In vitro potential of equine DEFA1 and eCATH1 as alternative antimicrobial drugs in rhodococcosis treatment

Abstract

Rhodococcus equi, the causal agent of rhodococcosis, is a severe pathogen of foals but also of immunodeficient humans, causing bronchopneumonia. The pathogen is often found together with Klebsiella pneumoniae or Streptococcus zooepidemicus in foals. Of great concern is the fact that some R. equi strains are already resistant to commonly used antibiotics. In the present study, we evaluated the in vitro potential of two equine antimicrobial peptides (AMPs), eCATH1 and DEFA1, as new drugs against R. equi and its associated pathogens. The peptides led to growth inhibition and death of R. equi and S. zooepidemicus at low micromolar concentrations. Moreover, eCATH1 was able to inhibit growth of K. pneumoniae. Both peptides caused rapid disruption of the R. equi membrane, leading to cell lysis. Interestingly, eCATH1 had a synergic effect together with rifampin. Furthermore, eCATH1 was not cytotoxic against mammalian cells at bacteriolytic concentrations and maintained its high killing activity even at physiological salt concentrations. Our data suggest that equine AMPs, especially eCATH1, may be promising candidates for alternative drugs to control R. equi in mono- and coinfections.

Fig 1

Impact of phospholipid membranes on the secondary structure of DEFA1 and eCATH1. The secondary structures of both DEFA1 (left) and eCATH1 (right) were investigated by CD spectroscopy in the absence (black lines) and presence (gray lines) of liposomes composed of negatively charged (PG, top) or electrostatically neutral (PC, bottom) phospholipids.

Fig 2

Salt dependence of the antimicrobial activity of eCATH1. The salt tolerance was tested against R. equi ATCC 33701 P+ by measuring the MBCs in the presence of 0, 50, 100, and 150 mM sodium chloride. (A) Mortality curves: values are expressed as the mean of three independent experiments ± standard error. (B) LD₉₀s and MBCs in the absence or presence of salt.

Fig 3

Scanning electron micrographs of R. equi ATCC 33701 P+ treated with DEFA1 or eCATH1. R. equi in mid-logarithmic-phase growth was incubated with 100 μg/ml of antimicrobial peptide eCATH1 or DEFA1 or with the peptide solvent (as a negative control) for 5 min. Observation at low (A) and high (B) magnifications.

Fig 4

Cytotoxic activity of DEFA1, eCATH1, and nisin. (A and B) Vero CCL-81 (A) and RK13 (B) cells were incubated with increasing amounts of DEFA1 (♦), eCATH1 (■), and nisin as a safe control (▲). Cytotoxicity was measured spectrophotometrically using the LDH release assay.