Male white grub beetles prefer the pheromone composition of young females in the field.

Females of the white grub beetle, Dasylepida ishigakiensis, release both (R)- and (S)-2-butanol as sex pheromones, but the males are only attracted to (R)-2-butanol. In laboratory-reared females, the proportion of the (R)-isomer decreased significantly as their calling opportunities increased and as they aged. We examined whether such qualitative changes also occur in field populations. We collected virgin females from the field and then trapped and analysed the volatiles emitted during their first and second callings. The ratio of (R)- to (S)-2-butanol (R/S) was 78:22 at the first calling, but shifted to 39:61 at the second calling. While investigating the composition of the female pheromones, the question arose as to whether the male preferences change in response to the shift in female pheromone composition. To answer this question, we observed the behaviour of young and old males in response to various R/S ratios as lures in the laboratory and in the field. In the flight tunnel assay of laboratory-reared individuals, young males touched female models with a 9:1 R/S ratio lure less than those with pure (R)-2-butanol; however, older males touched the two groups with equivalent frequency. In the field trap test, older males were much more attracted to (R)-2-butanol-scented lures. When we tested using lures with the same amount of (R)-2-butanol but added different amounts of the (S)-isomer, we found that increased levels of (S)-2-butanol resulted in lower attractiveness to males. (S)-2-butanol was confirmed to have an inhibitive activity in the attractiveness of (R)-2-butanol.

Mating disruption by a synthetic sex pheromone in the white grub beetle Dasylepida ishigakiensis (Coleoptera: Scarabaeidae) in the laboratory and sugarcane fields.

A serious sugarcane pest, Dasylepida ishigakiensis, remains in the soil during most of its life cycle except for a short period for mating. Mating disruption by an artificial release of the sex pheromone (R)-2-butanol (R2B), therefore, may be a feasible method to control this pest. We examined the effects of artificial release of R2B and its related compounds, (S)-2-butanol (S2B) and the racemic 2-butanol (rac-2B), on the mating success of this beetle both in the laboratory and in the field. In flight tunnel experiments, almost all males orientated towards a R2B-releasing source and 40% of them landed on the source. When the atmosphere was permeated with R2B, the frequency of males landing on the model was significantly reduced. Both rac-2B and S2B were less effective, but substantial reduction in landing success by males was achieved at higher rac-2B concentrations. R2B released from polyethylene dispensers in sugarcane plots greatly reduced not only the proportion of females mated with males but also the number of males caught by R2B-baited traps, indicating that male mate-searching behaviour was strongly affected by the released R2B. Similar inhibitory effects on male behaviour were also observed when tube- or rope-type dispensers released high rac-2B concentrations in the field. These results indicate that it would be highly possible to control D. ishigakiensis through the disruption of the sexual communication by releasing either synthetic R2B or rac-2B.

In vitro antibacterial activity of S-4661, a new parenteral carbapenem, against urological pathogens isolated from patients with complicated urinary tract infections.

The antibacterial activity of S-4661, a new parenteral carbapenem antibiotic, was assessed against the major urological pathogens isolated from patients with complicated urinary tract infections. S-4661 was slightly less active than imipenem and panipenem, but more active than meropenem and ceftazidime against Gram-positive bacteria. Against Gram-negative bacteria, S-4661 was similar to meropenem, similar to or more effective than imipenem, and more active than panipenem and ceftazidime. Thus S-4661 possesses potent and well-balanced wide-spectrum antibacterial activity against various urological pathogens.

Effect of moderate exercise on immune senescence in men.

The purpose of this study was to examine the relationship between active compared to inactive lifestyles and immunocompetence in men. Subjects, all male volunteers, regularly exercising moderately were separated into three age groups: young (20-39 years), middle-aged (40-59 years) and elderly (more than 60 years). Age-matched sedentary male subjects served as controls in each group. Immunological assessments were, total leucocyte count, lymphocyte subpopulation counts, natural killer cell activity and neutrophilic phagocytosis. Total leucocyte and T-cell (CD3+) counts were not significantly different among the groups. Among T-cell subsets, there was a slight increase in helper T-cell (CD3+CD4+) and a decrease in cytotoxic/suppressor T-cell (CD3+CD8+) concentrations in the older sedentary subjects, resulting in an age-associated significant increase in the CD4:CD8 ratio among those control groups. However, among the exerciser groups, no such increase and decrease in the T-cell subpopulations or an age-related increase of the CD4:CD8 ratio were observed. Considering the components of innate immunity, the concentration of NK-cells (CD16+CD56+) significantly increased in the elderly exercisers, compared to that of the age-matched control subjects, or of the young group. The phagocytotic activity of neutrophils showed an age-associated decline, but of lesser degree in the elderly exercisers than in the elderly controls. Taken together, these results suggest that habitual and moderate training in later life is associated with a lesser age-related decline in certain aspects of circulating T-cell function and innate immunity.

