Understanding the effect of silver exposure on P. aeruginosa biofilms

Abstract

Background. The longstanding belief of the rarity of silver resistance development has facilitated the widespread use of silver in wound dressings. However, studies have shown that the prolonged exposure of clinical samples under laboratory investigation can result in reduced silver susceptibility. Most of these prolonged exposures have been conducted on planktonic isolates, whereas biofilms are the predominant state in chronic wounds. This PhD thesis aims to assess the bacteriological effects of repeated passaging of bacterial biofilm to ionic silver. Methods. Monospecies biofilms of Pseudomonas aeruginosa (WIBG 2.2) were grown in MBEC devices and repeatedly passaged under two distinct silver nitrate concentrations setting: (i) a constant ionic silver concentration and (ii) dynamic concentrations, where the exposure concentration was determined before each passage. After the fifth passage, biofilm variants were extracted, cultured on agar and compared morphologically. New morphological variants were limited to biofilms exposed to dynamic concentrations. MICs and MBECs were performed to assess the silver sensitivity of these variants, as well as cross-resistance to antimicrobials utilizing the EUCAST zone of inhibition protocol. Biofilm biomass of P. aeruginosa silver-treated variants grown in an MBEC device was determined using crystal violet staining. Extracellular DNA (eDNA) within the biofilm was visualized and quantified utilizing confocal laser scanning microscopy. eDNA was further digested by Deoxyribonuclease-I to evaluate its role in silver recalcitrance and biofilm integrity. Given the generation of mucoid biofilm, alginate production was measured using a carbazole assay. Following the phenotypic changes, all variants underwent whole genome sequencing to detect associated mutations. Bacterial competitive fitness was assessed using a broth co-culture assay, and bacterial-host virulence was determined using Galleria mellonella survival model. Finally, the morphological changes of the biofilm-derived variants were monitored through planktonic passaging in the absence of silver exposure, with growth kinetics determined using a plate reader. Results. Following dynamic exposure to ionic silver, mucoid biofilms were generated after the fifth passage, exhibiting distinct colony morphology named SV, BV, and BWV. Control biofilms passaged in LB broth resulted in two distinct colony morphologies named SC and BC. The treated biofilm variants (SV and BV) and one of the control variants (SC) exhibited reduced colony morphology. SV and BV showed the highest MBEC values, 2227- fold greater than the parent strain and 32-fold greater than the control passage. Compared to the baseline (P0), SV and SC exhibited a highly significant increase in biofilm biomass (P<0.0001), while BV, BWV, and BC exhibited no significant changes. Increased eDNA disposition was observed exclusively within the SV biofilm architecture. Alginate production was highest in BV, followed by BWV and SV, while control biofilm variants (SC and BC) exhibited no significant increases compared to the parent strain. Silver sensitivity was restored by treating biofilms with DNAse I, although this treatment also significantly reduced the biofilm biomass. None of the bacterial variants showed altered virulence compared to the parent strain, although BV exhibited a reduced growth rate. Both SV and BV demonstrated higher competitive fitness than the parent strain. The adapted colony morphology was transient upon subsequent planktonic passage in the absence of ionic silver. SV's colony morphology remained stable until the 11th passage, after which it started to resemble the parent strain. Similarly, BV reverted to the parent morphology by the 10th passage, and BWV began reverting by the 5th passage. In controls, BC began reverting by the 3rd passage, while SC started reverting by the 9th passage. Conclusion: Silver-adapted strains gained resistance through various mechanisms within their sessile phenotype embedded in the complex and heterogenous biofilm ecosystem, including eDNA deposition and alginate production, accompanied by several mutations. These mutations were associated with transport activity, virulence, metabolism, transcription and regulatory functions.