ABSTRACT
In gram-negative bacteria, IS26 often exists in multidrug resistance (MDR) regions, forming a pseudocompound transposon (PCTn) that can be tandemly amplified. It also generates a circular intermediate called the "translocatable unit (TU)", but the TU has been detected only by PCR. Here, we demonstrate that in a Klebsiella pneumoniae MDR clone, mono- and multimeric forms of the TU were generated from the PCTn in a preexisting MDR plasmid where the inserted form of the TU was also tandemly amplified. The two modes of amplification were reproduced by culturing the original clone under antimicrobial selection pressure, and the amplified state was maintained in the absence of antibiotics. Mono- and multimeric forms of the circularized TU were generated in a RecA-dependent manner from the tandemly amplified TU, which can be generated in RecA-dependent and independent manners. These findings provide novel insights into the dynamic processes of genome amplification in bacteria.
Subject(s)
DNA Transposable Elements , Drug Resistance, Multiple, Bacterial , Klebsiella pneumoniae , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/drug effects , Drug Resistance, Multiple, Bacterial/genetics , DNA Transposable Elements/genetics , Rec A Recombinases/genetics , Rec A Recombinases/metabolism , Plasmids/genetics , Anti-Bacterial Agents/pharmacologyABSTRACT
BACKGROUND: MicroRNAs (miRNA) are expected as useful biomarkers for various diseases. We studied the pre-analytical factors causing variation in the analysis of miRNA. MATERIAL AND METHODS: Blood samples were collected from 25 healthy subjects. Plasma and serum were obtained from the same samples. The levels of miR-451, -16, -126, and -223 were analyzed using RT-qPCR. Cel-miR-39 was added as a spiked-in control in each sample. RESULTS: With the exception of miR-451, the levels of the miRNAs in plasma were higher than in serum. After high-speed centrifugation, the levels of miRNAs were almost equal between plasma and serum except for miR-451. Membrane filtration with 0.45 µm pore size reduced the levels of plasma miRNAs. The coagulation accelerators for serum processing did not affect the analysis of miRNA. The use of fraction containing particles of > 0.45 µm in size showed the inhibitory effect on the analysis of plasma miR-451. The RNase inhibitor was effective for protecting against the degradation of miRNAs. CONCLUSION: Plasma contains factors modifying miRNA profiles. The immediate processing of plasma with membrane filtration and RNase inhibitor may be a relevant method for achieving the stable analysis of miRNA.
