Hospital and urban wastewaters shape the structure and active resistome of environmental biofilms. wwBiofilms

We studied hospital and urban WW biofilms in situ and in an in vitro laboratory model to assess and compare their potential to actively disseminate and express ARGs. Biofilms were subjected to confocal microscopy to qualify and quantify their extracellular polymeric substances (EPSs), including polysaccharides, external DNA (eDNA), and lipids. We observed that antibiotic challenge had a differential impact on the EPS and the live-dead ratios of in vitro hospital and urban WW biofilms. Differential expression of a range of ARGs and MGEs, including those on plasmids, was assessed using a meta-transcriptomic approach. Expression of ARGs as well as MGEs, in situ, was 10-fold higher in hospital WW biofilms compared with urban, suggesting a higher frequency of ARG transfer might occur in hospital WW. In vitro data confirmed that the addition of ciprofloxacin significantly increased MGE expression in hospital but not in urban WW biofilms suggesting that the regulation of ARG and MGE expression is likely dependent on the structure and composition of the biofilm. The different responses to antibiotic pressure in hospital vs urban biofilms, coupled with differences in biofilm structure helps delineate distinct characteristics of hospital and urban WW biofilms.