Abstract
Gene transfer in biofilms is known to play an important role in antibiotic resistance dissemination. However, the process remains poorly understood. In this study, microfluidics with time-lapse imaging was used for real-time monitoring of plasmid-mediated horizontal gene transfer (HGT) in biofilms. Pseudomonas putida KT2440 harboring an antibiotic resistance plasmid RP4 was chosen as the donor while Escherichia coli and activated sludge bacteria were used as the recipient cells. Dynamic features of the transfer process, including the transfer rate, cell growth rate and kinetic changes of the transfer frequency, were determined. It was found that the routes for gene transfer strongly depend on the structure and composition of a biofilm. While intraspecies HGT is essential to initiate a transfer event, the secondary retransfer from transconjugants to the same species is more efficient and can cause cascading gene spread in single-strain biofilms. For the activated sludge biofilm, only small and scattered colonies formed and vertical gene transfer appears to be the dominant route after initial intraspecies transfer. Furthermore, more than 46% of genera in the activated sludge were permissive to plasmid RP4, many of which are associated with human pathogens. These phenomena imply early prevention and interruptions to biofilm structure could provide an effect way to inhibit rapid antibiotic resistance gene spread and reduce the likelihood of catastrophic events associated with antibiotic resistance.
Highlights
The rapid spread of antibiotic resistance has become a global crisis and imposes significant threats to human health and the natural environment
P. putida KT2440 donor and E. coli recipient cells were used as a model system to study the transfer of the broad-host-range plasmid RP4 in a single-strain recipient biofilm
By t = 8 h, the surface was almost fully occupied with cells, where P. putida colonies were in contact with E. coli cells (Figure 1a)
Summary
The rapid spread of antibiotic resistance has become a global crisis and imposes significant threats to human health and the natural environment. Plasmid-mediated conjugation can transfer DNA between genera and phyla.[4] Because of dense cell populations, conjugation in biofilms is generally high,[5] and was considered as a major pathway for spreading antibiotic resistance genes (ARG). Manipulation of bacteria at the single-cell level has become well established, leading to the discovery of a variety of new phenomena.[15−18] real-time imaging of in situ formation of biofilms on chip has been demonstrated.[19,20] These evidence suggest that microfluidics can provide a promising platform to investigate the dynamic process of ARG transfer within biofilms. The donor and recipient cells were first washed with PBS (three times) and diluted in LB or R2A medium to a concentration of ∼108 cells·mL−1 They were mixed at a ratio of 1:1 for on chip experiments. A uniform thickness of ∼18 μm was observed for the biofilms (SI Figure S3)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
