Middlebrook 7H10 Agar Research Articles

Use of BD BACTEC™ MGIT™ for the detection of non-tuberculous mycobacteria in sanitary water samples

IntroductionThe most commonly used method for the detection of non-tuberculous mycobacteria (NTM) is culture in BD BACTEC™ MGIT™ Mycobacteria Growth Indicator Tubes incubated in an automated growth detection reader BD BACTEC™ MGIT™ 960 Instrument. The system is currently validated for the detection of mycobacteria from clinical specimens but not environmental matrices.MethodsFrom November 2018 to December 2023, 1,369 sanitary water samples from 92 heater–cooler units (HCUs) and 747 sanitary water samples from 489 haemodialysis instruments (dialysis) were concentrated, decontaminated, and cultured on MGIT and solid Lowenstein–Jensen media to evaluate the presence of NTM. NTM-positive cultures (n = 261 HCUs and n = 20 dialysis) were purified by Middlebrook 7H11 agar plate subcultures and identified by MALDI-TOF mass spectrometry technology.ResultsThe purpose of this study was to evaluate the accuracy and reproducibility of the MGIT system on sanitary water from HCU and dialysis, using the two strains most frequently isolated on these devices as sources of NTM during the Emilia- Romagna surveillance programme: M. chimaera (79%) and M. saskatchewanense (100%), respectively. To evaluate the accuracy, sanitary water was spiked with M. chimaera and M. saskatchewanense at the theoretical concentrations of 100 and 10 CFU/mL, and all resulted positive in MGIT tubes. No significant changes in time to positivity were observed when MGIT tubes were inoculated with NTM at the theoretical concentrations of 10 and 100 CFU/mL on 3 consecutive days, indicating that the detection method is reproducible.DiscussionThe MGIT system is suitable for detecting the presence of NTM in sanitary water samples as it was capable of detecting up to 4 CFU/mL for both M. chimaera and M. saskatchewanense. Our results indicate that the MGIT system can be used for NTM detection not only for clinical samples but also for environmental matrices.

The role of rifampicin within the treatment of Mycobacterium avium pulmonary disease.

Rifampicin is recommended for the treatment of Mycobacterium avium complex pulmonary disease alongside azithromycin and ethambutol. We evaluated the azithromycin-ethambutol backbone with and without rifampicin in an intracellular hollow fiber model and performed RNA sequencing to study the differences in adaptation. In an in vitro hollow fiber experiment, we simulated epithelial lining fluid pharmacokinetic profiles of the recommended 3-drug (rifampicin, ethambutol, and azithromycin) or a 2-drug (ethambutol and azithromycin) treatment. THP-1 cells infected with M. avium ATCC700898 were exposed to these regimens for 21 days. We determined intra- and extra-cellular bacterial load- and THP-1 cell densities on days 0, 3, 7, 14, and 21, alongside RNA sequencing. The emergence of macrolide resistance was studied by inoculating intra- and extra-cellular fractions of azithromycin-containing Middlebrook 7H10 agar plates. Complete pharmacokinetic profiles were determined at days 0 and 21. Both therapies maintained stasis of both intra- and extra-cellular bacterial populations for 3 days, whilst regrowth coinciding with the emergence of a macrolide-resistant subpopulation was seen after 7 days. THP-1 cell density remained static. Similar transcriptional profiles were observed for both therapies that were minimally influenced by exposure duration. Transcriptional response was slightly larger during 2-drug treatment. Rifampicin did not add to the antimycobacterial effect to the 2-drug therapy or suppression of emergence resistance. RNA transcription was not greatly altered by the addition of rifampicin, which may be due to strong transcriptional influence of azithromycin and host cells. This questions the role of rifampicin in the currently recommended therapy. These findings should be confirmed in clinical trials.

Population pharmacokinetics and model-based dosing evaluation of bedaquiline in multidrug-resistant tuberculosis patients.

