In vitro propagation of the Garden Heliotrope, Valeriana officinalis L.: Influence of pre-chilling and light on seed germination.

Valeriana officinalis is an important medicinal herb commonly found in Kashmir valley. This study forms an important preliminary step for in-vitro micro propagation of V. officinalis from breaking the seed dormancy, inducing rapid seed germination and its subsequent micro propagation. We investigated the influence of pretreatment of V. officinalis seeds with reduced temperature and light on seed germination and in-vitro propagation. Culture of explants from cultivated seeds have demonstrated its potential for in vitro propagation and plantlet regeneration. Individual as well as combinations of treatments such as temperature and light availability influenced the germination of seeds variedly. Unchilled seeds of V. officinalis were given dip in GA3 (200 ppm) for 24, 48 and 120 h. Seeds treated with GA3 for 24 h and kept in darkness showed the best results, i.e. 48%. Seeds pretreated with GA3 for 120 h and incubated in dark showed 40% germination. Pre-chilling up to 72 h and kept in light showed maximum germination of 60% followed by 40% kept in darkness. Pre-chilling for 48 h resulted in 40 and 25% seed germination in light and darkness, respectively. GA3 pre-treatment for 72 h and 24 h pre chilling were most effective in inducing seed germination. Maximum shoot response was obtained on MS enriched with BAP (1mg/L) + IAA (0.1mg/L) combinations using shoot tips as explants. Multiple shoot regeneration from shoot apices was recorded on BAP (1mg/L) and BAP (1mg/L) + IAA (0.1mg/L).

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.

jefA (Rv2459), a drug efflux gene in Mycobacterium tuberculosis confers resistance to isoniazid & ethambutol.

Background & objectives: Drug efflux pumps have been contributing factor(s) in the development of multidrug resistance in various clinically relevant bacteria. During efflux pump gene expression studies on mycobacteria, we have found a previously uncharacterized open reading frame (ORF) Rv2459 to be overexpressed in drug stressed conditions. The objective of the present study was to investigate the role of this ORF as a drug efflux pump, which might add new information in our understanding about the alternative mechanisms of drug resistance in mycobacteria. Methods: The open reading frame Rv2459 of Mycobacterium tuberculosis encoding a probable drug efflux protein has been cloned using pSD5 E.coli-Mycobacterium shuttle vector and overexpressed in M. tuberculosis H37Rv. This ORF was named as jefA. Overexpression of this gene in clones has been verified by real-time reverse transcription PCR. Minimum inhibitory concentrations (MICs) of recombinant as well as non-recombinant clones were determined by resazurin microtitre assay plate method (REMA) with and without efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and verapamil. Results: In recombinant strains of M. tuberculosis, the overexpression of this gene led to an increase in MIC of anti-tubercular drugs isoniazid and ethambutol when tested by REMA. In the presence of CCCP and verapamil, the recombinant strains showed decrease in MIC for these drugs. Bioinformatic analysis has shown a close relation of JefA protein with drug efflux pumps of other clinically relevant bacteria. In homology derived structure prepared from nearest available model, it was observed that amino acids forming TMH 1, 8 and 11 participated in ethambutol specificity and those forming TMH 2, 7 and 10 participated in isoniazid specificity in JefA. Interpretation & conclusion: The increased transcription of jefA leads to increased resistance to ethambutol and isoniazid in M. tuberculosis via efflux pump like mechanism and contributes in the development of resistance to these drugs. JefA amino acid sequence is well conserved among clinically important bacterial genera, which further provides evidence of being a potent drug efflux pump. The involvement in drug resistance and very little homology with any of the human proteins makes JefA important to be included in the list of potential drug targets.

Anti-tuberculosis activity of selected medicinal plants against multi-drug resistant Mycobacterium tuberculosis isolates.

