A multichannel time-of-flight system for observation of energetic ions of multispecies generated from relativistic laser plasma.

A multichannel time-of-flight (TOF) system was constructed to observe the ions generated from relativistic laser plasma, where the ions have polychromatic energies and multiple species. The TOF system is composed of a ten-channel scintillation detector array and an electromagnet that generates a magnetic field of 0-1.24 T. The magnet field enables us to analyze protons, deuterons, and full-stripped carbon ions to 50, 25, and 150 MeV, respectively. The system experimentally identified protons of 0.27-1.6 MeV energy and ions of a half specific charge (deuterons of 0.3-0.8 MeV and full-stripped carbons of 1.8-4.8 MeV). The measured TOF values agree well with the calculated values within the designed accuracy; +/-2.5 ns for protons and +/-5 ns for the others (d or C(6+)) on each detector channel. Comparison of ion numbers detected by a track detector (CR-39) and the TOF system enabled us to obtain the number of ions detected on each scintillation counter with less than 16% error.

Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer.

We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells.

Antibacterial activities of new synthetic divalent cation chelators.

A series of new synthetic ligand compounds which chelate divalent cations was examined for the antibacterial activities of the compounds. Only 2 of 14 synthetic chelators, 9-trans-anthryl-1, 4, 8, 11-tetraaza-tetradecane (No. 6) and bis(2-pyridyl)methylamine (No. 13) showed antibacterial activities, whereas none of the diamines, hydrophilic triamines nor tetramines showed antibacterial activities. Chelators No. 6 and No. 13 inhibited the growth of both Gram-negative and -positive bacteria at doses of 25-200 microg/ml, comparable to those of common antibiotics such as polymixin B, fosfomycin and macrolides. Ethylenediaminetetraacetate (EDTA) potentiated these antibacterial activities, whereas an inhibitory effect of Mg2+ on the MICs of these chelators was observed. Moreover, these chelators enhanced the leakage of periplasmic beta-lactamase. It is therefore suggested that chelators No. 6 and No. 13 disrupt both the membranes and cytoplasmic functions of bacteria, resulting in cell death.

Four critical aspartic acid residues potentially involved in the catalytic mechanism of Escherichia coli K-12 WaaR.

Escherichia coli K-12 WaaR is a non-processive alpha-1,2 glucosyltransferase, involved in the synthesis of the R-core of lipopolysaccharide. WaaR possesses the four conserved structural regions I, II, III and IV, each presumably involved in the mechanistic function in catalysis. Regions I and III contain the pair of strictly conserved Asp residues. Asp-129, 131 (region I) and 215, 217 (region III) of WaaR were individually converted to Asn by the site-directed mutagenesis of the waaR gene. All mutated enzymes were inactive, supporting the model for an alpha-glycosyl transfer reaction where the pair of strictly conserved aspartic acid residues in regions I and III play a critical role in the catalytic function.

Conserved structural regions involved in the catalytic mechanism of Escherichia coli K-12 WaaO (RfaI).

Escherichia coli K-12 WaaO (formerly known as RfaI) is a nonprocessive alpha-1,3 glucosyltransferase, involved in the synthesis of the R core of lipopolysaccharide. By comparing the amino acid sequence of WaaO with those of 11 homologous alpha-glycosyltransferases, four strictly conserved regions, I, II, III, and IV, were identified. Since functionally related transferases are predicted to have a similar architecture in the catalytic sites, it is assumed that these four regions are directly involved in the formation of alpha-glycosidic linkage from alpha-linked nucleotide diphospho-sugar donor. Hydrophobic cluster analysis revealed a conserved domain at the N termini of these alpha-glycosyltransferases. This domain was similar to that previously reported for beta-glycosyltransferases. Thus, this domain is likely to be involved in the formation of beta-glycosidic linkage between the donor sugar and the enzyme at the first step of the reaction. Site-directed mutagenesis analysis of E. coli K-12 WaaO revealed four critical amino acid residues.

RobA-induced multiple antibiotic resistance largely depends on the activation of the AcrAB efflux.

RobA is a member of the XylS/AraC subfamily of DNA binding proteins, and when overexpressed, it induces multiple antibiotic resistance in Escherichia coli. In this study, we introduced a multicopy robA plasmid (pMEP1) and its derivative into OmpF mutants and an AcrAB-deficient mutant. We found that a decrease in susceptibility to multiple antibiotics in these OmpF mutants when pMEP1 was introduced did not depend on OmpF porin expression. Interestingly, a delta ompF mutant (TK007) became more sensitive when pMEP1 was introduced. Moreover, no effect of RobA on the induction of multiple antibiotic resistance in an acrA1- mutant was observed. Therefore, we conclude that the multiple antibiotic resistance induced by the overexpression of RobA largely depends on the activation of the AcrAB efflux, as well as the activation of micF.

