Middlebrook 7H10 Medium Research Articles

Evaluation of fetal bovine serum as a source of enrichment in synthetic culture medium for the isolation of Mycobacterium bovis

A Middlebrook 7H10 medium added with 0.2% glucose and Fetal Bovine Serum (FBS) at concentrations of 5, 10, and 15% was compared with a Middlebrook 7H11 medium enriched with commercial OADC to evaluate the growth of Mycobacterium bovis, strain AN5, in the dilutions 10-1, 10-2, 10-3 and 10-4 performed from the McFarland scale 1. The results demonstrated micro and macroscopic growth of mycobacteria in all concentrations of SFB two weeks after inoculation, and in greater numbers when compared to Middlebrook 7H11 supplemented with commercial OADC. Statistical analysis found a significant reduction in the Middlebrook 7H11 group when compared to the 15% SFB concentration group in dilutions 10-2, 10-3, and 10-4. The results presented allowed us to conclude that FBS can be used as a source of enrichment for a Middlebrook 7H10 Medium in a thin layer, for isolation of Mycobacterium bovis, with the advantage of being more accessible and cheaper than the commercial medium.

Performance of lipid fingerprint by routine matrix-assisted laser desorption/ionization time of flight for the diagnosis of Mycobacterium tuberculosis complex species

ObjectivesRapid detection of bacterial pathogens at species and sub-species levels is crucial for appropriate treatment, infection control, and public health management. Currently, one of the challenges in clinical microbiology is the discrimination of mycobacterial sub-species within the M. tuberculosis complex (MTBC). Our objective was to evaluate the ability of a biosafe mycobacterial lipid-based approach to identify MTBC cultures and sub-species. MethodsA blinded study was conducted using 90 mycobacterial clinical isolate strains comprising MTBC strains sub-cultured in Middlebrook 7H11 medium supplemented with 10% oleic-acid, dextrose, catalase growth supplement and incubated for up to 6 weeks at 37°C and using the following seven reference strains (M. tuberculosis H37Rv, M canettii, M. africanum, M. pinnipedii, M. caprae, M. bovis, and M. bovis BCG) grown under the same conditions, to set the reference lipid database and test it against the 90 MTBC clinical isolates. Cultured mycobacteria were heat-inactivated and loaded onto the matrix-assisted laser desorption/ionization target followed by the addition of the matrix. Acquisition of the data was performed using the positive ion mode. ResultsBased on the identification of clear and defined lipid signatures from the seven reference strains, the method that we developed was fast (<10 minutes) and produced interpretable profiles for all but four isolates, caused by poor ionization giving an n = 86 with interpretable spectra. The sensitivity and specificity of the matrix-assisted laser desorption/ionization time of flight were 94.4 (95% CI, 86.4–98.5) and 94.4 (95% CI, 72.7–99.9), respectively. ConclusionsMycobacterial lipid profiling provides a means of rapid, safe, and accurate discrimination of species within the MTBC.

Middlebrook 7h11 reduces invalid results and turnaround time of phenotypic drug-susceptibility testing of M. tuberculosis.

Phenotypic drug-susceptibility testing (pDST), which relies on growth inhibition in the drug-containing media, remains a challenge for fastidious Mycobacterium tuberculosis complex (MTBc) isolates due to insufficient growth on the growth controls (GC). Middlebrook 7H11 (M7H11) medium contains casein hydrolysate, which may favor the growth of such strains. In this study, we tested whether M7H11 reduces invalid results due to insufficient growth on the GCs and the turnaround time (TAT) of pDST for MTBc compared to Middlebrook 7H10 (M7H10) without affecting the accuracy of the pDST results and how it differs between rifampicin- and isoniazid-susceptible non multi-drug resistant (non-MDR), MDR and MDR with additional resistance to fluoroquinolones (Pre-XDR) MTBc isolates. We compared the proportions of invalid pDST results due to lack of growth on the GCs, TATs of valid parallel drug-susceptibility testings as an indicator of speed of MTBc growth, and colony-forming unit (CFU) count on the most diluted GC of the parallel pDSTs after equal incubation periods as an indicator of growth abundance on M7H11 and M7H10. We also analyzed the agreement between the pDST results of the same drug or drugs in the same drug class, tested in parallel on both media. For MDR and pre-XDR isolates, relative to M7H10, M7H11 significantly reduced the occurrence of invalid pDST results due to insufficient growth on the GCs (odds ratio [OR] = ∞ [95% confidence interval (CI) 1.9-∞], P = 0.004 for MDR, OR = ∞ [95% CI 3.3-∞], P = 0.0001 for pre-XDR) and the TAT of pDSTs (OR = 17 [95% CI 2.6-710.4], P = 0.0001 for MDR, OR = 9.3 [95% CI 4.0-26.5], P < 0.0001 for pre-XDR). The growth abundance of MTBc on M7H11 was significantly higher compared to M7H10 (17 CFU on M7H10 vs. 28 on M7H11), irrespective of drug-resistance profiles. The agreement between the pDST results between the two media was high (Cohen's k > 0.98). Our study findings suggest that M7H11 is preferred over M7H10 for pDSTs of MTBc isolates.

