Antimicrobial Efficacy of a Combination of Crocodile (Crocodylus siamensis) Leukocyte Extract and Hen Egg Lysozyme

Abstract

The combination of two or more natural antimicrobial substances is extensively used in clinical therapy. In this context, lysozymes represent an interesting group of naturally antimicrobial proteins, since they were found to display synergistic activity in combination with other antimicrobial substances. Furthermore, studies employing Crocodylus siamensis leukocyte extract (cLE) recently revealed the potent antimicrobial properties. However, potential synergistic interactions of cLE with other antimicrobials have not been reported to date. In this work, we were thus interested to investigate the synergy of cLE with hen egg lysozyme (hEL) in more detail. Employing a checkerboard technique, the combination of cLE and hEL in vitro showed partial synergy against foodborne V. cholerae (clinical isolation) with the fractional inhibitory concentration index (sFIC) value of 0.75. At the same concentration, a strong synergistic efficacy of the hEL-cLE combination was observed using time-kill assay. SEM images also suggest that the killing mechanism of the combination involves bacterial cell wall lysis and membrane damages. Additionally, in-vivo toxicity test of the combination in mice was performed. The results show that the hEL-cLE combination at 5 ´ sFIC neither induced significant modulation of most biological parameter levels in mice serum, nor affected the histopathological features of mice livers and kidneys. These observations provide clear evidence that the combination of hEL and cLE is essentially non-toxic and represents a promising target for development in clinical therapy from bacterial infection.