Nosocomial spread of cephem-resistant Escherichia coli strains carrying multiple Toho-1-like beta-lactamase genes.

Escherichia coli HKY56, which demonstrated resistance to various beta-lactams except carbapenems, was isolated from the throat swab of an inpatient in 1994. Conjugal transfer of cephem resistance from HKY56 to E. coli CSH2 was not successful. Three cefotaxime-resistant E. coli clones harboring plasmid pMRE001, pMRE002, or pMRE003, each of which carried a 3.4-, 5.8-, or 6.2-kb EcoRI fragment insert, respectively, were obtained from HKY56. Although restriction analysis suggested their different origins, these clones showed similar profiles of resistance to various beta-lactams. The sequence of 10 amino acid residues at the N terminus of beta-lactamase purified from E. coli HB101(pMRE001) was identical to that of Toho-1. This Toho-1-like beta-lactamase-1 (TLB-1) was able to hydrolyze cefoperazone and cefotaxime efficiently, but it failed to hydrolyze cephamycins. A Toho-1-specific DNA probe was hybridized with three distinct EcoRI fragments derived from the chromosomal DNA of strain HKY56, and these fragments corresponded to DNA inserts carried by pMRE001, pMRE002, and pMRE003, respectively. PCR and Southern hybridization analysis suggested that all six cephem-resistant E. coli strains, strains HKY273, HKY285, HKY288, HKY305, HKY316, and HKY335, which were isolated in 1996 at the same hospital where strain HKY56 had been isolated, also possessed multiple Toho-1-like beta-lactamase (TLB) genes, and the hybridization patterns obtained with the Toho-1-specific probe were quite similar among these six isolates. The DNA fingerprinting patterns observed by pulsed-field gel electrophoresis revealed that among the E. coli isolates tested, all isolates except HKY56 possessed a similar genetic background. These findings suggested that E. coli strains that carry chromosomally multiplied TLB genes may have been proliferating and transmitted among patients in the same hospital.

PCR detection of metallo-beta-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum beta-lactams.

We applied PCR to the rapid detection of the metallo-beta-lactamase gene, blaIMP, in clinically isolated gram-negative rods. A total of 54 high-level ceftazidime-resistant strains (MICs, > 128 micrograms/ml) were subjected to PCR analyses with the blaIMP-specific primers, since the blaIMP-bearing clinical isolates tested in our previous study always demonstrated high-level resistance to ceftazidime. Twenty-two blaIMP-positive strains including 9 Pseudomonas aeruginosa, 9 Serratia marcescens, 2 Alcaligenes xylosoxidans, 1 Pseudomonas putida, and 1 Klebsiella pneumoniae strains were newly identified from 18 different hospitals in Japan. These strains were mostly isolated from urine samples and showed high-level resistance to almost every cephem, while their levels of resistance to carbapenems were diverse. The PCR analyses with novel integrase gene-specific (intI3) and acc(6')-Ib gene-specific primers suggested that the integron structure found in a large plasmid harbored by S. marcescens AK9373 was also well conserved among blaIMP-positive strains. These results imply that the blaIMP gene cassettes have been dispersing into various gram-negative rods with the help of the newly identified integron element. Thus, the PCR-aided rapid detection will be helpful for the early recognition of emerging blaIMP-positive clinical isolates which demonstrate consistent resistance to beta-lactams.

Molecular aspects of high-level resistance to sulbactam-cefoperazone in Klebsiella oxytoca clinical isolates.

Nine Klebsiella oxytoca strains which demonstrated resistance to the combination of sulbactam and cefoperazone were isolated from geographically separate hospitals in Japan in 1995. Among them, K. oxytoca SB23 showed high-level resistance to sulbactam-cefoperazone (MIC > 128 micrograms/ml) and aztreonam (MIC, 128 micrograms/ml). The sulbactam-cefoperazone resistance was not transferred from strain SB23 to Escherichia coli CSH2 by conjugation, beta-Lactamase RbiA, produced by strain SB23, was purified, and the molecular mass was estimated to be 29 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic parameters for RbiA revealed that cefoperazone and aztreonam were hydrolyzed efficiently by this enzyme. Moreover, ceftazidime and imipenem were also hydrolyzed weakly by RbiA, although strain SB23 did not show any resistance to these agents. Clavulanate, sulbactam, and tazobactam failed to block the hydrolysis of cefoperazone by RbiA. The structural gene of RbiA (blaRBI) was cloned and sequenced, and the deduced amino acid sequence of RbiA demonstrated high-level similarities to those of the beta-lactamases found in K. oxytoca D488, E23004, and plasmid-mediated MEN-1, which have been classified into Bush functional group 2be. Although RbiA demonstrates high-level molecular similarity to the enzymes in group 2be, from an enzymological point of view, this enzyme might be differentiated from the enzymes in that group. Hybridization analysis revealed that beta-lactamase genes highly similar to blaRBI were generally encoded on the chromosome of the sulbactam-cefoperazone-resistant clinical isolates of K. oxytoca tested in the study, despite their different derivations. This observation suggests that sulbactam-cefoperazone-resistant A. oxytoca strains which produce RbiA-type beta-lactamases have been proliferating in many hospitals in Japan.

