Rapid detection of ethambutol-resistant Mycobacterium tuberculosis in clinical specimens by real-time polymerase chain reaction hybridisation probe method

Background: Early diagnosis of drug resistance (DR) to ethambutol (EMB) in tuberculosis (TB) remains a challenge. Simple and reliable method (s) are needed for rapid detection of DR Mycobacterium tuberculosis (MTB) in clinical specimens. Objectives: The aim of this study was to design fluorescence resonance energy transfer hybridisation probe-based real-time polymerase chain reaction (PCR) method for the early detection of EMB-resistant MTB direct from clinical sputa. Materials and Methods: Primers and probes were designed against 306 codon of embB gene which is commonly associated with EMB resistance. A comparative study was done between Lowenstein–Jenson (L–J) proportion and hybridisation probe-based real-time PCR method for susceptibility testing. DNA sequencing was used in nine representative isolates to validate the efficiency of real-time PCR method to detect emb306 mutation of MTB. Results: A total of 52 clinical sputum samples and corresponding culture isolates (from category II pulmonary TB cases) were included in this study. Out of 52 MTB isolates, 32 and 20 were resistant and susceptible to EMB, respectively, as determined by L–J proportion method. Real-time PCR showed 95% specificity, 75% sensitivity and 82.69% accuracy when compared with L–J proportion method. A 100% of concordance was observed by validating the real-time PCR results with DNA sequencing. Conclusions: Our real-time PCR hybridisation probe method promises for rapid detection of EMB-resistant MTB directly from clinical specimens. However, future studies and modifications of method by incorporating other potential loci along with targeted mutation (emb306) are still required to increase the sensitivity of method.

Analysis of expression profile of mce operon genes (mce1, mce2, mce3 operon) in different Mycobacterium tuberculosis isolates at different growth phases.

Background & objectives: Mycobacterium tuberculosis (M. tuberculosis) has four homologous mammalian cell entry (mce) operons (mce1-4) that encode exported proteins and have a possible role in the virulence mechanism of this pathogen. The expression of mce operon is considered to be complex and not completely understood. Although expression of mce operon at different in vitro growth phases has been studied earlier, its expression in different M. tuberculosis isolates under different growth phases is not yet studied. The present preliminary study was conducted on a limited number of isolates to know the trend of expression pattern of mce operon genes in different M. tuberculosis isolates under different growth stages. Methods: In this study, we monitored the transcriptional profile of selected mce operon genes (mce1A, mce1D, mce2A, mce2D, mce3A, mce3C) in different M.tuberculosis isolates (MDR1, MDR2, and sensitive isolate) at early exponential and stationary phases using real-time quantitative PCR. Results: The expression ratio of all selected mce operon genes in all M. tuberculosis isolates was reduced at the initial phase and increased substantially at a later phase of growth. Higher expression of mce1 operon genes was found in all M. tuberculosis isolates as compared to other mce operon genes (mce2 and mce3 operons) at stationary growth phase. Interpretation & conclusions: The higher expression of mce operon genes at stationary phase (as compared to early exponential phase) suggested growth phase dependent expression of mce operon genes. This indicated that the mce operon genes might have a role in M. tuberculosis survival and adaptation on the onset of adverse condition like stationary phase. Identification of differentially expressed genes will add to our understanding of the bacilli involved in adaptation to different growth conditions.

A pilot study to determine genetic polymorphism in Mycobacterium tuberculosis isolates in Central India.

This study was carried out to identify predominant spoligotypes responsible for transmission and prevalence of tuberculosis in central India since there is no data available about the genetic biodiversity of Mycobacterium tuberculosis isolates from patients with tuberculosis in this region. 35 strains of Mycobacterium tuberculosis were subjected to spoligotyping according to the standard protocol. A total of 25 strains out of the 35 (71.42%) could be grouped in to 6 clusters. The largest cluster comprised 8 isolates. Unique (Non-clustered) spoligotypes were seen in 10 isolates, Nine strains did not match the data base (Spol DB-4 data base). The results indicate that there may be a number of orphan strains unique to this geographical area. Further studies on a larger sample size derived from this area would help us delineate the epidemiology of Mycobacterium tuberculosis infection in this area.

