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In 1968, the agar dilution method was developed as an independent Japanese method for measuring the minimal inhibitory concentration (MIC) of antimicrobial agents. As this method differed in a few respects from the MIC measurement methods used in other countries, it was revised in 1981, by a committee headed by Susumu Mitsuhashi, and the revised method (Chemotherapy 29:76-79, 1981) has been used since then.In 1979, an agar dilution method for measuring the MIC of anaerobes was developed by a committee chaired by Nozomu Kosakai (Chemotherapy 27:559-561, 1979). In 1990, a committee headed by Sachiko Goto approved a broth microdilution method for nonfastidious bacteria (Chemotherapy 38:102-105, 1990). Later, a committee headed by Atsushi Saito examined media that would be suitable for nonfastidious bacteria and fastidious bacteria, and they endeavored to prepare a broth microdilution method for anaerobic bacteria. In this context, a new broth microdilution method was proposed at the 40th Annual Meeting of the Japanese Society of Chemotherapy (JSC) in Nagoya in 1992, and the proposal was adopted as the standard JSC method after some modification (Chemotherapy 41: 183-189, 1993).The agar dilution method has remained unrevised for approximately 20 years. A proposal to review this method was recently made, and the 2007 Committee on Antimicrobial Susceptibility Testing was formed, comprising the JSC members listed below. Under the auspices of this committee, the method revised in 1981 was reviewed in comparison to the international standard method (Clinical and Laboratory Standards Institute [CLSI] method).

Recently we have heard warnings of an outbreak of a highly pathogenic avian influenza virus (H5N1). Although, to prevent such infections we must prepare anti-viral drugs and type-specific vaccines against influenza, we need various simple and effective protection methods, such as the use of face masks for public health. Also, in any consideration of bacterial infections, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and multidrug-resistant Pseudomonas aeruginosa (MDRP) also pose serious concerns which must be addressed. I examined the antiviral activity of gentian violet (GV) and GV-dyed cloth against the influenza A (H1N1) virus. Time-kill studies were carried out, and the virus titer was determined based on the 50% tissue culture infective dose (TCID50). The minimum inhibitory concentrations (MICs) of GV against bacteria were also determined, and the killing activities of the GV-dyed cloth were judged from viable cell counts. GV immediately killed the influenza A virus and this was confirmed by electron microscopy. Moreover, cloth dyed with a combination of GV and copper showed not only excellent antiviral activity but also prominent bactericidal activities.

In general, the elderly show a significant age-related decline in their immune response, thus leading to an increased vulnerability to infections or to an increase in the occurrence of malignant tumors. In this study, we examined the effect of Hochu-ekki-to (HOT or TJ-41) on the immunological capacity of the elderly. A group of elderly patients complaining of general fatigue or weakness were orally administered 7.5 g of HOT everyday for at least 120 days (4 months), whereas another group of aged patients mainly complaining of a loss of appetite were daily given 7.5 g of Anchu-san (TJ-5) during the same period and served as a control group. From the immunological point of view, the total number of circulating leukocytes remained unchanged, during the observation period both in the HOT and Anchu-san groups, as well as the ratios between CD3(+) T and CD20(+) B cells and between CD4(+) T and CD8(+) T cells. In addition, no differences were observed in the expression of CD25 antigen, which represents an activated state of T cells. However, as verified on day 30 as well as on day 120 after the administration of HOT, the natural killer (NK) activity against K562 target cells was significantly enhanced, in comparison to the results on day 0 in the HOT group, as well as to that activity on days 0, 30 and 120 in the Anchu-san group. In addition, on days 30 and 120 in the HOT group, there was a significant increase in the serum IFN-gamma level, which is thought to be associated with the NK activity, whereas no significant changes in that level were observed in the Anchu-san group, during the study period. From these results, it may be concluded that the administration of HOT to elderly people may help them improve, at least to some degree, their immunological capacity.

