Drug resistance among extrapulmonary TB patients: Six years experience from a supranational reference laboratory.

BACKGROUND & OBJECTIVES: There is limited information available about the drug resistance patterns in extrapulmonary tuberculosis (EPTB), especially from high burden countries. This may be due to difficulty in obtaining extrapulmonary specimens and limited facilities for drug susceptibility testing. This study was undertaken to review and report the first and second-line anti-TB drug susceptibility patterns in extrapulmonary specimens received at the National Institute for Research in Tuberculosis (NIRT), Chennai, India, between 2005 and 2012. METHODS: Extrapulmonary specimens received from referring hospitals were decontaminated and cultured using standard procedures. Drug susceptibility testing (DST) for Mycobacterium tuberculosis was done by absolute concentration or resistance ratio methods for the first and the second line anti-TB drugs. RESULTS: Between 2005 and 2012, of the 1295 extrapulmonary specimens, 189 grew M. tuberculosis, 37 (19%) cases were multidrug resistant (MDR) while one was extensively drug resistant (XDR). Specimen-wise MDR prevalence was found to be: CSF-10 per cent, urine-6 per cent, fluids and aspirates-27 per cent, pus-23 per cent, lymph nodes-19 per cent. Resistance to isoniazid and ethionamide was found to be high (31 and 38%, respectively). INTERPRETATION & CONCLUSIONS: Drug resistance including MDR-TB was observed in a significant proportion of extrapulmonary specimens referred for DST. Access to culture and DST for extrapulmonary specimens should be expanded. Guidelines for MDR-TB management should have explicit sections on extra-pulmonary tuberculosis and training on laboratory techniques is urgently required.

Revisiting the susceptibility testing of Mycobacterium tuberculosis to ethionamide in solid culture medium.

BACKGROUND & OBJECTIVES: Increase in the isolation of drug resistant phenotypes of Mycobacterium tuberculosis necessitates accuracy in the testing methodology. Critical concentration defining resistance for ethionamide (ETO), needs re-evaluation in accordance with the current scenario. Thus, re-evaluation of conventional minimum inhibitory concentration (MIC) and proportion sensitivity testing (PST) methods for ETO was done to identify the ideal breakpoint concentration defining resistance. METHODS: Isolates of M. tuberculosis (n=235) from new and treated patients were subjected to conventional MIC and PST methods for ETO following standard operating procedures. RESULTS: With breakpoint concentration set at 114 and 156 µg/ml, an increase in specificity was observed whereas sensitivity was high with 80 µg/ml as breakpoint concentration. Errors due to false resistant and susceptible isolates were least at 80 µg/ml concentration. INTERPRETATION & CONCLUSIONS: Performance parameters at 80 µg/ml breakpoint concentration indicated significant association between PST and MIC methods.

Sputum culture conversion with moxifloxacin-containing regimens in the treatment of patients with newly diagnosed sputum-positive pulmonary tuberculosis in South India.

BACKGROUND: Rapid sputum culture conversion at 2 months indicates the sterilizing capacity and potential of regimens to shorten duration of tuberculosis treatment. We compared results of sputum culture conversion by moxifloxacin and control regimens and identified factors affecting sputum culture positivity after 2 months of treatment. METHODS: Human immunodeficiency virus-uninfected adults with newly diagnosed smear-positive pulmonary tuberculosis were randomized to receive a 3- or 4-month moxifloxacin regimen (moxifloxacin [M], isoniazid [H], rifampicin [R], pyrazinamide [Z], ethambutol [E]) or the control regimen (RHZE thrice weekly). Bacteriological assessments were done at 15, 30, 45, and 60 days of treatment. Because all patients in the moxifloxacin groups received 2 months of daily RHZEM, they were grouped together for analysis. Statistical methods included χ(2) test and logistic regression analysis. RESULTS: Sputum culture conversion was analyzed in 780 (616 in the moxifloxacin group and 164 in the control group) of 801 enrolled patients. Ninety-five percent of 590 patients in the moxifloxacin group and 81% of 151 patients in the control group had negative sputum cultures at month 2 (P < .001). The control regimen, age (≥35 years), initial sputum culture grade (2+ or 3+), and male sex were significantly associated with higher odds of positive sputum cultures at 2 months. CONCLUSIONS: A 5-drug daily regimen with moxifloxacin results in significantly higher sputum culture conversion in the first 2 months compared with a thrice-weekly, 4-drug regimen in patients with newly diagnosed sputum-positive pulmonary tuberculosis.

