Synthesis of new sugar derivatives and evaluation of their antibacterial activities against Mycobacterium tuberculosis.

A series of sugar derivatives (1-13) were synthesized and evaluated for antibacterial activity against Mycobacteriumtuberculosis (MTB), especially multi-drug resistant (MDR) MTB, and the structure-activity relationships of these compounds were studied. The results showed that the compound OCT313 (2-acetamido-2deoxy-beta-D-glucopyranosyl N,N-dimethyldithiocarbamate) (4) exhibited significant in vitro bactericidal activity, and that the dithiocarbamate group at C-1 position of the glucopyranoside ring was requisite for the antibacterial activity.

[Identification of 23 mycobacterial species by Invader assay with targeting 16S rRNA gene and ITS-1 region--comparison with DDH method in clinical isolates].

PURPOSE AND METHOD: The Invader assay was developed to identify 23 mycobacterial species using probes derived from the species-specific region of the 16S rRNA gene and the 16S-23S rRNA internal transcribed spacer 1 (ITS-1) region, with minor modifications of our previous study. In the present study, we compared the identification capability between the Invader assay and DNA-DNA hybridization (DDH) method. DDH is commonly used to identify non-tuberculosis mycobacterium in Japan and 636 clinical mycobacterial strains cultured on Ogawa slants were tested. RESULTS: The Invader assay could identify 615 (96.7%) of the 636 strains. The results contained 14 M.lentiflavum, 3 M. parascrofulaceum and 1 M. intermedium, which were undetectable with DDH method. On the other hand, DDH method could identify 580 (91.2%) strains with duplicate assay. Of 628 strains except 8 strains identified as a few species by Invader assay, 551 (87.7%) strains were identified as the same species by two methods. Discordant results were mainly recognized for the identification of M. gordonae, M. avium, M. lentiflavum and M. intracellurare. The results of other methods targeting 16S rRNA indicated correctness of the Invader assay. CONCLUSION: These results indicate that Invader assay could identify more correctly than DDH method and could identify about 97% of clinically important mycobacterium.

Biological and molecular characteristics of Mycobacterium tuberculosis clinical isolates with low-level resistance to isoniazid in Japan.

We reevaluated the BACTEC MGIT 960 antimicrobial susceptibility testing system (MGIT 960 AST) by using 1,112 isolates of Mycobacterium tuberculosis. When the results of MGIT 960 AST were compared with that of the proportion method using Ogawa medium (Ogawa PM), discrepant results were obtained for 30 strains with isoniazid, all resistant by MGIT 960 AST but susceptible by Ogawa PM. For 93% of the strains that produced discrepant results, the MIC was 0.4 or 0.8 microg/ml, showing resistance by the proportion method using Middlebrook agar plates. Furthermore, it was also established by analyses of the katG and inhA genes that strains resistant only by MGIT 960 AST have a low level of isoniazid (INH) resistance, indicating that MGIT 960 AST is a reliable method. Ninety-six strains were resistant to 0.1 microg/ml INH by MGIT 960 AST. When they were divided into three groups, Low-S (susceptible at 0.2 microg/ml), Low-R (resistant at 0.2 microg/ml), and High-R (resistant at 1.0 microg/ml), by Ogawa PM, 43.3% of the Low-S strains had mutations in the promoter region of inhA and no mutations were detected in katG codon 315, while 61.7% of the High-R strains had katG codon 315 mutations or a gross deletion of katG. These results suggest that mutations in inhA are associated with low-level resistance to INH and katG codon 315 mutations are associated with high-level resistance to INH. In addition, the analyses demonstrated some relationship of mutations in the inhA gene with ethionamide resistance for the Low-S strains, but not for the High-R strains.

[Comparative study of bactec MGIT 960 AST and conventional proportion method using Ogawa medium for the drug susceptibility testing of Mycobacterium tuberculosis to isoniazid].

