CmeABC, a resistance-nodulation-division (RND) type of efflux pump, contributes to Campylobacter resistance to a broad spectrum of antimicrobial agents and is also essential for Campylobacter colonization of the animal intestinal tract by mediation of bile resistance. As one of the main systems for Campylobacter adaptation to different environments, CmeABC is likely subject to control by regulatory elements. We describe the identification of a transcriptional repressor for CmeABC. Insertional mutagenesis of cmeR , an open reading frame immediately upstream of the cmeABC operon, resulted in overexpression of cmeABC , as determined by transcriptional fusion (P _cmeABC-lacZ ) and immunoblotting with CmeABC-specific antibodies. Overexpression of the efflux pump was correlated with a moderate increase in the level of resistance of the cmeR mutant to several antimicrobials. In vitro, recombinant CmeR bound specifically to the promoter region of cmeABC , precisely, to the inverted repeat sequences in the cmeABC promoter. A single nucleotide deletion between the two half sites of the inverted repeat reduced the level of CmeR binding to the promoter sequence and resulted in overexpression of cmeABC . Together, these findings indicate that cmeR encodes a transcriptional repressor that directly interacts with the cmeABC promoter and modulates the expression of cmeABC . Mutation either in CmeR or in the inverted repeat impedes the repression and leads to enhanced production of the MDR efflux pump.

Campylobacter includes a group of genetically diverse species causing a range of diseases in animals and humans. The bacterium is frequently associated with two economically important and epidemiologically distinct reproductive diseases in ruminants: enzootic infectious infertility in cattle owing to Campylobacter fetus subsp. venerealis and abortions in sheep, goats, and cattle. Septic abortion, usually epizootic in sheep, has been historically associated with C. fetus subsp. fetus and to a lesser extent with Campylobacter jejuni. However, there has been a dramatic species shift in the etiology of Campylobacter abortions in recent years: C. jejuni has now replaced C. fetus subsp. fetus as the predominant cause of sheep abortion in the United States, which appears to be driven primarily by clonal expansion of a hypervirulent tetracycline-resistant C. jejuni clone. Here we provide a review on the recent advances in understanding the pathobiology of Campylobacter infections in animals, with an emphasis on the diseases in ruminants, covering epidemiology, pathogenesis, genomics, and control measures.

The prevalence of fluoroquinolone (FQ)-resistant Campylobacter has become a concern for public health. To facilitate the control of FQ-resistant (FQ^R) Campylobacter, it is necessary to understand the impact of FQ^R on the fitness of Campylobacter in its natural hosts as understanding fitness will help to determine and predict the persistence of FQ^RCampylobacter. Previously it was shown that acquisition of resistance to FQ antimicrobials enhanced the in vivo fitness of FQ^RCampylobacter. In this study, we confirmed the role of the Thr-86-Ile mutation in GyrA in modulating Campylobacter fitness by reverting the mutation to the wild-type (WT) allele, which resulted in the loss of the fitness advantage. Additionally, we determined if the resistance-conferring GyrA mutations alter the enzymatic function of the DNA gyrase. Recombinant WT gyrase and mutant gyrases with three different types of mutations (Thr-86-Ile, Thr-86-Lys, and Asp-90-Asn), which are associated with FQ^R in Campylobacter, were generated in E. coli and compared for their supercoiling activities using an in vitro assay. The mutant gyrase with the Thr-86-Ile change showed a greatly reduced supercoiling activity compared with the WT gyrase, while other mutant gyrases did not show an altered supercoiling. Furthermore, we measured DNA supercoiling within Campylobacter cells using a reporter plasmid. Consistent with the results from the in vitro supercoiling assay, the FQ^R mutant carrying the Thr-86-Ile change in GyrA showed much less DNA supercoiling than the WT strain and the mutant strains carrying other mutations. Together, these results indicate that the Thr-86-Ile mutation, which is predominant in clinical FQ^RCampylobacter, modulates DNA supercoiling homeostasis in FQ^RCampylobacter.

