Multiplex PCR amplification followed by either agarose gel electrophoresis (PCR-AGE) or microchip electrophoresis (PCR-ME) was used to test a total of 120 fungal strains. The internal transcribed spacer 1 (ITS1) and ITS2 regions and the 5.8S ribosomal DNA (rDNA) region of the fungi were amplified by using universal primers ITS1 and ITS4. The ITS2 region was simultaneously amplified by using universal primers ITS3 and ITS4. Since Trichosporon asahi and T. asteroides showed similar lengths for two amplicons, 29 different gel patterns were demonstrated for 30 yeast species tested on the basis of differences in the lengths of one or two amplicons. Of 75 yeast isolates from clinical materials, 5 isolates (6.8%) which were incompletely identified or not identified by the phenotypic method were identified with our PCR-based method (2 isolates as Candida guilliermondii, 2 as C. krusei, and 1 as C. zeylanoides). No differences in discriminating power or sensitivity were observed between the PCR-AGE method and the PCR-ME method. These methods, prospectively applied to 24 yeast-positive blood culture bottles (16 patients), resulted in the correct detection of 24 yeast strains. In conclusion, multiplex PCR followed by electrophoresis seems to be a promising tool for the rapid identification of common and uncommon yeast strains from culture colonies and from yeast-positive blood culture bottles (5.5 h for the PCR-AGE method and 3 h for the PCR-ME method).

The structure of liquid Sn was studied by neutron scattering experiments in the widest temperature range that was ever performed. Though, on increasing temperature, the existence of the shoulder in the structure factor, S(Q), becomes less clear in the change of the overall shape of the S(Q), the structure related to this shoulder seems to be present even at 1873 K. The first-principle molecular-dynamics ͑FPMD͒ simulation was performed for the first time for liquid Sn by using the cell size of 64 particles. The calculated results well reproduced S(Q) obtained by the neutron experiments. The angle distribution, g (3) (,r c ), was evaluated for the angle between vectors from centered atom to other two atoms in spheres of cutoff radii r c 's. The g (3) (,r c ) shows that, with the decrease of r c from 0.4 to 0.3 nm, a rather sharp peak around 60°disappears and only a broad peak around 100°remains; the former peak may be derived from the feature of the closely packed structures and the latter one is close to the tetrahedral angle of 109°. In addition, the coordination number, n, of liquid Sn counted within the sphere of r c ϭ0.3 nm is found to be 2-3 and does not change with the increase of temperature even up to 1873 K. These facts indicate that at least the fragment of the tetrahedral unit may be essentially kept even at 1873 K for liquid Sn. For comparison, the FPMD simulation was performed for the first time also for liquid Pb. No sign of the existence of the tetrahedral structure was observed for liquid Pb. Unfortunately, the self-diffusion coefficients, D's, obtained from this FPMD for liquid Sn do not agree with those obtained by the microgravity experiments though the structure factors, S(Q)'s, are well reproduced. To remove the limitation of the small cell size of the FPMD, the classical molecular-dynamics simulations with a cell size of 2197 particles were performed by incorporating the present experimental structural information of liquid Sn. Obtained D's are in good agreement with the microgravity data.

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We report on a case of fungemia due to fluconazole-resistant Candida nivariensis (MIC, >128 g/ml). Internal transcribed spacer PCR followed by microchip gel electrophoresis with a blood culture that tested positive revealed a unique pattern different from those of other pathogenic yeasts. CASE REPORT

We have carried out ab initio molecular-dynamics simulations for liquid phosphorus under high temperature and high pressure in order to investigate the microscopic mechanism of the recently observed liquid-liquid phase transition of liquid phosphorus. We have successfully shown by our simulation that the structural phase transition with increasing pressure corresponds to the structural change from the molecular liquid composed of stable tetrahedral P4 molecules to the polymeric liquid with complex network structure and that the calculated structure factors are in good agreement with those obtained by the x-ray diffraction experiments. It is also found from our calculated electronic structure that this structural change gives rise to the nonmetal-metal transition, which is the transition from the nonmetallic molecular liquid to the metallic polymeric liquid.