OBJECTIVE: To evaluate the accuracy of drug susceptibility testing to isoniazid with BACTEC MGIT 960 (MGIT AST) comparing with the standard proportion method using Ogawa medium. METHOD: A total of 1109 M. tuberculosis strains, which were selected from the collection of RYOKEN drug resistance survey in 2002, were selected and subjected to the susceptibility testing to isoniazid using MGIT AST and 1% Ogawa standard methods. The results from MGIT AST were compared with the judicial diagnosis by Ogawa. The sensitivity to detect drug resistance, the specificity for susceptible strain, the efficiency of overall agreement, and kappa coefficient were calculated to evaluate the performance. The treatment process, outcome and prognosis were analysed for the patients on whom the tests showed discrepant results. RESULTS: Compared with the judicial results, the sensitivity, specificity, efficiency, and kappa coefficient of MGIT AST were 100%, 97.1%, 97.3%, and 0.798, respectively. The strains, which showed discrepant results between MGIT AST and Ogawa, were all susceptible by Ogawa and resistant by MGIT AST. A total of 11 out of 30 discrepant cases were followed clinically and no relapse cases were identified, irrespective of the modification of the treatment regimen. As for the proportion of primary INH drug resistance in the present study, it was 5.3% with MGIT AST but was 2.7% with Ogawa, and the difference was statistically significant (p = 0.005). DISCUSSION: The discrepancies on the results of drug susceptibility testing of M. tuberculosis strains to isoniazid between MGIT AST and 1% Ogawa proportion method have been reported. In the present study, the sensitivity, specificity, and overall efficiency of MGIT AST on the prevalent strains in Japan were all beyond 95%, and considered sufficient as the anti-tuberculosis drug susceptibility testing (AST), though 2.7% of discrepancy was observed. Even for the discrepant cases, there was no difference in the treatment outcome and prognosis. Thus, MGIT AST was confirmed as a reliable AST method comparable to Ogawa standard. However, MGIT AST might increase the proportion of INH resistance if it was used as a major AST method, compared with Ogawa.

Evaluation of major membrane protein-II as a tool for serodiagnosis of leprosy.

As serodiagnosis is the easiest way of diagnosing a disease, the utility of Mycobacterium leprae-derived major membrane protein-II (MMP-II), one of the immuno-dominant antigens, in the serodiagnosis of leprosy was examined. The percent positivity by an enzyme-linked immunosorbent assay for anti-MMP-II antibody was 82.4% for multi-bacillary leprosy, and the specificity of the test was 90.1%. For pauci-bacillary leprosy where cell-mediated immunity predominates, 39.0% showed positive results. These percentage values were significantly higher than these values obtained for existing phenolic glycolipid-I based methods, suggesting that MMP-II antibody detection would facilitate the diagnosis of leprosy.

Synthesis of new sugar derivatives from Stachys sieboldi Miq and antibacterial evaluation against Mycobacterium tuberculosis, Mycobacterium avium, and Staphylococcus aureus.

A series of sugar derivatives (7-14) were synthesized from stachyose, a sugar compound of Stachys sieboldi Miq, and evaluated for antibacterial activity against Mycobacterium tuberculosis, Mycobacterium avium, and Staphylococcus aureus, and their structure-activity relationships were studied. The results showed that the compound OCT359 (allyl O-(2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl)-(1-->6)-O-(2,3,4-tri-O-acetyl-alpha-D-galactopyranosyl)-(1-->6)-O-2,3,4-tri-O-acetyl-beta-D-glucopyranoside) (12) exhibited in vitro antibacterial activity. The allyl group at C-1 and the acetoxy groups of the manninotrioside were requisite for the antibacterial activity.

[BACTEC MGIT 960 system for drug susceptibility testing of Mycobacterium tuberculosis: a study using external quality assessment strains].

