Robert Colquhoun Shields
Extracellular DNA (eDNA) is a ubiquitous component of the extracellular matrix of microbial biofilms. It has a number of functions that include a role as an adhesin during biofilm attachment, and facilitating matrix stability in mature biofilms. Increasingly, deoxyribonuclease (DNase) enzymes have been shown to reduce the colonisation of many microbial biofilms, both bacterial and fungal. Biofilms are estimated to be responsible for around 60% of bacterial infections, including many chronic diseases. The aim of this work was to determine the role of eDNA in chronic mixed-species biofilm infections, and to explore the potential of DNase enzymes for biofilm control. This included three major areas of research, focusing on chronic rhinosinusitis, tracheoesophageal speech valves (TESVs), and dental plaque. An important aspect was to test the efficacy of a novel bacterial nuclease, NucB, isolated from a seaweed-associated strain of Bacillus licheniformis, against microbial biofilms. The colonisation of speech valves by micro-organisms was studied using scanning electron microscopy (SEM). In keeping with previous observations, these biofilms were co-aggregations of fungal and bacterial species. Using confocal laser scanning microscopy, eDNA was observed in the biofilm matrix. Extracellular DNA was extracted and quantified from TESV biofilms. All six biofilms studied had detectable nucleic acids, as measured by NanoDrop spectrophotometry. The eDNA was apparently heavily degraded, and produced smears by agarose gel electrophoresis. Nevertheless, eDNA appeared to be providing biofilm stability since micro-organisms were liberated from the surface of the valves following treatment with NucB in over 60% of the TESVs tested. To assess the role of eDNA in biofilms associated with chronic rhinosinusitis, ‘obstructive mucin’ and sinus mucosa biopsy samples collected during functional endoscopic sinus surgery at the Freeman Hospital, Newcastle, were analysed for the presence of biofilms and biofilm-forming micro-organisms. An average of 3.75 bacterial species per patient were cultured from obstructive mucin. The most commonly isolated micro-organisms were Staphylococcus aureus, coagulase-negative staphylococci and α-haemolytic streptococci. Micro-organisms were not detected by transmission electron microscopy of the obstructive mucin and this material appeared to originate through a host inflammatory response. However, bacteria were visualised on the surface of sinus mucosa using a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) universal bacteria probe. Twenty-four bacterial isolates were iv assessed for their ability to form biofilms in a microtitre plate model. All micro-organisms tested formed biofilms, and 14 of 22 were susceptible to NucB. In total, 15 of 24 microbial species produced eDNA that was detectable by agarose gel electrophoresis. By SEM, cellular colonisation was lower in treated samples and, in the case of Streptococcus constellatus FH20 stringy, matrix-like material was not present after DNase treatment. The role of eDNA in matrix stability and initial biofilm attachment was also studied in oral bacteria. Streptococcus gordonii DL1, Streptococcus mutans GS-5, Fusobacterium nucleatum 25586 and Actinomyces oris MG1 were examined using DNase treatment, CLSM, and eDNA extraction. Of these species, all except S. gordonii appeared to rely on high molecular weight (HMW) eDNA for biofilm attachment and biofilm stability. Although S. gordonii did not produce detectable HMW eDNA, nucleic acids were detectable by NanoDrop spectrophotometry. Furthermore, this species produces an extracellular nuclease which may degrade the HMW eDNA in the conditions used to culture biofilms. Interestingly, four S. mutans strains differed in their sensitivity to DNase treatment. Oral biofilms were also modelled in a BioFlux microfluidics device using flowing human saliva. Mixed-species biofilms and single species biofilms of S. mutans UA159 and S. gordonii DL1 were cultured using this technique, to determine whether this model would allow more realistic experiments for DNase testing. Finally, the extracellular nuclease, SsnA, of S. gordonii DL1 was characterised. A nuclease-deficient mutant did not produce extracellular nuclease activity on DNase Test agar or during a Forster Resonance Energy Transfer (FRET) assay. Nuclease activity was cell-wall-associated as predicted from the predicted amino acid sequence of SsnA. Allelic exchange mutagenesis determined that ssnA expression was regulated by CcpA in response to repressing sugars. However, in planktonic culture non-repressing carbon sources also inhibited enzyme activity during a FRET assay. Further experiments using acidic buffers replicated the inhibition of SsnA without the presence of sugars. SsnA was purified as a GST-tagged fusion protein in an Escherichia coli protein expression system, and had anti-biofilm activity against S. mutans GS-5. However, this species is strongly acidogenic and therefore it is hypothesised that although SsnA may be a competition biofilm factor, acid production by S. mutans may reduce its efficacy in vivo. In conclusion, this thesis has provided strong evidence for the role of eDNA in facilitating biofilm formation and mature biofilm stability of clinically relevant v biofilms. Nucleic acids were present in biofilms associated with a chronic infection, medical implant biofouling and dental plaque. A variety of DNase enzymes (NucB, DNase I, and SsnA) were capable of reducing biofilm colonisation. Given the adhesive function of eDNA in biofilm matrices it is proposed that DNase enzymes may be beneficial for controlling healthcare-related biofilms.