Culture-independent pilot study of microbiota colonizing open fractures and association with severity, mechanism, location, and complication from presentation to early outpatient follow-up

Abstract

Precise identification of bacteria associated with post-injury infection, co-morbidities, and outcomes could have a tremendous impact in the management and treatment of open fractures. We characterized microbiota colonizing open fractures using culture-independent, high-throughput DNA sequencing of bacterial 16S ribosomal RNA genes, and analyzed those communities with respect to injury mechanism, severity, anatomical site, and infectious complications. Thirty subjects presenting to the Hospital of the University of Pennsylvania for acute care of open fractures were enrolled in a prospective cohort study. Microbiota was collected from wound center and adjacent skin upon presentation to the emergency department, intraoperatively, and at two outpatient follow-up visits at approximately 25 and 50 days following initial presentation. Bacterial community composition and diversity colonizing open fracture wounds became increasingly similar to adjacent skin microbiota with healing. Mechanism of injury, severity, complication, and location were all associated with various aspects of microbiota diversity and composition. The results of this pilot study demonstrate the diversity and dynamism of the open fracture microbiota, and their relationship to clinical variables. Validation of these preliminary findings in larger cohorts may lead to the identification of microbiome-based biomarkers of complication risk and/or to aid in management and treatment of open fractures.

Keywords: 16S rRNA; bacteria; infection; microbiome; open fracture.

Figure 1. The changing relationships between open fracture wound and adjacent skin microbiota of 10 patients over time

PCoA plots representing the Bray-Curtis metric comparing beta diversity of open fracture and skin microbiota. Each color represents a different patient, while triangles and circles represent wound center and adjacent skin microbiota, respectively. Shown are the first two principle coordinates and the percent variation explained by each principle coordinate is indicated in parentheses by the axis. The two samples (open fracture wound and adjacent skin) for each patient at a given time point are connected by a line. An ANOSIM test was used to examine the association between swab location and the overall community composition; this association is significant (at P<0.05) only for the ER time point.

Figure 2. Alpha diversity and bacterial load of open fracture wound and adjacent skin

Alpha diversity is depicted as measured by Faith’s PD (A) and observed species-level OTUs (B). Bacterial load (C) is represented as estimates from quantitative PCR of the 16S rRNA gene. The upper and lower box hinges correspond to the first and third quartiles (25% and 75%), and the distance between the first and third quartiles is defined as the inter quartile range (IQR). Lines within the box depict median, and the whiskers extend to the highest and lowest values within 1.5 times the IQR. Outliers of the IQR are depicted with black dots above or below the whiskers. An asterisk (*) inside the box indicates significance of P<0.05 (Wilcoxon rank-sum test) between the adjacent skin and open fracture wound at the indicated time point. An asterisk (*) outside of the box indicates significance of P<0.05 (Wilcoxon rank-sum test) between the indicated time points.

Figure 3. Gram-positive and Gram-negative bacteria in the open fracture wound and on the adjacent skin

Open fracture wound relative abundance is shown in (A) and adjacent skin relative abundance is shown in (B). The upper and lower box hinges correspond to the first and third quartiles. Lines within the box depict median, and the whiskers extend to the highest and lowest values within 1.5 times the IQR. Outliers of the IQR are depicted with black dots above or below the whiskers. *P<0.05 (Wilcoxon rank-sum test).

Figure 4. Association of alpha diversity with open fracture characteristics

Faith’s PD comparing alpha diversity according to mechanism of injury (A), Gustilo-Anderson classification (B), whether or not the fracture healing process was complicated (C), and the anatomical location of the open fracture (D). The upper and lower box hinges correspond to the first and third quartiles. Lines within the box depict median, and the whiskers extend to the highest and lowest values within 1.5 times the IQR. Outliers of the IQR are depicted with black dots above or below the whiskers. *P<0.05 (Wilcoxon rank-sum test).