resistance led to a rare atypical phenotype of ciprofloxacin resistance but nalidixic acid sensitivity. Long-read sequencing predicted AMR genes were mainly located on the chromosome, and rarely on plasmids. Predictions from long- and short-read sequencing, respectively, often differed. AMR genes were often organized in multidrug resistance islands (MDRI) and partially located in proximity to transposase genes, suggesting main mobilization of resistance determinants is via natural transformation and transposition in Campylobacter. CONCLUSIONS: The results of this study suggest that there is frequent resistance gene duplication, mosaicism, and mutation leading to gene variation and truncation in Campylobacter strains that have not been reported in previous studies and are missing from databases. Furthermore, there is a need for deciphering yet unknown resistance mechanisms and resistance spread in thermotolerant Campylobacter spp. that may pose a challenge to global food safety.

Investigating bacteriophages as a novel multiple-hurdle measure against Campylobacter: field trials in commercial broiler plants.

Campylobacter mitigation along the food production chain is considered effective for minimizing the public health burden of human campylobacteriosis. This study is the first combining different measures in a multiple-hurdle approach, using drinking water additives and feed additives in single and combined application schemes in commercial broiler plants. Broiler chickens in the study groups were naturally contaminated with Campylobacter. Application of an organic acid blend via drinking water, consisting of sodium propionate, potassium sorbate, and sodium diacetate, resulted in significant reductions of up to 4.9 log10 CFU/mL in fecal samples and in cecal samples at slaughter. The application of a phage mixture, consisting of Fletchervirus phage NCTC 12673 and Firehammervirus phage vB_CcM-LmqsCPL1/1, resulted in reductions of up to 1.1 log10 CFU/mL in fecal samples 1 day after dosing. The sole administration of curcumin via feed resulted in small and inconsistent reductions. In the group receiving a combination of all tested measures, reductions of up to 1.1 log10 CFU/mL were observed. Based on the results of our field trials, it was shown that both the sole application and the combined application of mitigation measures in primary production can reduce the Campylobacter load in broiler chickens, while no synergism could be observed.

Prevalence and Antimicrobial Resistance of Campylobacter in Conventional and No Antibiotics Ever Broiler Farms.

Campylobacter is a leading cause of enteric disease worldwide. No antibiotics ever (NAE) poultry has become increasingly popular, yet little is known about the incidence and antimicrobial resistance (AMR) of Campylobacter in this production system. This study was conducted to determine the prevalence, concentration, and AMR of Campylobacter in conventional (CV) and NAE-raised broilers. Two CV and two NAE commercial broiler flocks were included in this study. Cecum (n = 420) and ileum (n = 420) of chickens were collected at different stages during the broiler grow-out phase and following transportation to the processing plant. Samples of litter (n = 24), feed (n = 24), and water (n = 24) were also collected. Screening for Campylobacter was conducted using real-time PCR assay, and enumeration was performed by direct plating on Campy Cefex agar. Campylobacter isolates were confirmed by real-time PCR, and antimicrobial susceptibility was evaluated following the National Antimicrobial Resistance Monitoring System (NARMS) methods. Whole Genome Sequencing (WGS) was used to identify AMR genes carried by the resistant isolates. Campylobacter prevalence reached 100% within the first 3 weeks of summer production under both NAE and CV rearing. A lower Campylobacter prevalence was detected in conventionally raised broilers during fall (P ≤ 0.05), yet no change in prevalence was observed in NAE birds (P > 0.05). Populations were high in the cecum, carrying an average of 6.6 Log10 CFU/g after transportation, and antimicrobial-resistant Campylobacter was isolated from CV broilers during the fall. Three isolates (1.2%), identified as C. coli, carrying the gyrA and tet(O) genes, exhibited simultaneous resistance to ciprofloxacin, tetracycline, and nalidixic acid. Results from this study can help identify important shifts in gut microbial community dynamics and Campylobacter prevalence associated with antibiotic administration within commercial poultry operations.

Identification of Campylobacter jejuni and Campylobacter coli genes contributing to oxidative stress response using TraDIS analysis.

BACKGROUND: Campylobacter jejuni and Campylobacter coli are the major causative agents of bacterial gastroenteritis worldwide and are known obligate microaerophiles. Despite being sensitive to oxygen and its reduction products, both species are readily isolated from animal food products kept under atmospheric conditions where they face high oxygen tension levels. RESULTS: In this study, Transposon Directed Insertion-site Sequencing (TraDIS) was used to investigate the ability of one C. jejuni strain and two C. coli strains to overcome oxidative stress, using H2O2 to mimic oxidative stress. Genes were identified that were required for oxidative stress resistance for each individual strain but also allowed a comparison across the three strains. Mutations in the perR and ahpC genes were found to increase Campylobacter tolerance to H2O2. The roles of these proteins in oxidative stress were previously known in C. jejuni, but this data indicates that they most likely play a similar role in C. coli. Mutation of czcD decreased Campylobacter tolerance to H2O2. The role of CzcD, which functions as a zinc exporter, has not previously been linked to oxidative stress. The TraDIS data was confirmed using defined deletions of perR and czcD in C. coli 15-537360. CONCLUSIONS: This is the first study to investigate gene fitness in both C. jejuni and C. coli under oxidative stress conditions and highlights both similar roles for certain genes for both species and highlights other genes that have a role under oxidative stress.