OBJECTIVE: To evaluate the performance of the BACTEC MGIT 960 system for drug susceptibility testing (MGIT AST) of Mycobacterium tuberculosis to isoniazid, rifampin, streptomycin and ethambutol. DESIGN: Fifty external quality assessment strains of M. tuberculosis provided by the Coordinating Centers of WHO/ IUATLD were tested by BACTEC MGIT 960 system, and the results were compared with the referee results of the WHO/IUATLD Supranational Reference Laboratory Network (SRLN). RESULTS AND CONCLUSION: Overall concordance rates of the results obtained by MGIT AST and the referee results of the SRLN were 97.3% for four first-line drugs. Agreement rates were particularly high for isoniazid, rifampin, and streptomycin (agreement rate of over 97%), but somewhat lower for ethambutol, which relates to a lower sensitivity of MGIT AST. Turnaround times from inoculation to drug susceptibility results ranged from 6 to 13 days for the MGIT AST system with a median time of 7 days; this contrasted with three weeks for the proportion method using Middlebrook 7H10 agar, indicating that MGIT AST system has the potential to consistently meet with the turnaround time guidelines suggested by the Centers for Disease Control and Prevention of the United States. These results demonstrate that the fully automated BACTEC MGIT 960 AST system is useful for the rapid diagnosis of drug resistant tuberculosis.

Simple fibroblast-based assay to test the pyrazinamide susceptibility of Mycobacterium tuberculosis.

A simple fibroblast-based assay (SFA) was found to be efficient in evaluating the susceptibilities of clinical isolates of Mycobacterium tuberculosis to pyrazinamide (PZA). Forty-five clinical isolates were examined. The MICs of PZA for susceptible strains in an SFA were between 3.13 and 12.5 microg/ml, and the MICs of PZA for resistant strains were more than 100 microg/ml.

Mutations including IS6110 insertion in the gene encoding the MPB64 protein of Capilia TB-negative Mycobacterium tuberculosis isolates.

A simple immunochromatographic assay, Capilia TB, using anti-MPB64 monoclonal antibodies, is a kit for discriminating between the Mycobacterium tuberculosis complex and mycobacteria other than tubercle bacilli. The sensitivity of the kit was estimated to be 99.2% (381 of 384 samples). The sequencing analysis revealed that all of the Capilia TB-negative isolates had mutations within the mpb64 gene, leading to the production of an incomplete protein as a result of a deletion of the C-terminal region of the protein.

[Standardization of laboratory tests for tuberculosis and their proficiency testing].

