Next-generation DNA sequencing of medically relevant biofilms

Background - A better understanding of the microbial communities in medical environments is crucial for improving human health, given the increasing problem of biofilm-related infections. With the rapidly expanding molecular methods opening new horizons in the study of the presence of microbial biofilm from samples collected within medical settings. The aims of this project were therefore to better understand the microbiome and the role of biofilm in (i) providing a protected source of pathogens that can cause healthcare-associated infections (HAIs), (ii) causing granulomatous reactions and its possible role in potentiating cancer, and (iii) chronic wound infections. Methods - We employed advanced molecular methods, including next-generation DNA sequencing, fluorescence in situ hybridisation (FISH) and real-time quantitative polymerase chain reaction (qPCR), along with confocal laser scanning and scanning electron microscopy, to investigate the project aims. Results(i) The majority of ICU surfaces sampled from three hospitals were contaminated with polymicrobial biofilms, which contained MDR strains, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE) and extended spectrum beta-lactamase (ESBL) Sphingomonas paucimobilis. Similarly, a high number of patient-ready endoscopes were contaminated with biofilms composed of potentially pathogenic Gram-negative bacteria, including Shigella dysenteriae, Escherichia coli and Klebsiella pneumonia. (ii) Soft-tissue fillers support the growth of Staphylococcus epidermidis biofilm in vitro. While soft-tissue filler clinical samples all demonstrated multi-species biofilm with a predominance of Pseudomonas, Staphylococcus and Propionibacterium. In breast implant-associated anaplastic large-cell lymphoma (BIA-ALCL) clinical samples, a high bacterial load, present as a biofilm was identified. Moreover, a distinct microbiome in BIA-ALCL specimens was identified, with a significantly greater proportion of Ralstonia spp. compared to non-tumour contracted capsules. (iii) Diabetic foot ulcer (DFU) clinical samples all contained biofilm, with multi-species communities comprising of both strict anaerobes and aerobic species. In addition, chronic DFUs were found to be associated with a highly polymicrobial microbiome with greater species richness and diversity. In the treatment of chronic non-healing DFUs complicated by biofilm, cadexomer iodine was found to significantly reduce the microbial load. However, short exposure times to topical antimicrobial solutions (commonly utilised by clinicians) were found to be ineffective against microbial biofilms in vivo. Conclusions - Using next-generation DNA sequencing it was possible to investigate the complex arrays of bacterial species residing in hospital surfaces, medical and implantable devices, and DFU tissue specimens. This provides significant new insights into the study of the microbiome in medical practice, which is crucial for improving human health.