In vitro susceptibility of bovine digital dermatitis treponemes to conventional and novel antimicrobial agents, and evidence of heavy metal resistance.

Abstract

OBJECTIVES: Bovine digital dermatitis (BDD) is a globally endemic, painful infectious foot disease of cattle; however, gaps in knowledge regarding treatment efficacy limit our ability to control it. Our objectives were to collect data on minimum inhibitory concentrations and minimum bactericidal concentrations (MICs and MBCs) for conventional and naturally derived therapeutic agents against specific Treponema strains important in BDD pathogenesis in the context of the need for responsible use of antimicrobials. Since antibacterial metals are widely used to control infectious lameness in ruminants, we also examine BDD treponeme susceptibility to heavy metals; and identify evidence of mechanisms for heavy metal resistance in BDD treponemes in comparison with other ruminant infectious lameness pathogens Fusobacterium necrophorum subsp. necrophorum and Dichelobacter nodosus. METHODS: We used a broth microdilution assay to collect MIC and MBC data for Treponema phagedenis (strain T320A) and Treponema pedis (strain T3552B) against the relevant antimicrobials. We used encoded protein sequences in a heavy metal resistance database (BacMet) together with comparative genomics to identify and compare metal resistance genes (MRGs) for T. phagedenis, T. pedis, F. necrophorum subsp. necrophorum and D. nodosus. RESULTS: BDD treponemes were susceptible to four of seven conventional antimicrobials tested: cefixime, ceftriaxone, linezolid and metronidazole. Six naturally derived therapeutic agents demonstrated intermediate susceptibility values and may be suitable as topical treatments. We identified reduced BDD treponeme susceptibility to zinc and nickel sulphate compared with copper sulphate, indicating evidence of heavy metal tolerance, and identified zinc and manganese MRGs (tro and mntA) in the treponeme genomes, with tro present across a range of treponemes but absent for F. necrophorum and D. nodosus. Comparative genomics of the tro operon highlighted further T. pedis genome features potentially related to heavy metal tolerance. CONCLUSIONS: We have identified several novel therapeutics with in vitro efficacy against BDD associated treponemes. Moreover, differences in presence of MRGs and in vitro susceptibility across lameness associated bacteria indicate heavy metal tolerance to zinc in BDD treponemes but not in other relevant ruminant lameness associated bacteria, potentially mediated by the tro metal resistance gene. These data appear to explain differences in clinical treatment responses to zinc sulphate observed for different ruminant infectious lameness diseases depending on presence or absence of treponemes and should help inform more tailored treatment approaches in the future.