Small noncoding RNAs (ncRNAs) are involved in many important physiological functions in pathogenic microorganisms. Previous studies have identified the presence of noncoding RNAs in the major zoonotic pathogen Campylobacter jejuni; however, few have been functionally characterized to date. CjNC110 is a conserved ncRNA in C. jejuni, located downstream of the luxS gene, which is responsible for the production of the quorum sensing molecule autoinducer-2 (AI-2). In this study, we utilized strand specific high-throughput RNAseq to identify potential targets or interactive partners of CjNC110 in a sheep abortion clone of C. jejuni. These data were then utilized to focus further phenotypic evaluation of the role of CjNC110 in motility, autoagglutination, quorum sensing, hydrogen peroxide sensitivity, and chicken colonization in C. jejuni. Inactivation of the CjNC110 ncRNA led to a statistically significant decrease in autoagglutination ability as well as increased motility and hydrogen peroxide sensitivity compared to the wild-type. Extracellular AI-2 detection was decreased in ΔCjNC110; however, intracellular AI-2 accumulation was significantly increased, suggesting a key role of CjNC110 in modulating the transport of AI-2. Notably, ΔCjNC110 also showed a decreased ability to colonize chickens. Complementation of CjNC110 restored all phenotypic changes back to wild-type levels. The collective results of the phenotypic and transcriptomic changes observed in our data provide valuable insights into the pathobiology of C. jejuni sheep abortion clone and strongly suggest that CjNC110 plays an important role in the regulation of energy taxis, flagellar glycosylation, cellular communication via quorum sensing, oxidative stress tolerance, and chicken colonization in this important zoonotic pathogen.

Fluoroquinolones, such as danofloxacin, are used to control bovine respiratory disease complex in beef cattle; however, little is known about their effects on gut microbiota and resistome. The objectives were to evaluate the effect of subcutaneously administered danofloxacin on gut microbiota and resistome, and the composition of Campylobacter in calves. Twenty calves were injected with a single dose of danofloxacin, and ten calves were kept as a control. The effects of danofloxacin on microbiota and the resistome were assessed using 16S rRNA sequencing, quantitative real-time PCR, and metagenomic Hi-C ProxiMeta. Alpha and beta diversities were significantly different (p < 0.05) between pre-and post-treatment samples, and the compositions of several bacterial taxa shifted. The patterns of association between the compositions of Campylobacter and other genera were affected by danofloxacin. Antimicrobial resistance genes (ARGs) conferring resistance to five antibiotics were identified with their respective reservoirs. Following the treatment, some ARGs (e.g., ant9, tet40, tetW) increased in frequencies and host ranges, suggesting initiation of horizontal gene transfer, and new ARGs (aac6, ermF, tetL, tetX) were detected in the post-treatment samples. In conclusion, danofloxacin induced alterations of gut microbiota and selection and enrichment of resistance genes even against antibiotics that are unrelated to danofloxacin.

Campylobacter jejuni is a zoonotic pathogen and is one of the leading causes of human gastroenteritis worldwide. C. jejuni IA3902 (representative of the sheep abortion clone) is genetically similar to C. jejuni W7 (representative of strain-type NCTC 11168), however, there are significant differences in the ability of luxS mutants of these strains to colonize chickens. LuxS is essential for the activated methyl cycle and generates homocysteine for conversion to L-methionine. Comparative genomics identified differential distribution of genes metA and metB, which function to convert homoserine for downstream production of L-methionine, between IA3902 and W7 which could enable a secondary pathway for L-methionine biosynthesis in W7ΔluxS but not in IA3902ΔluxS. To test the hypothesis that genes metA and metB contribute to L-methionine production and chicken colonization by Campylobacter, we constructed two mutants for phenotypic comparison: W7ΔmetABΔluxS and IA3902ΔluxS::metAB. Quantitative reverse transcription PCR and tandem mass spectrometry protein analysis were used to validate MetAB transcription and translation as present in IA3902ΔluxS::metAB and absent in W7ΔmetABΔluxS. Time-resolved fluorescence resonance energy transfer fluorescence assays demonstrated L-methionine and S-adenosyl methionine concentrations decreased in W7ΔmetABΔluxS and increased in IA3902ΔluxS::metAB. Assessment of chicken colonization revealed that IA3902ΔluxS::metAB partially rescued the colonization defect of IA3902ΔluxS, while W7ΔmetABΔluxS resulted in significantly decreased colonization compared to the wild type and W7ΔluxS. These results indicate that the ability to maintain L-methionine production in vivo, conferred by metA and metB in the absence of luxS, is critical for normal chicken colonization by C. jejuni.

Enrofloxacin is a fluoroquinolone drug used to prevent and control bovine respiratory disease (BRD) complex in multiple or single doses, ranging from 7.5 to 12.5 mg/kg body weight. Here, we examined the effects of high and low doses of a single subcutaneously injected enrofloxacin on gut microbiota and resistome in calves. Thirty-five calves sourced for this study were divided into five groups: control (n =7), two low dose groups (n = 14, 7.5 mg/kg), and two high dose groups (n = 14, 12.5 mg/kg). One group in the low and high dose groups was challenged with Mannheimia haemolytica to induce BRD. Both alpha and beta diversities were significantly different between pre- and post-treatment microbial communities (q < 0.05). The high dose caused a shift in a larger number of genera than the low dose. Using metagenomic ProxiMeta Hi-C, 32 unique antimicrobial resistance genes (ARGs) conferring resistance to six antibiotic classes were detected with their reservoirs, and the high dose favored clonal expansion of ARG-carrying bacterial hosts. In conclusion, enrofloxacin treatment can alter fecal microbiota and resistome irrespective of its dose. Hi-C sequencing provides significant benefits for unlocking new insights into the ARG ecology of complex samples; however, limitations in sample size and sequencing depth suggest that further work is required to validate the findings.

Bovine digital dermatitis (DD) is a contagious infectious cause of lameness in cattle with unknown definitive etiologies. Many of the bacterial species detected in metagenomic analyses of DD lesions are difficult to culture, and their antimicrobial resistance status is largely unknown. Recently, a novel proximity ligation-guided metagenomic approach (Hi-C ProxiMeta) has been used to identify bacterial reservoirs of antimicrobial resistance genes (ARGs) directly from microbial communities, without the need to culture individual bacteria. The objective of this study was to track tetracycline resistance determinants in bacteria involved in DD pathogenesis using Hi-C. A pooled sample of macerated tissues from clinical DD lesions was used for this purpose. Metagenome deconvolution using ProxiMeta resulted in the creation of 40 metagenome-assembled genomes with ≥80% complete genomes, classified into five phyla. Further, 1959 tetracycline resistance genes and ARGs conferring resistance to aminoglycoside, beta-lactams, sulfonamide, phenicol, lincosamide, and erythromycin were identified along with their bacterial hosts. In conclusion, the widespread distribution of genes conferring resistance against tetracycline and other antimicrobials in bacteria of DD lesions is reported for the first time. Use of proximity ligation to identify microorganisms hosting specific ARGs holds promise for tracking ARGs transmission in complex microbial communities.