Effect of efflux pump inhibitors on drug susceptibility of ofloxacin resistant Mycobacterium tuberculosis isolates.

BACKGROUND & OBJECTIVES: In drug resistant, especially multi-drug resistant (MDR) tuberculosis, fluoroquinolones (FQs) are used as second line drugs. However, the incidence of FQ-resistant Mycobacterium tuberculosis is rapidly increasing which may be due to extensive use of FQs in the treatment of various other diseases. The most important known mechanism i.e., gyrA mutation in FQ resistance is not observed in a significant proportion of FQ resistant M. tuberculosis isolates suggesting that the resistance may be because of other mechanisms such as an active drug efflux pump. In this study we evaluated the role of the efflux pumps in quinolone resistance by using various inhibitors such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP), 2,4-dinitrophenol (DNP) and verapamil, in clinical isolates of M. tuberculosis. METHODS: A total of 55 M. tuberculosis clinical isolates [45 ofloxacin (OFL) resistant and 10 ofloxacin sensitive] were tested by Resazurin microtitre assay (REMA) to observe the changes in ofloxacin minimum inhibitory concentration (MIC) levels in presence of efflux inhibitors as compared to control (without efflux inhibitor). RESULTS: The MIC levels of OFL showed 2-8 folds reduction in presence of CCCP (16/45; 35.5%), verapamil (24/45; 53.3%) and DNP (21/45; 46.6%) while in case of isolates identified as OFL sensitive these did not show any effect on ofloxacin MICs. In 11 of 45 (24.5%) isolates change in MIC levels was observed with all the three inhibitors. Overall 30 (66.6%) isolates had reduction in OFL MIC after treatment with these inhibitors. A total of eight isolates were sequenced for gyrA gene, of which, seven (87.5%) showed known mutations. Of the eight sequenced isolates, seven (87.5%) showed 2 to 8 fold change in MIC in presence of efflux inhibitors. INTERPRETATION & CONCLUSIONS: Our findings suggest the involvement of active efflux pumps of both Major Facilitator Super Family (MFS) family (inhibited by CCCP and DNP) and ATP Binding Cassette (ABC) transporters (inhibited by verapamil) in the development of OFL resistance in M. tuberculosis isolates. Epidemiological significance of these findings needs to be determined in prospective studies with appropriate number of samples/isolates.

Simultaneous ethambutol & isoniazid resistance in clinical isolates of Mycobacterium tuberculosis.

BACKGROUND & OBJECTIVE: There is a need to understand the nature of drug resistance patterns and predictors of emergence of drug resistance in Mycobacterium tuberculosis. There could be common factors/mechanisms for resistance to the drugs, isoniazid and ethambutol, both acting on cell wall. The present study was conducted to analyze the antimycobacterial susceptibility patterns of M. tuberculosis isolates to determine the minimum inhibitory concentrations (MICs) of ethambutol for M. tuberculosis; and to find out possible association of ethambutol resistance with isoniazid resistance. METHODS: A total of 380 M. tuberculosis isolates were tested for their susceptibilities to ethambutol at 2, 4, 6 microg/ml, isoniazid at 1 microg/ml and rifampicin at 64 microg/ml using MIC method. RESULTS: 44.21, 24.73 and 14.21 per cent isolates were resistant to ethambutol at concentrations of 2, 4 and 6 microg/ml respectively. At 6 microg/ml of ethambutol concentration, 85.18 per cent ethambutol resistant isolates were resistant to isoniazid also. At the same ethambutol concentration a fraction of 28.75 per cent isoniazid resistant isolates were ethambutol resistant. INTERPRETATION & CONCLUSION: Ethambutol resistance was accompanied with isoniazid resistance in a large percentage of isolates whereas ethambutol resistance was weakly linked with multidrug resistance. On the other hand, association between isoniazid and ethambutol resistance was weak showing one way linkage.

Simultaneous use of two PCR systems targeting IS6110 and MPB64 for confirmation of diagnosis of tuberculous lymphadenitis.

PCR has emerged as a powerful technique for detection of various pathogens including Mycobacterium tuberculosis. In present study, eighty one samples of lymph node biopsies from clinically suspected cases of tuberculous lymphadenitis were examined for AFB, culture on Löwenstein Jensen medium and simultaneous use of two PCRs targeting IS6110 and MPB64. Positivity with M. tuberculosis culture and AFB was 13.6% and 28.4% respectively. All samples culture positive for nontuberculous mycobacteria were negative by both PCR systems. Higher proportion of positive results were observed with PCR targeting IS6110 by which 56 of 81 (69.1%) samples showed positive results as compared to PCR targeting MPB64 by which 39 of 81 (48.2 %) samples showed positive results. When combined, 63 out of 81 (77.8%) samples were detected positive for M. tuberculosis DNA. However, 7/81 (8.6 %) samples remained negative by IS6110 but positive by MPB64 method. Thus our data suggest that the use of one additional PCR (other than IS6110 system) can reduce false negativity of PCR results in the samples harboring zero copy of IS6110 element which is known to exist in Indian population.

