Absolute calibration of imaging plate for GeV electrons.

An imaging plate has been used as a useful detector of energetic electrons in laser electron acceleration and laser fusion studies. The absolute sensitivity of an imaging plate was calibrated at 1 GeV electron energy using the injector Linac of SPring-8. The sensitivity curve obtained up to 100 MeV in a previous study was extended successfully to GeV range.

Development of a novel matrix-assisted laser desorption/ionization time-of-flight mass spectrum (MALDI-TOF-MS)-based typing method to identify meticillin-resistant Staphylococcus aureus clones.

BACKGROUND: Mass spectrum analysis enables species- and subspecies-level identification, and can be used as an epidemiological tool in outbreak management. However, its reliability at clonal level has yet to be established. AIM: To establish a matrix-assisted laser desorption/ionization time-of-flight mass-spectrum-based method that enables bacterial clone identification with accuracy equivalent to pulsed-field gel electrophoresis/phage open-reading frame typing (PFGE/POT). METHODS: Meticillin-resistant Staphylococcus aureus (MRSA) was used in this study. Mass spectra were obtained from a standard strain of S. aureus (ATCC29213) and 57 clinically isolated strains, categorized according to POT. Peaks associated with MRSA clone identification (N = 67) were extracted. Based on this peak information, the feasibility of MRSA clone identification was examined by cluster analysis. FINDINGS: In addition to the 58 strains used for peak extraction, mass spectrum analysis of 24 clinically isolated outbreak strains revealed that peak data could be used for successful identification of clones. These typing results were fully consistent with the PFGE and POT results. CONCLUSION: This novel method enables simple and rapid typing with accuracy equivalent to PFGE/POT. This method would be suited to rapid outbreak analysis, offering accurate information to combat infectious diseases.

UCN-01, an anti-tumor drug, is a selective inhibitor of the conventional PKC subfamily.

A selective PKC inhibitor, UCN-01, was shown to exhibit anti-tumor activity in vitro and in vivo. We investigated UCN-01 with respect to isozyme-specific PKC inhibition using purified recombinant or rabbit brain PKC isozymes, cPKC alpha, beta and gamma, nPKC delta, epsilon and eta, and a PKC zeta. Of the PKC isozymes examined, cPKC alpha was inhibited by UCN-01 most effectively (Ki = 0.44 nM), suggesting cPKC alpha is the prime candidate for the physiological target of UCN-01. The Ki values of UCN-01 estimated from Dixon plots for cPKC isozymes are approximately 1 nM, whereas the Ki values for nPKC isozymes are about 20 nM. Moreover, the Ki value for aPKC zeta is 3.8 microM. Thus, UCN-01 discriminates between PKC subfamilies. In addition, the inhibitory effects of staurosporine, H7, and calphostin C on aPKC zeta were examined and compared with those for cPKC alpha.

Detection Methods of Glycopeptide-Resistant Staphylococcus aureus I : Susceptibility Testing.

The breakpoint for resistance to vancomycin for Staphylococcus aureus is a minimum inhibitory concentration (MIC) of greater than 8 µg/mL (1). Isolation of the first strain of MRSA resistant to vancomycin (VRSA) Mu50 was made from a Japanese surgical patient with a wound infection who had failed with vancomycin therapy (2). A strain has been described that is heterogeneously resistant to vancomycin (Mu3 strain), and it was found to be susceptible to vancomycin (MIC 2 µg/mL by NCCLS criteria), but there were cells within the Mu3 population that resisted a vancomycin concentration of up to 9 µg/mL) (1). Most of the clinical S. aureus strains having reduced susceptibility to glycopeptide antibiotics are heterogeneous in their phenotypic resistance expression. The strains contain small subpopulations of cells that have different levels of glycopeptide resistance. They are designated hetero-resistant strains, and are defined by the population analysis (see below). MIC or paper disc susceptibility tests cannot detect heteroresistant strains. It would appear that heterogeneously resistant VRSA is a preliminary stage that allows development into full resistance upon further exposure to vancomycin. Therefore, it seems reasonable to include VRSA and hetero-VRSA as possible risk factors for vancomycin therapeutic failure in MRSA infection.

Detection Methods for Glycopeptide-Resistant Staphylococcus aureus II : Cell Wall Analysis.

