Ariaki Nagayama

The effects of vancomycin and beta-lactam antibiotics on vancomycin-resistant Staphylococcus aureus

To the Editor: First Sieradzki et al.1 and then Climo et al.2 presented evidence that combinations of vancomycin and β-lactam antibiotics were synergistic in vitro and in vivo against vancomycin-re...

Emergence of vancomycin resistance during therapy against methicillin-resistant Staphylococcus aureus in a burn patient—importance of low-level resistance to vancomycin

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.

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.

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).

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 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.

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.

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.

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.

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 β-lactam antibiotics show antagonism have recently emerged, and these strains are called β-lactaminduced 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.

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.