Background & objectives: Emergence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) strains of Mycobacterium tuberculosis has further complicated the problem of tuberculosis (TB) control. Medicinal plants offer a hope for developing alternate medicines for the treatment of TB. The present study was done to evaluate in vitro anti-tubercular activity of five medicinal plants viz., Acalypha indica, Adhatoda vasica, Allium cepa, Allium sativum and Aloe vera. Methods: Aqueous extracts of leaves of A. indica, A. vasica, bulbs of A. cepa, cloves of A. sativum and pure gel of A. vera leaves, were tested in vitro for their activity against two MDR isolates (DKU-156 and JAL-1236), reference susceptible strain M. tuberculosis H37Rv as well as rapid grower mycobacterial pathogen M. fortuitum (TMC-1529) using Lowenstein Jensen (L-J) medium and colorimetric BacT/ALERT 3D system. Activity in L-J medium was evaluated by percentage inhibition which was calculated by mean reduction in number of colonies on extract containing as compared to extract free controls. Results: Extracts of all the five plants A. indica, A. vasica, A. cepa, A. sativum and A. vera exhibited anti-tuberculosis activity in L-J medium, the proportion of inhibition of these plants extract in respect mentioned above is 95, 32, 37, 72, 32 per cent, respectively for MDR isolate DKU-156 and 68, 86, 79, 72, 85 per cent, respectively for another MDR isolate JAL-1236, while for sensitive M. tuberculosis H37Rv, inhibition was found to be 68, 70, 35, 63 and 41 per cent, at 4 per cent v/v concentration in L-J medium. There was no inhibition against rapid grower M. fortuitum (TMC-1529). In BacT/ALERT also, extracts of these plants showed significant inhibition against M. tuberculosis. Interpretation & conclusions: Our findings showed that all these plants exhibited activity against MDR isolates of M. tuberculosis. While the anti-TB activity of A. vera, A. vasica and A. sativum against MDR isolates confirm earlier results, activity of the extracts of A. indica and A. cepa is reported for the first time. Further studies aimed at isolation and identification of active substances from the extracts which exhibited promising activities, need to be carried out.

In vitro effect of fl uoroquinolones against Mycobacterium tuberculosis isolates from Agra & Kanpur region of north India.

Background & objectives: Fluoroquinolones (FQs) are important drugs used for treatment of drug resistant tuberculosis and are also now being considered as fi rst line drugs to shorten the duration of treatment of tuberculosis (TB). In order to fi nd out useful FQs for treatment of tuberculosis, the comparative effi cacy of fi ve FQs, namely, ofl oxacin (OFL), ciprofl oxacin (CIP), sparfl oxacin (SPX), gatifl oxacin (GAT) and levofl oxacin (LEVX) was studied against Mycobacterium tuberculosis (MTB) isolates obtained from both treated and untreated patients from Agra and Kanpur regions of north India. Methods: A total of 162 MTB isolates [including 110 MTB isolates obtained from untreated patients (Cat-I) and 52 isolates from treated patients (Cat-II)] were tested for their susceptibilities to FQs using standard minimum inhibitory concentration (MIC) method on Löwenstein-Jensen medium. Results: Keeping in view the therapeutically achievable drug levels, it was found that in Cat-I 97.2 per cent (107/110) isolates were sensitive to GAT, 89 per cent (98/110) to LEVX at 1 μg/ml whereas 92.7 per cent (102/110) isolates were inhibited by OFL at 2 μg/ml and 73.6 per cent (81/110) to SPX at 0.5 μg/ml. Only 63.6 per cent (70/110) isolates were found to be sensitive to CIP at 2 μg/ml which increased to 89 per cent (98/110) at 4 μg/ml (higher than achievable peak serum level). On the other hand, among 52 isolates for Cat-II, 37 (71.2%) were found to be sensitive to GAT and 33 (63.5%) to LEVX at 1 μg/ml concentration, 28 (53.8%) to SPX at 0.5 μg/ml whereas 33 (63.5%) and 24 (46.2%) isolates were found to be sensitive to OFL and CIP at 2 μg/ml, respectively. Interpretation & conclusions: It appears that GAT has higher activity against MTB isolates followed by OFL, LEVX and SPX whereas CIP showed the lowest activity. GAT was also found to be the most effective FQ against multi-drug resistant (MDR) isolates both from Cat-I and Cat-II patients. Thus, except CIP, other FQs showed potential to be included in the treatment regimens of tuberculosis including MDR-TB.

