Macroscale spatial variation in chronic wound microbiota: a cross-sectional study.

Controlling for sample site is considered to be an important aspect of chronic wound microbiological investigations; yet, macroscale spatial variation in wound microbiota has not been well characterized. A total of 31 curette samples were collected at the leading edge, opposing leading edge, and/or center of 13 chronic wounds. Bacterial community composition was characterized using a combination of 16S rRNA gene-based pyrosequencing; heat map display; hierarchical clustering; nonmetric multidimensional scaling; and permutation multivariate analysis of variance. A total of 58 bacterial families and 91 bacterial genera were characterized among the 13 wounds. While substantial macroscale spatial variation was observed among the wounds, bacterial communities at different sites within individual wounds were significantly more similar than those in different wounds (p=0.001). Our results support the prevalent opinion that controlling for sample site may improve the quality of wound microbiota studies; however, the significant similarity in bacterial communities from different sites within individual wounds indicates that studies failing to control for sampling site should not be disregarded based solely on this criterion. A composite sample from multiple sites across the surface of individual wounds may provide the most robust characterization of wound microbiota.

Community analysis of chronic wound bacteria using 16S rRNA gene-based pyrosequencing: impact of diabetes and antibiotics on chronic wound microbiota.

BACKGROUND: Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota. METHODOLOGY/PRINCIPAL FINDINGS: We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds--approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007) and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics. CONCLUSIONS/SIGNIFICANCE: The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure--reducing some bacteria while selecting for others.