Determination of Anti-tuberculosis activity of Psychotria sarmentosa, Aponogeton crispus and two species of Pleurotus mushrooms

Tuberculosis (TB) is a chronic disease caused by Mycobacterium tuberculosis (Mtb) complex. The global TB epidemic has been aggravated by the emergence of disease outbreaks caused by multi-drug resistant and extensively drug-resistant strains. The aim of the present study was to evaluate the in-vitro, anti-TB activity of leaves of Psychotria sarmentosa, Aponogeton crispus and the mushrooms Pleurotus ostreatus and Pleurotus cystidiosus found in Sri Lanka. Leaves of Psychotria sarmentosa, Aponogeton crispus and the mushrooms; Pleurotus ostreatus and P. cystidiosus were dried until a constant weight and 120 g each were taken to prepare crude extracts with distilled water (1.9 L) by heating at a moderate temperature and the final volume was reduced to 240 ml. Freeze dried aqueous extracts were incorporated in Middle Brook 7H11 medium (1mg/ml) using pour plate method.Two ten-fold dilutions (10-2 and 10-4) of standard H37Rv Mtb suspensions were inoculated on Middle Brook 7H11 media with the crude extracts. The plates were incubated at 37 0C for 4 weeks until visible appearance of Mtb colonies. The inhibitory effect of each extract was calculated by the mean reduction of number of colonies on extract containing medium compared to extract-free control medium. Accordingly, the highest mean percentage inhibition was shown by P. sarmentosa (71.0 %). The mean percentage inhibition exerted by A. crispus, P. ostreatus and P. cystidiosus were 46.0 %, 43.4 % and 39.5 % respectively. Therefore, freeze-dried aqueous extract from leaves of P. sarmentosa has certain activity against the tested standard mycobacterial strain and has a potential to be used as an anti-TB drug component.

Testing Susceptibility of &lt;i&gt;M. tuberculosis&lt;/i&gt; to Second Line Anti-Tuberculosis Drugs Using the XDR Test in Clinical Trials and International Professional Testing Cycles

The objective of the study: to evaluate the commercial XDR test for susceptibility testing of M. tuberculosis to second line anti-tuberculosis drugs in clinical trials and as part of annual professional testing cycles coordinated by the World Health Organization (WHO).Subjects and Methods. Cultures of M. tuberculosis (n = 90) freshly isolated on egg media from clinical samples collected in tuberculosis patients were tested using the Bactec MGIT 960 system and the XDR test under identical conditions. Well-studied strains of M. tuberculosis (n = 216) obtained from the WHO supranational laboratories were repeatedly cultured on Middlebrook 7H10 medium before the study. The drug susceptibility of the cultures was assessed using the XDR test by the nitrate reductase method.Results. A high concurrence (96.7-100%) of the results was shown when testing susceptibility of 90 M. tuberculosis isolates to kanamycin, amikacin, capreomycin and ofloxacin using the XDR test and the Bactec MGIT 960 system with comparable test periods. The use of the XDR test for drug susceptibility testing of 216 M. tuberculosis strains in eleven annual professional testing cycles coordinated by the WHO supranational laboratories provided the results consistent with the consensus one for kanamycin, capreomycin, ofloxacin and amikacin in 98.6, 99.4, 99.4, and 99.0% of cases, respectively. For moxifloxacin and levofloxacin additionally incorporated to the XDR test, completely identical results were obtained.