Vancomycin resistance of Mu50 (VRSA) and Mu3 (hetero-VRSA) is associated with the changes in cell-wall synthesis. Therefore, the analysis of cellwall synthesis and cell-wall composition study is of cardinal importance for the understanding of this resistance mechanism. The rate of the cell-wall synthesis can be evaluated by measuring the rate of up-take of (14)C-N-acetylglucosamine into the cell, since more than 95% of (14)C-N-acetylglucosamine is known to be incorporated into the cell wall (1). Murein monomer precursor (UDP-N-acetylmuramyl-pentapeptide) (MMP) is an important intermediate substrate of peptidoglycan synthesis, detection, and quantitation of MMP is useful for the investigation of how the cell-wall synthesis system is altered in S. Aureus in association with glycopeptide resistance. For example, the cytoplasmic pool size of MMP is several times greater in Mu50 and Mu3 than in vancomycin-susceptible S. Aureus strains (2).

Phosalacine, a new herbicidal antibiotic containing phosphinothricin. Fermentation, isolation, biological activity and mechanism of action.

Phosalacine, a new herbicidal antibiotic containing phosphinothricin was isolated from the culture filtrate of a soil isolate Kitasatosporia phosalacinea KA-338. It was a water soluble, amphoteric compound obtained as an amorphous powder (C14H28N3O6P, MW 365). The antibiotic exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria and some fungi on a minimal medium and the activity was reversed by L-glutamine. It also showed herbicidal activity against alfalfa. It is suggested that phosalacine was decomposed to provide phosphinothricin after its incorporation into microbial or plant cells, and exhibited the antimicrobial and herbicidal activities by inhibiting glutamine synthetase with phosphinothricin although phosalacine itself hardly inhibited the enzyme.

Tazobactam-piperacillin compared with sulbactam-ampicillin, clavulanic acid-ticarcillin, sulbactam-cefoperazone, and piperacillin for activity against beta-lactamase-producing bacteria isolated from patients with complicated urinary tract infections.

The antibacterial activity of tazobactam-piperacillin was compared with that of sulbactam-ampicillin, clavulanic acid-ticarcillin, sulbactam-cefoperazone and piperacillin against beta-lactamase-producing bacteria isolated from patients with complicated urinary tract infections. Tazobactam-piperacillin showed a broad antibacterial spectrum against gram-negative and gram-positive bacteria. The minimum inhibitory concentrations (MIC90) of tazobactam-piperacillin were 6.25 micrograms/ml against Escherichia coli, 1.56 micrograms/ml against Proteus mirabilis, 3.13 micrograms/ml against Proteus vulgaris, 6.25 micrograms/ml against methicillin-susceptible Staphylococcus aureus, and 6.25 micrograms/ml coagulase-negative methicillin-susceptible staphylococci. Against all beta-lactamase-producing bacteria tested the antibacterial activity of tazobactam-piperacillin was at least 4- to 64-fold stronger than that of piperacillin, clavulanic acid-ticarcillin, and sulbactam-ampicillin, and similar to or greater than that of sulbactam-cefoperazone except for E. coli.

[Combination effect of teicoplanin and various antibiotics against hetero-VRSA and VRSA].

Eleven clinical strains of MRSA which were detected as heterogeneously-resistant to vancomycin (hetero-VRSA) on Mu3-medium (a newly devised hetero-VRSA detecting medium) were subjected to a study to explore the therapeutic possibility of combination therapy. Combination effects of teicoplanin with six different beta-lactam antibiotics (imipenem, panipenem, meropenem, flomoxef, sulbactam/ampicillin, cefoselis), arbekacin, and minocycline were evaluated on the strains of Mu3, Mu50 and the above 11 strains. Combination of teicoplanin with five beta-lactam antibiotics individually (except for cefoselis) showed a synergistic effect, while that with cefoselis showed synergistic or additive effect. Neither indifference nor antagonism effect was observed in combination of seicoplanin with beta-lactam antibiotics on these MRSA strains. The degree of synergistic effect in combination with teicoplanin was the strongest in imipenem, followed by panipenem > meropenem > flomoxef > sulbactam/ampicillin > cefoselis in this order. The average FIC index of the beta-lactam antibiotics against these strains was 0.113, 0.124, 0.163, 0.230, 0.264 and 0.388, respectively. Arbekacin and minocycline showed variable of effects in combination with teicoplanine. In the case of arbekacin, the ratio of synergy, addition, indifference, and antagonism were 30.8, 30.8, 0 and 38.4%, respectively, and in the case of minocycline, they were 15.4. 7.7, 0 and 76.9%, respectively. Vancomycin activity against hetero-VRSA and VRSA is antagonized with beta-lactam antibiotics, while teicoplanin activity is synergistic or additive. It is known that MRSA is relatively easy to emerge resistance to teicoplanin. Therefore, teicoplanin is not desirable for a monotherapy. However, in a combination with beta-lactam antibiotics, teicoplanin appeared to be a promising agent for the treatment of MRSA infection.

