Fluoroquinolone (FQ) resistance in a major foodborne bacterial pathogen, Campylobacter jejuni, derived from cattle has recently become prevalent and poses a significant public health concern. However, the underlying factors for this increase are not entirely clear. To evaluate the effect of enrofloxacin treatment on FQ-resistance development in C. jejuni, 35 commercial calves were equally divided into five groups (Groups 1–5) and were orally inoculated with FQ-susceptible (FQ-S) C. jejuni. Eight days later, Groups 4 and 5 were challenged with Mannheimia haemolytica via a transtracheal route to induce a respiratory disease; after 8 days, Groups 2, 3, 4, and 5 were injected subcutaneously with enrofloxacin (7.5 mg/kg for Groups 2 and 4, and 12.5 mg/kg for Groups 3 and 5). Colonization levels by FQ-resistant (FQ-R) and FQ-S Campylobacter in rectal feces were determined via differential culture throughout the experiment. Before oral inoculation with C. jejuni, only five calves were naturally colonized by Campylobacter, four of which were also colonized by FQ-R C. jejuni (three in Group 1 and one in Group 3). Soon after the oral inoculation, almost all calves in the groups became stably colonized by FQ-S C. jejuni (~3–6 log10 CFU/g), except that the four calves that were pre-colonized before inoculation remained positive with both FQ-R and FQ-S C. jejuni. Following enrofloxacin administration, C. jejuni colonization declined sharply and rapidly in all treated groups to undetectable levels; however, the vast majority of the animals were recolonized by C. jejuni at comparable levels 72 h after the treatment. Notably, no FQ-R C. jejuni was detected in any of the calves that received enrofloxacin, regardless of the drug dose used or disease status of the animals. The lack of detection of FQ-R C. jejuni was likely due to the localized high concentration of the antibiotic in the intestine, which may have prevented the emergence of the FQ-R mutant. These findings indicate that single-dose enrofloxacin use in cattle poses a low risk for selection of de novo FQ-R mutants in C. jejuni.

Campylobacter is a major food safety concern and is transmitted mainly via poultry meat. We previously found that some commercial broiler farms consistently produced Campylobacter-negative flocks while others were consistently Campylobacter-positive for consecutive production cycles although the farms operated under similar management practices. We hypothesized that this difference in Campylobacter colonization might be associated with the gut microbiota composition. To address this, six commercial broiler farms were selected based on their Campylobacter status (three negative and three positive) to evaluate the microbiota differences between each farm category. For each farm on each production cycle (2–3 cycles), 40 ceca collected from five-week-old broilers were processed for microbiota analysis via 16S rRNA gene sequencing. Cecal microbiota species richness, phylogenetic diversity, community structure, and composition of Campylobacter-positive farms were noticeably different from those of Campylobacter-negative farms. Rikenella, Methanocorpusculum, Barnesiella, Parasutterella, and Helicobacter were significantly more abundant among Campylobacter-positive farms. In contrast, Ruminococcaceae, Streptococcus, Escherichia, Eggerthellaceae, Lactobacillus, Monoglobus, and Blausia were more abundant in Campylobacter-negative farms. Eggerthellaceae, Clostridia, Lachnospiraceae, Lactobacillus, Monoglobus, and Parabacteroides were significantly negatively correlated with Campylobacter abundance. These findings suggest that specific members of cecal microbiota may influence Campylobacter colonization in commercial broilers and may be further explored to control Campylobacter in poultry.

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...

To aid development of phage therapy against Campylobacter, we investigated the distribution of the clustered regularly interspaced short palindromic repeats (CRISPR) systems in fluoroquinolone (FQ)-resistant Campylobacter jejuni. A total of 100 FQ-resistant C. jejuni strains from different sources were analyzed by PCR and DNA sequencing to determine resistance-conferring mutation in the gyrA gene and the presence of various CRISPR systems. All but one isolate harbored 1–5 point mutations in gyrA, and the most common mutation was the Thr86Ile change. Ninety-five isolates were positive with the CRISPR PCR, and spacer sequences were found in 86 of them. Among the 292 spacer sequences identified in this study, 204 shared 93–100% nucleotide homology to Campylobacter phage D10, 44 showed 100% homology to Campylobacter phage CP39, and 3 had 100% homology with Campylobacter phage CJIE4-5. The remaining 41 spacer sequences did not match with any phages in the database. Based on the results, it was inferred that the FQ-resistant C. jejuni isolates analyzed in this study were potentially resistant to Campylobacter phages D10, CP39, and CJIE4-5 as well as some unidentified phages. These phages should be excluded from cocktails of phages that may be utilized to treat FQ-resistant Campylobacter.

Nucleic acid tests have been widely used for diagnosis of diseases by detecting the relevant genetic markers that are usually amplified using polymerase chain reaction (PCR). This work reports the use of a plasmonic device as an efficient and low-cost PCR thermocycler to facilitate nucleic acid-based diagnosis. The thermoplasmonic device, consisting of a one-dimensional metal grating, exploited the strong light absorption of plasmonic resonance modes to heat up PCR reagents using a near-infrared laser source. The plasmonic device also integrated a thin-film thermocouple on the metal grating to monitor the sample temperature. The plasmonic thermocycler is capable of performing a PCR amplification cycle in approximately 2.5 minutes.We successfully demonstrated the multiplex and real-time PCR amplifications of the antibiotic resistance genes using the genomic DNAs extracted from Acinetobacter baumannii, Klebsiella pneumonia, Escherichia coli, and Campylobacter.