On the Consequences of Poorly Defined Breakpoints for Rifampin Susceptibility Testing of Mycobacterium tuberculosis Complex.

In a recent report of a systematic review of critical concentrations (CCs), the World Health Organization (WHO) lowered the rifampin (RIF) CC for antimicrobial susceptibility testing (AST) of the Mycobacterium tuberculosis complex using Middlebrook 7H10 medium and the Bactec Mycobacterial Growth Indicator Tube (MGIT) 960 system from 1 to 0.5 μg/ml. The previous RIF CC for 7H10 had been in use for over half a century. Because it had served as the de facto reference standard, it contributed to the endorsement of inappropriately high CCs for other AST methods, including the U.S. Food and Drug Administration (FDA)-approved MGIT system. Moreover, this resulted in confusion about the interpretation of seven borderline resistance mutations in rpoB (i.e., L430P, D435Y, H445L, H445N, H445S, L452P, and I491F). In this issue of the Journal of Clinical Microbiology, Shea et al. (J Clin Microbiol 59:e01885-20, 2021, https://doi.org/10.1128/JCM.01885-20) provide evidence that the CC endorsed by the Clinical and Laboratory Standards Institute for the Sensititre MYCOTB system, which is not FDA approved but is CE-IVD marked in the European Union, is likely also too high. These findings underscore the importance of calibrating AST methods against a rigorously defined reference standard, as recently proposed by the European Committee on Antimicrobial Susceptibility Testing, as well as the value of routine next-generation sequencing for investigating discordant AST results.

Licensed Bacille Calmette-Guérin (BCG) formulations differ markedly in bacterial viability, RNA content and innate immune activation

BackgroundBacille Calmette-Guérin (BCG), the live attenuated tuberculosis vaccine, is manufactured under different conditions across the globe generating formulations that may differ in clinical efficacy. Innate immune recognition of live BCG contributes to immunogenicity suggesting that differences in BCG viability may contribute to divergent activity of licensed formulations. MethodsWe compared BCG-Denmark (DEN), -Japan (JPN), -India (IND), -Bulgaria (BUL) and -USA in vitro with respect to a) viability as measured by colony-forming units (CFU), mycobacterial membrane integrity, and RNA content, and b) cytokine/chemokine production in newborn cord and adult peripheral blood. ResultsUpon culture, relative growth was BCG-USA > JPN ≫ DEN > BUL = IND. BCG-IND and -BUL demonstrated >1000-fold lower growth than BCG-JPN in 7H9 medium and >10-fold lower growth in commercial Middlebrook 7H11 medium. BCG-IND demonstrated significantly decreased membrane integrity, lower RNA content, and weaker IFN-γ inducing activity in whole blood compared to other BCGs. BCG-induced whole blood cytokines differed significantly by age, vaccine formulation and concentration. BCG-induced cytokine production correlated with CFU, suggesting that mycobacterial viability may contribute to BCG-induced immune responses. ConclusionsLicensed BCG vaccines differ markedly in their content of viable mycobacteria possibly contributing to formulation-dependent activation of innate and adaptive immunity and distinct protective effects.

Evaluation of nitrate reductase assay in 7H11 agar for diagnosis of multi-drug-resistant tuberculosis in eastern Nepal

