The streptococcal phage SA2 and B30 endolysins act synergistically and kill mastitis causing streptococci in milk

Authors: Schmelcher, Mathias; Pohl, Calvin; Donovan, David

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/25/2015
Publication Date: 4/21/2015
Citation: Schmelcher, M., Pohl, C.S., Donovan, D.M. 2015. The streptococcal phage SA2 and B30 endolysins act synergistically and kill mastitis causing streptococci in milk. Applied and Environmental Microbiology. 99(20):8475-86.

Interpretive Summary: Problem Bovine mastitis, an inflammation of the mammary gland in cows, is currently the most costly disease in dairy industry, resulting in annual costs between $1.7 billion and $2 billion in the United States alone. The most relevant causative agents of mastitis include the three streptococcal species S. agalactiae, S. dysgalactiae, and S. uberis. Currently, there is no effective treatment for bovine mastitis. Conventional use of broad range antibiotics is successful in only about half of the cases and may contribute to development of antibiotic resistance. Therefore, alternative antimicrobials for the control of these pathogens are needed. Accomplishment In this study, the streptococcal phage 'SA2 and B30 endolysins were biochemically characterized and shown to exhibit lytic activity against live streptococcal cells in vitro under physico-chemical conditions present in cows milk. When applied in combination, the two enzymes were demonstrated to exhibit a strong synergistic effect, killing streptococci even more efficiently than when used alone. Both enzymes were also shown to be active in cows milk against all three relevant streptococcal species, and to also reduce bacterial numbers in milk more efficiently when used in combination. Contribution of Accomplishment to Solving the Problem These findings provide new evidence that bacteriophage endolysins have a high potential as alternative antimicrobials for the control of pathogenic bacteria such as streptococci. It is widely accepted that the chance of resistance development against phage endolysins is lower than for antibiotics. Despite efforts to find them, bacterial strains resistant to phage lysins have not been reported. The findings that the 'SA2 and B30 endolysins kill their target bacteria in a synergistic manner and are active against all three relevant streptococcal species in whole cows milk makes them promising new antimicrobials for the treatment of bovine mastitis.

Technical Abstract: Bovine mastitis results in billion dollar losses annually in the United States alone. Among the most relevant causative agents of this disease are members of the genus Streptococcus, particularly the species S. agalactiae (Group B Streptococcus; GBS), S. dysgalactiae (Group C; GCS), and S. uberis. Conventional treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistant strains of pathogens. Bacteriophage endolysins present a new promising source of antimicrobials against these pathogens. In this work, we biochemically characterized the endolysins of the streptococcal phages 'SA2 and B30, examining the influence of salt concentration, pH, and bivalent metal cations on their in vitro lytic activity against S. dysgalactiae. Both enzymes exhibited near-optimum activities at an ionic strength, pH conditions, as well as a Ca2+ concentration consistent with milk. When applied in combination, the 'SA2 and B30 lysins were found to exhibit a synergistic effect against streptococci. Using a novel plate lysis checkerboard assay, the sum of the fractional inhibitory concentrations of both enzymes against S. dysgalactiae was determined to be <0.5, indicating strong synergy. Furthermore, we examined the activity of both lysins against different streptococcal species in whole milk. The addition of 100 µg/ml B30 or 'SA2 lysin to milk samples inoculated with 103 cfu/ml of S. dysgalactiae resulted in an instantaneous drop in cell concentrations and kept them ~2.5 log units (for B30) or >3.5 log units (for 'SA2) below those of the control samples during an incubation period of three hours at 37°C. For 'SA2, concentrations as low as 1 µg/ml were sufficient to keep cell numbers in milk below those of the control. Both proteins were also shown to be active against S. dysgalactiae at higher inoculation levels (5 x 106 cfu/ml), and against S. agalactiae and S. uberis. In a proof-of-concept experiment, we demonstrated that the effect of 'SA2 and B30 in combination against S. dysgalactiae in milk was greater than theoretically expected for an additive action, suggesting that the synergy between these enzymes also holds true in a milk environment. Overall, our results with the B30 and 'SA2 lysins illustrate the high potential of these enzymes as antimicrobials for the treatment of bovine mastitis.