[Comparison of the antimicrobial susceptibility testing with three automated systems for MRSA, VISA, ESBL-producing Escherichia coli and Klebsiella pneumoniae].

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 ß-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.

Effects of two basidiomycete species on interleukin 1 and interleukin 2 production by macrophage and T cell lines.

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.

Final report from the Committee on Antimicrobial Susceptibility Testing, Japanese Society of Chemotherapy, on the agar dilution method (2007).

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).

The antagonistic effects of a combination of vancomycin and minocycline in Staphylococcus aureus with heterogeneous resistance to vancomycin.

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.

[Evaluation of rapid antimicrobial susceptibility test for Staphylococcus aureus by chemiluminescent assay and its application for screening of beta-lactam antibiotic induced vancomycin-resistant MRSA].

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.

Effects of levofloxacin and doxycycline on interleukin-6 production of Chlamydia trachomatis-infected human synovial fibroblasts.

BACKGROUND: A large amount (80,000-100,000 pg/ml) of interleukin-6 (IL-6) was detected in cultures of human synoviocytes infected with Chlamydia trachomatis. In this study, we investigated the effect of antibiotics on the IL-6 production of C. trachomatis-infected human fibroblast-like synovial cells (HFLS). METHODS: The minimum inhibitory concentrations of levofloxacin and doxycycline against C. trachomatis were determined in either HFLS or HeLa 229 cells. The number of live Chlamydia was also examined. IL-6 in the supernatants of infected cultures was quantified by capture ELISA. RESULTS: The production of IL-6 was suppressed to as low as 1,800 pg/ml in the infected HFLS treated with levofloxacin or doxycycline immediately or early after infection. In HFLS treated with levofloxacin and doxycycline, the IL-6 levels decreased to 37,000 and 21,000 pg/ml, respectively, 48 h after infection, and levofloxacin was thus found to be less effective than doxycycline. In addition, the number of viable C. trachomatis in the infected cultures treated with levofloxacin 24 h after infection was higher than when treated with doxycycline. CONCLUSIONS: The early administration and proper selection of antibiotics is important for the suppression of inflammatory cytokine production. These findings indicate that antibiotic therapy will not only work in treating infections but might also be useful in treating reactive arthritis secondary to the infection.

Inactivation of influenza A virus by gentian violet (GV) and GV-dyed cotton cloth, and bactericidal activities of these agents.

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.

Investigation of beta-lactam antibiotic-induced vancomycin-resistant MRSA (BIVR).

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; P < 0.001; 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.

In vitro assessment of the APTIMA Combo 2 assay for the detection of Chlamydia trachomatis using highly purified elementary bodies.

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.

Antibacterial activity of crude extracts from Mexican medicinal plants and purified coumarins and xanthones.

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.

[Molecular epidemiologic analysis of nosocomocal infection due to TEIC resistant MRSA strains].

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.

A rapid antimicrobial susceptibility test based on chemiluminescence assay and its application to screening of genotypes in vancomycin-resistant enterococci.

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.

[The isolation frequency and antimicrobial susceptibility of Haemophilus influenzae isolated in Saga University 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.

Rapid detection and differentiation method of VanA, VanB and VanC phenotypes in vancomycin-resistant enterococci.

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.

Effect of a traditional Japanese herbal medicine, hochu-ekki-to (Bu-Zhong-Yi-Qi Tang), on immunity in elderly persons.

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.

Method of detecting beta-lactam antibiotic induced vancomycin resistant MRSA (BIVR).

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.

Infection of human fibroblast-like synovial cells with Chlamydia trachomatis results in persistent infection and interleukin-6 production.

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.

[Antibacterial activity of oral Cephems against various clinically isolated strains].

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) were < or = 0.03 microgram/mL against all strains of S. pyogenes. The MICs of cefteram (CFTM) and cefditoren (CDTR) were < or = 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 were < or = 1 microgram/mL of BLNAR (15 strains). However there of CFDN and CPDX were 8 micrograms/mL and CCL were > or = 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.