BackgroundEmergence of multi-drug-resistant tuberculosis is a serious challenge for successful global tuberculosis control. Early diagnosis of drug-resistant tuberculosis by direct nitrate reductase assay (NRA) aids in appropriate treatment and reduction in disease transmission, particularly in countries with high tuberculosis burden. The aim of this study was to evaluate the performance of NRA for direct detection of resistance to rifampicin and isoniazid in Mycobacterium tuberculosis in laboratories with limited resources.MethodsFifty-eight new smear-positive sputum samples were processed as per the guidelines of revised national tuberculosis control program, India. The performance of NRA on middlebrook 7H11 agar was evaluated for detection of rifampicin and isoniazid resistance directly on smear-positive sputum specimens, and the results were compared with conventional proportion method. Sensitivity and specificity of the test were compared with the gold standard proportion method. Mc Nemar chi-square test was used to find out the significant difference between two methods.ResultsDirect NRA for detection of rifampicin resistance was 85.7% sensitive and 100% specific, whereas sensitivity and specificity of isoniazid resistance were 87.5% and 100%, respectively. Agreement between NRA and proportion method was 98% for both the drugs. The mean days of drug susceptibility testing results were 19.3 days for NRA and 72 days for conventional proportion method. The results of NRA were available in 21 days for 83% of the samples.ConclusionsDirect NRA on middlebrook 7H11 medium is a highly sensitive, reliable, and significantly faster method to perform drug susceptibility testing. It has the potential to be implemented for rapid detection of multi-drug-resistant tuberculosis against insufficient resources.

A protocol for culturing environmental strains of the Buruli ulcer agent, Mycobacterium ulcerans

Contaminations and fastidiousness of M. ulcerans may have both hamper isolation of strains from environmental sources. We aimed to optimize decontamination and culture of environmental samples to circumvent both limitations. Three strains of M. ulcerans cultured onto Middlebrook 7H10 at 30 °C for 20 days yielded a significantly higher number of colonies in micro-aerophilic atmosphere compared to ambient atmosphere, 5% CO2 and anaerobic atmosphere. In a second step, we observed that M. ulcerans genome uniquely encoded chitinase, fucosidase and A-D-GlcNAc-diphosphoryl polyprenol A-3-L-rhamnosyl transferase giving M. ulcerans the potential to metabolize chitine, fucose and N-acetyl galactosamine (NAG), respectively. A significant growth-promoting effect of 0.2 mg/mL chitin (p < 0.05), 0.01 mg/mL N-acetyl galactosamine (p < 0.05), 0.01 mg/mL fucose (p < 0.05) was observed with M. ulcerans indicating that NAG alone or combined with fucose and chitin could complement Middlebrook 7H10. Finally, the protocol combining 1% chlorhexidine decontamination with micro-aerophilic incubation on Middlebrook 7H10 medium containing chitin (0.2%), NAG (0.01%) and fucose (0.01%) medium and auto-fluorescence detection of colonies allowed for the isolation of one mycolactone-encoding strain from Thryonomys swinderianus (aulacode) feces specimens collected near the Kossou Dam, Côte d’Ivoire. We propose that incubation of chlorhexidine-decontaminated environmental specimens on Middlebrook 7H10-enriched medium under micro-aerophilic atmosphere at 30 °C may be used for the tentative isolation of M. ulcerans strains from potential environmental sources.

ЭКСПЕРИМЕНТАЛЬНОЕ СРАВНЕНИЕ АЭРОЗОЛЬНОГО МЕТОДА ПРИ ДЕЗИНФЕКЦИИ ВОЗДУХА И ПОВЕРХНОСТЕЙ, КОНТАМИНИРОВАННЫХ M. TUBERCULOSIS

