Biofilm prevalence and microbial characterisation in chronic wounds in a Sri Lankan cohort

Abstract

Biofilms have been associated with chronic wound infections in diabetic patients. The study assessed the occurrence of biofilms in chronic diabetic wounds (CDWs) in a Sri Lankan cohort. Tissue specimens collected during surgical debridement were analysed by quantitative differential viable counting, scanning electron microscopy (SEM), fluorescence insitu hybridization (FISH) and light microscopy with Gram and Haematoxylin-Eosin staining. All specimens harboured >5·0 log₁₀ CFU per g bacteria and 2-9 distinct species per specimen were recovered from twenty wounds by culture. The most frequently isolated bacterium was Pseudomonas spp. (12/20;60%). Strict anaerobes were isolated from 10/20 specimens. Gram and Haematoxylin-Eosin staining showed aggregated micro-colonies, embedded in the wound tissue bed (20/20) but the exopolymer matrix was not visible in all samples (13/20). Fluorescence microscopy using a eubacteria-specific FISH probe indicated the presence of bacterial aggregates within the deep layers of the wound tissues (20/20). SEM revealed the presumptive architecture of matrix-embedded microbial clusters (20/20). The approximate diameter of bacterial aggregates in tissues ranged between 12 and 400 µm. Bacterial infiltration into the internal portions of the tissues was apparent using FISH, Gram, and Haematoxylin-Eosin staining. All CDWs carried biofilm-specific morphological features. FISH was more specific than SEM and indicated the presence of microcolonies within deeper tissues.

Keywords: Gram staining; Haematoxylin-Eosin staining; biofilm; diabetic chronic wounds; fluorescence in situ hybridization (FISH); scanning electron microscopy (SEM).