Exploring novel antibiotic and enzyme combinations to improve the treatment options for biofilm-associated diabetic foot infections

Staphylococcus aureus isolates collected from the wounds of a cohort of patients presenting at University Hospital Galway (UHG) with diabetic foot infections were shown to produce a mix of polysaccharide and protein type biofilms in vitro. Several of the MSSA isolates also demonstrated the ability to switch from a polysaccharide to protein mechanism of biofilm formation. This latter phenotype is more commonly associated with MRSA isolates following growth in culture media supplemented with glucose. Hyperglycaemia is common in patients with diabetes mellitus and may be accompanied by increased levels of glucose in the wound milieu, which in turn would promote protein-adhesin type biofilm formation. Several coagulase-negative staphylococci including two methicillin-resistant Staphylococcus epidermidis isolates, which are often commonly overlooked in chronic wounds, also formed robust biofilms in vitro suggesting they were clinically significant in the pathogenesis of the wound infections from which they were isolated. In a related collaborative study with colleagues at the University of Liverpool on chronic infections in patients with cystic fibrosis, twelve MRSA isolates were collected sequentially over a period of four years from the lungs of a single patient. All of these isolates formed robust protein-type biofilms in vitro. Isolate LCF269, which was the fourth sequential strain isolated from the patient produced approximately 4X more biofilm than the other isolates. Whole genome sequencing analysis revealed that LCF269 and subsequent isolates were genetically different to the first three isolates. Furthermore LCF269 was found to contain 6 synonymous mutations in sdrC, which encodes a cell wall anchored protein previously implicated in the biofilm phenotype. Interestingly, two of the clinical DFI-associated MSSA isolates were shown to be very similar to one another. Both demonstrated the ability to switch between the protein and polysaccharide type biofilm production and both possessed similar antimicrobial susceptibility profiles. Upon taxonomic classification these were the only two isolates collected from the patients at UHG shown to share a top common hit during sequence similarity searches using NCBI BLAST. These isolates were collected from two separate patients presenting at UHG within one week of each other raising the possibility that similar MSSA strains are circulating among patients attending this clinic. Antibiotic doses up to 1000X the minimum inhibitory concentration were required to inactivate biofilm bacteria, even when the drugs were used in combination, reflecting the high tolerance of biofilms to antimicrobial drugs. The most effective combination therapy identified in this study comprised rifampicin which targets bacterial transcription, gentamicin which targets translation and the clinically used enzymatic debridement agent trypsin. Exposure of biofilms to this triple combination for 48 hours resulted in a > 3 log (99.9%) reduction in the number of colony forming units for all of the DFD staphylococcal isolates tested, surpassing the proposed threshold for clinical significance. Moreover, this triple combination was also highly effective against S. aureus and Pseudomonas aeruginosa mixed species biofilms using a chronic wound biofilm model at clinically achievable drug and enzyme concentrations. The rifampicin/gentamicin/trypsin combination represents a promising new therapeutic strategy for the treatment of biofilm-associated diabetic foot infections.

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University of Galway

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Attribution-NonCommercial-NoDerivatives 4.0 International

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University of Galway Theses (PhD Theses)