Minimum inhibitory concentrations (MICs) of vancomycin (VCM) and teicoplanin (TEIC) were measured using a novel susceptibility test based on the chemiluminescence assay (CA) method (Rapid-Lumi Eiken; Eiken Chemicals, Tokyo, Japan) against 33 strains in total: 7, 5, and 10 strains of which are VCM-resistant enterococci (VRE) with vanA, vanB, and vanC genes, respectively, and the other 11 strains are vancomycin-susceptible enterococci (VSE). The results were in good accordance with the values determined by the standard broth dilution method approved by the National Committee for Clinical Laboratory Standards (NCCLS): i.e., 88% (29/33) of consistency for VCM and 97% (32/33) for TEIC, respectively. In addition, genotypes in VRE strains (vanA, vanB, vanC-1, and vanC-2/3 genes) were properly estimated from the results of the CA method and the NCCLS interpretive categories, even though the incubation time was very short (2-4 h). In conclusion, it was found that the new method is reliable and rapid to detect VRE strains in clinical laboratories.

Despite the fact that the combination of vancomycin and a beta-lactam antibiotic are known to act synergistically on vancomycin-susceptible Staphylococcus aureus (VSSA), some MRSA have emerged showing antagonism to the combination of vancomycin and a beta-lactam antibiotic. These MRSA are called beta-lactam antibiotic-induced vancomycin resistant MRSA (BIVR). A method based on this antagonistic phenomenon has been devised to detect BIVR strains. The method inhibits the VSSA strain but allows the BIVR strain to grow. Forty-six commercially available beta-lactam antibiotics induced the vancomycin-resistance. Using this detection method, 717 MRSA clinical isolates obtained from eight institutes throughout Japan were thus screened and 6.3% of these were detected as BIVR when judged at 48 h.

Objectives: Staphylococcus aureus with low-level resistance to vancomycin (VLSA) which could develop into vancomycin-resistant S. aureus (VRSA) is most important. However, VLSA is difficult to detect by standard laboratory methods. We describe here improved methods to detect VLSA. Methods: Three methicillin-resistant S. aureus (MRSA) strains, designated Fu6, Fu10, and Fu18, were sequentially isolated from the burn wound site of a patient, during vancomycin therapy. The properties of these strains were compared with those of reference strains Mu3 and Mu50 (previous resistant isolates from other patients). Results: The isolated strains, Fu10 and Fu18, had identical phenotypes and genotypes. The vancomycin resistance of Fu10 was equivalent to that of strain Mu3, whereas Fu18 had much higher vancomycin resistance than Fu10 and Mu3, although reaching the level of Mu50. Fu18 showed similar growth to Mu50 on gradient gels and on Mu3 medium. Conclusions: Our data indicate that the VLSA developed vancomycin resistance during exposure to vancomycin in vivo. The population analysis of tested VLSA and vancomycin intermediately resistant S. aureus (VISA) indicates that a penem at relatively low concentrations induced a significant increase in the number of vancomycin-resistant subpopulations. Furthermore, we confirmed that gradient gel analysis and Mu3 medium are simple and useful methods for the detection of VLSA judged as VSSA by its conventional MIC alone.

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.

スーパーマーケットと鮮魚店で市販されていた刺身 (180検体) について, 黄色ブドウ球菌の汚染状況を平板培養法と増菌培養法の2つの検出法を用いて比較検討した.平板培養法において, 黄色ブドウ球菌は180検体から51検体 (28.3%) が検出された.一方, 増菌培養法では180検体から111検体 (61.7%) が検出され, 平板培養法で黄色ブドウ球菌が陰性と判定された129検体から, さらに60検体において黄色ブドウ球菌が検出された.分離された黄色ブドウ球菌111株中60株 (54.1%) がエンテロトキシンを産生しており, その中で60株中38株 (63.3%) がエンテロトキシンA型を産生していた.また, 増菌培養法でさらに検出された60株中35株 (58.3%) がエンテロトキシン産生株であり, 増菌培養法によってエンテロトキシン産生株の検出も増加した.これらの結果は市販刺身が黄色ブドウ球菌によって高率に汚染されていること, さらに, 黄色ブドウ球菌を検体から確実に検出するには平板培養法だけでは不十分であり, 増菌培養法が必要であることを示唆している.

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.

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 μg/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 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 do

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.