Aflatoxin contamination in stored rice variety PAU 201 collected from Punjab, India.

Background & objectives: The present study was carried out on stored rice variety PAU 201 in Punjab that was not permitted for milling and public distribution due to the presence of damaged grains at levels exceeding the regulatory limits of 4.75 per cent. The aim of the study was to determine fungal and aflatoxin contamination in the rice samples to assess hazard from the presence of damaged grains. Presence of iron in discoloured rice grains was also assessed. Methods: Stored samples of paddy of PAU 201 rice variety were collected from six districts of Punjab, milled and analysed for presence of fungal and aflatoxin contamination. Scanning electron microscopy (SEM), energy dispersive X-ray (EDX) analysis and Prussian blue staining was used to determine fungal spores and presence of iron, respectively. Results: Aflatoxin analysis of rice samples indicated that none exceeded the Food Safety and Standards (Contaminants, Toxins and Residues) Regulations, 2011 tolerance limit of 30 μg/kg and majority of the samples had levels <15 μg/kg. The proportion of damaged grains exceeding the limit of 5 per cent was observed in 85.7 per cent of the samples. SEM and Prussian blue staining and EDX analysis of black tipped and pin point damaged rice grains did not show presence of fungal structures and presence of iron. Interpretation & conclusions: The results of the study indicated that the stored rice samples did not pose any health concern with respect to aflatoxin contamination as per the criteria laid down by the Food Safety and Standards Authority of India.

Mycobacterium indicus pranii as stand-alone or adjunct immunotherapeutic in treatment of experimental animal tuberculosis.

Background & objectives: Mycobacterium w (M.w) is a saprophytic cultivable mycobacterium and shares several antigens with M. tuberculosis. It has shown good immunomodulation in leprosy patients. Hence in the present study, the efficacy of M.w immunotherapy, alone or in combination with multi drug chemotherapeutic regimens was investigated against drug sensitive M. tuberculosis H37Rv and three clinical isolates with variable degree of drug resistance in mice. Methods: BALB/c mice were infected with M. tuberculosis H37Rv (susceptible to all first and second line drugs) and three clinical isolates taken from the epository of the Institute. The dose of 200 bacilli was used for infection via respiratory route in an aerosol chamber. Chemotherapy (5 days/wk) was given one month after infection and the vaccinated group was given a dose of 1×107 bacilli by subcutaneous route. Bacterial load was measured at 4 and 6 wk after initiation of chemotherapy. Results: M.w when given along with chemotherapy (4 and 6 wk) led to a greater reduction in the bacterial load in lungs and other organs of TB infected animals compared to. However, the reduction was significantly (P<0.05) more in terms of colony forming units (cfu) in both organs (lungs and spleen). Conclusion: M.w (as immunomodulator) has beneficial therapeutic effect as an adjunct to chemotherapy.

ICMR-DBT Guidelines for Evaluation of Probiotics in Food.

There has been an increased influx of probiotic products in the Indian market during the last decade. However, there has been no systematic approach for evaluation of probiotics in food to ensure their safety and efficacy. An initiative was, therefore, taken by the Indian Council of Medical Research (ICMR) along with the Department of Biotechnology (DBT) to formulate guidelines for regulation of probiotic products in the country. These guidelines define a set of parameters required for a product/strain to be termed as ‘probiotic’. These include identification of the strain, in vitro screening for probiotic characteristics, animal studies to establish safety and in vivo animal and human studies to establish efficacy. The guidelines also include requirements for labeling of the probiotic products with strain specification, viable numbers at the end of shelf life, storage conditions, etc., which would be helpful to the consumers to safeguard their own interest.

