Antibacterial and Antibiofilm Efficacy of Endolysin LysEf-9 and Crude Bacteriocin Against Multidrug and Phage-Resistant Enterococci.

A gene encoding endolysin LysEf-9 from the phage PEF9, a lytic enterococcal phage, was cloned and overexpressed in Escherichia coli. The expressed LysEf-9 protein with a concentration of 62.5 μg/mL reduced the turbidity and viability of E. faecalis JCM 7783 after 3 h of incubation. LysEf-9 showed stability at different pH (2-12) and withstood different temperature treatments up to 60 °C. In addition, it showed a wide spectrum against Enterococci. Phage vB_EfKS5 with antibacterial and antibiofilm properties against E. faecalis was isolated previously. The phage-resistant mutant was isolated and used as an indicator strain in the present study. A bacteriocin-producing E. faecalis strain was also isolated, and the cell-free supernatant (crude bacteriocin) was partially purified by ammonium sulfate precipitation. The crude bacteriocin showed a wide spectrum against Enterococcus spp. and lysed the phage-resistant mutant. Using LysEf-9 with the crude bacteriocin reduced the viable count of E. faecalis JCM 3378 by 2.45 log after 24 h. In addition, LysEf-9 with phage vB_EfKS5 retarded the growth of the phage-resistant mutant. The antibiofilm efficacy of LysEf-9 and crude bacteriocin was also determined. LysEf-9 lowered the viable count of E. faecalis JCM 7783, E. faecium No. 26, and the phage-resistant mutant in the 48-h biofilm by 3 log, 2 log, and 1.5 log, respectively. The combination of LysEf-9 with crude bacteriocin reduced the total viable count of the indicator strains by at least 3 logs. This study shows the strong antibacterial and antibiofilm activity of LysEf-9, and the activity was enhanced when used with crude bacteriocin or phage.