We assessed the C. trachomatis antibody assay kit HITAZYME (Hitachi Chemical Co., Ltd.) using clinical specimens. This kit is based on an enzyme immunoassay (EIA) which utilized purified Chlamydia trachomatis outer membrane antigen as the solid phase antigen. Twenty-nine untreated male urethritis patients, 816 pregnant housewives, 188 cervicitis patients, and 76 pelvic inflammatory disease patients were tested. Agreement between the HITAZYME test and antigen detection in infected area was assessed, and a comparison was made with IPAzyme (a commercially available indirect immunoperoxidase assay kit). 1) Summary of HITAZYME and IPAzyme IgA: Agreement between the two assays was relatively good, i.e., 82.6% (916/1109). However, 5.5% (61/1109) were HITAZYME (-), IPAzyme (+), and 11.9% (132/1109) were HITAZYME (+), IPAzyme (-). Thus, in quite a few cases the results did not agree. IgG: Agreement between the two assays was 73.7% (817/1109). Agreement was relatively low, 24.4% (271/1109) were HITAZYME (-), IPAzyme (+). 2) In the cases of disagreement, more specific Western blot analysis was performed to check the reactivity of the anti-C. trachomatis antibody. When IgA was used, agreement between HITAZYME and Western blot analysis was 69.6% (16/23), and agreement between IPAzyme and Western blot analysis was 30.4% (7/23), whereas when IgG was used, agreement between HITAZYME and Western blot analysis was 80.0% (12/15), and agreement between IPAzyme and Western blot analysis was 20.0% (3/15). There was significantly greater agreement with HITAZYME than with IPAzyme. In other words, HITAZYME had greater specificity when reacted with C. trachomatis antigen than IPAzyme. 3) The IgA antibody-positive rate in antigen (+) cases (male urethritis: 72.7%, pregnant housewives: 65.7%, cervicitis: 70.3%, pelvic inflammatory disease: 70.0%) was significantly (p < 0.01) higher than in antigen (-) cases (male urethritis: 16.7%, pregnant housewives: 13.6%, cervicitis: 22.6%, pelvic inflammatory disease: 30.4%). Therefore, IgA antibody can serve as a suitable indicator for active infection. 4) The IgG antibody-positive rate in antigen (-) female cases was 15.5% using HITAZYME and significantly (p < 0.01) lower than with IPAzyme. HITAZYME had greater specificity than IPAzyme. In conclusion, HITAZYME has relatively good sensitivity and specificity. Moreover, since it is an EIA assay, it allows objective evaluation of results. It permits processing of a large number of specimens because it is easy to perform. Thus, HITAZYME is a superior antibody assay for C. trachomatis. It can be used when antigen tests are difficult to perform. It is strongly anticipated that HITAZYME will be able to be used clinically as a screening test.

Some automated systems of the identification and susceptibility for microorganisms are used and prevail in hospital laboratories. One of the most serious problems is to perform accurate susceptibility testing for low-level resistant organisms, while antibiotic resistant microbes are increasing in medical fields. To evaluate automated machines for the susceptibility testing, several antibiotic resistant organisms were examined by plural technicians in some laboratories. Each strain of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycinintermediate S. aureus (VISA), extended-spectrum β-lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae was tested by three automated systems of WalkAway, VITEK2/VITEK2 compact and Phoenix for susceptibility. The results for antibiotics generated by the systems were compared to those generated by reference methods according to CLSI guidelines. The results of WalkAway, VITEK2/VITEK2 compact, and Phoenix demonstrated 92%, 91%, and 96% of reproducibilities, 92%, 94%, and 91% of MIC agreements, 0.5%, 0.8%, and 0.3% of very major error (VME) and 0.3%, 1.4%, and 2.3% of major error (ME), respectively. All automated systems had a high reproducibility even under the performance of plural technicians, although the differences of VMEs and MEs were observed among the systems. From these data, the automated systems for antimicrobial susceptibility testing were more useful for the detection of antibiotic resistant organisms by understanding the characteristics of each system.

Two basidiomycete species, Lentinus edodes mycelia (LEM) and Cordyceps sinensis (CS) were examined for induction of cytokines in murine macrophage cell line R309 (R309) and T cell line LBRM-33 1A5 (1A5). When lipopolysaccharide (LPS)-activated R309 were exposed to the extracts of basidiomycetes, R309 induced significant levels of interleukin 1 (IL-1). Interleukin 2 (IL-2) induction was recognized in 1A5 cultures in the presence of IL-1 and phytohemagglutinin (PHA). However, no enhancement of IL-2 production by these basidiomycetes was discerned in 1A5 cultures with IL-1 and PHA, i.e., direct action of basidiomycetes was not found on IL-2 production of 1A5. PHA-stimulated 1A5 exposed to basidiomycetes induced IL-2 without IL-1 when co-cultured with LPS-activated R309 as a source of IL-1. Effects of basidiomycetes on IL-2 production in 1A5 seemed to be caused through their action on macrophages. The induction of IL-2, Th1 type cytokine in T lymphocyte, is a significant finding since basidiomycetes, taken as a dietary supplement for immuno-suppressed patients, especially cancer patients, would be helpful in improving their immune activity against cancer.

