Journal Articles
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Item Microtiter Screening Reveals Oxygen-Dependent Antimicrobial Activity of Natural Products Against Mastitis-Causing Bacteria(Frontiers Media S.A., 2019-08-28) Ferguson SA; Menorca A; Van Zuylen EM; Cheung C-Y; McConnell MA; Rennison D; Brimble MA; Bodle K; McDougall S; Cook GM; Heikal A; Debnath AIn this study we investigated the influence of oxygen availability on a phenotypic microtiter screen to identify new, natural product inhibitors of growth for the bovine mastitis-causing microorganisms; Streptococcus uberis, Staphylococcus aureus, and Escherichia coli. Mastitis is a common disease in dairy cattle worldwide and is a major cause of reduced milk yield and antibiotic usage in dairy herds. Prevention of bovine mastitis commonly relies on the application of teat disinfectants that contain either iodine or chlorhexidine. These compounds are used extensively in human clinical settings and increased tolerance to chlorhexidine has been reported in both Gram-positive and Gram-negative microorganisms. As such new, non-human use alternatives are required for the agricultural industry. Our screening was conducted under normoxic (20% oxygen) and hypoxic (<1% oxygen) conditions to mimic the conditions on teat skin and within the mammary gland respectively, against two natural compound libraries. No compounds inhibited E. coli under either oxygen condition. Against the Gram-positive microorganisms, 12 inhibitory compounds were identified under normoxic conditions, and 10 under hypoxic conditions. Data revealed a clear oxygen-dependency amongst compounds inhibiting growth, with only partial overlap between oxygen conditions. The oxygen-dependent inhibitory activity of a naturally occurring quinone, β-lapachone, against S. uberis was subsequently investigated and we demonstrated that this compound is only active under normoxic conditions with a minimum inhibitory concentration and minimum bactericidal concentration of 32 μM and kills via a reactive oxygen species-dependent mechanism as has been demonstrated in other microorganisms. These results demonstrate the importance of considering oxygen-availability in high-throughput inhibitor discovery.Item Antimicrobial susceptibilities in dairy herds that differ in dry cow therapy usage(Elsevier Inc and the Federation of Animal Science Societies (Fass) Inc on behalf of the American Dairy Science Association, 2021-08) McDougall S; Penry J; Dymock DIntramammary infusion of antimicrobials at the end of lactation (dry cow therapy; DCT) is a central part of mastitis control programs and is one of the major indications for antimicrobial use in dairy cows. However, with increasing focus on prudent use of antimicrobials and concerns about emergence of antimicrobial resistance, the practice of treating every cow at the end of lactation with DCT is in question. This cross-sectional, observational study determined the minimum inhibitory concentrations (MIC) of 10 antimicrobials for coagulase-negative staphylococci (CNS), Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis isolates from milk samples from dairy cows with somatic cell counts >200,000 cells/mL in herds that had been organic for >3 yr (n = 7), or had used either ampicillin-cloxacillin DCT (n = 11) or cephalonium DCT (n = 8) in the preceding 3 yr. The organic herds were certified under the United States Department of Agriculture National Organic Program, meaning that there was no blanket DCT, and minimal use of antimicrobials in general, with a loss of organic status of the animal if treated with antimicrobials. Breakpoints (where available) were used to categorize isolates as resistant, intermediate, or susceptible to antimicrobials. The MIC distributions of isolates from different herd types were compared using binomial or multinomial logistic regression. Of 240 CNS isolates, 12.9, 0.8, 7.1, 32.6, and 1.2%, were intermediate or resistant to ampicillin, cephalothin, erythromycin, penicillin, and tetracycline, respectively. Of 320 Staph. aureus isolates, 29.0, 2.5, 1.2, and 34.9% were intermediately resistant or resistant to ampicillin, penicillin, erythromycin, and oxacillin, respectively. Of 184 Strep. uberis