Identification of environmental mycobacteria isolated from Agra, north India by conventional & molecular approaches.

Background & objectives: Several environmental mycobacteria have been shown to be important human pathogens linked to immunomodulation especially in relation to effect on vaccination. Hence identification of mycobacteria to the species level is not only relevant to patient management but also to understand epidemiology of mycobacterial diseases and effect on vaccination. We undertook this study to assess the usefulness of various conventional and molecular methods in identification of environmental mycobacterial species from Agra, north India. Methods: One hundred nineteen isolates of environmental mycobacteria were grown from 291 (116 soil and 175 water) samples. These isolates were identified by standard biochemical tests, and a simple, rapid and cost-effective in-house developed gene amplification restriction analysis targeting 16S-23S rRNA spacer and flanking region and 16S rRNA sequencing. Results: Biochemical tests could clearly identify only 68.1 per cent (81/119) of isolates to species level. An in-house developed gene amplification - restriction analysis method could confirm the identity of 102 of 119 (85.7%) isolates and the remaining 17 isolates (14.3%) were confirmed by 16S rRNA sequencing also. These 119 environmental mycobacterial isolates, included several potentially pathogenic species such as M. fortuitum, M. chelonae, M. avium, M. marinum, M. manitobense, M. kansasii and others belonged to nonpathogenic species, M. terrae, M. smegmatis and M. flavescens. M. chelonae was isolated from water samples only whereas M. fortuitum was isolated from both water as well as soil samples. Interpretation & conclusion: The in-house developed gene amplification restriction analysis method though failed to accurately identify 14.3 per cent of isolates, facilitated rapid differentiation of most of environmental mycobacteria including potential pathogens from this area and thus would have diagnostic potential in cases with NTM infections. This combination strategy using PCR-RFLP and 16S rRNA sequencing may be useful for characterization of mycobacteria from similar environmental settings from other parts of world.

Serological diagnosis of tuberculosis.

OBJECTIVE: To evaluate the efficacy of ELISA for the detection of IgG antibodies against antigen 85 complex (Ag 85 complex) of Mycobacterium tuberculosis. METHODS: Children of either sex, 0-18 years of age, attending the outpatient department and admitted in the casualty and wards of the Department of Pediatrics, S.N. Medical College, Agra, were included in present study. The study was carried out on children with pulmonary and CNS tuberculosis along with matching controls (83 cases and 32 controls). Informed consents of their parents or guardians were taken. They were subjected to clinical examination, relevant laboratory investigations, tuberculin test and chest radiograph. Relevant body fluids were subjected to bacteriological tests; ELISA was applied to serum samples for detection of IgG antibodies against antigen 85 complex (Ag85). The result of ELISA was compared with bacteriological tests [Ziehl Neelson (ZN) staining for acid-fast bacilli, culture on Lowenstein Jensen (LJ) medium and culture on BacT/Alert 3D system]. RESULTS: ELISA tests showed a significantly higher sensitivity (59.1%) as compared with LJ medium culture method (19.3%), BacT/Alert 3D system (24.1%) and ZN staining (16.9%) in all patients (p<0.001). Specificity of ELISA test was 71.9%. CONCLUSION: In view of the convenience, low cost and good sensitivity, ELISA tests have a promising future in the diagnosis of childhood tuberculosis.

Isolation of Mycobacterium bovis & M. tuberculosis from cattle of some farms in north India--possible relevance in human health.