Minimum inhibitory concentrations (MICs) of vancomycin (VCM) and teicoplanin (TEIC) were measured using a novel susceptibility test based on the chemiluminescence assay method (CA) (Rapid-Lumi Eiken; Eiken Chemicals, Tokyo, Japan) against 84 strains of Staphylococcus aureus, consisting of 82 strains of methicillin-resistant S. aureus (MRSA) from clinical isolated, S. aureus Mu3 involving beta-lactam antibiotic induced vancomycin (VCM) resistant MRSA (BIVR) and methicillin-susceptible S. aureus ATCC 29213. The results were in good accordance with the values determined by Clinical and Laboratory Standards Institute (CLSI): i.e., 100% (84/84) of consistency for VCM and 95% (80/84) for TEIC, respectively. In addition, BIVR strains were properly estimated from the results of the CA method and using the BIVR detection method with Mu3 agar (Mu3 Agar method), even though the incubation times was very short (2-4 h). In conclusion, it was found that the new method is reliable and rapid to detect BIVR strains in clinical laboratories.

Some methicillin-resistant Staphylococcus aureus (MRSA) strains in which combinations of vancomycin (VCM) and beta-lactam antibiotics show antagonism have recently emerged, and these strains are called beta-lactam-induced VCM-resistant MRSA (BIVR). We examined whether various antibiotics exhibited an antagonistic effect with VCM when used against Mu3 and Fu10 (representative BIVR strains), using a simple agar disc method. Chloramphenicol, tetracyclines, macrolides, and lincosamides showed an antagonistic effect with VCM. We attempted to elucidate the antagonistic mechanism of a combination of VCM and minocycline (MINO) in BIVR strains. We determined the rates of autolysis, autolytic activities, and the change in morphology of Mu3 treated with a combination of VCM and MINO. We observed that Mu3 grown in a combination of VCM and MINO showed increasing rates of autolysis, and lower minimal bacteriolytic enzyme dose (MBD) values compared with Mu3 grown in VCM alone, but no cell wall thickening was observed. Taken together, these results suggest that cell wall thickening may not be essential in the increased resistance of BIVR strains. Our present data therefore suggest that these combination therapies of VCM with tetracyclines should be adopted with great care in order to prevent VCM treatment failure.

The Gen-Probe APTIMA Combo 2 assay has previously been reported to have a high sensitivity. The end point of this assay was evaluated using highly purified chlamydial elementary bodies (EBs). The performance of the APTIMA Combo 2 assay was compared with a commercially available PCR kit, AMPLICOR Chlamydia trachomatis. The number of inclusions of C. trachomatis at the end point of the APTIMA Combo 2 assay was 0.005 inclusion-forming units (i.f.u.) ml−1, which was equivalent to 0.008 EBs per assay. The end point of the AMPLICOR kit was 5 i.f.u. ml−1 (equivalent to 0.5 EB per assay). The efficacy of the AMPLICOR C. trachomatis assay was inhibited by phosphate or Fe2+ ion, while these had no effect on the APTIMA Combo 2 assay. In conclusion, the APTIMA Combo 2 assay appears to have a greater sensitivity than the AMPLICOR C. trachomatis assay for detection of C. trachomatis, while demonstrating few problems with inhibitory substances such as phosphate and Fe2+ ion.

We performed epidemiologic study of 109 strains of methicillin resistant Staphylococcus aureus (MRSA) which were detected in our hospital. Of these strains, 6 strains showed resistant to Teicoplanin (TEIC) which MIC level were between 4 to 8microg/mL. All of them showed some phenotype, such as type II of coagulase, type A of enterotoxin, and were producing TSST-1. Genotype analysis by PFGE also showed that those strains ware identical. From analyzing the spreading rout of these TEIC resistant MRSA, we speculate that they first were in ICU ward, then spread all over the hospital carried by the stuff cross-working ICU and other units of hospital.

