Experimental treatment of Staphylococcus aureus bovine intramammary infection using a guanine riboswitch ligand analog

摘要

Staphylococcus aureus is a leading cause of intramammary infections (IMI). We recently demonstrated that Staph. aureus strains express the gene guaA during bovine IMI. This gene codes for a guanosine monophosphate synthetase and its expression is regulated by a guanine riboswitch. The guanine analog 2,5,6-triaminopyrimidine-4-one (PCI) is a ligand of the guanine riboswitch. Interactions between PCI and its target result in inhibition of guanosine monophosphate synthesis and subsequent death of the bacterium. The present study describes the investigational use of PCI for therapy of Staph. aureus IMI in lactating cows. The in vitro minimal inhibitory concentration of PCI ranged from 0.5 to 4 μg/mL for a variety of Staph. aureus and Staphylococcus epidermidis strains and required a reducing agent for stability and full potency. A safety assessment study was performed, whereby the healthy quarters of 4 cows were infused with increasing doses of PCI (0, 150, 250, and 500 mg). Over the 44 h following infusions, no obvious adverse effect was observed. Ten Holstein multiparous cows in mid lactation were then experimentally infused into 3 of the quarters with approximately 50 cfu of Staph. aureus strain SHY97-3906 and infection was allowed to progress for 2 wk before starting PCI treatment. Bacterial counts reached then about 10~3 to 10~4 cfu/mL of milk. Infected quarters were treated with 1 of 3 doses of PCI (0, 250, or 500 mg) after each morning and evening milking for 7 d (i.e., 14 intramammary infusions of PCI). During the treatment period, milk from PCl-treated quarters showed a significant reduction in bacterial concentrations. However, this reduction of Staph. aureus count in milk was not maintained during the 4 wk following the end of the treatment and only 15% of the PCl-treated quarters underwent bacteriological cure. The somatic cell count and the quarter milk production were not affected by treatments. Although bacterial clearance was not achieved following treatment with PCI, these results demonstrate that the Staph. aureus guanine riboswitch represents a relevant and promising drug target for a novel class of antibiotics for the animal food industry.