[In vitro susceptibility of Streptococcus agalactiae clinical isolates to beta-lactam antibiotics].

The susceptibility of Streptococcus agalactiae (S. agalactiae) clinical isolates of Juntendo University Urayasu Hospital, and type strain ATCC 13813 to beta-lactam antimicrobial agents was evaluated by means of macro-broth dilution MIC determination, killing kinetics and population analysis. When 10(6) cells of S. agalactiae were inoculated and cultured in Todd-Hewitt broth containing two-fold serial dilutions of penicillin, the viable cell count showed that about 10(2) cells survived irrespective of the penicillin concentration which ranged from 0.063 to 128 micrograms/ml. The result indicated that S. agalactiae had tolerance to penicillin (MICs were around 0.063 microgram/ml). Furthermore, the S. agalactiae strains were found to have a paradoxical response to penicillin in an acidic condition (pH 5.5). When the cell counts were performed at pH 5.5, about 10(2) cells survived at penicillin concentrations from 0.016 to 0.125 microgram/ml, while about 10(4) cells survived at the concentrations of 1 to 8 micrograms/ml. The antibiotic tolerance and paradoxical effects of S. agalactiae were also observed in killing kinetics. The ATCC 13,813 and 10 out of 11 clinical strains showed slow response to penicillin-mediated killing at pH 7.8 and ATCC 13,813 and one of the clinical strains showed a reduced response with increase in penicillin concentration at pH 5.5. These results suggested that the tolerance and paradoxical effect of S. agalactiae cells to beta-lactam antibiotics may be one of the reasons for frequent re-colonization of S. agalactiae at the time of delivery after the chemophylaxis in the 2nd trimester.

[Evaluation of reduced vancomycin susceptibility of MRSA strain Mu50 with various conditions of antibiotic susceptibility tests].

We evaluated a clinical strain of methicillin-resistant Staphylococcus aureus (MRSA), Mu50, for vancomycin susceptibility. Mu50 was isolated from a patient with infection of a surgical incision site which resisted vancomycin therapy. Mu50 showed a decrease in susceptibility to vancomycin, but this strain did not carry vanA or vanB or vanC genes as judged from PCR amplification. MICs of vancomycin against Mu50 and vancomycin-susceptible S. aureus FDA209P, S. aureus ATCC-29213, and MRSA H-1 were 8, 1, 1, and 1 microgram/ml, respectively by agar dilution and macro-broth dilution methods according to NCCLS. MIC values with agar dilution method using MHA + 20% horse serum, HIA, and BHIA agreed with the MIC values with micro- and macro-broth dilution method. Population analysis revealed that vancomycin concentration required for inhibition of ca. 10(7) cells of Mu50, S. aureus FDA209P, S. aureus ATCC29213, and MRSA H-1 were 36, 2, 2, and 2 micrograms/ml, respectively. These results showed that the activity of vancomycin against Mu50 was at least 8-fold decreased compared to that against S. aureus FDA209P, S. aureus ATCC29213, and MRSA H-1.

[Investigation of the antibacterial activity of faropenem against Streptococcus pneumoniae].

We evaluated the antibacterial activity of faropenem against penicillin-susceptible Streptococcus pneumoniae (PSSP) and penicillin-resistant S. pneumoniae (PRSP). It was shown that the minimum inhibitory concentrations against 90% of the clinically isolated strains (MIC90) of faropenem, penicillin G, cefaclor, cefcapene, and cefditoren against PSSP were 0.032, 0.063, 2, 0.25, and 0.125 micrograms/ml, respectively. While those against PRSP were 0.5, 2, > 128, 1, and 1 micrograms/ml, respectively. Furthermore, we evaluated the bactericidal activity, at the level of 1/4, 1, and 4 MIC, of faropenem and the above four reference antibacterial agents against PSSP and PRSP. Against PSSP No. 127, a sensitive strain to both penicillin G and cefcapene, faropenem showed almost the same bactericidal activity as those of reference agents. Against PSSP No. 108, a penicillin-susceptible and cephem-resistant strain, and PRSP No. 57, a resistant strain to both of penicillin and cephem, faropenem of 1 MIC showed bactericidal activity, but reference agents needed 4 MIC to show bactericidal activity.