BACKGROUND & OBJECTIVE: Infection due to Mycobacterium bovis typically occurs in cattle and animals transmit infection to each other. The choice of appropriate clinical specimen is very important for isolation of M. bovis and M. tuberculosis from cattle. The present study reports the isolation of M. tuberculosis and M. bovis from different types of specimens from cattle suspected to be suffering from tuberculosis in certain organized cattle farms in north India. METHODS: A total of 768 specimens (heparinized or EDTA containing blood (162), fine needle aspirates from prescapular lymph gland (PSLG,160), milk (154), pharyngeal swab (PhS, 98), rectal pinch (RP, 97) and faecal sample (97) from 161 cattle of organized cattle farms in north India suspected to be suffering from tuberculosis were analyzed. After decontamination by modified Petroff's method isolation of M.tuberculosis complex was done on Lowenstein-Jensen medium (with and without pyruvate). The culture isolates were identified as M. tuberculosis and M. bovis on the basis of biochemical tests. RESULTS: A total of 54 M. tuberculosis complex isolates were obtained, of them 40 were identified as M.bovis and 14 as M. tuberculosis. M.bovis were isolated from 12 of 38 animals in group A (Tuberculin +ve with signs of tuberculosis), 7 of 37 animals in group B (Tuberculin +ve and apparently healthy), 9 of 21 group C animals in (Tuberculin -ve with clinical signs of tuberculosis), 4 of 26 animals in group D (Tuberculin -ve and apparently healthy), 4 of 27 group E animals (having non-mycobacterial infection) and 4 of 12 animals in group F (having clinical signs such as debilitated condition, cough, decreasing milk production, etc). Maximum number of M. bovis (19/40, 47.5%) and M. tuberculosis (5/14, 35.7%) isolates were grown from prescapular lymph gland biopsy (PSLG) followed by blood from which 9/40 (22.5%) M. bovis and 4/14 (28.5%) M. tuberculosis were isolated. M. bovis [6/40(15%)] and M. tuberculosis [4/14(28.5%)] were also isolated from milk. Only 3/40 (7.5%) isolates of M.bovis could be isolated from 97 rectal pinch followed by 98 pharyngeal swab 2/40 (5%) and 97 fecal samples 1/40 (2.5%) while 1/14 (7.1%) M.tuberculosis isolates were obtained from pharyngeal swab. INTERPRETATION & CONCLUSION: Among the samples analyzed, PSLG was found to be most suitable specimen for isolation of M. tuberculosis complex from cattle and is thus of diagnostic importance. M. bovis in milk indicates the need to investigate the transmission to human in such settings. Isolation of M. bovis and/or M. tuberculosis from apparently healthy cattle indicates sub-clinical infection in the herd. Further, isolation of a significant number of M. tuberculosis from cattle suggests possible human-to-cattle transmission which need to be confirmed by prospective studies including tools like DNA fingerprinting.

Molecular typing of Mycobacterium tuberculosis isolates from different parts of India based on IS6110 element polymorphism using RFLP analysis.

BACKGROUND & OBJECTIVE: IS 6110 based typing remains the internationally accepted standard and continues to provide new insights into the epidemiology of Mycobacterium tuberculosis. The aim of the study was to characterize M. tuberculosis isolates obtained from different parts of India based on IS6110 element polymorphism using restriction fragment length polymorphism (RFLP) analysis. METHODS: RFLP was analyzed among 308 isolates of M. tuberculosis deposited in the Mycobacterial Repository Centre, Agra, from different parts of India. DNAs isolated from these strains were restricted with Pvu II, transferred on to nylon membrane and hybridized with a PCR amplified DIG-labeled 245 bp IS6110 probe. RESULTS: Based on the copy number, M. tuberculosis isolates were classified into four groups, (i) lacking IS6110 element; (ii) low copy number (1-2); (iii) intermediate copy number (3-5); and (iv) high copy number (6-19). Copy number higher than 19 however was not observed in any of the isolates studied. At the national level, 56 per cent of the isolates showed high copy number of IS6110, 13 per cent showed intermediate copy number, 20 per cent showed low copy number, whereas 11 per cent isolates lacked IS6110 element. At the regional level, there was not much difference in the RFLP profiles of isolates (IS6110 copy numbers/patterns) from different parts of the country. INTERPRETATION & CONCLUSION: IS6110 DNA based fingerprinting could be a potentially useful tool for investigating the epidemiology of tuberculosis in India.