Isolation frequency and antimicrobial susceptibility of Haemophilus influenzae isolated in Saga University hospital from October 2002 to September 2003 were investigated. Out of 155 H. influenzae strains subjected 77 were isolated from pediatrics specimens. beta-Lactamase negative ampicillin (ABPC)-resistant H. influenzae (BLNAR), against which MICs of ABPC were higher than 4 microg/mL, were 32 strains (20.6%), and it became 63 strains (41.3%) when Low-BLNAR, against which MICs of ABPC were higher than 2 microg/mL, were included. beta-Lactamase positive ABPC-resistant H. influenzae (BLPAR) were 8 strains (5.2%). Although those BLNAR were also resistant to variety of beta-lactams, fluoroquinolones and other antibiotics were not affected by the level of ABPC-resistance. Resistant strains of BLPAR against SBT/ABPC, a combination of a beta-lactamase inhibitor, were detected but all of them were sensitive to TAZ/PIPC, an another combination. Those strains were able to be considered as beta-lactamase positive amoxicillin-clavulanate resistant H. influenzae (BLPACR). PIPC, TAZ/PIPC, CTRX, CDTR, MEPM, LVFX and CPFX showed good activity among tested antibiotics.

We could not detect hetero-vancomycin-intermediate resistant Staphylococcus aureus (hetero-VISA), according to the definition of hetero-VISA, from the clinical isolates of 140 methicillin-resistant S. aureus (MRSA) strains. However, 15 beta-lactam antibiotic-induced vancomycin-resistant MRSA (BIVR) strains were detected from the same strains. We screened 1882 MRSA clinical isolates obtained in 2002 from 21 institutes throughout Japan. The detection rate of blood-isolated BIVR was 12.6% (19/151), and that of nonblood-isolated BIVR was 4.9% (85/1731; P0.001; chi2 test). Uridine-diphosphate-N-acetylmuramyl-L: -alanyl-D: -isoglutamyl-L: -lysine, used as the peptidoglycan material of S. aureus, showed the same results as beta-lactam antibiotics in BIVR.

We determined the antibacterial activities of oral Cephems against isolated from the patients with the respiratory infections, the urinary tract infections, and infections in the obstetrics field of an adult and a child, during the period from 2002 to 2003; Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Branhamella catarrhalis, Klebsiella pneumoniae and Escherichia coli of 40 strains of each, and Peptostreptococcus spp. 22 strains. S. pneumoniae and H. influenzae strains that resistant is regarded were collected mainly, penicillin-intermediate S. pneumoniae (PISP), penicillin-resistant S. pneumoniae (PRSP) and beta-lactamase negative ampicillin-resistant H. influenzae (BLNAR) strains. The MICs of Cephems except cefaclor (CCL) wereor = 0.03 microgram/mL against all strains of S. pyogenes. The MICs of cefteram (CFTM) and cefditoren (CDTR) wereor = 0.0125 microgram/mL activity against 7 strains penicillin-susceptible S. pneumoniae (PSSP). However the MIC90s of cefditoren (CDTR) was 1 microgram/mL, cefteram (CFTM), and cefcapene (CFPN) were 2 micrograms/mL against PISP and PRSP, were higher than those of other drugs, but showed slightly higher than PSSP. The MIC90s of Cephems. were 0.5-4 micrograms/mL against strains of E. coli. The MIC90s of CFTM was 0.5 microgram/mL, and CDTR, CFPN were 1 microgram/mL against E. coli were higher than those of other drugs. The four strains of E. coli however were highly-resistant which MIC90s of CCL were more than 32 micrograms/mL were obtains. Furthermore it is necessary to pay much attention to the trend of resistant such as E. coli of Cephems. Although all strains showed resistant to AMPC, MIC90 of Cephems were 0.25-1 microgram/mL, good activities against K. pneumoniae. Against beta-lactamase negative ampicillin-susceptible H. influenzae (BLNAS) 23 strains the MIC90s of CCL and other Cephems were 64 micrograms/mL and 0.25-8 micrograms/mL. The MIC90s of CDTR and CFTM wereor = 1 microgram/mL of BLNAR (15 strains). However there of CFDN and CPDX were 8 micrograms/mL and CCL wereor = 16 micrograms/mL. Two strains which were produced beta-lactamase were highly--ABPC resistant. Although B. catarrhalis all strains were produced beta-lactamase and Cephems except for CCL showed better susceptibility than AMPC. The MIC90s of Cephems were 0.25-2 micrograms/mL against Peptostreptococcus spp.