[A novel method of detecting Staphylococcus aureus heterogeneously resistant to vancomycin (hetero-VRSA)].

We have developed a new method of detecting clinical S. aureus strains possessing heterogeneous resistance to vancomycin (hetero-VRSA). The method exploits characteristic antagonism between vancomycin and beta-lactam antibiotics against the strain Mu3, a representative hetero-VRSA. The method comprises of the following procedures: 1) overnight culture of bacteria is streaked on brain heart infusion agar containing 4 micrograms/ml of vancomycin, 2) paper discs impregnated with any one of beta-lactam antibiotics are placed onto the plate, and 3) the plate is incubated at 37 degrees C and the cell growth was observed after 24 h and 48 h of incubation. The Mu3 cells were grown only around the beta-lactam discs due to the antagonism between vancomycin and beta-lactam against Mu3 cells. A total of 321 MRSA clinical strains isolated in 1995 and 1996 from 12 medical facilities in Japan were tested with this method. A total of 39 strains (12.1%; 24 h) and 67 strains (20.96%; 48 h) grew around the beta-lactam discs. All the 10 strains (representing 10 facilities), tested with the analysis of resistant subpopulations to vancomycin, showed similar heterogeneous patterns as observed with Mu3. The method was considered as a convenient and rapid substitute for the population analysis, the authentic method for the detection of hetero-VRSA strains.

[Increase of non-amidated muropeptides in the cell wall of vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50].

The mechanism of resistance was studied with vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50. It was demonstrated that the incorporation of 14C-N-acetylglucosamine into the cell wall of Mu50 was not suppressed in the presence of 8 microliters/ml of vacomycin, whereas it was completely suppressed in vancomycin-susceptible strains FDA209P and H-1. Increased binding of vancomycin to the wall of Mu50 was observed compared to the control strains: 1.7 x 10(16) (Mu50), 6.1 x 10(15) (209P), and 6.7 x 10(15) (H-1) vancomycin molecules/mg cell wall, respectively. Remarkable proportion of the cell-wall component muropeptides were non-amidated in the cell wall of Mu50. In concordance with this phenomena, peptidoglycan cross-linkage decreased strikingly in the Mu50 strain. Free D-Ala-D-Ala residues at the end of muropeptides in the pre-existing cell wall generated by decreased cross-linkage seems to account for increased vancomycin binding. The increase of vancomycin-resistance level is presumably caused by sequestration of vancomycin molecules from primary target point on cell membrane. It was considered that at least two phenotypic changes are required for the vancomycin resistance in the Mu50 strain. First, as we have described previously, is the activated cell wall synthesis, and second, the reduction of cross-linkage of peptidoglycan by production of non-amidated muropeptide precursors.

[In vitro susceptibilities of clinical isolates of coagulase-negative staphylococci to glycopeptide antibiotics].

Two hundred and thirteen clinical strains of coagulase-negative staphylococci isolated in Japan between 1980 and 1997 were analyzed for glycopeptide susceptibility by determining MIC using both Mueller-Hinton agar (MHA) and Brain Heart Infusion agar (BHIA) plates. Of 37 Staphylococcus epidermidis strains isolated between 1980 and 1981, all were susceptible to vancomycin and teicoplanin on both MHA and BHIA. However, of 122 isolates of Staphylococcus epidermidis isolated between 1994 and 1997, 1 (0.8%) was intermediate to vancomycin on MHA and 39 (32%) were intermediate on BHIA, while 3 (2.5%) and 27 (22.1%) were intermediate or resistant to teicoplanin on MHA and BHIA, respectively. It was demonstrated that the susceptibilities of the strains in 1990s to vancomycin and teicoplanin were significantly decreased compared with those in 1980s. Population analysis was performed with six strains each of Staphylococcus epidermidis and Staphylococcus haemolyticus (three with vancomycin MIC > or = 8 micrograms/ml and three with vancomycin MIC < or = 4 micrograms/ml using BHIA). The population curves of the Staphylococcus epidermidis strains showed a homogeneous pattern of susceptibility. Whereas, those for two Staphylococcus haemolyticus strains (vancomycin MIC = 8 micrograms/ml using BHIA) showed a typical heterogeneous pattern. Vancomycin-resistant mutants (MIC > or = 32 micrograms/ml) were obtained with a high frequency of 10(-4)-(-5) from the strains by one-step selection with 16 micrograms/ml of vancomycin.

