Exploration of Bacterial Re-Growth as In Vitro Phenomenon Affecting Methods for Analysis of the Antimicrobial Activity of Chimeric Bacteriophage Endolysins

Drug alternatives to combat methicillin-resistant Staphylococcus aureus (MRSA) in human and animal healthcare are urgently needed. Recently, the recombinant bacteriophage endolysins, PRF-119 and its successor substance HY-133, have proven to be highly active against various S. aureus clonal lineages and to exhibit a very rapid bactericidal effect when standard methods for susceptibility testing are applied. Along with subsequent growth curve experiments, a re-growth phenomenon was observed in vitro necessitating its clarification for the assessment of the agent's stability and activity as well as for methodological aspects of endolysin testing in general. Distinct in vitro parameters were comparatively examined applying also scanning electron microscopy, fluorescence assays and SDS-PAGE analysis. The shape and material of the culture vessels as well as the shaking conditions were identified as factors influencing the in vitro stability and activity of HY-133. The highest function maintenance was observed in plain centrifuge tubes. Based on this, the conditions and parameters of assays for testing the antimicrobial activities of phage endolysins were determined and adjusted. In particular, shear forces should be kept to a minimum. Our results form the basis for both future test standardization and re-growth-independent experiments as prerequisites for exact determination of the antimicrobial activities of engineered endolysins.

Automated summary

Recombinant bacteriophage endolysins PRF-119 and its successor HY-133 have been found to be highly active against various MRSA clonal lineages. However, in vitro re-growth phenomenon was observed, necessitating clarification for the assessment of the agent's stability and activity as well as for methodological aspects of endolysin testing. Factors such as the shape and material of culture vessels and shaking conditions were identified to influence the in vitro stability and activity of HY-133. The findings provide a basis for future standardization and accurate determination of the antimicrobial activities of engineered endolysins.