The objective of the study: to analyze efficiency of an aerosol method of M. tuberculosis deactivation in the air and on surfaces versus the conventional methods of the disinfectants' application. Subjects and Methods. The article describes the evaluation of efficiency of the aerosol method of M. tuberculosis, H37Rv strain, deactivation on surfaces (tested objects made of linoleum) and in the air using the disinfectant of Green Dez based on chlorine dioxide versus deactivation through wiping and irrigation. The efficiency of disinfectant was tested by the device of 099С А4224 manufactured by Glas-Col, USA, using the air sampler of PU-1B, Russia. The Mobile Hygienic Center (MNC), Russia, was used for application of the disinfectant, wiping and irrigation was done using the disperser of Avtomaks AO-2, Russia. The bacterial aerosol was generated in the Glass-Col chamber with the concentration 5 ± 2.5 × 102 CFU/cm3, by spraying the suspension of M. tuberculosis, H37Rv strain. After that, the disinfectant spray was supplied to the chamber, where linoleum objects were placed horizontally on a variety of surfaces. In order to evaluate efficiency of surface treatment by wiping, the test objects were wiped with a tissue, soaked with the solution of Green Dez, based on consumption of 100-150 ml/m2. In 15, 30 and 60 minutes, the samples of inactivated M. tuberculosis aerosol were collected using an aspirator, chambers with test objects were closed and placed in the vent hood. To monitor efficiency of disinfection of the test object surfaces, the rinse blanks were done by wiping the surface with a sterile gauze wad, soaked with 0.5% of sodium thiosulfate solution. The samples of deactivated aerosol and rinse blanks from the surfaces of test objects were put into Petri dishes with Middlebrook 7H11 medium. The cultures were incubated in the thermostat at the temperature of 37 ± 1° C for 10-21 days, and the number of colonies was counted. Sterile water was used instead of disinfectant for control tests treating the tests objects contaminated in the same manner. Results. The good disinfection properties of Green Dez aerosol were proved during the simulation of M. tuberculosis, H37Rv strain, disinfection on the surfaces and in the air. The efficiency analysis of the aerosol method of disinfection proved the reduction of disinfectant consumption by 4-5 times versus wiping and irrigation, due to this the aerosol treatment can be regarded as a safer and more environmentally friendly method. The finely dispersed disinfectant aerosol, produced by MNC, effectively decontaminated the whole space, including the remote and hard-to-reach places. Full automation of disinfection implies no human presence providing high safety of disinfection.

Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration

Environmental exposure is considered to be responsible for nontuberculous mycobacterial infections in humans. To facilitate the isolation of mycobacteria from soil, Middlebrook 7H10 agar was optimized as an enhanced selective medium by increasing the concentration of malachite green. A series of modified Middlebrook 7H10 agar media with malachite green concentrations ranging from 2.5 to 2500 mg/L was evaluated using 20 soil samples decontaminated with 3% sodium dodecyl sulfate plus 2% NaOH for 30 min. Among these modified Middlebrook 7H10 media, the medium with malachite green at a concentration of 250 mg/L, i.e., at the same concentration as in Löwenstein–Jensen medium, was the most effective in terms of the number of plates with mycobacterial growth. This medium was further evaluated with 116 soil samples. The results showed that 87.1% (101/116) of the samples produced mycobacterial growth, and 15 samples (12.9%) produced no mycobacterial growth. Of the plates inoculated with the soil samples, each in duplicate, 5.2% (12/232) showed late contamination. In total, 19 mycobacterial species were isolated, including seven (36.8%) rapidly growing mycobacteria and 12 (63.2%) slowly growing mycobacteria. Our results demonstrate that the modified Middlebrook 7H10 agar with 250 mg/L malachite green is useful for the primary isolation of nontuberculous mycobacteria from soil.

Influence of cultivation conditions on the proteomic profile of Mycobacterium tuberculosis H37RV

Comparative proteomic profiling of M. tuberculosis H37Rv strains cultured on two different nutrient media, Levenstein-Jensen and Middlebrook 7H11, was performed using a label-free LC-MS/MS approach. It was shown that results obtained from two media possessed high convergence. The only difference was observed in the representation of fumarate reductase FrdB, its abundance was higher in the mycobacterial cells cultured on Levenstein-Jensen medium. The correlation analysis of biological repeats revealed the high convergence of the results obtained from Middlebrook 7H11 medium. Thus, we can conclude that the use of the Middlebrook 7H11 medium is most appropriate in the scientific laboratory.

Influence of the incubation conditions on culture media to optimize primary isolation of Mycobacterium bovis

