Abstract

We investigated the evolutionary importance of cryptic prophage elements in a blaNDM-1-bearing plasmid by exploring the effect of prophage regions on survival against antibiotic stress. While analysing a plasmid harboring an NDM-1-encoding gene in Klebsiella pneumoniae from South Korea, we found a prophage region within the plasmid. We constructed single-prophage knockout (KO) mutants by gene replacement. The intact plasmid and plasmids with deleted prophages were conjugated into Escherichia coli DH5α. Growth rate and antibiotic susceptibility were determined, and survival rates of strains were evaluated in the presence of antibiotics, such as imipenem, amikacin, gentamicin, cefotaxime, and piperacillin/tazobactam. A transcriptional response of sigma factor-coding genes (rpoS and rpoE) and reactive oxygen species (ROS)-related genes from different operons (soxS, fumC, oxyR, and katE) to a sub-inhibitory concentration of aminoglycosides was monitored by quantitative real-time polymerase chain reaction (qRT-PCR). The prophage region consists of four cryptic prophages of 16,795 bp and 19 coding DNA sequences. An Escherichia coli transconjugant carrying the plasmid with intact prophages showed increased survival during treatment with various antibiotics, including imipenem and amikacin; however, transconjugants carrying this plasmid with a single-prophage KO did not. mRNA expression analyses revealed that sigma factor proteins (rpoB and rpoE) were highly upregulated by antibiotics. We propose that cryptic prophages in the antibiotic resistance plasmid may contribute to adaptation of the bacterial host to antibiotic stress. We are concerned that the combination of prophages with a drug resistance plasmid helps drug-resistant bacteria in a hostile environment and accelerates their dissemination.

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