<b>Characterization of Gram-positive Bacteria Isolated from Gallstones of Iraqi Patients</b>

Background: Gallstone disease is a major global health problem, the prevalence of which depends on metabolic, genetic, and infectious factors. Recent reports point out that Gram-positive bacteria, mainly Staphylococcus aureus and Enterococcus species, are also involved in the development of gallstones via enzyme action and biofilm formation. Objectives: To characterize Gram-positive bacteria within gallstones from Iraqi patients, evaluate their biofilm-forming capacity, and analyze the relationship between bacterial colonization, gallstone type, as well as cholesterol levels. Methods: A total of 100 gallstones were obtained from 100 patients undergoing elective cholecystectomy between October 2024 and March 2025. Stones were aseptically processed for bacterial isolation and identification using selective culture media and the VITEK® 2 Compact System. Serum cholesterol levels were determined by enzymatic colorimetric assay. Biofilm formation was quantified via the 96-well microtiter plate method, and statistical correlations between gallstone type, cholesterol level, and bacterial presence were analyzed. Chi-square assessed the association between gallstone type and bacterial count, while Mann-Whitney U and Kruskal-Wallis tested differences in study variables. Results: Cholesterol stones were more prevalent than pigment and mixed stones (57%, 40%, 3%), respectively. Bacterial growth was observed in 43% of gallstones, with Enterococcus species (31 isolates) predominating over S. aureus (12 isolates). Species-level identification revealed E. faecalis (n= 16), E. faecium (n= 9), and E. gallinarum (n= 6), marking the first reported isolation of E. gallinarum from gallstones. Cholesterol concentrations were significantly higher in sterile stones (median 235 mg/dl) compared to bacteria-positive stones (173-186 mg/dl). Biofilm analysis showed all S. aureus isolates as strong producers, whereas E. faecalis exhibited predominantly moderate-to-strong formation, while E. faecium and E. gallinarum displayed weaker capacities. Conclusion: There is a significant interplay between microbial colonization and gallstone composition. Strong biofilm-producing Gram-positive bacteria, particularly S. aureus and E. faecalis, may facilitate stone maturation and persistence. The novel isolation of E. gallinarum expands the spectrum of biliary microbiota.