Phage Lytic Protein LysRODI Prevents Staphylococcal Mastitis in Mice

Abstract

Phage lytic proteins are promising antimicrobials that could complement conventional antibiotics and help to combat multi-drug resistant bacteria that cause important human and animal infections. Here, we report the characterization of endolysin LysRODI (encoded by staphylophage phiIPLA-RODI) and its application as a prophylactic mastitis treatment. The main properties of LysRODI were compared with those of endolysin LysA72 (encoded by staphylophage phiIPLA35) and the chimeric protein CHAPSH3b (derived from the virion-associated peptidoglycan hydrolase HydH5 and lysostaphin). Time-kill experiments performed with Staphylococcus aureus and Staphylococcus epidermidis demonstrated that the killing rate of LysRODI and CHAPSH3b is higher than that of LysA72 (0.1 μM protein removed 10 7 CFU/ml of S. aureus in 30 min). Of note, all proteins failed to select resistant mutants as bacterial exposure to sub-lethal concentrations of the proteins did not alter the MIC values. Additionally, LysRODI and CHAPSH3b were non-toxic in a zebrafish embryo model at concentrations near the MIC (0.5 and 0.7 μM, respectively). Moreover, these two proteins significantly reduced mortality in a zebrafish model of systemic infection. In contrast to LysRODI, the efficacy of CHAPSH3b was dosedependent in zebrafish, requiring higher-dose treatments to achieve the maximum survival rate. For this reason, LysRODI was selected for further analysis in mice, demonstrating great efficacy to prevent mammary infections by S. aureus and S. epidermidis. Our findings strongly support the use of phage lytic proteins as a new strategy to prevent staphylococcal mastitis.

Characterization of Clinical MRSA Isolates from Northern Spain and Assessment of Their Susceptibility to Phage-Derived Antimicrobials

“…To date, several works have demonstrated the high in vitro and in vivo activity of endolysins against S. aureus isolates from different origins, including clinical staphylococci (revised by Gutiérrez et al [45]). The lytic protein used in this study, LysRODI, had been previously shown to be highly active against different staphylococcal strains [24]. Along the same lines, the data presented here indicates that all clinical MRSA isolates were susceptible to LysRODI, although to different degrees.…”

“…Our laboratory has already demonstrated the potential of several phage endolysins to remove S. aureus cells from different environments. For example, various endolysins and chimeric proteins showed lytic activity against S. aureus cells in pasteurized milk [25,26] and biofilms [24,27,28]. Moreover, we showed that subinhibitory doses of some endolysins can inhibit biofilm formation in S. aureus through the downregulation of autolysin-encoding genes [28].…”

“…Characterization of phiIPLA-RODI revealed that it is a broad-host-range phage, able to infect several species belonging to the Staphylococcus genus [23]. Moreover, genomic analysis of this phage led to the identification of an endolysin, LysRODI, which contains two catalytic domains (CHAP and amidase-2 domain) and one cell wall-binding domain (CBD), SH3b [23,24]. Our laboratory has already demonstrated the potential of several phage endolysins to remove S. aureus cells from different environments.…”

“…More recently, we showed the ability of some of these proteins (LysRODI, LysA72 and CHAPSH3b) to remove staphylococcal strains in vivo. Indeed, LysRODI and CHAPSH3b were non-toxic in a zebrafish embryo model and significantly reduced mortality in a zebrafish model of systemic infection [24]. Moreover, LysRODI demonstrated great efficacy in preventing mammary infections by S. aureus and S. epidermidis in a mouse model of mastitis [24].…”

“…Indeed, LysRODI and CHAPSH3b were non-toxic in a zebrafish embryo model and significantly reduced mortality in a zebrafish model of systemic infection [24]. Moreover, LysRODI demonstrated great efficacy in preventing mammary infections by S. aureus and S. epidermidis in a mouse model of mastitis [24]. However, most of these studies were carried out with MSSA strains.…”