Reactivity of a dual amplified chlamydia immunoassay with different serovars of Chlamydia trachomatis.

A study was undertaken with different serovars (D, E, F, L2, MoPn) of Chlamydia trachomatis to determine the analytical sensitivity of a new dual amplified immunoassay (IDEIA PCE Chlamydia) for detecting chlamydial lipopolysaccharide. IDEIA PCE Chlamydia incorporates a polymer conjugate consisting of multiple copies of antibody and enzyme molecules to provide signal amplification. The test was also assessed with different protein A producing strains of Staphylococcus aureus in order to assess whether the use of a multiple antibody conjugate increased nonspecific binding. The detection limits varied for each serovar with a detection limit of 38 IFU/ml obtained with serovar F and 237 IFU/ml obtained with serovar D. The incorporation of the polymer conjugate resulted in a 2-5 fold increase in analytical sensitivity compared to an earlier version of the test using a conventional conjugate. No increase in cross reactivity with protein A producing strains of S. aureus was obtained. The new dual amplified test format offers potential as a sensitive low-cost screening assay for C. trachomatis infections.

Minimum inhibitory and minimal lethal concentration against Chlamydia trachomatis dependent on the time of addition and the duration of the presence of antibiotics.

The purpose of this study was to investigate the properties of several antimicrobial agents found to be effective against Chlamydia trachomatis and to verify the eradication therapy schedule. The in vitro activities of two quinolones (sparfloxacin, ofloxacin), of three macrolides (azithromycin, erythromycin, clarithromycin) and of a tetracycline (doxycycline) against C. trachomatis were evaluated by several methods for the determination of the minimum inhibitory concentration (MIC) and minimal lethal concentration (MLC). MLC of azithromycin was only 2 times higher than that of MIC. On the other hand, MLCs of other antibiotics were 4-16 times higher than their respective MICs. When all antimicrobial agents were added to the infected culture at different times, we found that the quinolones even at a concentration of 64 microg/ml could not inhibit the formation of inclusion if they were added after 20 h from the start of infection. The corresponding period for macrolides and doxycycline was 24 h. When the antibiotics were removed at 8 h after the start of the infection, all antibiotics except azithromycin and clarithromycin were needed at a concentration much higher than their MLCs to inhibit the formation of inclusion. We consider macrolides, especially azithromycin, to be an excellent anti-C. trachomatis drug because of its lower MICs and MLCs values which were also closer together.

The inhibitory effect of phosphate on the ligase chain reaction used for detecting Chlamydia trachomatis.

AIMS: To examine the detection limit of the ligase chain reaction kit for Chlamydia trachomatis, to study the inhibitory effect of phosphate on the ligase chain reaction, and to clarify the mechanism of inhibition. METHODS: Three reference serovars of C trachomatis--D/UW-3/Cx, F/UW-6/Cx, and L2/434/Bu--were used to test the sensitivity of the chlamydia ligase chain reaction. Comparison was made of the inhibition by phosphate before and after DNA amplification. Phosphate in up to 2.4 mM concentration was added to specimens of C trachomatis serovar D (1 to 50 inclusion forming units (IFU)/reaction) before DNA amplification to examine the concentration dependency of phosphate inhibition of the ligase chain reaction. RESULTS: The detection limits were 0.6 IFU/reaction for serovar D/UW-3/Cx and F/UW-6/Cx, and 0.4 IFU/reaction for L2/434/Bu. Phosphate inhibited the ligase chain reaction only when it was added before the amplification stage. The specimens containing chlamydia at 1 to 50 IFU/reaction were negative when the concentration of phosphate added at the prethermocycle stage was more than 1.2 mM. CONCLUSIONS: Ligase chain reaction analysis is a reliable method of diagnosing C trachomatis infection because of its high sensitivity. It would be clearly superior to the currently used methods if the problem of inhibitors could be eliminated. The mechanism of inhibition of the ligase chain reaction by phosphate was thought to be blockade of the amplification of the target DNA. The efficacy of the ligase chain reaction could be inhibited by phosphate in the urine, so duplicate dilution analysis of some negative specimens should be useful.

[Basic evaluation of Chlamydia antigen detection by EIA using a dual amplification enhanced immunoassay method].