Rapid identification of mycobacteria by gene amplification restriction analysis technique targeting 16S-23S ribosomal RNA internal transcribed spacer & flanking region.

BACKGROUND & OBJECTIVE: Conventional identification of a clinical isolate of mycobacteria primarily based on culture characteristics and biochemical tests needs several weeks and may remain inconclusive. This study was undertaken to develop a new rapid method to identify the mycobacterial isolates at species level by gene amplification restriction analysis using primers encoding 16S-23S rRNA internal transcribed spacer (ITS) region and flanking parts of the 16S as well as 23S rRNA gene. METHODS: This system is based on the amplification of approximately 1.8 kb fragment encoding 16S-23S rRNA spacer region and flanking parts of the 16S as well as 23S rRNA gene. This assay was applied on 13 reference strains and 480 clinical isolates of mycobacteria to validate the technique. Restriction was carried out with three restriction endonucleases Hha I, Hinf I and Rsa I. RESULTS: Distinct gene amplification restriction analysis patterns were obtained by restriction of amplicons with three distinct restriction endonucleases (Hha I, Hinf I and Rsa I) which could differentiate various mycobacterial species. INTERPRETATION & CONCLUSION: Restriction patterns with the enzymes used in this study could clearly distinguish Mycobacterium tuberculosis complex from other non chromogenic clinically important species M. avium and M. intracellulare. Results indicated this assay to be a simple, rapid and reproducible method to identify clinically relevant mycobacteria.

Applications of real-time PCR technology to mycobacterial research.

Real-time polymerase chain reaction (PCR) is being used worldwide for various research purposes. In this review we discuss the principles of real-time PCR, different methodologies and chemistries available and their applications in mycobacterial research. This technology allows for the direct detection of PCR product during the exponential phase of the reaction. Being a very powerful, accurate, rapid and sensitive method, this technique holds immense promise for detection of mycobacteria, differentiation of mycobacterial species, quantification of mycobacterial load and detection of drug resistance in mycobacterial infection.

Partial purification and cytotoxic effects of Salmonella proteinous moieties on chick embryo fibroblasts.

Salmonella enterica serovars, viz., S. Weltevreden, S. Typhimurium, S. Gallinarum and S. Bareilly were treated with cephotaxime to release of intracellular proteins. The cephotaxime extract (CE) was salt precipitated with ammonium sulphate (45-70%) and dialyzed, and denoted as precipitated dialyzed proteins (PDP). Further, both CE and PDP of Salmonella Weltevreden and PDP of rest of the serovars were subjected to gel filtration using Sephacryl S-200HR. Different fractions along with CE and PDP were studied for their cytotoxicity using chicken embryo fibroblast (CEF). All the CE and PDP exerted cytotoxic effects, characterized by rounding, detachment, shrinkage and clumping of cells with syncytia formation. Also, the fractions eluted in the 2nd and 3rd peaks through Sephacryl S-200HR column invariably had cytotoxic activity. It was concluded that in place of Vero cell line, CEF cells could also be used to test cytotoxicity.

Detection of Mycobacterium leprae DNA from soil samples by PCR targeting RLEP sequences.

Despite near elimination of leprosy as a public health problem, several problems in leprosy still remain. These include early detection, determining efficacy of the treatment and differentiating relapses from re-infection. These aspects have important impact on the patients undergoing treatment and also have a bearing on understanding transmission dynamics in the community. While early diagnosis and management do not need major technological inputs, various reports have suggested that M. leprae is found in the environment and may have a role in continued transmission of disease. In earlier studies from other parts of world the presence of M. leprae DNA in the environment has been investigated both by microbiological and molecular studies. In the present study, an attempt was made to extract M. leprae DNA from soil samples, which were collected from eighteen different locations including 3 from our Institute area and 15 from different villages of Ghatampur area. We optimized a protocol for the extraction of DNA and amplified a fragment of M. leprae using specific primers targeting RLEP sequences. It was found that 33.3% of these soil samples collected from areas inhabited by leprosy cases gave positive result for M. leprae specific DNA. The utility of this method needs to be explored on a larger scale to establish the presence of M.leprae in the environment, and its role in the spread of the disease.