Role of embCAB gene mutations in ethambutol resistance in Mycobacterium tuberculosis isolates from India.

In the present study, ethambutol (EMB) resistance-associated mutations were characterised in the embCAB genes of clinical isolates of Mycobacterium tuberculosis (MTB) collected in India. Thirty MTB isolates were tested for their susceptibility to first-line antitubercular drugs using the Löwenstein-Jensen proportion method, and EMB minimum inhibitory concentrations of MTB isolates were determined by the resazurin microtitre assay. Sequencing of various regions of the embCAB genes was performed to identify EMB resistance-associated mutations. Mutations of embB306 were detected in 15 of 23 EMB-resistant MTB isolates. Three EMB-resistant isolates had mutations at codon 270 of the embC gene, two of which also harboured embB306 mutations. No mutation was identified in the embA gene. All seven EMB-sensitive MTB isolates had the wild-type embCAB sequence. In summary, embB306 mutations were associated with EMB resistance, and mutation at codon 270 of the embC gene may contribute to high-level EMB resistance in some MTB isolates.

Determination of ethambutol MICs for Mycobacterium tuberculosis and Mycobacterium avium isolates by resazurin microtitre assay.

OBJECTIVES: To test susceptibilities of Mycobacterium tuberculosis (MTB) isolates to ethambutol by the Löwenstein-Jensen (LJ) proportion method and resazurin microtitre assay (REMA) and to evaluate REMA for the determination of ethambutol MICs for MTB and Mycobacterium avium isolates. METHODS: A total of 50 MTB and 20 M. avium isolates were tested to determine the MICs of ethambutol by REMA and agar dilution method. MTB isolates were also tested by the LJ proportion method. RESULTS: REMA provided ethambutol susceptibility results for all the isolates within 8-9 days. For MTB isolates, REMA showed 96.7% sensitivity, 100.0% specificity and 98.0% accuracy when LJ proportion results were taken as 'gold standard'. For both MTB and M. avium isolates, the MICs determined by REMA were lower than those determined in agar medium, indicating that MIC values determined by REMA are closer to the actual MICs for the isolates. CONCLUSIONS: REMA can be used as a rapid and inexpensive method for mycobacterial drug susceptibility testing against ethambutol. In comparison with the agar method, the MICs determined by REMA can more accurately be correlated with achievable plasma concentrations of antimycobacterial agents.

Comparative evaluation of Löwenstein-Jensen proportion method, BacT/ALERT 3D system, and enzymatic pyrazinamidase assay for pyrazinamide susceptibility testing of Mycobacterium tuberculosis.

Pyrazinamide (PZA) is an important first-line antituberculosis drug because of its sterilizing activity against semidormant tubercle bacilli. In spite of its very high in vivo activity, its in vitro activity is not apparent unless an acidic environment is available, which makes PZA susceptibility testing difficult by conventional methods. The present study was, therefore, planned to assess the performance of the colorimetric BacT/ALERT 3D system and compare the results with those from conventional tests, i.e., the Löwenstein-Jensen (LJ) proportion method (pH 4.85) and Wayne's pyrazinamidase (PZase) assay, using 107 clinical isolates. The concordance among all of these tests was 89.71% after the first round of testing and reached 92.52% after resolution of the discordant results by retesting. Prolonged incubation of the PZase tube for up to 10 days was found to increase the specificity of the PZase test. The concordances between LJ proportion and BacT/ALERT 3D, LJ proportion and the PZase assay, and BacT/ALERT 3D and the PZase assay were found to be 99.06%, 93.46%, and 92.52%, respectively. Using the LJ results as the gold standard, the sensitivities of BacT/ALERT 3D and the PZase assay were 100 and 82.85%, respectively, while the specificity was 98.61% for both of the tests. The difference between the sensitivities of BacT/ALERT 3D and the PZase assay was significant (P = 0.025). The mean turnaround times for the detection of resistant and susceptible results by BacT/ALERT 3D were 8.04 and 11.32 days, respectively. While the major limitations associated with the PZase assay and the LJ proportion method are lower sensitivity in previously treated patients and a longer time requirement, respectively, the BacT/ALERT 3D system was found to be rapid, highly sensitive, and specific.