Chlamydia trachomatis is one of the important pathogens of STD in our country. Therefore, rapid accurate, reliable and convenient tests for its detection are required. So far, IDEIA Chlamydia has been employed as a useful diagnostic kit. Now, IDEIA PCE Chlamydia, applied as a dual amplification EIA method, has been developed. In our present studies, the sensitivity, reproducibility, cross reactivity, and reliability of IDEIA PCE Chlamydia were investigated and compared with those of IDEIA Chlamydia and LCR Chlamydia. The sensitivity of IDEIA PCE Chlamydia showed 2.4 x 10(2) IFU/ml for C. trachomatis D, 1.2 x 10(2) IFU/ml for C. trachomatis E, 3.8 x 10 IFU/ml for C. trachomatis F, and 1.25 x 10(2) IFU/ml for C. trachomatis L2. With regard to reproducibility, more than 2.4 x 10(2) IFU/ml of all strains of C. trachomatis and negative samples gave highly reproducible values. Though no cross reactivity was recognized among three strains of Staphylococcus aureus with concentrations of more than 10(9) IFU/ml, non-heated samples of over 10(6) CFU/ml showed cross reactivity. In our observations, phosphate, Mg2+, Ca2+, and Fe3+ inhibited the efficacy of both IDEIA and IDEIA PCE Chlamydia. Ca2+ per se could be an inhibitor in the case of urine samples analyzed by IDEIA and IDEIA PCE Chlamydia. These results indicate that IDEIA PCE Chlamydia kit for detection of C. trachomatis may be clinically useful because of its improved sensitivity over IDEIA Chlamydia and its invariable specificity and reliability.

Transgenic potato expressing a double-stranded RNA-specific ribonuclease is resistant to potato spindle tuber viroid.

We have produced transgenic potato lines expressing the yeast-derived double-stranded RNA-specific ribonuclease pac1. Five lines of pac1 potato (Solanum tuberosum L., cultivar Russet Burbank) challenged with potato spindle tuber viroid (PSTVd) suppressed PSTVd infection and accumulation. All of the progeny potato tubers produced by resistant plants were also free of PSTVd. Because the pac1 gene product digested PSTVd in vitro, double-stranded regions in PSTVd molecule and/or replicative intermediates may be targeted by pac1 gene product in the transgenic potato plant.

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.

Binding of complement C3 to Klebsiella pneumoniae treated with sub-MIC cefodizime and chemiluminescence response of human polymorphonuclear leukocytes.

The binding of complement C3 to the cell surface of Klebsiella pneumoniae exposed to human serum complement after treatment with or without sub-MIC of antibiotics was examined by double diffusion immunoprecipitation against anti-human complement C3, and the production of oxygen-derived radicals by human polymorphonuclear leukocytes stimulated by complement-opsonized K. pneumoniae after treatment with or without sub-MIC of antibiotics was measured using the chemiluminescence (CL) assay. Complement C3 bound to the cell surface of K. pneumoniae treated with cefodizime was detected after exposure to human serum complement. The CL response induced by complement-opsonized bacteria after treatment with cefodizime was much higher than the response induced by nontreated bacteria or complement-opsonized bacteria after treatment with other antibiotics. These findings indicate that treatment with sub-MIC cefodizime make K. pneumoniae more susceptible to opsonization by complement and promotes the specific phagocytosis mediated by complement receptors.

Distribution of a methicillin-resistance gene in urinary isolates of methicillin-resistant staphylococci examined by enzymatic detection of the polymerase chain reaction.

We tried to examine the susceptibility to various antimicrobial agents and to detect the mec A gene using enzymatic detection of the polymerase chain reaction in methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA) and Staphylococcus epidermidis isolated from patients with complicated urinary tract infections (UTIs). All the strains of MRSA and MSSA showed a low sensitivity to imipenem (IPM), ceftazidime (CAZ), flomoxef (FMOX), amikacin (AMK), ciprofloxacin (CPFX) and ofloxacin (OFLX). Although all the strains of MRSA had the mec A gene, none of the MSSA strains had it. 74% of S. epidermidis had the mec A gene and strains resistant to methicillin were seen in 72% of them. The mec A-positive S. epidermidis showed a lower susceptibility to IPM, CAZ, FMOX, AMK, CPFX and OFLX than the mec A-negative strains. These results suggest that methicillin resistance was due to the mec A gene in MRSA and methicillin-resistant S. epidermidis (MRSE), and that MRSEs were very common among the bacteria causing complicated UTI. When we try to control nosocomial infections due to MRSA, it should also be noted that MRSE can be a reservoir of the mec A gene.

Effects of sub-minimal inhibitory concentrations of antimicrobial agents on the cell surface of Klebsiella pneumoniae and phagocytic killing activity.

