Bovine mastitis is an inflammation of the udder tissue parenchyma that causes pathological changes in the glandular tissue and abnormalities in milk leading to significant economic losses to the dairy industry across the world. Mammary pathogenic Escherichia (E.) coli (MPEC) is one of the main etiologic agents of acute clinical mastitis in dairy cattle. MPEC strains have virulence attributes to resist the host innate defenses and thrive in the mammary gland environment. The association between specific virulence factors of MPEC with the severity of mastitis in cattle is not fully understood. Furthermore, the indiscriminate use of antibiotics to treat mastitis has resulted in antimicrobial resistance to all major antibiotic classes in MPEC. A thorough understanding of MPEC’s pathogenesis and antimicrobial susceptibility pattern is required to develop better interventions to reduce mastitis incidence and prevalence in cattle and the environment. This review compiles important information on mastitis caused by MPEC (e.g., types of mastitis, host immune response, diagnosis, treatment, and control of the disease) as well as the current knowledge on MPEC virulence factors, antimicrobial resistance, and the dilemma of MPEC as a new pathotype. The information provided in this review is critical to identifying gaps in knowledge that will guide future studies to better design diagnostic, prevent, and develop therapeutic interventions for this significant dairy disease.

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.

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.

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 leading cause of foodborne gastroenteritis. Recent studies have indicated a rise in fluoroquinolone-resistant (FQ-R) Campylobacter in cattle, where FQ is used to control bovine respiratory disease (BRD). To assess the effect of danofloxacin treatment on the development of FQ-resistance in C. jejuni, 30 commercial calves were divided into Group 1, Group 2, and Group 3 (n = 10), and were all inoculated orally with FQ-susceptible (FQ-S) C. jejuni; seven days later, Group 3 was challenged with transtracheal Mannheimia haemolytica, and one week later, Group 2 and Group 3 were injected subcutaneously with danofloxacin. Rectal feces were collected to determine relative percentages of FQ-R Campylobacter via culture. Before oral inoculation with C. jejuni, 87% of calves were naturally colonized by FQ-R C. jejuni. Two days after the inoculation, FQ-R C. jejuni decreased substantially in the majority of calves. Within 24 h of danofloxacin injection, almost all C. jejuni populations shifted to an FQ-R phenotype in both FQ-treated groups, which was only transitory, as FQ-S strains became predominant during later periods. Genotyping indicated that the spike seen in FQ-R C. jejuni populations following the injection was due mainly to enrichment of preexisting FQ-R C. jejuni, rather than development of de novo FQ resistance in susceptible strains. These results provide important insights into the dynamic changes of FQ-resistant Campylobacter in cattle in response to FQ treatment.

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.