Phage lysin to control the overgrowth of normal flora in processed sputum samples for the rapid and sensitive detection of Mycobacterium tuberculosis by luciferase reporter phage assay.

BACKGROUND: Phage lysin, extracted from three bacteriophages was used in place of antibiotics to control the overgrowth of normal flora in processed sputum samples leading to the sensitive detection of Mycobacterium tuberculosis using diagnostic luciferase reporter phage assay (DLRPA). METHODS: A total of 129 sputum samples were processed by modified Petroff's method. Two Lowenstein Jensen slopes were inoculated from the processed sputum deposit thus obtained. The remaining deposits were transferred to 7 ml of Middlebrook 7H9 complete medium supplemented with phage lysin and incubated at 37°C. DLRPA was done using phAE129 at days 7, 9, 14 and 21. At the end of day 21, the samples were centrifuged and the pellets were inoculated on to 2 more LJ slopes to validate DLRPA results. RESULTS: The sensitivity and specificity of DLRPA in detecting M. tuberculosis from sputum specimens was 90% and 81% respectively compared to conventional LJ culture. The agreement between the methods was 87%. The rate of contamination for DLRPA using phage lysin was 9.3%. CONCLUSION: Phage lysin can be used to decontaminate sputum samples for the detection of M. tuberculosis by DLRPA directly from processed sputum specimens.

Modification of proportion sensitivity testing method for ethionamide.

Standardized methodology for drug susceptibility testing of second line drugs is vital for treatment of multi/extensively drug resistant tuberculosis. Discrepancy between laboratory methods and clinical interpretation is well established for bacteriostatic drugs such as ethionamide. Optimization of the standard proportion sensitivity testing (PST) method for ethionamide was under taken in 235 Mycobacterium tuberculosis isolates from new and previously treated pulmonary tuberculosis patients. An additional higher concentration of 57 µg/ml was evaluated against at the standard 40 µg/ml concentration in PST method. Performance parameters and agreement between the two drug concentrations was higher indicating the efficiency of PST method at its present format at 40 µg/ml and additional higher concentration of 57 µg/ml as an alternative when required.

Antibacterial and Anti-HIV Metabolites from Marine Streptomyces albus MAB56 Isolated from Andaman and Nicobar Islands, India.

Marine-derived actinobacteria have tremendous potential to produce novel metabolites with diverse biological activities. The Andaman coast of India has a lot of microbial diversity, but it is still a relatively unknown ecology for isolating novel actinobacteria with beneficial bioactive compounds. We have isolated 568 actinobacterial strains from mangrove rhizosphere sediments and sponge samples. Crude extracts from 75 distinct strains were produced by agar surface fermentation and extracted using ethyl acetate. In the disc diffusion method, 25 actinobacterial strains showed antimicrobial activity; notably, the strain MAB56 demonstrated promising broad-spectrum activity. Strain MAB56 was identified as Streptomyces albus by cultural, microscopic, and molecular methods. Conditions for bioactive metabolites from MAB56 were optimized and produced in a lab-scale fermenter. Three active metabolites (C1, C2, and C3) that showed promising broad-spectrum antimicrobial activity were isolated through HPLC-based purification. Based on the UV, FT-IR, NMR, and LC-MS analysis, the chemical nature of the active compounds was confirmed as 12-methyltetradecanoic acid (C1), palmitic acid (C2), and tridecanoic acid (C3) with molecular formulae C14H28O2, C16H32O2, and C13H26O2, respectively. Interestingly, palmitic acid (C2) also exhibited anti-HIV activity with an IC50 value of < 1 µg/ml. Our findings reveal that the actinobacteria from the Andaman marine ecosystems are promising for isolating anti-infective metabolites.

In-vivo studies on Transitmycin, a potent Mycobacterium tuberculosis inhibitor.

This study involves the in-vitro and in-vivo anti-TB potency and in-vivo safety of Transitmycin (TR) (PubChem CID:90659753)- identified to be a novel secondary metabolite derived from Streptomyces sp (R2). TR was tested in-vitro against drug resistant TB clinical isolates (n = 49). 94% of DR-TB strains (n = 49) were inhibited by TR at 10µg ml-1. In-vivo safety and efficacy studies showed that 0.005mg kg-1 of TR is toxic to mice, rats and guinea pigs, while 0.001mg kg-1 is safe, infection load did not reduce. TR is a potent DNA intercalator and also targets RecA and methionine aminopeptidases of Mycobacterium. Analogue 47 of TR was designed using in-silico based molecule detoxification approaches and SAR analysis. The multiple targeting nature of the TR brightens the chances of the analogues of TR to be a potent TB therapeutic molecule even though the parental compound is toxic. Analog 47 of TR is proposed to have non-DNA intercalating property and lesser in-vivo toxicity with high functional potency. This study attempts to develop a novel anti-TB molecule from microbial sources. Though the parental compound is toxic, its analogs are designed to be safe through in-silico approaches. However, further laboratory validations on this claim need to be carried out before labelling it as a promising anti-TB molecule.