[Effect of arbekacin on the production of toxic shock syndrome toxin 1 by methicillin-resistant Staphylococcus aureus].

The effect of arbekacin (ABK), vancokmycin (VCM) and teicoplanin (TEIC) on the production of toxic shock syndrome toxin-1 (TSST-1) by methicillin-resistant Staphylococcus aureus was examined. In logarithmic-phase cultures, ABK, VCM and TEIC inhibited TSST-1 production by 85, 10 and 25%, respectively, at the concentration of one-fourth the each MIC. In stationary-phase cultures, ABK inhibited TSST-1 production by 50% or 90% compared with the control at the concentration of 4.0 micrograms/ml or 5.0 micrograms/ml respectively. VCM and TEIC did not inhibit TSST-1 production at the concentration of 8.0 micrograms/ml or lower. In human blood cultures, TSST-1 production was inhibited by ABK by 50% at 0.04 microgram/ml (1/256 of Cmax), but not inhibited by VCM and TEIC at the concentration of 1/16 of Cmax or lower. It has been already known that ABK has higher bactericidal activity than VCM and TEIC. ABK combined the inhibition of TSST-1 production with high bactericidal activity in both bacterial growth phases, and therefore ABK should be considered for the treatment of TSST-1-mediated MRSA-infection.

[Mechanism of vancomycin resistance in MRSA strain Mu50].

The mechanism of vancomycin resistance in methicillin-resistant Staphylococcus aureus (MRSA) Mu50 was investigated. More than 3 times increase of the incorporation of 14C-GluNAc into the cell wall of Mu50 was observed compared to those of vancomycin-susceptible strains FDA209P, H-1, LR5P1. The amount of cytoplasmic murein-monomer precursor increased more than 3 times in Mu50 compared to those of control strains. There was an increased production of PBP1, PBP2, and PBP2', which were 1.51, 17.2, and 7.06 times greater, respectively, in Mu50 than those in H-1, and 2.38, 4.46, and 1.96 times greater respectively, than those in LR5P1. By transmission electromicrograph, it was shown that the cell wall of Mu50 was twice thicker than that of LR5P1. Increase of tightly-bound vancomycin to the cell wall fraction was observed in Mu50 when compared to those in FDA209P and H-1 strains. From these results, the increase of the vancomycin targets, free D-Ala-D-Ala residues in the cell wall, in number, due to the activated cell wall synthesis, and/or decrease of the cross-linkage of the cell wall was suggested to be the mechanism of vancomycin resistance in the Mu50 strain.

[Molecular design of anti-MRSA drugs].

The true nature of resistance of methicillin-resistant Staphylococcus aureus (MRSA) is penicillin-binding protein 2' (PBP2'). Affinities of almost all beta-lactam antibiotics to PBP2' were very low. Therefore, MRSA which produces PBP2' shows resistance to all beta-lactam antibiotics. However, PBP2' has a different affinity to each beta-lactam antibiotic. For this reason, we thought that some derivatives of beta-lactam compounds could have high affinity to PBP2'. Accordingly, we developed cephem compounds which are more stabile and safe than previous penicillin and carbapenem compounds. Firstly, we investigated the side chain at C-7 position on 2-thioisocephem skeletal. Hydroxyimino-aminothiazol at C-7 position on 2-thioisocephem skeletal had the strongest activity against MRSA. Secondly, we investigated the linkage styles at C-3 position on 2-thioisocephem skeletal which were methylene, vinyl, and propylene. The compound of vinyl linkage style at C-3 position on 2-thioisocephem skeletal showed high activity against MRSA. Finally, we investigated 1-thiocephem, 2-thioisocephem, and 2-oxaisocephem as cephem-skeletals. Simultaneously, we studied C-3 linkage styles which were methylene, vinyl, and propylene. From these results, we found out that the compound of hydroxyiminoaminothiazol at C-7 position and vinyl linkage style at C-3 position on 1-thiocephem skeletal has superb activity against MRSA.