Thirty-two extracts from 22 Mexican medicinal plants of 15 different families were assayed to determine their antibacterial activity against Escherichia coli and Staphylococcus aureus. Seventeen plants showed antibacterial activity, while five plants showed no activity against both bacteria. All of the extracts showed higher activity against Staphylococcus aureus (methicillin-sensitive and methicillin-resistant) than against Escherichia coli, except one. Among the plants examined, Bursera simaruba (L.) Sarg. (Burseraceae), Haematoxylum brasiletto H. Karst. (Fabaceae), Calophyllum brasiliense Cambess. (Clusiaceae), and Mammea americana L. (Clusiaceae) were highly active against Staphylococcus aureus. Coumarins (mammea A/BA and mammea A/AA) and xanthones, namely jacareubin and 1,3,5,6-tetrahydroxy-2-(3,3-dimethylallyl) xanthone, were isolated as the principle compounds from the last two plants.

We developed a simple method that can replace the polymerase chain reaction (PCR) to distinguish between vancomycin-resistant enterococci (VRE) with the vanA, vanB and vanC genes. The method is based on induction of teicoplanin resistance by vancomycin in vanB-VRE, while the two compounds have a synergistic effect in vanC-VRE. In addition, vanA-VRE shows resistance to both vancomycin and teicoplanin, and both the compounds can induce resistance to vanA-VRE. Utilising these properties, we attempted to develop a simple method to distinguish between vanA, vanB and vanC. We compared our simple method with the PCR method in 43 strains of vanA-VRE, 35 strains of vanB-VRE and 37 strains of vanC-VRE. The results were 100% consistent with that obtained by PCR.

Recent studies have shown that the urogenital pathogen Chlamydia trachomatis to be a major bacterium triggering reactive arthritis (ReA), and is able to induce interleukin-6 (IL-6) production in human fibroblast-like synovial cells (FSC) in vitro. In the present study, we examined the correlation between IL-6 production and multiplication of chlamydia in FSC. All FSC from five patients secreted highly increased quantities of IL-6 in a dose-dependent and time-dependent fashion. Heat and UV inactivated chlamydia failed to enhance production of IL-6. When azithromycin was added to infected cultures of FSC at 0 or 48 h after infection, the level of IL-6 production was very low. Transmission electron microscopy of such infected cultures revealed many abnormal forms of chlamydia within the inclusions in FSC. From one step-growth curve experiments, it was suggested that C. trachomatis hardly multiplied in FSC. In contrast, in C. trachomatis infected HeLa 229 cells, chlamydia multiplied as usual, but little IL-6 production were found. These observations indicated that live chlamydia and the persistence of chlamydia may be essential for stimulating the synthesis of IL-6 in FSC.

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.

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.

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.

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.

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.

We examined the responsiveness of two factor-dependent macrophage cell lines, BDM-1 and its subclone, BDM-1W3, to bacterial lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) for their growth. LPS inhibited the M-CSF-dependent proliferation of BDM-1 cells but it had no effect on the proliferation of BDM-1W3 cells. LPS promoted DNA synthesis and supported the cell viability in the absence of CSFs in BDM-1 and BDM-1W3 cells, suggesting that the intracellular signals are transduced from the interaction of LPS with LPS-binding sites in BDM-1W3 cells as well as in BDM-1 cells. IFN-gamma inhibited the proliferation of BDM-1 and BDM-1W3 cells. However, BDM-1 cells were more susceptible to the inhibitory effect of IFN-gamma than BDM-1W3 cells. In contrast to LPS and IFN-gamma, TNF-alpha did not inhibit the proliferation of BDM-1 and BDM-1W3 cells. These cell lines should be useful for studying the regulatory mechanisms in CSF-dependent macrophage proliferation.