The isolation of Mycobacterium bovis is critical to a surveillance system for bovine tuberculosis based on detection of lesions in abattoirs. Thus, four solid culture media and three incubation conditions were investigated to elucidate which combination overcomes the others by assessing growth, time to the first appearance of colonies and their number. Ninety-seven samples of granulomatous lesions were submitted to the decontamination procedure by 1-hexadecylpyridinium chloride at 0.75% w/v, and inoculated on two egg-based media, Stonebrink’s (ST) and Löwenstein-Jensen’s with sodium pyruvate (LJp), and two agar-based media, tuberculosis blood agar (B83) and Middlebrook 7H11 medium (7H11). Each medium was incubated at 37°C for 90 days in three incubation conditions: in air, in air containing 10% carbon dioxide (CO2), and in air in slopes closed with burned hydrophobic cotton and subsequently plugged with a cork to create a microaerophilic atmosphere. The colonies appeared faster and in higher number when incubated in air containing 10% CO2 (p &lt; 0.01), independent of media. B83 showed a faster growth and detected more isolates at 30 days of incubation, when compared to ST (0.0178), LJp (p &lt; 0.0001) and 7H11 (p &lt; 0.0001), though there was no difference between B83, ST and LJp at 60 and 90 days of incubation. 7H11 presented the lowest number of isolates (p &lt; 0.0001) and a longer period for the appearance of the first colony (p &lt; 0.001). According to our findings, the concomitant use of ST and B83 media incubated in air containing 10% CO2 increases the isolation of M. bovis in a shorter period of time, which improves bovine tuberculosis diagnosis.

Characterization of rpoB Gene Mutations in Clinical Isolates of Mycobacterium TB

SESSION TITLE: Tuberculosis SESSION TYPE: Original Investigation Poster PRESENTED ON: Wednesday, October 26, 2016 at 01:30 PM - 02:30 PM PURPOSE: 1) to evaluate the phenotypic characteristics of the specific mutation site, 2) to compare the performance of phenotypic and genotypic (molecular) susceptibility test for rifampin (RIF), 3) to define mutation sites which are correlated with RIF resistance levels and rifabutin (RBU) resistance. METHODS: Conventional phenotypic drug susceptibility testing (DST) in Middlebrook 7H10 medium, rpoB gene sequencing for 1kb around RIF resistance determining region (RRDR), and molecular DST (GenoType® MTBDRplus assay, MTBDRplus) were performed for 674 clinical isolates of M. tuberculosis which were obtained from the patients who had failed with first-line therapy. RESULTS: Based on conventional phenotypic DST 502 (74.5%) isolates were resistant to RIF. The rpoB gene sequencing showed 491 isolates possessed 517 mutation sites in the RRDR. The most frequent mutated site was codon 531 (185/517, 35.7%). Among the RIF-resistant isolates having the mutations at codon 533, most of the isolates (89%) showed minimal inhibitory concentration (MIC) lower than 16 ug/ml, suggesting that the mutation at codon 533 may not be associated with a high level of RIF resistance. Moreover, mutations at codon 533 (44/64, 68.8%) were frequently found in RIF-resistant and RBU-susceptible isolates. Sensitivity and specificity of the the rpoB gene sequencing and MTBDRplus were 93.4%, 91.4% and 91.4%, 95.1% respectively. CONCLUSIONS: Mutations at codon 533 were frequently found in clinical isolates of M. tuberculosis which were RIF-susceptible, low level resistant or RBU-susceptible. CLINICAL IMPLICATIONS: Physicians can expect phenotypic RIF- susceptible or RBU-susceptible despite of rpoB gene mutation if the mutation site is codon 533. DISCLOSURE: The following authors have nothing to disclose: Yangjin Jegal, Jihee Jung, Nackmoon Sung No Product/Research Disclosure Information

Comparative study of Mycobacterium bovis primary isolation methods

For the definitive diagnosis of bovine tuberculosis, isolation of the etiologic agent is required. However, there is no consensus on the best methodology for isolation of Mycobacterium bovis in Brazil. This study evaluated the most used decontaminants and culture media in the country, in order to identify the best combination for the Brazilian samples. Three decontaminants – 2% sodium hydroxide (w/v), 0.75% hexadecylpiridinium chloride (w/v) and 5% sulphuric acid (v/v) and four culture media – 7H11 Middlebrook with additives and OADC supplement “A” (7H11 A), the same media with another supplement trademark (7H11 B), tuberculosis blood agar (B83) and Stonebrink's medium were compared. Regarding the isolation, there were no significant differences between the decontaminants and media combinations, except 7H11A combined to any decontaminant. However, the mean colonies score was significantly greater when the samples were decontaminated with 5% sulphuric acid and inoculated in 7H11 B or SB, without significant difference between them, although colonies appeared earlier on 7H11B than on SB. The trademark of OADC supplement influenced the isolation rate and the number of isolated colonies in Middlebrook 7H11. An incubation time of four weeks was required to detect all positive samples in 7H11 B after decontamination with 5% sulphuric acid but there was an increase in the number of colonies until the sixth week of incubation. Overall, the best strategy for the primary isolation of M. bovis from Brazilian samples was the decontamination with 5% sulphuric acid (final concentration) and inoculation in Middlebrook 7H11 medium formulated with OADC supplement “B”.

