Human breast milk microbiota is essential for infant immune system development, maturation and
protection against infection. However, there is scarce information on the fluid’s microbiological
composition from Malaysia. The objective of the study was to isolate, identify and characterise commensal
bacterial population present in human breast milk from Malaysia. One hundred bacteria were isolated
from the human breast milk of healthy lactating women (n=30). After preliminary screening, 20 isolates
were characterised using both phenotypic and molecular techniques. The results indicated that most
frequently identified bacteria in this study were E. faecalis and S. hominis. These organisms alongside E.
cloacae were all metabolised D-Maltose, Sucrose, D-Turanose, α-D-Glucose, D-Fructose, D-Mannose,
D-Galactose, D-sorbitol and D-Mannitol and were able to grow at pH 5 and 6, 1% sodium lactate, 1%,
2% and 8% NaCl. BLAST showed over 99% similarity to those deposited in Genbank. Phylogeneticrelatedness
was depicted using neighbour-joining method and had two clades with 100% bootstrap. These
findings provided insight into the nature, characteristics and also phylogenetic-relatedness of bacteria
present in human milk from Malaysia. Isolation and identification of commensal bacteria from human
milk are considered the first step for future studies on the benefit of these organisms towards human health.