Metagenomic analysis permitting identification of the minority bacterial populations in the oral microbiota

AbstractIn recent years, genetic techniques have been applied for the analysis of constitutive bacterial species of the oral microbiota. To analyze a wide variety of bacterial species simultaneously, universal PCR amplification using primers targeting the same conserved area of 16S rRNA is commonly used. However, because of PCR competition when using this strategy, only the DNA of the predominant bacterial species is amplified at the expense of the DNA of the minority bacterial species. Hence, many reports using 16S rRNA gene amplification as the first step have an inbuilt bias towards analyzing only predominant organisms. In order to remove excess predominant bacterial DNA before applying PCR amplification, we developed a new technique “DNA deduction,” which allowed us to determine the diversity of the bacterial flora of the mouth, including minority species that may well have been overlooked so far. DNA was extracted from healthy human saliva samples and the 16S rRNA gene was PCR-amplified both before and after applying DNA deduction. Amplicons were sequenced using a 454 FLX titanium pyrosequencer. In total, we collected 264,000 sequence reads. These data included 9 phyla, 16 classes, 26 orders, 55 families, and 111 genera (OUT was defined within 3% genetic difference). Using this technique, we detected approximately 29% more types of microbes than those detected from the same sample without using DNA deduction. From these data, we conclude that the DNA deduction technique will lead to a better understanding of the diversity of the human oral microbiota

Efflux-mediated fluoroquinolone resistance in the multidrug-resistant Pseudomonas aeruginosa clinical isolate PA7: identification of a novel MexS variant involved in upregulation of the mexEF-oprN multidrug efflux operon

The emergence of multidrug-resistant Pseudomonas aeruginosa has become a serious problem in medical settings. P. aeruginosa clinical isolate PA7 is resistant to fluoroquinolones, aminoglycosides, and most -lactams but not imipenem. In this study, enhanced efflux-mediated fluoroquinolone resistance of PA7 was shown to reflect increased expression of two resistance nodulation cell division (RND) -type multidrug efflux operons, mexEF-oprN and mexXY-oprA. Such a clinical isolate has rarely been reported because MexEF-OprN-overproducing mutants often increase susceptibility to aminoglycosides apparently owing to impairment of the MexXY system. A mutant of PA7 lacking three RND-type multidrug efflux operons (mexAB-oprM, mexEF-oprN, and mexXY-oprA) was susceptible to all anti-pseudomonas agents we tested, supporting an idea that these RND-type multidrug efflux transporters are molecular targets to overcome multidrug resistance in P. aeruginosa. mexEF-oprN-upregulation in P. aeruginosa PA7 was shown due to a MexS variant harboring the Valine-155 amino acid residue. This is the first genetic evidence shown that a MexS variant causes mexEF-oprN-upregulation in P. aeruginosa clinical isolates

Dynamic nuclear polarization and Knight shift measurements in a breakdown regime of integer quantum Hall effect

Nuclear spins are polarized electrically in a breakdown regime of an odd-integer quantum Hall effect (QHE). Electron excitation to the upper Landau subband with the opposite spin polarity flips nuclear spins through the hyperfine interaction. The polarized nuclear spins reduce the spin-splitting energy and accelerate the QHE breakdown. The Knight shift of the nuclear spins is also measured by tuning electron density during the irradiation of radio-frequency magnetic fields.Comment: 3 pages, 2 figures, EP2DS-1

Coherent manipulation of nuclear spins in the breakdown regime of integer quantum Hall states

We demonstrate a new method for electrical manipulation of nuclear spins utilizing dynamic nuclear polarization induced by quantum Hall effect breakdown. Nuclear spins are polarized and detected through the hyperfine interaction between a nuclear spin system and a two-dimensional electron system located at an interface of GaAs/AlGaAs single heterostructure. Coherent oscillations between the nuclear-spin quantum states are observed by measuring the longitudinal voltage of the conductor.Comment: 4 pages, 2 figure