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.

Campylobacter is a major foodborne pathogen in humans and a significant cause of abortion in sheep. Although ruminants are increasingly recognized as important reservoirs for Campylobacter species, limited information is available about the molecular epidemiology and antimicrobial resistance (AMR) profiles of sheep Campylobacter. Here, we describe a two-trial study that examined Campylobacter profiles in sheep and determined whether in-feed tetracycline (TET) influenced the distribution and AMR profiles of Campylobacter. Each trial involved 80 commercial sheep naturally infected with Campylobacter: 40 of these sheep were medicated with tetracycline in feed, while the other 40 received feed without antibiotics. Fecal and bile samples were collected for the isolation of Campylobacter. The bacterial isolates were analyzed for antimicrobial susceptibility and genotypes. The results revealed that 87.0% and 61.3% of the fecal and bile samples were positive for Campylobacter (Campylobacter jejuni and Campylobacter coli), with no significant differences between the medicated and nonmedicated groups. All but one of the tested Campylobacter isolates were resistant to tetracycline. Although fluoroquinolone (FQ) resistance remained low in C. jejuni (1.7%), 95.0% of the C. coli isolates were resistant to FQ. Genotyping revealed that C. jejuni sequence type 2862 (ST2862) and C. coli ST902 were the predominant genotypes in the sheep. Feed medication with tetracycline did not affect the overall prevalence, species distribution, and AMR profiles of Campylobacter, but it did increase the total Campylobacter counts in bile and gallbladder. These findings identify predominant Campylobacter clones, reveal the high prevalence of FQ-resistant C. coli, and provide new insights into the epidemiology of Campylobacter in sheep. IMPORTANCE Campylobacter is a major cause of foodborne illness in humans, and antibiotic-resistant Campylobacter is considered a serious threat to public health in the United States and worldwide. As a foodborne pathogen, Campylobacter commonly exists in the intestinal tract of ruminant animals, such as sheep and cattle. Results from this study reveal the predominant genotypes and high prevalence of tetracycline (TET) and fluoroquinolone (FQ) resistance in sheep Campylobacter. The finding on fluoroquinolone resistance in sheep Campylobacter is unexpected, as this class of antibiotics is not used for sheep in the United States, and it may suggest the transmission of fluoroquinolone-resistant Campylobacter from cattle to sheep. Additionally, the results demonstrate that in-feed medication with tetracycline increases Campylobacter counts in gallbladders, suggesting that the antibiotic pro-on July 11, 2020 by guest http://aem.asm.org/ Downloaded from on July 11, 2020 by guest http://aem.asm.org/ Downloaded from the extent of FQ resistance in sheep Campylobacter and determine the effect of in-feed tetracycline on the prevalence, species distribution, and antimicrobial resistance profiles of Campylobacter in sheep. RESULT...

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Small non-coding RNAs (sRNAs) are important players in modulating gene expression in bacterial pathogens, but their functions are largely undetermined in Campylobacter jejuni , an important cause of foodborne gastroenteritis in humans. In this study, we elucidated the functions of sRNA CjNC140 and its interaction with CjNC110, a previously characterized sRNA involved in the regulation of several virulence phenotypes of C. jejuni . Inactivation of CjNC140 increased motility, autoagglutination, L-methionine concentration, autoinducer-2 production, hydrogen peroxide resistance, and early chicken colonization, indicating a primarily inhibitory role of CjNC140 for these phenotypes. Apart from motility, all these effects directly contrasted the previously demonstrated positive regulation by CjNC110, suggesting that CjNC110 and CjNC140 operate in an opposite manner to modulate physiologic processes in C. jejuni . RNAseq and northern blotting further demonstrated that expression of CjNC140 increased in the absence of CjNC110, while expression of CjNC110 decreased in the absence of CjNC140, suggesting a possibility of their direct interaction. Indeed, electrophoretic mobility shift assay demonstrated a direct binding between the two sRNAs via GA- (CjNC110) and CU- (CjNC140) rich stem-loops. Additionally, RNAseq and follow-up experiments identified that CjNC140 positively regulates p19 , which encodes a key iron uptake transporter in Campylobacter . Furthermore, computational analysis revealed both CjNC140 and CjNC110 are highly conserved in C. jejuni, and the predicted secondary structures support CjNC140 as a functional homolog of the iron regulatory sRNA, RyhB. These findings establish CjNC140 and CjNC110 as a key checks-and- balances mechanism in maintaining homeostasis of gene expression and optimizing phenotypes critical for C. jejuni pathobiology. IMPORTANCE Gene regulation is critical to all aspects of pathogenesis of bacterial disease, and small non-coding RNAs (sRNAs) represent a new frontier in gene regulation of bacteria. In Campylobacter jejuni , the role of sRNAs remains largely unexplored. Here, we investigate the role of two highly conserved sRNAs, CjNC110 and CjNC140, and demonstrate that CjNC140 displays a primarily inhibitory role in contrast to a primarily activating role for CjNC110 for several key virulence-associated phenotypes. Our results also revealed that the sRNA regulatory pathway is intertwined with the iron uptake system, another virulence mechanism critical for in vivo colonization. These findings open a new direction for understanding C. jejuni pathobiology and identify potential targets for intervention for this major foodborne pathogen.