Rifampicin resistance mutations in the 81bp RRDR of rpoB gene in Mycobacterium tuberculosis clinical isolates using Xpert MTB/RIF in Khyber Pakhtunkhwa, Pakistan: a retrospective study.

BackgroundMulti-drug resistant tuberculosis (MDR-TB) is a major public health problem especially in developing countries. World Health Organization (WHO) recommends use of Xpert MTB/RIF assay to simultaneously detecting Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance. The primary objective of this study was to determine the frequency of MDR-TB in patients suspected to have drug resistance in Khyber Pakhtunkhwa. The frequency of probes for various rpoB gene mutations using Xpert MTB/RIF assay within 81 bp RRDR (Rifampicin Resistance Determining Region) was the secondary objective.MethodsA total of 2391 specimens, received at Programmatic Management of Drug Resistant TB (PMDT) Unit, Lady Reading Hospital (LRH) Peshawar, Pakistan, between October 2011 and December 2014, were analyzed by Xpert MTB/RIF test. MTB positive with rifampicin resistance were further analyzed to first line anti-mycobacterial drug susceptibility testing (DST) using middle brook 7H10 medium. The data was analyzed using statistical software; SPSS version 18.ResultsOut of 2391 specimens, 1408 (59 %) were found positive for MTB and among them, 408 (29 %) showed rifampicin-resistance with four different rpoB gene mutations within 81 bp RRDR. The frequency of various probes among RIF-resistant isolates was observed as: probe E, in 314 out of 408 isolates; B, 44 out of 408; A, 5 out of 408; D, 34 out of 408; and probe C was observed among 6 out of 408 RIF-resistant isolates. The probe A&B and E&D mutation combination was found in only 1 isolate in each case, while B&D mutation combination was detected among 3 out of 408 RIF-resistant isolates.ConclusionsHence, it is concluded from our study on a selected population, 29 % of patients had MDR-TB. Probe E related mutations (also known as codon 531and 533) were the most common rpoB genetic mutation [314 (77 %)], acknowledged by Xpert MTB/RIF assay. Least mutation was detected within the sequence 511 (1.2 %).

Inverse correlation between salt tolerance and host-adaptation in mycobacteria.

BackgroundThe genus Mycobacterium includes host-adapted organisms regarded as obligate and opportunistic pathogens and environmental organisms. Factors contributing to this wide range of adaptations are poorly known.ResultsWe studied the salt tolerance of 46 Mycobacterium species of medical interest. Representative strains of the Mycobacterium tuberculosis complex, Mycobacterium avium complex, Mycobacterium chelonae-abscessus complex, Mycobacterium ulcerans, Mycobacterium marinum, Mycobacterium lentiflavum, Mycobacterium fortuitum and Mycobacterium conceptionense were inoculated on Middlebrook 7H10 medium supplemented with 0–10 % sodium chloride. Colonies were counted after 2–4 week incubation at the appropriate 30–37 °C temperature depending on the tested strain. Further comparative genomics was done on 15 Mycobacterium strains representing the spectrum of salt-tolerance of mycobacteria. Based on the results the different species were grouped according to their salt tolerance into a “salt-sensitive” group (growth up to ≤3 % salt) containing the M. tuberculosis complex, Mycobacterium chelonae, Mycobacterium lentiflavum, Mycobacterium ulcerans and Mycobacterium marinum; a “salt-intermediate” group (growth between 4 and 6 % salt) comprising Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera and a “salt-resistant” group (growth up to >6 %) comprising Mycobacterium homonissuis, Mycobacterium bolettii, Mycobacterium fortuitum and Mycobacterium conceptionense. Genomic analysis revealed that 290 genes were unique to species belonging to the salt-sensitive group; and that 15 % were annotated as being functionally associated with the ESX secretion systems Pro-Glu and Pro–Pro-Glu family proteins.ConclusionsIn this work we found an inverse correlation between salt tolerance and host adaptation. We thus propose that salinity is one of the multiple factors determining the ecological niches of mycobacteria.

