The effect of sub-inhibitory concentrations of antibiotics on the regulation of eDNA in Staphylococcal biofilms

Abstract

Biofilms are complex communities of microbial cells encased in a self-produced extracellular matrix which are associated with recurrent infections in clinical settings and are difficult treat. Many microorganisms including Staphylococcus species form biofilms. The difficulty of treating biofilms is due to tolerance phenotypes which include reduced growth rate, the extracellular matrix reducing antibiotic penetration and adaptive responses to environmental stresses. Sub-inhibitory concentrations of antibiotics have been reported to influence eDNA-dependent biofilm formation in species of S. aureus with poor biofilm forming ability in an eDNA-dependent manner. However, gene expression studies into the regulation of eDNA on strains which respond have not been conducted. The aim of this study was to evaluate whether there is a link between a positive biofilm response to sub-inhibitory concentrations of antibiotics and up-regulation of genes involved in cell lysis and DNA degradation. A number of strains of Staphylococcus aureus were screened for their ability to form a biofilm. Those which showed poor biofilm forming ability were exposed to further analysis at sub-inhibitory concentrations of oxacillin and vancomycin. Two strains, S. aureus WSU2 and WSU3 showed a response to 1/2 MIC of vancomycin and 1/4 MIC of oxacillin respectively. S. aureus WSU2 showed a 1.8 times increase in eDNA with a similar increase in biofilm production whilst S. aureus WSU3 showed a doubling of eDNA and biofilm production. Reverse Transcription Real Time PCR was used to analyse the expression of genes which have been linked to cell death and lysis in S. aureus. These genes included cidA and lrgA, which act on murein hydrolases, the autolysin atlA, and the staphylococcal nuclease genes nuc 1 and nuc 2. The polysaccharide gene, icaA was also measured to see if it was linked to increased biofilm production in the test strains. There were small changes in gene regulation for both S. aureus WSU2 and WSU3 for all genes tested. S. aureus WSU2 treated with vancomycin showed different expression of genes compared to S. aureus WSU3 treated with oxacillin. In most cases the nucleases genes were unaffected, however in S. aureus WSU3 the nuclease genes were found to be significantly downregulated. The autolysin gene was significantly upregulated 1.6 fold in S. aureus WSU2 treated with 1/2 MIC vancomycin. This upregulation of the autolysin was consistent with the increase in eDNA observed for the strain. This effect was also observed when 24 hour biofilms were exposed to a single 90 minute antibiotic treatment. The gene expression changes observed in this study, although are less than 2-fold, do appear to show a consistent trend and suggest that atlA could play an important role in eDNA-dependent biofilm formation. With further investigation, the link between gene expression and eDNA-dependent biofilm formation under sub-inhibitory doses of antibiotics can be further understood. The increase in biofilm formation and alteration of eDNA within the biofilm matrix can potentially impact S. aureus biofilm-associated infections. Therefore, further studies may give a better understanding of the effect of sub-inhibitory antibiotic exposure on biofilm formation which could lead to improved treatment strategies.

Date of Award	2017
Original language	English