Characterization of mycobacteria isolated from bovines by PRA-targetting hsp 65 gene region.

Bovine tuberculosis caused by the bacterium Mycobacterium bovis is a major infectious disease of animals and has zoonotic importance for humans. Even though the incidence is believed to be very low in India, human tuberculosis caused by M. bovis has been increasingly recognized in many other countries of the world. As differentiation of mycobacterial species take long time, a method for the rapid identification of mycobacteria isolated from bovine samples to the species level was used, which is based on polymerase chain reaction (PCR) of the gene encoding for the 65-kD protein followed by restriction analysis. The method involves restriction enzyme analysis of PCR products obtained with primers common to all mycobacteria and generate M. tuberculosis complex specific pattern. PRA was performed on 33 bovine isolates of which 90.9% (30/33) isolates were identified clearly as M. tuberculosis complex, M. fortuitum, M. phlei and M. smegmatis using restriction enzyme Hae III.

The paradox of pyrazinamide: an update on the molecular mechanisms of pyrazinamide resistance in Mycobacteria.

Pyrazinamide (PZA) is an important front line anti-tuberculosis drug because of its sterilizing activity against semi-dormant tubercle bacilli. In spite of its remarkable role in shortening the treatment duration from 9 months to 6 months when used in combination with Rifampicin and Isoniazid, PZA remains a difficult paradox because of its incompletely understood mode of action and mechanism of resistance. PZA is a nicotinamide analog prodrug which is converted into the active bactericidal form pyrazinoic acid by the bacterial enzyme pyrazinamidase (PZase). PZA does not appear to have a specific cellular target and instead, exerts its bactericidal effect by disrupting the membrane energetics and acidification of cytoplasm. Majority (72-97%) of PZA-resistant isolates of M. tuberculosis exhibit mutations in their pncA gene or upstream area leading to loss of PZase activity. A wide diversity of pncA mutations scattered along the entire length of pncA gene is unique to PZA resistance. However, PZA resistant isolates with normal PZase activity and wild type pncA sequences have also been reported in several studies which indicate that alternate mechanisms of PZA resistance exist. Investigations into these mechanisms would be useful in developing alternative diagnostic/therapeutic measures. This review presents the update of various mechanisms of PZA resistance in different mycobacteria with special emphasis on mode of action of PZA and mechanisms of resistance in Mycobacterium tuberculosis.

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.

Estimation of efflux mediated multi-drug resistance and its correlation with expression levels of two major efflux pumps in mycobacteria.

Multidrug resistance has been posing an increasing problem in the treatment of tuberculosis. Mutations in the genomic targets of drugs have been identified as the major mechanism behind this resistance. However, high degree of resistance in some isolates towards major drugs like rifampicin, isoniazid, ethambutol and streptomycin can not be explained solely on the basis of mutations. Besides this, certain other mechanisms like efflux pumps have also been considered as alternative mechanisms in the drug resistant isolates where there is no mutation and these mechanisms are specially important for drug resistance in non-tuberculous mycobacteria (NTM). In this study, we have estimated efflux pump mediated drug resistance in different mycobacterial species with the help of efflux pump inhibitors. All major anti-tuberculous drugs have been shown to be extruded by efflux pumps and the degree to which these drugs are extruded, vary in different mycobacterial species and isolates. The correlation of this resistance with functional activity of two major efflux pump genes pstB and Rv1258c was also assessed by reverse transcription PCR. Besides the significant role of these pumps observed, other efflux pumps, present in mycobacteria, may also be involved in drug resistance and need to be investigated.