Microwave-assisted synthesis, molecular docking and antitubercular activity of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives.

Based on bioisosteric similarities with isoniazid, a series of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives has been designed. The target compounds have been synthesized by multicomponent reaction which involves one-pot organic reactions using ethylcyanoacetate, urea/thiourea and arylaldehydes in presence of ethanolic K(2)CO(3). Two methodologies, conventional and microwave-assisted, have been adopted for the synthesis. The later strategy gave high yields in less than 10 min as compared to long hours using the former approach. Molecular docking of the target compounds into the enzyme Mycobacterium tuberculosis enoyl reductase (InhA) revealed important structural information on the plausible binding interactions. Major binding interactions were found to be of dispersion type (residues Tyr158, Ile215, Met103 and Met199) and a hydrogen bond with Tyr158. Binding poses of the all the compounds were energetically favorable and showed good interactions with the active site residues. Few selected compounds were also evaluated for antitubercular activity in vitro against drug-sensitive M. tuberculosis H37Rv strain and clinically isolated S, H, R and E resistant M. tuberculosis by luciferase reporter phage (LRP) assay method. Some compounds displayed promising antimycobacterial activity comparable or less than the standard drugs isoniazid and rifampicin.

Consistency of standard laboratory strain Mycobacterium tuberculosis H 37 Rv with ethionamide susceptibility testing.

Drug susceptibility pattern of standard Mycobacterium tuberculosis strain H 37 Rv showed discrepancy in minimum inhibitory concentration method for ethionamide and consistent results were obtained for the other second line drugs namely, kanamycin and ofloxacin. It is, therefore, necessary to revisit the susceptibility testing method for ethionamide for effective clinical management of patients with drug resistant tuberculosis.

Phage lysin to supplement phagebiotics to decontaminate processed sputum specimens.

The overgrowth of normal flora escaping the action of sputum processing chemicals is the major problem in broth-based tuberculosis (TB) detection systems. The use of phages to control the overgrowth of normal flora in processed sputum samples has already been established. Phage lysin and its supplementation to phagebiotics for the effective control of normal flora in sputum specimens were evaluated. Crude lysin was prepared from phage host mixture using standard procedures. About 120 sputum samples processed with 4% NaOH were collected and used to evaluate the effect of lysin, phagebiotics and phagebiotics supplemented with lysin on the overgrowth of normal flora. The effect of phagebiotics and lysin on the growth and retrieval of Mycobacterium tuberculosis was studied by conventional methods and the luciferase reporter phage (LRP) assay. Lysin alone and phagebiotics supplemented with lysin arrested the growth of normal flora in a significantly greater number of samples than phagebiotics alone. Lysin and phagebiotics did not show any inhibitory activity on M. tuberculosis. The use of antibiotics can be replaced by lysin or phagebiotics supplemented with lysin to control the overgrowth of normal flora in processed sputum samples without hampering the viability of M. tuberculosis.

Novel 1,3,5-triphenyl-2-pyrazolines as anti-infective agents.

Sixteen 1,3,5-triphenyl-2-pyrazolines were synthesized and their anti-infective activities (against Mycobacterium tuberculosis H(37)Rv, six bacterial and four fungal strains) were tested. Only compound with SO(2)CH(3) in the para position of the A-ring was active against the tubercular strain at 100 microg/ml concentration. All compounds showed good anti infective activity against Escherichia coli and poor activity against Staphylococcus aureus. Compounds 4, 12, 13 and 14 exhibited reasonable activity against all the organisms tested (<0.309 microM except against S. aureus. The activity of these compounds correlated with their lipophilic/hydrophilic nature. Compounds 4, 10 and 16 showed very good activity (>88% reduction) against four fungi studied at 2mg/ml. All these compounds possess halogen substitutions. Compound 11 showed very high activity (>90%) against three fungi. Majority of the compounds showed more than 90% inhibition against one or two fungi. Since pyrazolines are reported to inhibit the activity of p-glycoprotein, they may prevent drug resistance developed by microorganism.

Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform.

Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 "phamilies" of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three-encoding tape-measure proteins, lysins, and minor tail proteins-are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education.