Explanations of proper collection procedures are imperative for accurate laboratory analysis. The quality of specimens collected and the proper transport of those specimens to the laboratory are critical to successful isolation of etiological agents. Most mycobacteria grow at a relatively slow rate. Therefore, the acid-fast smear plays an important role in the early diagnosis of mycobacterial infection. There are several methods of determining the acid-fast nature of an organism. In the carbolfuchsin procedures (Ziehl-Neelsen, Kinyoun), acid-fast organisms appear red, and in the fluorochrome procedures (auramine O, auramine-rhodamine, acridine orange), the acid-fast organisms fluoresce yellow to orange. Fluorochrome-stained slides may be directly restained with any of the carbolfuchsin staining procedures. This may be done to confirm a positive fluorochrome slide and to study organism morphology. In the last 10 years, there were many advances in the culture examinations of mycobacteria. The newly developed Mycobacteria Growth Indicator Tube (MGIT), BacT/Alert, ESP Myco, MB Redox and KRD "Nichi B", biphasic Septi-Chek AFB and Myco-Acid, and radiometric BACTEC 460TB systems based on liquid media, proved to be significantly better than the egg-based solid media for the isolation of mycobacteria from clinical specimens. In addition to liquid-based medium, agar (Middlebrook 7H10 or 7H11)- or egg (Ogawa or Löwenstein-Jensen)-based media should be used in the primary isolation of mycobacteria. To identify mycobacteria, conventional biochemical tests are traditionally used. Key test can be used to identify species, or further preliminary grouping may be used. Other approaches to identifying some species of mycobacteria are available. They include niacin accumulation, p-nitrobenzoic acid and p-nitro-alpha-acetylamino-beta-hydroxypropiophenone tests for discrimination of the Mycobacterium tuberculosis complex from mycobacteria other than M. tuberculosis (MOTT); DNA probe methods for identification or confirmation of the M. tuberculosis complex, M. avium complex, M. kansasii, and M. gordonae; DNA-DNA hybridization method for identification of 22 Mycobacterium species; and gas-liquid chromatography or high performance liquid chromatography analyses for recognizing the patterns of the mycobacterial cell wall fatty acids or mycolic acids. The advantages of the last four methods are that they are capable of providing definitive identification within 2 to 5 h after adequate growth. Capilia TB is the newly developed immunochromatographic assay for rapid discrimination between the M. tuberculosis complex and MOTT bacilli. The kit can be easily used for rapid identification of the M. tuberculosis complex in combination with the culture systems based on liquid media. In addition, Capilia TB could correctly detect the M. tuberculosis complex from mixed cultures with the M. tuberculosis complex and MOTT bacilli. The WHO/IUATLD supranational reference laboratory (SRL) network was created in 1994, to ascertain the accuracy of the susceptibility test methods used in different laboratories across the world, and to allow comparability of the surveillance data gathered in countries participating in the Global Project on Anti-tuberculosis Drug Resistance Surveillance. Today, the network has evolved and 23 SRLs actively participate. Results of five rounds of proficiency testing in the SRL network suggest that performance of the network has improved substantially through the years. This progress has been particularly evident for streptomycin and ethambutol sensitivity, which was very low in the first rounds of proficiency testing. In 1998 sensitivity for these two drugs was higher than 95%. For isoniazid and rifampin, sensitivity has been consistently high since the beginning of the Global Project. This indeed reflects the enhanced efforts made by the SRLs to improve their individual performance.

Heterogeneity of RNA polymerase gene (rpoB) sequences of Mycobacterium gordonae clinical isolates identified with a DNA probe kit and by conventional methods.

In a previous study, we have evaluated genetic identification by using the rpoB gene, which was recently introduced by Kim et al. (J. Clin. Microbiol. 39:2102-2109, 2001; J. Clin. Microbiol. 37:1714-1720, 1999). In this process, we examined the rpoB gene heterogeneity of clinical isolates identified as Mycobacterium gordonae with the conventional biological and biochemical tests and/or a commercially available DNA probe kit. Sequencing of the rpoB gene of 34 clinical isolates revealed that M. gordonae clinical isolates were classified into four major clusters (A, B, C, and D). Interestingly, organisms belonging to cluster D (15 isolates) did not hybridize with M. gordonae ATCC 14470 and specifically possessed urease activity. Therefore, it could be considered to be a novel mycobacterium. The identification of M. gordonae is known to have ambiguous results sometimes. On the other hand, identification of clinical isolates seems to be inconvenient and unsuitable because of a more than 99% 16S rRNA gene similarity value between clusters. These findings suggest that the existence of M. gordonae-like mycobacteria that share similar biochemical and biological characteristics with the 16S rRNA gene of an M. gordonae type strain but less similarity at the genomic DNA level may have complicated the identification of M. gordonae in many laboratories. Furthermore, compared with hsp65 PCR restriction analysis (PRA), rpoB PRA would have the advantage of producing no ambiguous results because of the intracluster homogeneity of the rpoB gene. In this case, rpoB would provide clearer results than hsp65, even if PRA analysis was used. We demonstrated that these M. gordonae-like mycobacteria were easily distinguished by PRA of the rpoB sequence. Additionally, the significance of this M. gordonae-like cluster may help to establish the comparison between the M. gordonae isolates from a clinical specimen and an infectious process in a given patient and to determine the true incidence of infection with this microorganism.