Modeling of cerebral tuberculosis in BALB/c mice using clinical strain from patients with CNS -TB infection

Background: This study describes development of a TBM animal model in mice using C3 strain isolated from CSF of TBM patients which can be used to study pathogenesis of C3 strain in mice. Methods & Materials: Female BALB/c mice aged 6–10 weeks were housed in a BSL- 3 facility. Two groups of mice (n = 12) were challenged intravenously through tail vein with 2 × 107 of C3 strain. Control group of mice (n = 10) was separately maintained and injected with sterile saline. Mice were killed at 30 and 50 days after development of infection, for estimating number of mycobacteria colonizing in brain,lungs and for histopathological changes. The mycobacterial burden was determined by plating and counting the number of CFUs. Serially diluted homogenates of individual lungs and brains were plated onto Middlebrook 7H11 medium. CFU's were counted after 3–4 weeks of infection. Histological sections were stained using hematoxylin and eosin. Results: Mice infected with C3 strain showed prominent edema of the brain in left hemisphere at 30 days, which increased at later stage compared to control mice. Histopathological examination of brain showed swelling of neurons along with lymphocytic infiltration which was progressively more at 50 days. The lung section at 30 days revealed >40 lession in lung parenchyma which improved at 50 days, with lession reduced to <20. Brain and lungs of control mice showed no major changes. Infection with C3 strain showed significant levels of mycobacterial load in brains with progress in infection. Analysis of CFU count revealed significantly high load in lungs (6.80 ± 0.1) at 30 days post infection, however the mycobacterial burden gradually decreased in lungs (5.8 ± 0.2) with progress in infection at 50 days. Analysis of CFU load in brain showed significantly high mycobacterial load at 50 days (4.0 ± 0.2) compared to infection at 30 days (5.0 ± 0.1). No mycobacterial load was observed in brains and lungs of control mice.C3 strain infection was associated with reduced survival-40% and high mortality rate -60%compared to control mice. Conclusion: Our present study demonstrated that intravenous inoculation by C3 strain from TBM patient's leads to progressive dissemination and development of TBM disease in mice.

Detection of drug resistance in Mycobacterium tuberculosis.

Detection of drug resistance in Mycobacterium tuberculosis.

Tropical Plant Extracts Modulating the Growth of Mycobacterium ulcerans.

Mycobacterium ulcerans, the etiologic agent of Buruli ulcer, has been detected on aquatic plants in endemic tropical regions. Here, we tested the effect of several tropical plant extracts on the growth of M. ulcerans and the closely related Mycobacterium marinum. M. ulcerans and M. marinum were inoculated on Middlebrook 7H11 medium with and without extracts from tropical aquatic plants, including Ammannia gracilis, Crinum calamistratum, Echinodorus africanus, Vallisneria nana and Vallisneria torta. Delay of detection of the first colony and the number of colonies at day 7 (M. marinum) or day 16 (M. ulcerans) were used as endpoints. The first M. ulcerans colonies were detected at 8 ± 0 days on control Middlebrook 7H11 medium, 6.34 ± 0.75 days on A. gracilis-enriched medium (p<0.01), 6 ± 1 days on E. africanus- and V. torta-enriched media (p<0.01), 6 ± 0 days on V. nana-enriched medium (p<0.01) and 5.67 ± 0.47 days on C. calamistratum-enriched medium (p<0.01). Furthermore, the number of detected colonies was significantly increased in C. calamistratum- and E. africanus-enriched media at each time point compared to Middlebrook 7H11 (p<0.05). V. nana- and V. torta-enriched media significantly increased the number of detected colonies starting from day 6 and day 10, respectively (p<0.001). At the opposite, A. gracilis-enriched medium significantly decreased the number of detected colonies starting from day 8 PI (p<0.05). In conclusion, some aquatic plant extracts, could be added as adjuvants to the Middlebrook 7H11 medium for the culturing of M. marinum and M. ulcerans.