Mouse macrophage BAM3 cells produced colony-stimulating factors (CSFs) after stimulation with bacterial lipopolysaccharide (LPS). By assaying the CSF using various interleukin 3-dependent cell lines, it was shown that most of the CSFs produced by BAM3 cells were granulocyte CSF (G-CSF). The granulocyte-macrophage CSF (GM-CSF) gene was also expressed in BAM3 cells after stimulation with LPS. When BAM3 cells were fused with the mouse renal adenocarcinoma cell line RAG which does not produce G-CSF, two of four hybrid cell lines constitutively produced large quantities of G-CSF. About 300 bp of the promoter region of mouse G-CSF chromosomal gene was inserted upstream of the Escherichia coli chloramphenicol acetyltransferase gene, and introduced into BAM3, RAG and hybrid cells. The G-CSF promoter was activated by stimulation with LPS, in BAM3 cells, but was inert in RAG cells. On the other hand, there was significant constitutive CAT activity in the hybrid cells.

Three macrophage cell lines from bone marrow cells of C3H/HeN mice were isolated by successive transfer of the cells in culture with L-cell-conditioned medium (LCM) or WEHI-3 cell-conditioned medium (WEHI-3CM). These cell lines, which express Fc receptors, are involved in Fc-mediated phagocytosis and possess nonspecific esterase activity. Two (BDM-1 and BDM-2) of three cell lines show dependency for growth on either macrophage colony-stimulating factor (M-CSF) (CSF-1) or granulocyte-macrophage colony-stimulating factor (GM-CSF) and do not respond to interleukin 3 (IL-3). The third clone (BDM-3) proliferates in response to IL-3 as well as to GM-CSF and weakly responds to M-CSF and to interleukin 4 (IL-4). GM-CSF, in combination with the suboptimal concentration of M-CSF, acted synergistically on the proliferation of BDM-1 cells. The tumor-promoting phorbol diester, 12-o-tetradecanoyl-phorbol-13-acetate (TPA) also acted synergistically with the three CSFs (IL-3, GM-CSF, and M-CSF) to stimulate the proliferation of BDM-1 cells. The synergistic effect was observed when cells were pretreated with TPA and subsequently stimulated with IL-3. The calcium ionophore A23187 enhanced the proliferation of BDM-1 cells costimulated with TPA and IL-3. These factor-dependent macrophage cell lines should be useful for studying signal transduction mechanisms in the regulation of cell growth.

The interaction of herpes simplex virus type 1 (HSV-1) with murine macrophage cell lines was examined. The cell lines appeared to be moderately permissive for HSV-1 replication, though the yield of the virus was limited compared with that in Vero cells. Furthermore, the murine macrophage cell line SL-1, bearing Ia antigen, was persistently infected with HSV-1 for over one year, and was designated SL-1/KOS. Persistent infection could not be established in an Ia antigen-negative macrophage cell line, SL-4. In the SL-1/KOS culture, there was a small number of infected cells as revealed by infectious center assay. Treatment with monoclonal antibody against HSV-1 cured the persistent infection. Therefore maintenance of the persistent infection is considered to be due to a carrier culture consisting of a minority of infected cells and a majority of uninfected cells. In the SL-1/KOS cultures a low level of interferon (IFN) was found. When a large amount of exogenous recombinant murine IFN-beta (10(5)-10(6) international units/ml) was added to the culture, virus production diminished to undetectable levels. These results suggest that IFN plays an important role in the maintenance of persistent infection. In long-term persistently infected cultures, syncytium formation appeared and the virus from such cultures had a different DNA structure from that of the virus originally used for infection as revealed by restriction endonuclease analysis.

After treatment of HeLa and L cells with vinblastine sulfate the material of microtubules (tubulin) was reorganized into (a) large paracrystals (PC) of tightly packed tubules; (b) smaller aggregates of tubules with greater diameter whose walls are constituted from well defined, helically arranged morphological subunits; and (c) microtubules associated with helices of polyribosomes of uniform size. All of these structures survived disruption of cellular membranes by means of a nonionic detergent. Following a thorough stripping of membranes there remained a subcellular fraction sedimenting at 1,500 g for 15 min, in which were contained nuclei, centrioles, and the above mentioned microtubular elements, maintained as a complex of organelles by an interconnecting network of 80 Å microfibrils. As a result of membrane disruption it was possible to localize precisely in the electron microscope the binding of ferritin antibody conjugates. Specific labeling at the surface of PC and microtubule aggregates could be demonstrated. This result was substantiated by means of the immunoperoxidase method of labeling the PC. A concentrated deposit of ferritin was also found in the vicinity of centrioles and related structures, the annuli of the nuclear pore complex and the annulate lamellae. However, the specificity of the label on these organelles remains questionable because ferritin, albeit in lower concentration, was also present on them in control preparations reacted with preimmune sera.

