Escherichia coli and its lipopolysaccharide modulate in vitro Candida biofilm formation

Demystification of microbial behaviour in mixed biofilms could have a major impact on our understanding of infectious diseases. The objectives of this study were to evaluate in vitro the interactions of six different Candida species and a Gram-negative coliform, Escherichia coli, in dual-species biofilms, and to assess the effect of E coli LPS on Candida biofilm formation. A single isolate of E coli ATCC 25922 and six different species of Candida, Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019 and Candida dubliniensis MYA-646, were studied using a standard biofilm assay. Each Candida species was co-cultured with E. coli on a polystyrene surface and biofilm formation was quantified by a c.f.u. assay. The biofilm was then analysed by Live/Dead staining and fluorescence microscopy (confocal laser-scanning microscopy, CLSM), whilst scanning electron microscopy (SEM) was employed to visualize the biofilm architecture. The effect of E coli LPS on Candida biofilm cell activity at defined time intervals was assessed with an XTT reduction assay. A significant quantitative reduction in c.f.u. counts of C. tropicalis (after 90 min), C. parapsilosis (after 90 min and 24 h), C. krusei (after 24 h) and C. dubliniensis (after 24 and 48 h) was noted on incubation with E. coli in comparison with their monospecies biofilm counterparts (P <0.05). On the other hand, a simultaneous and significant reduction in E coli cell numbers occurred on co-culture with C. albicans (after 90 min), and an elevation of E coli cell numbers followed co-culture with C. tropicalis (after 24 h) and C. dubliniensis (after 24 h and 48 h) (P <0.05). All quantitative findings were confirmed by SEM and CLSM analyses. By SEM observation, dual-species biofilms demonstrated scanty architecture with reduced visible cell counts at all stages of biofilm development, despite profuse growth and dense colonization in their single-species counterparts. Significantly elevated metabolic activity, as assessed by XTT readings, was observed in E coli LPS-treated C. tropicalis and C. parapsilosis biofilms (after 48 h), whilst this had the opposite effect for C. dubliniensis (after 24 h) (P <0.05). These data indicate that E coli and Candida species in a mixed-species environment mutually modulate biofilm development, both quantitatively and qualitatively, and that E coli LPS appears to be a key component in mediating these outcomes.