Recombinant of the Staphylococcal Bacteriophage Lysin CHAP(k) and Its Elimination against Streptococcus agalactiae Biofilms

Bovine mastitis is the most important infectious disease, causing significant losses in the dairy industry, in which Streptococcus agalactiae is a major pathogen. In this study, lysin CHAP(k), derived from bacteriophage K, was expressed heterogeneously, and its antimicrobial and anti-biofilm effects against S. agalactiae isolated from bovine mastitis were further analyzed. CHAP(k) was expressed in Escherichia coli BL21 (DE3), in which the purified yield of CHAP(k) was up to 14.6 mg/L with the purity of 95%. Time-killing kinetic curves showed that CHAP(k) fastly killed S. agalactiae in TSB medium and in milk within 25 min (by 3.3 log(10) CFU/mL and 2.4 log10 CFU/mL, respectively). Observation of scanning electron microscope (SEM) showed cells wrinkled and ruptured after the treatment of CHAP(k). CHAP(k) effectively inhibited early biofilms by 95% in 8 x MIC, and eradicated mature biofilms by 89.4% in 16 x MIC. Moreover, CHAP(k) killed 99% bacteria in mature biofilms. Confocal laser scanning microscopy (CLSM) also demonstrated the potent antimicrobial and anti-biofilm action of CHAP(k). It was firstly demonstrated CHAP(k) had the characters of inhibition/elimination of S. agalactiae biofilms and killing the bacteria in biofilms. CHAP(k) has the potential to develop a new antibacterial agent for mastitis treatment of S. agalactiae infections.