Changes in the phagocytic killing activity, capsule structure, and physicochemical properties such as the hydrophobicity and charge of the cell surface were studied in Klebsiella pneumoniae treated with sub-minimal inhibitory concentrations (MICs) of various antimicrobial agents. The phagocytic killing activity of macrophages was enhanced by penicillins, cephems, and monobactam in the absence of antibodies specific to the capsule or complement. No enhancement was observed with new quinolones, aminoglycosides, macrolide, or carbapenem. The thickness of the capsule structure was considerably reduced after the treatment with penicillins, cephems, and monobactam compared with the untreated control, and it was slightly reduced by new quinolones. No changes were observed in the capsule structure with aminoglycosides, macrolide, and carbapenem. The hydrophobicity on the cell surface of the bacteria was considerably increased after the treatment with penicillins, cephems, and monobactam compared with the control, slightly increased with new quinolones and carbapenem, and not changed with aminoglycosides and macrolide. The negative charge of the cell surface of the bacteria was reduced by penicillins, cephems, and monobactam compared with the control. It was slightly reduced by new quinolones and carbapenem but was not reduced by aminoglycosides and macrolide. These findings suggest that sub-MIC beta-lactam drugs such as penicillins, cephems, and monobactams cause thinning of the capsule of K. pneumoniae with increases in the hydrophobicity and decreases in the negative charge of the cell surface, which reduces the physical repulsion between the K. pneumoniae and phagocytes and enhances the sensitivity of the bacteria to phagocytic killing activity.

Mechanism of enhancement of bactericidal activity of phagocytes against Klebsiella pneumoniae treated with subminimal inhibitory concentrations of cefodizime.

The effects of a sub-MIC of cefodizime on the morphology of the capsular structures and on the surface physicochemical properties, such as hydrophobicity and charge, of encapsulated Klebsiella pneumoniae were studied. The enhancement of bactericidal activity of macrophages against bacteria treated with sub-MICs of antibiotics was evaluated as the killing index. Cefodizime treatment gave the highest value of 32. Electron microscope observations revealed that the capsular material layer of cefodizime-treated K. pneumoniae was markedly thinner (32 nm) than that of untreated bacteria (160 nm) or bacteria treated with other antibiotics (75-90 nm). Contact angle measurement revealed that the surface of cefodizime-treated K. pneumoniae was more hydrophobic than that of untreated bacteria or bacteria treated with other antibiotics. Furthermore, the negative charge of the surface of K. pneumoniae decreased significantly with cefodizime treatment compared with the surface of untreated bacteria. These findings suggest that the treatment of K. pneumoniae with a sub-MIC of cefodizime reduced the thickness of the capsular material layer and that these changes increased the surface hydrophobicity of the bacteria and decreased the negative charge of the bacterial surface to render K. pneumoniae more susceptible to phagocytic activity by reducing the physical repulsion between the bacteria and phagocytes.

In vitro and in vivo enhancement of bactericidal activity of phagocytes against Klebsiella pneumoniae treated with subminimal inhibitory concentrations of cefodizime.

The effects of a subminimal inhibitory concentration (sub-MIC) of cefodizime on the bactericidal activity of phagocytes against encapsulated Klebsiella pneumoniae were studied in an in vitro system using an established mouse macrophage cell line, and in an in vivo system using mice in which many phagocytes were induced in the peritoneal cavity. In the in vitro system, the bactericidal activity of mouse macrophages against K. pneumoniae treated with a sub-MIC of cefodizime was significantly enhanced, and was greater than that of cefotaxime or cefoperazone. Significantly more bacteria treated with a sub-MIC of cefodizime were killed by serum complement than those treated with cefotaxime or cefoperazone. In the in vivo system, cefodizime-treated bacteria were phagocytosed and killed by phagocytes in the mouse peritoneal cavity, whereas, cefotaxime- and cefoperazone-treated and untreated bacteria were hardly phagocytosed at all or killed by phagocytes in the mouse peritoneal cavity, and bacterial regrowth was observed 24 h after bacterial challenge. Furthermore, the virulence of K. pneumoniae in mice was reduced more by treatment with cefodizime than with cefotaxime or cefoperazone. These findings indicate that K. pneumoniae treated with a sub-MIC of cefodizime become more susceptible to the bactericidal activity of phagocytes both in vitro and in vivo. This provides evidence that cefodizime at a sub-MIC may act together with the phagocytes against bacterial infections.

Changes of surface hydrophobicity and charge of Staphylococcus aureus treated with sub-MIC of antibiotics and their effects on the chemiluminescence response of phagocytic cells.

The effects of the sub-MIC of antibiotics on the surface hydrophobicity and charge of Staphylococcus aureus were examined by the contact angle method and by microscopic electrophoresis, and the production of oxygen-derived radicals by mouse peritoneal macrophages was measured by a luminol-chemiluminescence assay. The treatment of the bacterial cells with antibiotics induced an increase in hydrophobicity and a decrease in the negative charge of the bacterial surface. The chemiluminescence of the macrophages stimulated by S. aureus treated with antibiotics was significantly higher than that obtained with the untreated bacterial cells. These findings suggest that the antibiotics caused an increase in the hydrophobicity and a decrease in the negative charge of the surface of S. aureus, resulting in the enhancement of nonopsonic phagocytosis of S. aureus by macrophages.