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.

Campylobacter jejuni continues to be a major public health issue worldwide. Poultry are recognized as the main reservoir for this foodborne pathogen. Implementing measures to decrease C. jejuni colonization on farms has been regarded as the most effective strategy to control the incidence of campylobacteriosis. The probiotics supplementation has been regarded as an attractive approach against C. jejuni in chickens. Here the inhibitory effects of one probiotic B. velezensis isolate CAU277 against C. jejuni was evaluated in vitro and in vivo. The in vitro antimicrobial activity showed that the supernatant of B. velezensis exhibited the most pronounced inhibitory effects on Campylobacter strains compared to other bacterial species. When co-cultured with B. velezensis, the growth of C. jejuni reduced significantly from 7.46 log10 CFU/mL (24 h) to 1.02 log10 CFU/mL (48 h). Further, the antimicrobial activity of B. velezensis against C. jejuni remained stable under a broad range of temperature, pH, and protease treatments. The in vivo experiments demonstrated that oral administration of B. velezensis significantly reduced the colonization of C. jejuni by 2.0 log10 CFU/g of feces in chicken cecum at 15 d postinoculation. In addition, the supplementary of B. velezensis significantly increased microbial species richness and diversity in chicken ileum, especially enhanced the bacterial population of Alistipes and Christensenellaceae, and decreased the existence of Lachnoclostridium. Our study presents that B. velezensis possesses antimicrobial activities against C. jejuni and promotes microbiota diversity in chicken intestines. These findings indicate a potential to develop an effective probiotic additive to control C. jejuni infection in chicken.

The foodborne pathogenic bacterium Campylobacter jejuni utilizes chemotaxis to assist in the colonization of host niches. A key to revealing the relationship among chemotaxis and pathogenicity is the discovery of signaling molecules perceived by the chemoreceptors. The C. jejuni chemoreceptor Tlp11 is encoded by the highly infective C. jejuni strains. In the present study, we report that the dCache-type ligand-binding domain (LBD) of C. jejuni ATCC 33560 Tlp11 binds directly to novel ligands methyl pyruvate, toluene, and quinoline using the same pocket. Methyl pyruvate elicits a strong chemoattractant response, while toluene and quinoline function as the antagonists without triggering chemotaxis. The sensory LBD was used to control heterologous proteins by constructing chimeras, indicating that the signal induced by methyl pyruvate is transmitted across the membrane. In addition, bioinformatics and experiments revealed that the dCache domains with methyl pyruvate-binding sites and ability are widely distributed in the order Campylobacterales. This is the first report to identify the class of dCache chemoreceptors that bind to attractant methyl pyruvate and antagonists toluene and quinoline. Our research provides a foundation for understanding the chemotaxis and virulence of C. jejuni and lays a basis for the control of this foodborne pathogen.

Campylobacter jejuni is recognized as a significant foodborne pathogen, and recent studies have indicated a rising trend of aminoglycosides resistance gene aph(2″)-If among C. jejuni isolates from food-producing animals in China. However, systematic information about aph(2″)-If-positive C. jejuni from food-producing animals and other sources worldwide based on whole-genome analysis remains a knowledge gap. In this study, we aimed to analyze the worldwide distribution, genetic environment and phylogenetic tree of aph(2″)-If by utilizing Whole Genome Sequencing (WGS) data obtained, coupled with information in the GenBank database. A total of 160C. jejuni isolates in the GenBank database and 14C. jejuni isolates in our laboratory carrying aph(2″)-If gene were performed for further analysis. WGS analysis revealed the global distribution of aph(2″)-If among C. jejuni from 6 countries. Multilocus Sequence Typing(MLST) results indicated that 70 STs were involved in the dissemination of aph(2″)-If, with ST10086 being the predominant ST. Whole-genome Multilocus Sequence Typing(wg-MLST) analysis according to times, countries, and origins of C. jejuni isolation further demonstrated a close relationship between aph(2″)-If carrying C. jejuni isolates from farm and food. The findings also revealed the existence of 32 distinct types of genetic environments surrounding aph(2″)-If among these isolates. Notably, Type 30, characterized by the arrangement ISsag10-deoD-ant(9)-hp-hp-aph(2″)-If, emerged as the predominant genetic environment. In conclusion, our analysis provides the inaugural perspective on the worldwide distribution of aph(2″)-If. This resistance gene demonstrates horizontal transferability and regional diffusion in a clonal pattern. The close association observed among aph(2″)-If-positive C. jejuni strains isolated from poultry, food, and clinical environments underscores the potential for zoonotic transmission from these isolates.

BACKGROUND: To establish a method to induce Campylobacter jejuni colonization in the intestines of C57BL/6 mice through antibiotic-induced microbiome depletion.
RESULTS: Fifty-four female C57BL/6 mice were divided into the normal, control, and experimental groups. The experimental group was administered intragastric cefoperazone sodium and sulbactam sodium (50 mg/mL) for 2 days; then, the experimental and control mice were intragastrically administered 200 µL C. jejuni, which was repeated once more after 2 days. Animal feces were collected, and the HipO gene of C. jejuni was detected using TaqMan qPCR from day 1 to day 14 after modeling completion. Immunofluorescence was used to detect intestinal C. jejuni colonization on day 14, and pathological changes were observed using hematoxylin and eosin staining. Additionally, 16S rDNA analyses of the intestinal contents were conducted on day 14. In the experimental group, C. jejuni was detected in the feces from days 1 to 14 on TaqMan qPCR, and immunofluorescence-labeled C. jejuni were visibly discernable in the intestinal lumen. The intestinal mucosa was generally intact and showed no significant inflammatory-cell infiltration. Diversity analysis of the colonic microbiota showed significant inter-group differences. In the experimental group, the composition of the colonic microbiota differed from that in the other 2 groups at the phylum level, and was characterized by a higher proportion of Bacteroidetes and a lower proportion of Firmicutes.
CONCLUSIONS: Microbiome depletion induced by cefoperazone sodium and sulbactam sodium could promote long-term colonization of C. jejuni in the intestines of mice.

