Abstract
Antibiotic resistance is currently a major global public health issue. In particular, the emergence and transfer of antibiotic resistance genes (ARGs) is a matter of primary concern. This study presented a method for ruling out the transfer of naked DNA (plasmid RP4 lysed from donor cells) during the cell-to-cell conjugation, using a modified “U-tube”. A series of gene transfer assays was conducted in both flask and modified U-tube, using Pseudomonas putida KT2440 (P. putida (RP4)) harboring the RP4 plasmid as the donor strain, Escherichia coli (E. coli, ATCC 25922) in pure culture as sole recipient, and bacteria from reclaimed water microcosms as multi-recipients. The verification experiments showed that the U-tube device could prevent direct contact of bacteria without affecting the exchange of free plasmid. In the experiments involving a sole recipient, the transconjugants were obtained in flask samples, but not in modified U-tube. Furthermore, in experiments involving multi-recipients, transfer of naked DNA in the modified U-tube accounted for 5.18% in the transfer frequency of the flask transfer experiment. The modified U-tube proved to be useful for monitoring the interference of naked DNA in the research of conjugative transfer and calculating the exact conjugative transfer rate. This device is identified as a promising candidate for distinguishing different gene transfers in practical application because of its convenient use and easy and simple manufacture.
Highlights
In recent years, antibiotic resistance has received increasing attention as a major global public health issue [1,2,3]
antibiotic resistant genes (ARGs) can transfer among bacteria by vertical gene transfer (VGT) and horizontal gene transfer (HGT) [10,11]
All samples for verification tests were subjected to polymerase chain reaction (PCR) analysis to test the presence of plasmid
Summary
Antibiotic resistance has received increasing attention as a major global public health issue [1,2,3]. One of the greatest concerns about the presence of antibiotics in the environment is the emergence and dissemination of antibiotic resistant genes (ARGs). To illustrate the severe risk of ARGs, the World Health Organization named the World Health Day in 2011 as “Antimicrobial. Increasing ARGs including genes that confer resistance to sulfonamides, β-lactams, trimethoprim, tetracyclines, aminoglycosides, macrolides, and quinolones [4,5,6,7,8,9] have been detected in various environments. ARGs can transfer among bacteria by vertical gene transfer (VGT) and horizontal gene transfer (HGT) [10,11]. HGT facilitates the direct transfer of DNA between microorganisms, and it is regarded as the major evolutionary force that
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