Simple fibroblast-based assay for screening of new antimicrobial drugs against Mycobacterium tuberculosis.

In this study, we propose a simple and reproducible host-cell-based assay for the screening of antimycobacterial drugs that is suitable for drug discovery. The method evaluates both antimycobacterial activity of the drugs and their cytotoxicity to host cells. The basis of this simple fibroblast-based assay (SFA) is that cells of human lung fibroblast cell line MRC-5, which are highly sensitive to mycobacterial cytotoxicity, are killed by virulent Mycobacterium tuberculosis strain H(37)Rv bacilli in response to the viability of bacilli. Clinically used antimycobacterial drugs inhibited the mycobacterial cytotoxicity to MRC-5 cells in a dose-dependent manner. MICs of isoniazid, streptomycin, rifampin, and ethambutol determined by this SFA (0.428, 1.816, 0.013, and 3.465 microg/ml, respectively) were within 1 log of MICs determined by the broth dilution test (BDT) using Middlebrook 7H9 medium. The MIC of pyrazinamide, which exhibits bactericidal activity only at a high dose by BDT (1,231 microg/ml at pH 6.6 and 492 microg/ml at pH 5.8), was 3.847 microg/ml in the modified method of SFA. On the other hand, sodium azide, a toxic agent for both mammalian cells and bacteria, exhibited cytotoxicity to fibroblasts at a dose lower than that required to inhibit mycobacterial growth. Thus, this fibroblast-based method enabled us to evaluate both antibacterial activity of drugs and their cytotoxicity to human cells within a short period of time.

rpoB genotypes of Mycobacterium tuberculosis Beijing family isolates from East Asian countries.

The 81-bp region of the rpoB gene in 66 Rif(r) Mycobacterium tuberculosis isolates from China, Japan, Korea, and Taiwan was analyzed. Twelve single-nucleotide substitutions in the rpoB gene were detected. The most prevalent mutations were at Ser-531 (52%), Asp-516 (17%), and His-526 (11%). Mutations were not found in seven (11%) of the isolates. Higher mutation rates in 50 Beijing family isolates were found than in other isolates for mutations at Asp-516 (18 and 12.5%, respectively) and His-526 (12 and 6.3%, respectively). The different rates of mutation may reflect the choice of rifamycin analogs.

Evaluation of BACTEC MGIT 960 PZA medium for susceptibility testing of Mycobacterium tuberculosis to pyrazinamide (PZA): compared with the results of pyrazinamidase assay and Kyokuto PZA test.

The fully automated BACTEC MGIT 960 PZA medium for susceptibility testing of Mycobacterium tuberculosis to pyrazinamide (PZA) was evaluated using 101 Mycobacterium tuberculosis clinical isolates. The results obtained with the system were compared with those of the pyrazinamidase (PZase) assay and the Kyokuto PZA test based on a broth culture, which is commercially available in Japan. The overall concordance rate was 90.1% (91/101) among the three methods in the initial test. The concordance rates between the BACTEC MGIT 960 PZA medium vs the PZase assay, the BACTEC MGIT 960 PZA medium vs the Kyokuto PZA test, and the PZase assay vs the Kyokuto PZA test were 93.1, 91.1, and 96.0%, respectively. On the repeat test of the 10 strains with discrepant results among the three methods, the concordance rates reached over 97% between each of the two systems. The results of the repeat test were confirmed by MIC testing and sequencing analysis of the pncA gene encoding PZase of M. tuberculosis. The mean turnaround times from incubation for PZA susceptibility testing were almost similar for the two methods based on liquid media, the BACTEC MGIT 960 PZA medium and the Kyokuto PZA test (7.7 and 7.4 days, respectively). These results indicate that both methods based on liquid media, the fully automated BACTEC MGIT 960 PZA medium and the Kyokuto PZA test for susceptibility testing to PZA, are useful for rapid diagnosis of PZA resistant tuberculosis.