isolates, 1.1, 25.0, 1.6, and 1.6% were intermediately resistant or resistant to erythromycin, penicillin, pirlimycin, and tetracycline, respectively. Generally, the MIC of CNS and streptococcal isolates from organic herds were lower than isolates from herds using DCT. However, the differences in MIC distributions occurred at MIC below clinical breakpoints, so that the bacteriological cure rates may not differ between isolates of differing MIC. Bimodal distributions of MIC for ampicillin and penicillin were found in Staph. aureus isolates from organic herds, suggesting that isolates with a higher MIC are a natural part of the bacterial population of the bovine mammary gland, or that isolates with higher MIC have persisted within these organic herds from a time when antimicrobials had been used. Given these observations, further work is required to determine if exposure to DCT is causally associated with the risk of elevated MIC, and whether reduction or removal of DCT from herds would reduce the risk of elevated MIC of mastitis pathogens.Item The Genetic Relatedness and Antimicrobial Resistance Patterns of Mastitis-Causing Staphylococcus aureus Strains Isolated from New Zealand Dairy Cattle(MDPI (Basel, Switzerland), 2021-11-22) Greening SS; Zhang J; Midwinter AC; Wilkinson DA; McDougall S; Gates MC; French NP; Butaye PStaphylococcus aureus is one of the leading causes of bovine mastitis worldwide and is a common indication for use of antimicrobials on dairy farms. This study aims to investigate the association between on-farm antimicrobial usage and the antimicrobial resistance (AMR) profiles of mastitis-causing S. aureus. Whole-genome sequencing was performed on 57 S. aureus isolates derived from cows with either clinical or subclinical mastitis from 17 dairy herds in New Zealand. The genetic relatedness between isolates was examined using the core single nucleotide polymorphism alignment whilst AMR and virulence genes were identified in-silico. The association between gene presence-absence and sequence type (ST), antimicrobial susceptibility and dry cow therapy treatment was investigated using Scoary. Altogether, eight STs were identified with 61.4% (35/57) belonging to ST-1. Furthermore, 14 AMR-associated genes and 76 virulence-associated genes were identified, with little genetic diversity between isolates belonging to the same ST. Several genes including merR1 which is thought to play a role in ciprofloxacin-resistance were found to be significantly overrepresented in isolates sampled from herds using ampicillin/cloxacillin dry cow therapy. Overall, the presence of resistance genes remains low and current antimicrobial usage patterns do not appear to be driving AMR in S. aureus associated with bovine mastitis.Item Therapy of Subclinical Mastitis during Lactation(MDPI (Basel, Switzerland), 2022-02-07) McDougall S; Clausen LM; Hussein HM; Compton CWR; Krömker VThis study tested the hypothesis that increasing the duration and/or frequency of antimicrobial treatment of subclinical mastitis would result in a higher bacteriological cure rate. Glands with a positive California mastitis test (CMT) from cows with an elevated somatic cell count (>500,000 cells/mL) that had an intramammary infection were randomly assigned at cow level to no treatment (Control; n = 80 glands), intramammary infusion of 200 mg cloxacillin sodium on three occasions at 48 h intervals (3 × 48 h; n = 273 glands), five occasions at 24 h intervals (5 × 24 h; n = 279 glands), or on five occasions at 48 h intervals (5 × 48 h; n = 72 glands). Glands were resampled at 21 (±3) and 28 (±3) days after initiation of treatment. The gland-level cure rate for any pathogen was 5/80 (6.2%), 139/173 (49.8%), 172/297 (61.6%) and 58/72 (80.6%) for Control, 3 × 48 h, 5 × 24 h and 5 × 48 h, respectively. The cure rate for major pathogens (defined as Staphylococcus aureus or Streptococcus spp.) was 4/52 (7.7%), 84/197 (42.6%), 96/183 (52.5%) and 36/48 (75%) for Control, 3 × 48 h, 5 × 24 h and 5 × 48 h, respectively. We conclude that treatment was superior to no treatment, and bacteriological cure rate was higher with the 5 × 24 h protocol than for the 3 × 48 h protocol and was higher with the 5 × 48 h than the 5 × 24 h protocol.