Abstract
Staphylococcus aureus causes various infections in humans and animals, the skin being the principal reservoir of this pathogen. The widespread occurrence of methicillin-resistant S. aureus (MRSA) limits the elimination and treatment of this pathogen. Phage lytic proteins have been proven as efficient antimicrobials against S. aureus. Here, a set of 12 engineered proteins based on endolysins were conceptualized to select the most optimal following a stepwise funnel approach assessing parameters including turbidity reduction, minimum inhibitory concentration (MIC), time-kill curves, and antibiofilm assays, as well as testing their stability in a broad range of storage conditions (pH, temperature, and ionic strength). The engineered phage lysins LysRODIΔAmi and ClyRODI-H5 showed the highest specific lytic activity (5 to 50 times higher than the rest), exhibited a shelf-life up to 6 months and remained stable at temperatures up to 50°C and in a pH range from 3 to 9. LysRODIΔAmi showed the lower MIC values against all staphylococcal strains tested. Both proteins were able to kill 6 log units of the strain S. aureus Sa9 within 5 min and could remove preformed biofilms (76 and 65%, respectively). Moreover, LysRODIΔAmi could prevent biofilm formation at low protein concentrations (0.15–0.6 μM). Due to its enhanced antibiofilm properties, LysRODIΔAmi was selected to effectively remove S. aureus contamination in both intact and disrupted keratinocyte monolayers. Notably, this protein did not demonstrate any toxicity toward human keratinocytes, even at high concentrations (22.1 μM). Finally, a pig skin ex vivo model was used to evaluate treatment of artificially contaminated pig skin using LysRODIΔAmi (16.5 μg/cm2). Following an early reduction of S. aureus, a second dose of protein completely eradicated S. aureus. Overall, our results suggest that LysRODIΔAmi is a suitable candidate as antimicrobial agent to prevent and treat staphylococcal skin infections.
Highlights
Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogenic bacterium for both humans and animals worldwide
Twelve new phage lytic proteins were designed by combination of catalytic and cell wall-binding (CBD) domains from the previously characterized endolysins LysH5 (53.7 kDa), LysA72 (53.2 kDa), LysRODI (54.5 kDa), LysC1C (53.2 kDa), and the bacteriocin lysostaphin (28.1 kDa)
The development of bacterial resistance after repeated use of some decontamination compounds is an issue of major concern, and is of paramount importance in the search for alternative antimicrobials
Summary
Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogenic bacterium for both humans and animals worldwide. Bacterial carriage is an important risk factor for nosocomial and surgical site infections (Wertheim et al, 2004; Wertheim and Vos, 2005). Recent data show that MRSA decolonization decreases the risk of surgical site infections by approximately 39% in total knee and hip arthroplasty surgery (Sadigursky et al, 2017). Mupirocin has been usually prescribed as nasal ointment to remove S. aureus from the nose. Recolonization by this bacterium was observed after treatment, mainly due to the selection of bacterial resistance to this antibiotic (Coates et al, 2009)
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