Understanding the Effects of Topical Antibacterials on Wound Bacteria

Abstract

Background. Silver compounds are commonly used in wound dressings to control bacterial growth. Although sporadic cases of reduced silver susceptibility have been described, the routes of adaptation, particularly in sessile growth modes are not well understood. Methods. An agar-based diffusion system was used to expose wound isolates to sub-inhibitory concentrations of ionic silver over ten passages (P10) and again following ten additional passages in silver-free media (X10). Test bacteria were as follows: Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli and Bacteroides fragilis. Susceptibility to silver and antibiotics in planktonic and biofilm growth modes was assessed using standard methods. In addition, bacterial growth kinetics, biofilm formation ability and Galleria mellonella pathogenesis assay were determined following silver exposure. Passaged isolates exhibiting greater than 5-fold change in sensitivity to ionic silver when compared to parent strains were further characterised using molecular methods and subjected to biofilm imaging using laser scanning confocal microscope (LSCM). P. aeruginosa grown in sessile form was passaged through sub-inhibitory concentrations of cationic silver using an MBEC assayTM and changes in colony morphology and silver susceptibility assessed. In addition, whole genome sequencing was performed to detect mutations in all biofilm-derived variants. Results. Gram-positive bacteria exhibited a 4-fold decrease in susceptibility to ionic silver following passaging (P10). One strain of S. aureus showed non pigmented colonies, impaired haemolysin and DNase activities following silver exposure and exhibited reduced virulence in a Galleria assay. A reduction in haemolysis was also shown in an MRSA P10 bacterium. In contrast, S. epidermidis showed increased virulence and haemolysis activity compared to the respective parent strain. Adaptations in Gram-negative wound isolates were limited to one strain of P. aeruginosa (P10), which underwent a 768-fold reduction in silver susceptibility when grown in sessile form and exhibited a highly mucoid phenotype, and increased virulence in vivo. In addition, silver-adapted mutants of this bacterium formed thicker biofilm with a complex eDNA structure that was dispersed following DNase treatment. When passaged in sessile form, biofilms developed a similar pattern of silver insusceptibility and biofilm specific variants were identified. Genome analysis of these variants identified point mutations within the sulfonate ABC transporter substrate-binding protein, transcriptional regulator and another within a nuclease coding gene. These mutations could play an important role in the virulence and biofilm formation ability of silver treated P. aeruginosa biofilms. In the absence of silver exposure, full or partial reversion to baseline susceptibilities was observed in all bacteria. Conclusion. Together, these data suggest that repeated exposure of wound isolates to silver was associated with transient reductions in silver susceptibility, changes in virulence and biofilm susceptibility. Interestingly, significantly reduced susceptibility to silver in adapted P. aeruginosa was occurred only when grown only as biofilm. This contributes to our knowledge of the role of biofilms in biocide tolerance because the majority of previous studies focus on planktonic cultures.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Mcbain (Supervisor) & Gavin Humphreys (Supervisor)