A cellular automata model to investigate the role of small colony variants in the antibiotic resistance of a polymicrobial biofilm

Small Colony Variants are frequently isolated from many infections and are thought to be associated with persistent and relapsing nature of the infections despite of antibiotic therapy. To know the influence of SCVs on the antibiotic resistance of a biofilm their detection, isolation and subsequent characterization is important. As the response of biofilm to antibiotics is heterogeneous, dynamics of spatial distribution of SCVs has to be studied which is experimentally challenging. We developed a three-dimensional individual-based cellular automata model of a two-species cystic fibrosis lung infection bacterial biofilm to investigate the influence of phenotypic switching from wild-type S. aureus to small colony variants (SCVs) and on its response to treatment with antibiotics. Our model clearly predicted that the SCV+ biofilm was more resistant compared to the SCV biofilm. For the treated SCV+ biofilm, the fraction of SCVs increases with both treatment time, and the bulk antibiotic concentration indicating that the biofilm upregulates transformation of wild-type S. aureus to SCVs as a defense against antibiotic stress. This is a consequence of the spatial distribution of species in the SCV+ biofilm i.e., top layers are rich in S. aureus, P. aeruginosa is localized in the lower regions of the biofilm and hence HQNO availability is highest in this region. Consequently, at the start of treatment, the biofilm consists of two layers, based on the concentration of SCVs: the top layer containing very few SCVs, and the lower layer containing a high concentration of SCVs which here is termed as the region of maximal influence. As region of maximal influence is in the lower layers, SCVs benefit P. aeruginosa more than S. aureus. When treated with the same antibiotic concentration (Cab,bulk = 8 gm' 3), difference in response between the SCV+ and SCV' biofilms, quantified here in terms of biomass and spatial distribution of dead cells, antibiotics is shown after an initial treatment phase (~15 h), validating the idea of region of maximal influence. Furthermore, with treatment time, biofilm thickness decreases, with HQNO accumulating in the biofilm, and the proportion of S. aureus at any given layer decreases, whereas that of P. aeruginosa remains virtually constant thereby increasing local HQNO concentration per S. aureus cell leading to the higher number of SCVs