Effect of pH on activities of novel beta-lactamases and beta-lactamase inhibitors against these beta-lactamases.

The effects of acidic conditions on activities of seven beta-lactamases--TEM-1 (class A), KOXY (class A), IMP-1 (class B), AmpC (class C), MOX-1 (class C), OXA-5 (class D), and PSE-2 (class D)--and their inhibitors were measured. The enzymatic activities of KOXY, IMP-1, and MOX-1 at pH 5.8 were slightly lower than those at pH 7.5. However, the activities of PSE-2 and OXA-5 were greatly reduced at pH 5.8. All of the beta-lactamase inhibitors tested had poorer inhibitory activities at pH 5.8 than at pH 7.5 except clavulanic acid for TEM-1.

A novel integron-like element carrying the metallo-beta-lactamase gene blaIMP.

A plasmid-mediated metallo-beta-lactamase gene was cloned from a carbapenem-resistant Serratia marcescens strain, AK9373. The metallo-beta-lactamase gene was identical to the blaIMP, and it was located in the space between an integrase-like gene and an aac(6')-Ib-like gene. The deduced amino acid sequence for the putative integrase gene showed considerable identity (60.9%) to that of the Escherichia coli integrase reported. Sequences similar to the GTTRRRY and an atypical 59-base element containing a 67-bp inverted repeat sequence, which were peculiar to the integrase-dependent recombination, were also conserved in the flanking regions of the blaIMP gene. These findings imply that the metallo-beta-lactamase gene in S. marcescens AK9373 is carried by a novel integron-like element that is mediated by a transferable large plasmid.

Plasmid-mediated dissemination of the metallo-beta-lactamase gene blaIMP among clinically isolated strains of Serratia marcescens.

The distribution of strains producing metallo-beta-lactamase among 105 strains of Serratia marcescens was investigated. All of these strains were isolated in seven general hospitals located in Aichi Prefecture, Japan, from April to May 1993. Southern hybridization analysis suggested that four S. marcescens strains, AK9373, AK9374, AK9385, and AK9391, had a metallo-beta-lactamase genes similar to the blaIMP gene found by our laboratory (E. Osano, Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, H. Ito, F. Yoshimura, and N. Kato, Antimicrob. Agents Chemother. 38:71-78, 1994), and these four strains showed resistance to carbapenems as well as to the other broad-spectrum beta-lactams. In particular, strains AK9373, AK9374, and AK9391 showed an extraordinarily high-level resistance to imipenem (MICs, > or = 64 micrograms/ml), whereas strain AK9385 demonstrated moderate imipenem resistance (MIC, 8 micrograms/ml). The imipenem resistance of AK9373 was transferred to Escherichia coli CSH2 by conjugation with a frequency of 10(-5). The DNA probe of the blaIMP gene hybridized to a large plasmid (approximately 120 kb) transferred into the E. coli transconjugant as well as to the large plasmids harbored by AK9373. On the other hand, although we failed in the conjugational transfer of imipenem resistance from strains AK9374, AK9385, and AK9391 to E. coli CSH2, imipenem resistance was transferred from these strains to E. coli HB101 by transformation. A plasmid (approximately 25 kb) was observed in each transformant which acquired imipenem resistance. The amino acid sequence at the N terminus of the enzyme purified from strain AK9373 was identical to that of the metallo-beta-lactamase IMP-1. In contrast, strains ES9348, AK9386, and AK93101, which were moderately resistant to imipenem (MICs, > or = 4 to < or = 8 micrograms/ml), had no detectable blaIMP gene. As a conclusion, 19% of clinically isolated S. marcescens strains in Aichi Prefecture, Japan, in 1993 were resistant to imipenem (MICs, > or = 2 micrograms/ml), and strains which showed high-level imipenem resistance because of acquisition of a plasmid-mediated blaIMP-like metallo-beta-lactamase gene had already proliferated as nosocomial infections, at least in a general hospital.