Antibiotic resistance, particularly to fluoroquinolones and macrolides, in the major foodborne pathogen Campylobacter is considered a serious threat to public health. Although ruminant animals serve as a significant reservoir for Campylobacter, limited information is available on antibiotic-resistant Campylobacter of bovine origin. Here, we analyzed the antimicrobial susceptibilities of 320 C. jejuni and 115 C. coli isolates obtained from feedlot cattle farms in multiple states in the U.S. The results indicate that fluoroquinolone resistance reached to 35.4% in C. jejuni and 74.4% in C. coli, which are significantly higher than those previously reported in the U.S. While all fluoroquinolone resistant (FQ^R) C. coli isolates examined in this study harbored the single Thr-86-Ile mutation in GyrA, FQ^RC. jejuni isolates had other mutations in GyrA in addition to the Thr-86-Ile change. Notably, most of the analyzed FQ^RC. coli isolates had similar PFGE (pulsed field gel electrophoresis) patterns and the same MLST (multilocus sequence typing) sequence type (ST-1068) regardless of their geographic sources and time of isolation, while the analyzed C. jejuni isolates were genetically diverse, suggesting that clonal expansion is involved in dissemination of FQ^RC. coli but not C. jejuni. These findings reveal the rising prevalence of FQ^RCampylobacter in the U.S. and provide novel information on the epidemiology of antibiotic-resistant Campylobacter in the ruminant reservoir.

Antimicrobial resistance (AMR) of bacterial pathogens is an emerging public health threat. This threat extends to pets as it also compromises our ability to treat their infections. Surveillance programs in the United States have traditionally focused on collecting data from food animals, foods, and people. The Veterinary Laboratory Investigation and Response Network (Vet-LIRN), a national network of 45 veterinary diagnostic laboratories, tested the antimicrobial susceptibility of clinically relevant bacterial isolates from animals, with companion animal species represented for the first time in a monitoring program. During 2017, we systematically collected and tested 1968 isolates. To identify genetic determinants associated with AMR and the potential genetic relatedness of animal and human strains, whole genome sequencing (WGS) was performed on 192 isolates: 69 Salmonella enterica (all animal sources), 63 Escherichia coli (dogs), and 60 Staphylococcus pseudintermedius (dogs). We found that most Salmonella isolates (46/69, 67%) had no known resistance genes. Several isolates from both food and companion animals, however, showed genetic relatedness to isolates from humans. For pathogenic E. coli, no resistance genes were identified in 60% (38/63) of the isolates. Diverse resistance patterns were observed, and one of the isolates had predicted resistance to fluoroquinolones and cephalosporins, important antibiotics in human and veterinary medicine. For S. pseudintermedius, we observed a bimodal distribution of resistance genes, with some isolates having a diverse array of resistance mechanisms, including the mecA gene (19/60, 32%). The findings from this study highlight the critical importance of veterinary diagnostic laboratory data as part of any national antimicrobial resistance surveillance program. The finding of some highly resistant bacteria from companion animals, and the observation of isolates related to those isolated from humans demonstrates the public health significance of incorporating companion animal data into surveillance systems. Vet-LIRN will continue to build the infrastructure to collect the data necessary to perform surveillance of resistant bacteria as part of fulfilling its mission to advance human and animal health. A One Health approach to AMR surveillance programs is crucial and must include data from humans, animals, and environmental sources to be effective.

Campylobacterinfection is a leading cause of ovine abortion worldwide. Historically, genetically diverseCampylobacter fetusandCampylobacter jejunistrains have been implicated in such infections, but since 2003 a highly pathogenic, tetracycline-resistantC. jejuniclone (named SA) has become the predominant cause of sheep abortions in the United States. Whether clone SA was present in earlier U.S. abortion isolates (before 2000) and is associated with sheep abortions outside the United States are unknown. Here, we analyzed 54C. jejuniisolates collected from U.S. sheep abortions at different time periods and compared them with 42C. jejuniisolates associated with sheep abortion during 2002 to 2008 in Great Britain, using multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and array-based comparative genomic hybridization (CGH). Although clone SA (ST-8) was present in the early U.S. isolates, it was not as tetracycline resistant (19% versus 100%) or predominant (66% versus 91%) as it was in the late U.S isolates. In contrast,C. jejuniisolates from Great Britain were genetically diverse, comprising 19 STs and lacking ST-8. PFGE and CGH analyses of representative strains further confirmed the population structure of the abortion isolates. Notably, the Great Britain isolates were essentially susceptible to most tested antibiotics, including tetracycline, while the late U.S. isolates were universally resistant to this antibiotic, which could be explained by the common use of tetracyclines for control of sheep abortions in the United States but not in Great Britain. These results suggest that the dominance of clone SA in sheep abortions is unique to the United States, and the use of tetracyclines may have facilitated selection of this highly pathogenic clone.