Title: Antimicrobials for mastitis causing pathogens that are refractory to resistance development Authors
Submitted to: BARC Poster Day
Publication Type: Abstract Only
Publication Acceptance Date: April 15, 2009
Publication Date: April 23, 2009
Citation: Becker, S.C., Don, S., Baker, J.R., Foster Frey, J.A., Pritchard, D.G., Donovan, D.M. 2009. Antimicrobials for mastitis causing pathogens that are refractory to resistance development. BARC Poster Day. Interpretive Summary: Staphylococci are gram positive pathogens that have large negative impacts on both agriculture and human health. Staphylococci and streptococci are leading causes of bovine mastitis, a 2 billion dollar a year loss, accounting for more than 40% of all cases in one study. Traditional treatments for mastitis, most commonly antibiotics, have been shown to be only marginally effective. Bacteriophage endolysins are a potential source of narrow spectrum antibiotics for treatment of antibiotic resistant pathogens. Accomplishments: We have determined the cut sites of each active domain and further dissected LysK to determine the CHAP domain of LysK appears to be the crucial element in promoting exolysis, but the presence of a SH3b cell wall binding domain greatly increases the efficiency of lysis. Additionally we have determined that fusing a staphylococcal cell wall binding domain targets the streptococcal lytic domain from the Lambda SA2 phage endolysin to staphylococcal cell lysis with a 10X increased heterologous lytic activity. Contribution of Accomplishment to Solving the Problem: The findings of this work identify the necessary domains of these phage endolysins for optimal lytic activity and will aid scientists in the development of novel fusion antimicrobials.
Technical Abstract: Staphylococci and streptococci are both human and agricultural pathogens responsible for >50% of clinical mastitis incidents (resulting in losses to the dairy industry greater than $2 billion annually). The rise in bacterial resistance to antibiotics world-wide has precipitated the search for alternatives to broad range antimicrobials, and efforts to identify those that are refractory to resistance development. Peptidoglycan hydrolases (including bacteriophage endolysins) are one such group of novel antimicrobials. These enzymes cause cell lysis by degrading cell wall peptidoglycan, are often genus specific, and no bacteria have been identified that can evade lysis by their bacteriophage endolysin (despite efforts to identify them). Lysins are modular in structure, often with multiple lytic domains and a cell wall binding domain. We have shown that fusing domains from multiple enzymes, generates chimeric molecules that maintain their parental specificities and lytic activities. Here we describe fusion antimicrobials that each lyse mastitis causing staphylococci with three unique and synergistic lytic activities. Because no bacterium can resist three simultaneous lytic activities, we predict these fusions will be refractory to resistance development. The fusion proteins will be examined in multiple bactericidal assays, and for their efficacy against multiple pathogens. In an effort to further improve our engineered constructs, we have collated, and compared all staphylococcal SH3b cell wall binding domains. Through heterologous fusion to a streptococcal endolysin lytic domain, we have demonstrated the relative efficacy of bacteriocin- and endolysin-derived cell wall binding domains. The staphylococcal cell wall binding domains can re-direct the streptococcal lytic domain to kill S. aureus, with a 10X increase in lytic activity over the wild type streptococcal enzyme. Interestingly, the resultant heterologous fusion also maintains a high level of anti-streptococcal lytic activity, indicating that the SH3b domain is not the only factor that contributes to lytic specificity. These studies have resulted in two provisional patents.