Tape measure protein having MT3 motif facilitates phage entry into stationary phase cells of Mycobacterium tuberculosis.

Tape measure protein (TMP) having MT3 motif in mycobacteriophage TM4 genome has been reported to enable the phage infection of Mycobacterium smegmatis during stationary phase. In the present work looking at eight additional mycobacteriophage genomes by in silico analysis, six of them have been found to possess MT3 motif in TMP. The absence of MT3 motif in Che12 and D29 probably makes them incapable of infecting stationary phase cells of Mycobacterium tuberculosis which was experimentally evaluated by the performance of respective luciferase reporter phage constructs developed from the parental phages Che12, D29 and TM4.

Construction and evaluation of luciferase reporter phages for the detection of active and non-replicating tubercle bacilli.

The luciferase reporter phages (LRP) show great promise for diagnostic mycobacteriology. Though conventional constructs developed from lytic phages such as D29 and TM4 are highly specific, they lack sensitivity. We have isolated and characterized Che12, the first true temperate phage infecting M. tuberculosis. Since the tuberculosis (TB) cases among HIV infected population result from the reactivation of latent bacilli, it would be useful to develop LRP that can detect dormant bacteria. During dormancy, pathogenic mycobacteria switch their metabolism involving divergent genes than during normal, active growth phase. Since the promoters of these genes can potentially function during dormancy, they were exploited for the construction of novel mycobacterial luciferase reporter phages. The promoters of hsp60, isocitrate lyase (icl), and alpha crystallin (acr) genes from M. tuberculosis were used for expressing firefly luciferase gene (FFlux) in both Che12 and TM4 phages and their efficiency was evaluated in detecting dormant bacteria from clinical isolates of M. tuberculosis. These LRP constructs exhibited detectable luciferase activity in dormant as well as in actively growing M. tuberculosis. The TM4 ts mutant based constructs showed about one log increase in light output in three of the five tested clinical isolates and in M. tuberculosis H37Rv compared to conventional lytic reporter phage, phAE129. By refining the LRP assay format further, an ideal rapid assay can be designed not only to diagnose active and dormant TB but also to differentiate the species and to find their drug susceptibility pattern.

Pot method for storage & detection of acid-fast bacilli from sputum samples.

BACKGROUND & OBJECTIVES: Sputum acid-fast bacilli (AFB) microscopy services are not available in all health facilities. Alternative procedures are needed to transport sputum samples to the diagnostic centres for detection of AFB. The objective of the present study was to evaluate sputum smears made by pot-method with the direct smears made immediately after sputum collection by Ziehl-Neelsen (ZN) method. METHODS: Ninety three sputum samples from 49 pulmonary tuberculosis suspects were studied. Their direct smears (ZN smears) were stained by hot ZN method. The samples were then mixed with phenol ammonium sulphate basic fuchsin solution and stored at ambient conditions. The smears (pot smears), made on day 7, were then, decolourized and counter-stained for detection of AFB (pot method). The ZN and pot smears were read blind. After excluding 18 samples for various reasons, the results of pot and ZN smears of 63 samples from smear positive (2 of 3 direct smears were positive) and 12 from smear negative (3 of 3 direct smears were negative) patients were analysed. ZN method was the gold standard. RESULTS: Pot and ZN smears were positive in 61 of 63 samples from smear-positive patients and negative in 11 of 12 smear-negative patients (kappa = 0.87). The sensitivity and specificity of pot method were 96.8 and 91.7 per cent respectively. INTERPRETATION & CONCLUSION: Sputum samples can be stored for up to seven days in the sputum container with phenol ammonium sulphate basic fuchsin solution. However, a comprehensive study needs to be done confirm the accuracy of the pot method for storage and transportation of sputum to microscopy centres for detection of AFB.

Phenol ammonium sulphate basic fuchsin staining of sputum in pot for the detection of acid-fast bacilli.

BACKGROUND & OBJECTIVES: Improper practices of making direct smears of sputum for detection of acid-fast bacilli (AFB) and of disposing sputum cups are hazardous. The present study was undertaken with the objective to stain sputum samples in their containers by 'phenol (10%) ammonium sulphate (4%) basic fuchsin (2%) solution' and to decolourize and counterstain their smears for detection of AFB- (henceforth called pot method) and to compare the smear results of pot method with the standard Ziehl-Neelsen (ZN) method. METHODS: A total of 575 selected sputum samples from pulmonary tuberculosis patients were stained by the standard ZN and pot methods and the proportions of AFB positive smears were compared. RESULTS: Of the 575 samples, 126 were AFB positive for both the staining methods and the difference was not statistically significant. Pot method missed 9 ZN positive smears (8 scanty and one 1+) and ZN method missed 9 pot positive smears (9 scanty) and the difference was not significant. High grade smears (3+) were seen more in pot method (42) than in ZN method (25) and the difference was significant. INTERPRETATION & CONCLUSION: Our findings showed that pot method was comparable to standard ZN method and had many advantages. Pot method can be explored further for the detection of AFB in sputum samples obtained from pulmonary tuberculosis suspects.