Microbiological evaluation of a newly designed dental air-turbine handpiece for anti-cross contaminations.

The effectiveness of a newly developed anti-cross contamination device for a dental air-turbine handpiece was tested. The handpiece with or without the anti-cross contamination device was contaminated with two bacterial strains, Staphylococcus aureus and Streptococcus mutans, as well as two bacteriophage strains, T2 and MS2. After contamination with these microorganisms, the handpieces were disinfected with glutaraldehyde or replaced with newly autoclaved ones. Residual microorganisms inside the handpiece or an air/water supply hose line were collected and counted after overnight cultivation. The anti-cross contamination device effectively reduced the contamination level of an air-turbine handpiece to that of the negative control. No microbial contamination in the air/water supply hose line was detected with this device.

Bactericidal action of tachyplesin I against oral streptococci.

Tachyplesin I, a polycationic antimicrobial peptide isolated from hemocytes of horseshoe crabs, kills bacteria by disrupting the membrane potential of the cytoplasmic membrane. The present study shows that, among 36 oral streptococcal strains, 12 of 21 Streptococcus sanguis, 3 Streptococcus mutans, 9 Streptococcus salivarius and 3 Streptococcus milleri strains were susceptible to tachyplesin I, whereas 9 S. sanguis strains were resistant. Interestingly, these resistant strains include the clinical isolates from both Kawasaki disease and Behçet patients. According to the time-kill study, tachyplesin I inhibited irreversibly the growth of S. sanguis, S. mutans and S. salivarius strains within 20 min and an S. milleri strain within 80 min. Although it has been suggested that Escherichia coli cultured in rich media were more susceptible to tachyplesin I, the present results show that only 3 S. milleri strains were more sensitized to tachyplesin I in a glucose-supplemented medium, and other tested strains were not. Similarly, only 4 strains were more resistant to tachyplesin I in saline than these were in a rich medium.

Bacteriological evaluation of a new air turbine handpiece for preventing cross-contamination in dental procedures.

An autoclavable air turbine handpiece, Air Flushing Clean System (AFCS) (Osada Electric Co., Ltd., Tokyo, Japan) was developed for use in dentistry with the objective of reducing cross-contamination. Its potential for bacterial contamination was investigated in vitro using two bacterial strains (Streptococcus mutants ATCC 25175 and Staphylococcus aureus FDA 209 P). In theory, this device should prevent cross-contamination of the internal water and air lines of the handpiece, by maintaining an internal positive pressure even after the turbine is stopped. In the present study, this AFCS device was found to reduce the bacterial contamination within the air turbine handpiece more effectively than the conventional handpiece used according to accepted protocol. The reduction of such contamination by the AFCS is in keeping with the recent objective of the American Dental Association to reduce cross-contamination during dental procedures.

Lidocaine hydrochloride and acetylsalicylate kill bacteria by disrupting the bacterial membrane potential in different ways.

Lidocaine hydrochloride (LH), a local anesthetic, and acetylsalicylate (AcSAL), show antibacterial activity for both gram-negative and gram-positive bacteria. Kinetic studies indicated that antibacterial activity of LH was different from that of AcSAL. A subinhibitory concentration of LH and AcSAL enhanced the sensitivity of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa to novobiocin and nalidixic acid. The synergistic effect of AcSAL with novobiocin and nalidixic acid was higher than that of LH. The effect of both drugs on the membrane potential of inner membrane was also studied using inverted membrane vesicles of bacteria. Both LH and AcSAL depolarized the membrane potential after the vesicles were energized with nicotinamide adenine dinucleotide. However, unlike AcSAL, pre-treatment of vesicles with LH had no effect on the generation of membrane potential. These results suggest that depolarization of the cytoplasmic membrane, preceded by the permeabilization of the outer membrane for gram-negative bacteria, is associated with antibacterial activity of LH and AcSAL. The difference in actions of LH and AcSAL was discussed.

Quantitative fluorographic detection of 3H and 14C on two-dimensional thin-layer chromatographic sheets by an ultra-high-sensitivity TV camera system.

A method for quantitative detection of 3H and 14C on thin layers is described. After impregnation of the TLC sheet with 50% 2,5-diphenyloxazole-tetrahydrofuran, quantitative imaging of the distribution of weak beta-ray-emitting isotopes on the chromatogram was carried out at room temperature by using a TV camera system, which consisted of a two-stage microchannel plate image intensifier, a low-lag vidicon, and an image processor. The method is applicable for 14C- and 3H-labeled samples on TLC sheets (10 X 10 cm) emitting more than 0.17 and 7.5 Bq/mm2, respectively. The method is rapid and has a dynamic range far greater than that of film.