Culture Positivity Reflects Quantitative Bacterial Bioburden in Open Fractures

Infection following high-energy open fractures remains a major challenge. Open fractures are exposed to environmental and skin pathogens, which, along with traumatized tissue and implanted hardware, create a favorable environment for infection. 24-83% of open fractures yield positive culture results. Despite culture-positive wounds being at higher risk for infection, culture data are rarely used in clinical decision-making. Culture-positivity in open fractures is believed to indicate higher wound bioburden, but this hypothesis remains unconfirmed. This study aimed to correlate culture positivity and quantitative wound bioburden using next-generation sequencing (NGS) and to characterize patterns of bioburden and bacterial diversity amongst patients with open fracture and Fracture Related Infection. We hypothesized that higher wound bioburden would be associated with culture positivity.

This secondary analysis of the METRC Bioburden Study included 78 patients with severe open tibia fractures who underwent standardized tissue sampling at definitive wound closure and at later fracture site revision surgery. DNA extracted from intraoperative tissue was analyzed via 16S rRNA gene amplicon sequencing. Quantitative bioburden metrics included bacterial read percentage and bacteria-to-human read ratio (BHR). Associations between sequencing-based bioburden, culture results, and clinical infection (CDC-defined) were evaluated using Mann- Whitney U tests, McNemar's chi-squared tests, and unsupervised clustering analyses.

Culture-positive samples exhibited significantly higher bacterial read percentages (17.5 ± 26.7% vs. 0.8 ± 4.7%, p = 8×10□□) and higher log(BHR) values (1.73 ± 10.35 vs. 0.06 ± 0.52, p = 3×10□□) than culture-negative samples. Optimal thresholds yielded 65% sensitivity/94% specificity (bacterial reads) and 89% sensitivity/69% specificity (BHR) for predicting culture positivity. Neither baseline culture nor NGS positivity predicted later infection. However, follow-up samples from patients with established infection showed significantly higher bioburden and distinct microbiome structures. Clustering analyses identified two infection-associated microbial profiles: one with high bioburden and high diversity (polymicrobial infection) and one with low bioburden and low diversity (single-dominant pathogen).

Culture positivity in open fractures correlates strongly with increased bacterial bioburden, indicating that positive cultures may primarily reflect microbial load rather than specific pathogens. Quantitative NGS-based measures, including bacterial read proportion and BHR, may offer a biologically grounded approach to assess wound contamination and may inform infection risk. Distinct bioburden-diversity patterns suggest heterogeneous infection phenotypes that may require tailored surgical and antimicrobial strategies.