Acetate Kinase (AcK) is Essential for Microbial Growth and Betel-derived Compounds Potentially Target AcK, PhoP and MDR Proteins in M. tuberculosis, V. cholerae and Pathogenic E. coli: An in silico and in vitro Study.

BACKGROUND: Mycobacterium tuberculosis, Vibrio cholerae, and pathogenic Escherichia coli are global concerns for public health. The emergence of multi-drug resistant (MDR) strains of these pathogens is creating additional challenges in controlling infections caused by these deadly bacteria. Recently, we reported that Acetate kinase (AcK) could be a broad-spectrum novel target in several bacteria including these pathogens. METHODS: Here, using in silico and in vitro approaches we show that (i) AcK is an essential protein in pathogenic bacteria; (ii) natural compounds Chlorogenic acid and Pinoresinol from Piper betel and Piperidine derivative compound 6-oxopiperidine-3-carboxylic acid inhibit the growth of pathogenic E. coli and M. tuberculosis by targeting AcK with equal or higher efficacy than the currently used antibiotics; (iii) molecular modeling and docking studies show interactions between inhibitors and AcK that correlate with the experimental results; (iv) these compounds are highly effective even on MDR strains of these pathogens; (v) further, the compounds may also target bacterial two-component system proteins that help bacteria in expressing the genes related to drug resistance and virulence; and (vi) finally, all the tested compounds are predicted to have drug-like properties. RESULTS AND CONCLUSION: Suggesting that, these Piper betel derived compounds may be further tested for developing a novel class of broad-spectrum drugs against various common and MDR pathogens.

In silico analysis of mycobacteriophage Che12 genome: characterization of genes required to lysogenise Mycobacterium tuberculosis.

Che12 is a temperate Chennai phage infecting Mycobacterium tuberculosis. The nucleotide sequence of the 52,047 bp linear double stranded DNA genome has a GC content of 62.9% with 70 putative ORFs identified. Functions are assigned to 24 genes based on the similarity of the predicted products to known proteins. Che12 genome is highly similar to mycobacteriophage L5 and D29 genomes. The overall genome similarity of Che12 to L5 is 82.5% and D29 is 81.5%. The genes attributing to lysogeny such as integrase, excisionase and repressor protein are identified. The attachment site of Che12 genome attP is homologous to attB sites of Mycobacterium smegmatis and M. tuberculosis. Similarities between certain phage gene products are noted, in particular, the terminases, DNA primase and endonucleases. The complete sequence clarifies the overall transcription map of Che12 and the positions of elements involved in the maintenance of lysogeny.

Phage cocktail to control the exponential growth of normal flora in processed sputum specimens grown overnight in liquid medium for rapid TB diagnosis.

The mechanical pressure exerted during centrifugation and the chemical pressure experienced when sputum specimens are processed, leave the tubercle bacilli in the sputum unsuitable for rapid detection especially in phage based assays. Thus, growing Mycobacterium tuberculosis in broth, at least overnight, is mandatory for allowing the tubercle bacilli to recoup. During this time the surviving colonizing flora grow faster and overgrow tubercle bacilli interfering with TB diagnosis. In the present study normal flora surviving the action of 4% NaOH was isolated and characterized. Phages capable of killing 14 different species representing this normal flora were isolated from soil and sewage samples and characterized. A novel and bio-friendly approach to treat sputum samples with a cocktail of three phages capable of killing most of the 14 representative organisms and not infecting mycobacteria is explored to control the overgrowth of colonizing bacteria in broth culture. While 26 of the 100 sputum samples processed by modified Petroff's procedure showed growth of colonizing flora on blood agar, all of them when grown in broth overnight showed mixed, confluent growth. The addition of phagebiotics controlled them all, showing a significant reduction in colony forming units but resulting in few discrete colonies in 54 samples. Isolation of phages capable of controlling these surviving organisms and including them in the phagebiotics mixture should lead to the control of colonizing bacteria effectively.