OBJECTIVE: To investigate cases of five Campylobacter jejuni outbreaks and describe laboratory characteristics of these infections.
METHODS: Whole-genome sequencing and conventional methods were combined to thoroughly investigate the outbreaks, and data of contemporaneous sporadic cases was included for comparison.
RESULTS: Seven sequence types (STs) of C. jejuni caused 83 cases, including ST9079 which recurred across 2 years. Trace-back investigation could not identify any food items of infection but detected identical campylobacters from food contacts. Phylogenetic analysis unveiled genetic closeness between outbreak strains and some concurrent sporadic strains, indicating local campylobacteriosis may not be wholly sporadic but rather a series of linked cases. Virulence genes disclosed species/case-specific signatures to differentiate outbreaks from truly non-outbreak strains. Resistance to fluoroquinolones and/or macrolides was prevalent (90.8%, 108/119), with a noteworthy portion exhibiting multidrug resistance (31.1%, 37/119). Five types of plasmids were harbored among outbreak isolates, of which one plasmid harboring anti-stress and resistant genes was rarely found in C. jejuni.
CONCLUSIONS: This is the first reported sequential outbreak of C. jejuni in China. Our observations help to define the genomic landscape and antimicrobial resistance patterns of Campylobacter, emphasizing the need for a broader \'One Health\' perspective to combat the threats posed by campylobacteriosis.

Recombination of short DNA fragments via horizontal gene transfer (HGT) can introduce beneficial alleles, create genomic disharmony through negative epistasis, and create adaptive gene combinations through positive epistasis. For non-core (accessory) genes, the negative epistatic cost is likely to be minimal because the incoming genes have not co-evolved with the recipient genome and are frequently observed as tightly linked cassettes with major effects. By contrast, interspecific recombination in the core genome is expected to be rare because disruptive allelic replacement is likely to introduce negative epistasis. Why then is homologous recombination common in the core of bacterial genomes? To understand this enigma, we take advantage of an exceptional model system, the common enteric pathogens Campylobacter jejuni and C. coli that are known for very high magnitude interspecies gene flow in the core genome. As expected, HGT does indeed disrupt co-adapted allele pairings, indirect evidence of negative epistasis. However, multiple HGT events enable recovery of the genome\'s co-adaption between introgressing alleles, even in core metabolism genes (e.g., formate dehydrogenase). These findings demonstrate that, even for complex traits, genetic coalitions can be decoupled, transferred, and independently reinstated in a new genetic background-facilitating transition between fitness peaks. In this example, the two-step recombinational process is associated with C. coli that are adapted to the agricultural niche.IMPORTANCEGenetic exchange among bacteria shapes the microbial world. From the acquisition of antimicrobial resistance genes to fundamental questions about the nature of bacterial species, this powerful evolutionary force has preoccupied scientists for decades. However, the mixing of genes between species rests on a paradox: 0n one hand, promoting adaptation by conferring novel functionality; on the other, potentially introducing disharmonious gene combinations (negative epistasis) that will be selected against. Taking an interdisciplinary approach to analyze natural populations of the enteric bacteria Campylobacter, an ideal example of long-range admixture, we demonstrate that genes can independently transfer across species boundaries and rejoin in functional networks in a recipient genome. The positive impact of two-gene interactions appears to be adaptive by expanding metabolic capacity and facilitating niche shifts through interspecific hybridization. This challenges conventional ideas and highlights the possibility of multiple-step evolution of multi-gene traits by interspecific introgression.

Campylobacter jejuni (C. jejuni) is a common pathogen that often causes diarrhea, loss of appetite, and even enteritis in domestic cats, affecting their growth and development, especially in kittens under 6 months of age. Oral passive immunization with chicken yolk antibody Y has been proved effective for the treatment of gastrointestinal pathogen infections due to its high specificity. In this study, C. jejuni was isolated from diarrheal cat feces, and the specific egg yolk antibody Y against C. jejuni was demonstrated to effectively inhibit its proliferation in vitro experiments. To evaluate the effect of anti-C. jejuni IgY, the mouse C. jejuni infection model was established and it was found that IgY could alleviate C. jejuni-induced clinical symptoms. Consistent with these results, the reduction of pro-inflammatory factors and intestinal colonization by C. jejuni in the IgY-treated groups, especially in the high dose group. We then evaluated the protective effect of IgY on young Ragdoll cats infected with C. jejuni. This specific antibody reduced the rate of feline diarrhea, protected the growth of young cats, inhibited systemic inflammatory hyperactivation, and increased fecal short-chain fatty acid concentrations. Notably, IgY may have a protective role by changing intestinal amino acid metabolism and affecting C. jejuni chemotaxis. Collectively, specific IgY is a promising therapeutic strategy for C. jejuni-induced cat diarrhea.

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.

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.