The effects of sub-minimum inhibitory concentrations (sub-MICs) of monobactams (aztreonam and AMA1080) on the host-parasite relationship were studied in an in vitro system using an established mouse macrophage cell line. The presence of sub-MICs aztreonam or AMA1080 enhanced significantly the macrophage bactericidal activity against Escherichia coli S615, Pseudomonas aeruginosa K1, Klebsiella pneumoniae 12 and Serratia marcescens US5. Even four times the MIC of monobactams had no direct effect on macrophages. A synergistic bactericidal effect against E. coli was also observed with sub-MICs of monobactams and lysozyme or macrophage lysate. Furthermore, E. coli treated with sub-MICs of aztreonam was more sensitive to two bactericidal macrophage products, hydrogen peroxide and superoxide anion. These results suggest that the effects of monobactams are exerted on bacteria and not on macrophages; sub-inhibitory levels of monobactams may alter the bacterial cell rendering it more susceptible to bactericidal substances released by macrophages, thus favouring phagocytosis and killing by macrophages. Electron microscopic observations support these conclusions. This study provides evidence that monobactams at sub-MICs may work in partnership with host defenses against Gram-negative bacterial infections.

A new procedure for the rapid purification of vinblastine-induced microtubular paracrystals is described. The paracrystals constitute about 2 per cent of the total protein in L cells. They contain a labile ATPase with a Michaelis constant of 1.0 × 10 -4 . In the immunofluorescence test, sera prepared against paracrystals reacted positively with intracellular paracrystals and the mitotic apparatus. Antisera against paracrystals from a malignant murine cell also labeled the mitotic apparatus of HeLa and chicken embryonic cells, revealing the presence of a common or related antigen in the mitotic apparatus of these higher vertebrates.

The influence of rifampicin on the biogenesis of vaccinia virus was examined by combined biochemical and electron microscopic procedures. Penetration, uncoating, DNA replication and the assembly of membranes and immature particles proceeded in the presence of the antibiotic. However, when wild-type virus was used for inoculation virus maturation as well as the induction of nucleotide phosphohydrolase, RNA polymerase, and two DNases (four virus-associated enzymes), was blocked by rifampicin. By contrast infection with a drug-resistant mutant of vaccinia elicited the synthesis of these enzymes and mature progeny were formed. After washing out rifampicin additional synthesis of RNA and protein were required for the completion of wild-type virus development. These observations indicate that transcription of certain early functions can occur from the parental genome but the integration of late functions within virus membranes is essential for the completion of maturation. The sites of rifampicin action at the synthetic level in the vaccinia and bacterial systems are discussed.

Colony-stimulating factors (CSFs) produced by two simian virus 40(SV40) transformed macrophage cell lines (BAM1 and BAM3), and three hybrids (HM3-11, HM3-12, and HM3-14) derived from fusion between BAM3 and a Chinese hamster cell line (hs222-16) were examined. HM3-11 and HM3-14 produce two molecular species of CSF, which are not found in the conditioned media from cultures of BAM1 and BAM3 or lipopolysaccharide (LPS), phorbolmyristate-acetate (PMA), and zymosan-stimulated BAM3. HM3-12, which is classified into another group in terms of CSF secretion, does not produce these two CSFs. On the basis of various criteria, one of these CSF species (peak 1-CSF) was characterized as a macrophage-colony-stimulating factor (M-CSF). The other CSF (peak 2-CSF) induced a group of bone marrow cells in granulocytes and macrophages as well as growth of a mast cell line, IC2. This CSF has an apparent molecular weight of 18,000, estimated by SDS-polyacrylamide gel electrophoresis. Unlike interleukin 3 (IL3) from WEHI-3 cells, the growth factor activity of peak 2-CSF binds to DEAE-Sephacel. Thus, peak 2-CSF is similar to a granulocyte-macrophage colony-stimulating factor (GM-CSF) rather than to IL3. The anti L cell CSF serum does not inhibit the CSF activity in Chinese hamster fibroblast conditioned medium, and the IC2 cells do not respond to Chinese hamster lung conditioned medium (CHLCM), suggesting that peak 1- and peak 2-CSF are of mouse origin.