PCR analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) followed by microchip gel electrophoresis (MGE) was evaluated for its usefulness in identification of staphylococci. Forty ITS PCR patterns were demonstrated among 228 isolated colonies of Staphylococcus aureus: 26 patterns for methicillin-susceptible S. aureus (MSSA; 91 strains), 11 patterns for methicillin-resistant S. aureus (MRSA; 99 strains), and 3 patterns for both MSSA and MRSA (38 strains). Thirty-seven control strains of coagulase-negative staphylococci (CNS) representing 16 species showed unique ITS PCR patterns (24 patterns) at the species and subspecies levels: two patterns for S. caprae, S. cohnii, S. haemolyticus, and S. saprophyticus; three patterns for S. lugdunensis; four patterns for S. capitis; and one pattern for each of the other CNS species. The combined PCR-MGE method was prospectively adapted to the positive blood culture bottles, and this method correctly identified MSSA and MRSA in 102 (89%) of 114 blood cultures positive for S. aureus on the basis of the ITS PCR patterns. Eight ITS PCR patterns were demonstrated from 166 blood culture bottles positive for CNS. The most frequent CNS species isolated from blood cultures were S. epidermidis (76%), S. capitis (11%), and S. hominis (8%). Overall, all 280 blood culture bottles shown to contain a single Staphylococcus species by routine phenotypic methods were correctly identified by the PCR-MGE method at the species level, whereas the organism failed to be identified in 8 culture bottles (3%) with mixed flora. The PCR-MGE method is useful not only for rapid identification (ϳ1.5 h) of staphylococci in positive blood culture bottles, but also for strain delineation of S. aureus.The coagulase-positive species Staphylococcus aureus has been well documented as an opportunistic human pathogen. Infections produced by S. aureus are often acute and pyogenic and, if left untreated, can spread to surrounding tissue or other organs. Identification of S. aureus in blood cultures begins with presumptive identification of gram-positive cocci in clusters (GPCC) in gram-stained smears of blood culture bottles, signaling a positive result, whereas final identification must await subculture and overnight incubation. Direct identification of methicillin-resistant S. aureus (MRSA) from GPCC-positive blood culture bottles may provide important diagnostic information, which would allow the selection of an appropriate course of treatment in a timely manner. A variety of rapid identification systems for direct identification of S. aureus from positive blood culture bottles have been evaluated, and although the overall specificity is excellent, a wide range of sensitivities have been reported (2, 32). Molecular techniques, such as gene probe hybridization assay (16), DNA sequencing of rRNA genes (25), fluorescence in situ hybridization (23), and PCR (18,19,35), have also been reported for the identification of S. aureus directly from positive blood cultures. Although these methods provide same-da...

Multidrug efflux pumps contribute to the resistance of Escherichia coli against many antibiotics and biocides. Here, we report that the CRP regulator modulates multidrug resistance in E. coli through repression of the genes encoding the MdtEF multidrug efflux pump. Screening of mutants for ability to increase beta-lactam resistance in E. coli led to the identification of a mutation in crp, which codes for the major global regulator of catabolite-sensitive operons. Deletion of crp significantly increased the resistance of the E. coli strain to oxacillin, azithromycin, erythromycin and crystal violet. The increase in drug resistance caused by crp deletion was completely suppressed by deletion of the multifunctional outer membrane channel gene tolC. TolC interacts with different drug efflux pumps. Among the twenty drug efflux pumps in E. coli, quantitative real-time PCR analysis showed that CRP repressed the expression of mdtEF. Deletion of mdtEF completely suppressed CRP-modulated multidrug resistance. Therefore, in addition to its role in catabolite control, CRP contributes to multidrug resistance in E. coli. Our results indicate that the CRP regulator modulates multidrug resistance in E. coli by repressing expression of the MdtEF multidrug efflux pump.

The expression of MdtEF, a multidrug exporter in Escherichia coli, is positively controlled through multiple signaling pathways, but little is known about signals that induce MdtEF expression. In this study, we investigated compounds that induce the expression of the mdtEF genes and found that out of 20 drug exporter genes in E. coli, the expression of mdtEF is greatly induced by N-acetyl-D-glucosamine (GlcNAc). The induction of mdtEF by GlcNAc is not mediated by the evgSA, ydeO, gadX, and rpoS signaling pathways that have been known to regulate mdtEF expression. On the other hand, deletion of the nagE gene, encoding the phosphotransferase (PTS) system for GlcNAc, prevented induction by GlcNAc. The induction of mdtEF by GlcNAc was also greatly inhibited by the addition of cyclic AMP (cAMP) and completely abolished upon deletion of the cAMP receptor protein gene (crp). Other PTS sugars, glucose and Dglucosamine, also induced mdtEF gene expression. These results suggest that mdtEF expression is stimulated through catabolite control.