In vitro studies on quinolone-resistance in Pseudomonas aeruginosa have identified and characterized new mechanisms such as multidrug efflux systems, MexA-MexB-OprM, MexC-MexD-OprJ and MexEMexF-OprN which express nalB, nfxB and nfxC mutations, respectively, on the chromosome. Each system, which is organized by a inner membrane component (MexB, MexD or MexF), a outer membrane component (OprM, OprJ or OprN) and a periplasmic component (MexA, MexC or MexE), extrudes actively antibiotic molecules which permeate into the cells and contributes to multidrug resistance in P. aeruginosa. Of these systems, only MexAB-OprM is slightly expressed in the wild type strain and contributes to the intrinsic multidrug resistance. To overcome multidrug resistance in P. aeruginosa, we must investigate the functional and molecular analyses of these efflux systems.

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Gonococcal fluoroquinolone-resistance is now a significant problem in Japan. Mutation at serine-91 in the gonococcal gyrA gene is commonly identified in quinolone resistant isolates. We found a new method for direct detection of gyrA mutation in Neisseria gonorrhoeae in clinical isolates using pin-point sequencing. A wild strain of N. gonorrhoeae (T-25) highly susceptible to fluoroquinolones (FQs) and five mutant strains resistant to FQs were used as the test strains. The T-25 strain and five mutant strains of N. gonorrhoeae and other species of bacteria (α-hemolytic Streptococcus, coagulase (-) Staphylococcus and Escherichia coli) were each suspended in sterile distilled water at 10⁴-10⁵CFU/ml. PCR-amplification of the gyrA gene fragment of N. gonorrhoeae was performed with a codon 91 adjacent primer. Aliquots of PCR products were used for detection of single base substitution at codon 91 by pin-point sequencing. The signals of the fluorescein-labeled products were detected by ELISA. From 56 clinical specimens, this method was successfully applied to detect the fluoroquinolone-esistant cells of N. gonorrhoeae. There was one exception. These results suggest that this method is useful for the detection of fluoroquinoloneresistant strains in N. gonorrhoeae from clinical specimens directly.

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Recently, decreased susceptibility to vancomycin (VCM) among the clinical strains of methicillin resistant Staphylococcus aureus (MRSA) has been reported in Japan. To investigate VCM susceptibility among the clinical MRSA strains isolated in our hospital, we determined the MIC of VCM and performed a population analysis. A total of 116 strains, including 80 MRSA isolated from August 1996 to May 1997 (MRSA-A group), 16 MRSA isolated in 1990 when VCM was not available for MRSA infections (MRSAB group), and 20 methicillin-sensitive S. aureus (MSSA) isolated August 1996 to May 1997 (MSSA group), were analyzed. The MIC of VCM in all 96 MRSA strains was less than 4 μg/ml. The strains in the MRSA-A group tended to have a higher MIC than those in the MRSA-B group, although no statistically significant difference was seen. Population analysis revealed that 71 (88.8%) or 12 (15%) of 80 strains in the MRSA-A group, 15 (93.8%) or 4 (25%) of 16 strains in the MRSA-B group, and 19 (95%) or 6 (30%) of 20 strains in the MSSA group contained subclones that could grow on agar plates containing 4 or 8μg/ml of VCM, respectively. The strains in the MRSA-A group tended to form more colonies on agar plates containing VCM compared with those in the other two groups. The results of the present study suggest decreased susceptibility to VCM among MRSA strains in our hospital in recent years.

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Vancomycin susceptibilities of three methicillin-esistant Staphylococcus aureus (MRSA) strains Mu 3, Mu 50, H-1 and a methicillin-usceptible S. aureus strain ATCC 6538 P were compared by determining MIC and population analysis, which showed that their MIC values were 1, 8, 0.5 and 0.5μg/ml, respectively, while the vancomycin concentrations required to inhibit 1×10⁷ cells of these strains were 10, 13, 4, and 3μg/ml, respectively. Population analysis of Mu 3 showed a typical hetero-ype vancomycin resistance. The profiles of ¹⁴C-N-acetylglucosamine incorporation into the cell demonstrated that the incorporation to Mu 3 cells was 2.6 and 14.4-old higher than those to H-1 and ATCC 6538 P, respectively, and was 0.8-old lower than that to Mu 50 cells. In vitro synergy studies with a combination of vancomycin and aminobenzylpenicillin, cefpirome, ceftizoxime or aminobenzylpenicillin/sulbactam demonstrated an antagonism for the heterogeneous vancomycin-esistant S. aureus (hetero-VRSA) Mu 3, with a fractional inhibitory concentration (FTC) index of >2. Subsequently, a hetero-VRSA detection method was developed by means of detecting the antagonism vancomycin and ceftizoxime. 1, 673 clinical MRSA strains isolated from 1995 through 1996 in sixteen medical facilities all over Japan were screened for hetero-VRSA, which showed that the frequency of hetero-VRSA was 6.5% among the all MRSA clinical isolates.

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We have reported that long term clarithromycin (CAM) therapy improves the survival time of patients with non-small cell lung cancer. In the present study, we examined peripheral blood mononuclear cells for changes in cytokine mRNA by RT-PCR before and after CAM therapy. The study included 15 patients with unresectable non-small cell lung cancer. Before CAM therapy, 13 patients received basic therapy consisting of chemotherapy, radiotherapy or both. Two patients received no basic therapy. Interleukin-10 (IL-10), Interleuikin-12 (11-12), Interferon-gamma (IFN-γ) mRNA were measured before and at one and three months after starting CAM therapy. IL-12 and IFN-γ mRNA were significantly increased, and IL-10m RNA was decreased. The results suggest that CAM exhibits an antitumor effect that promotes Thlymphocytes shows a Th 1-like cytokine production.

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A case of intestinal tuberculosis showing small intestinal obstruction is presented. The patient was a 42-year-old male who had intermittent lower abdominal pain. Following a barium enema and an upper gastrointesinal series during remission, multiple stenotic sites were detected from the jejunum to the ascending colon. With the abdominal pain reappearing and increasing, laparotomy was undertaken, which revealed multiple stenotic involvement of the small and large bowel. Small intestinal segmental resections were performed (a total length of 175cm), with a bypass procedure through the terminal ileum to the transeverse colon. Frozen sections of the intestinal and lymphnode specimens showed positive acid-fast bacilli in tuberculous granulomas. Cultures of the operative specimens were positive. Fibrotic consolidations were detected on chest X ray. Smears of the sputa were negative on admission. Afterwards, however, acid-fast bacilli were detected on bronchoalveolar lavage on POD 21. The operative time course was not eventful and antituberculous chemotherapy was performed after surgery. As of April, 1996, the patient was well.

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