Molecular typing of Mycobacterium leprae strains from northern India using short tandem repeats.

Background & objectives: Due to the inability to cultivate Mycobacterium leprae in vitro and most cases being paucibacillary, it has been difficult to apply classical genotyping methods to this organism. The objective of this study was therefore, to analyze the diversity among M. leprae strains from Uttar Pradesh, north India, by targeting ten short tandem repeats (STRs) as molecular markers. Methods: Ninety specimens including 20 biopsies and 70 slit scrappings were collected in TE buffer from leprosy patients, who attended the OPD of National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Tajganj, Agra, and from villages of Model Rural Health Research Unit (MRHRU) at Ghatampur, Kanpur, Uttar Pradesh. DNA was extracted from these specimens and ten STRs loci were amplified by using published and in-house designed primers. The copy numbers were determined by electrophoretic mobility as well as sequence analysis. Phylogenetic analysis was done on variable number of tandem repeats (VNTRs) data sets using start software. Results: Diversity was observed in the cross-sectional survey of isolates obtained from 90 patients. Allelic index for different loci was found to vary from 0.7 to 0.8 except for rpoT for which allelic index was 0.186. Similarity in fingerprinting profiles observed in specimens from the cases from same house or nearby locations indicated a possible common source of infection. Such analysis was also found to be useful in discriminating the relapse from possible reinfection. Interpretation & conclusions: This study led to identification of STRs eliciting polymorphism in north Indian strains of M. leprae. The data suggest that these STRs can be used to study the sources and transmission chain in leprosy, which could be very important in monitoring of the disease dynamics in high endemic foci.

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.

Mycobacterium avium bacteremia and dual infection with mycobacterium avium and mycobacterium wolinskyi in the gut of an aids patient – First case report.

An AIDS patient was admitted to a tertiary care hospital in central India with fever, weight loss, breathlessness, night sweats, diarrhoea, BMI 14kg/m2, Hemoglobin 8gm% and CD4 counts 120 cells/cumm. His blood culture by BACTEC 460 TB system revealed Mycobacterium avium bacteremia and stool culture grew Mycobacterium avium and mycobacterium wolinskyi.

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.

A simple and rapid method of sample preparation from culture filtrate of M. tuberculosis for two-dimensional gel electrophoresis

Sample preparation for Two-dimensional gel electrophoresis (2DE) is tedious and not sufficient to provide a comparative profile of secreted proteins for various strains of M. tuberculosis. High lipid content in mycobacteria limits the use of common methods as it can hinder the 2DE run. This study highlights the significance of SDS-TCA procedure over common used methods for the preparation of sample from culture filtrate as well as other proteinaceous fluids.

Comparative growth pattern of multi drug resistance versus susceptible isolates of Mycobacterium tuberculosis in mice lungs.

Background & objectives: Rise in prevalence of multi-drug resistance (MDR) in tubercle bacilli is a serious cause of concern. As mutations with two house keeping genes rpoB and katG are associated with resistance to two important anti-tubercular drugs rifampicin and isoniazid respectively, there is a need to understand the growth kinetics of organisms with such mutated genes in experimental animals. This study was undertaken to study the growth kinetics of susceptible as well multi-drug resistance Mycobacterium tuberculosis isolates in mice. Methods: Two MDR (having mutations in rpoB and catG) and two drug susceptible isolates of M. tuberculosis along with H37Rv were grown in mice after aerogenic infection. Results: The MDR isolates grew slowly up to 3 wk though the growth was significantly different from sensitive strains. However, after 3 wk, the growth in sensitive as well MDR strains was similar, suggesting that even the mutations in the MDR strains did not have any impact on the growth kinetics. Interpretation & conclusions: The effect of mutations in other parts of these genes need to be studied. Retention of property of MDR strains to establish infection after aerogenic infection has epidemiological significance in terms of the transmission of MDR tuberculosis.

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.