AimsA novel antibacterial peptide from Crocodylus siamensis haemoglobin hydrolysate (CHH) was characterized for antimicrobial activity. Methods and ResultsCHHs were hydrolysed for 2h (2h-CHH), 4h (4h-CHH), 6h (6h-CHH) and 8h (8h-CHH). The 8h-CHH showed antibacterial activity against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa at concentrations of 20, 20, 20 and 10mgml(-1) (w/v) respectively. Fluorescent microscopy revealed that the 8h-CHH had bactericidal activity against E.coli and P. aeruginosa. -galactosidase assay supported by RT-qPCR demonstrated that the 8h-CHH resulted in differential expression of genes involved in iron homeostasis (ftnA and bfd) and oxidative stress (sodA, soxR and oxyR). Siderophore assay indicated that the 8h-CHH also impaired siderophore production with diminished expression of pvdF. This pattern of gene expression suggests that the 8h-CHH triggers the release of free ferric ions in the cytoplasm. However, decreased expression of genes associated with the SOS response (recA and lexA) in combination with neutral comet revealed that no DNA damage was caused by 8h-CHH. Membrane permeabilization assay indicated that 8h-CHH caused membrane leakage thought to mediate the antibacterial and iron-stress responses observed, due to loss of regulated iron transport. The novel active peptide from 8h-CHH was determined as QAIIHNEKVQAHGKKVL (QL17), with 41% hydrophobicity and +2 net charge. ConclusionsThe QAIIHNEKVQAHGKKVL fragment of C. siamensis haemoglobin is antibacterial via a mechanism that likely relies on iron dysregulation and oxidative stress which results in bacterial death. Significance and Impact of the StudyWe have described for the first time, a novel peptide derived from C. siamensis haemoglobin hydrolysate that has the potential to be developed as a novel antimicrobial peptide.