A novel microfluidic system enables visualization and analysis of antibiotic resistance gene transfer to activated sludge bacteria in biofilm

Highlights: • Microfluidics enabled close-to-nature culture of activated sludge biofilms. • Flow cytometry analysis revealed high occupancy of transconjugants. • Transfer frequency per donor and per receptor indicated distinct transfer kinetics. • Proteobacteria were highly abundant and susceptible to receive plasmids. Antibiotic resistance genes (ARGs) in environment have become a growing public concern, due to their potential to be obtained by pathogens and their duplication along cell division. Horizontal gene transfer (HGT) was reported to be responsible for ARGs dissemination in microbes, but the HGT feature in environmental biofilm was still unclear due to insufficient assay tools. To address this challenge, we applied a novel microfluidic system to cultivate thin biofilm by continuous supply of nutrients and close contact between cells. Resembling the living state of biofilm in open environment, this chip visualized the transfer of ARG-encoded plasmids RP4 and pKJK5 to the receptors, e.g., activated sludge bacteria. The average plasmid transfer frequency per receptor (T/R) from RP4-hosted Pseudomonas putida KT2440 to activated sludge bacteria was quantified to be 2.5 × 10⁻³ via flow cytometry, and T/R for pKJK5-hosted Escherichia coli MG1655 was 8.9 × 10⁻³, while the corresponding average frequencies per donor (T/D) were diverse for the two host strains as 4.3 × 10⁻³ and 1.4 × 10⁻¹ respectively. The difference between T/R and T/D was explained by the plasmid transfer kinetics, implying specific purposes of the two calculations. Finally, we collected the transconjugants by fluorescent activated cell sorting and further sequenced their 16S rDNA. Bacteria from phyla Proteobacteria and Firmicutes were found more susceptible to be transconjugants than those from Bacteroidetes. Our work demonstrated that microfluidic system was advantageous in biofilm HGT study, which can provide more insights into environmental ARG control.