Aims: Bedaquiline is now recommended to all patients in the treatment of multidrug-resistant tuberculosis (MDR-TB) using standard dosing regimens. As the ability to measure blood drug concentrations is very limited, little is known about drug exposure and treatment outcome. Thus, this study aimed to model the population pharmacokinetics as well as to evaluate the currently recommended dosage. Methodology: A bedaquiline population pharmacokinetic (PK) model was developed based on samples collected from the development cohort before and 1, 2, 3, 4, 5, 6, 8, 12, 18, and 24h after drug intake on week 2 and week 4 of treatment. In a prospective validation cohort of patients with MDR-TB, treated with bedaquiline-containing standardized regimen, drug exposure was assessed using the developed population PK model and thresholds were identified by relating to 2-month and 6-month sputum culture conversion and final treatment outcome using classification and regression tree analysis. In an exploratory analysis by the probability of target attainment (PTA) analysis, we evaluated the recommended dosage at different MIC levels by Middlebrook 7H11 agar dilution (7H11). Results: Bedaquiline pharmacokinetic data from 55 patients with MDR-TB were best described by a three-compartment model with dual zero-order input. Body weight was a covariate of the clearance and the central volume of distribution, albumin was a covariate of the clearance. In the validation cohort, we enrolled 159 patients with MDR-TB. The 7H11 MIC mode (range) of bedaquiline was 0.06mg (0.008-0.25mg/L). The study participants with AUC0-24h/MIC above 175.5 had a higher probability of culture conversion after 2-month treatment (adjusted relative risk, aRR:16.4; 95%CI: 5.3-50.4). Similarly, those with AUC0-24h/MIC above 118.2 had a higher probability of culture conversion after 6-month treatment (aRR:20.1; 95%CI: 2.9-139.4), and those with AUC0-24h/MIC above 74.6 had a higher probability of successful treatment outcome (aRR:9.7; 95%CI: 1.5-64.8). Based on the identified thresholds, simulations showed that the WHO recommended dosage (400mg once daily for 14 days followed by 200mg thrice weekly) resulted in PTA >90% for the majority of isolates (94%; MICs ≤0.125mg/L). Conclusion: We established a population PK model for bedaquiline in patients with MDR-TB in China. Based on the thresholds and MIC distribution derived in a clinical study, the recommended dosage of bedaquiline is sufficient for the treatment of MDR-TB.

Comparative diagnosis of bovine tuberculosis using single intradermal cervical tuberculin technique, conventional methods, enzyme-linked immunosorbent assay, and the gamma-interferon assay.

Background and Aim:Bovine tuberculosis (TB) is a zoonotic disease that causes huge economic losses. This study aimed to compare the result obtained from the single intradermal test, conventional methods (culture and microscopy), gamma-interferon (IFN-γ) assay, and indirect enzyme-linked immunosorbent assay (ELISA) to diagnose bovine TB.Materials and Methods:This study evaluated 2913 animals from milk farms in Cairo, El-Sharkia, and El-Qalyubia Governorates by single intradermal cervical tuberculin technique (SICTT), ELISA, and IFN-γ assay.Results:Of the 2913 dairy cows surveyed, 3.7% yielded positive results. Culture prepared samples on Lowenstein-Jensen and Middlebrook 7H10 agar media yielded 52 (1.85%) isolates of Mycobacterium spp. from 2805 milk samples that yielded negative tuberculin reactions and 56 (51.85%) isolates of Mycobacterium spp. were recovered from 108 lymph node samples from positive cases. ELISA analysis of the sera of 108 positive SICTT reactors revealed that 94 (87.03%) and 97 (89.81%) animals were positive for bovine purified protein derivative (PPD-B) antigen and commercial polypeptide antigen, respectively. IFN-γ assays were performed on whole blood samples collected from positive SICTT reactors and showed that 103 (95.37%) animals were positive.Conclusion:M. tuberculosis complex may be isolated from raw milk and not all infected animals shed mycobacterial bacilli in their milk. The use of polypeptide antigen in ELISA provides better diagnostic efficacy than PPD-B antigen. The IFN-γ assay is more sensitive than both SICTT and ELISA. It should be used in parallel with SICTT to allow the detection of more positive animals before they become a source of infection to other animals and humans.

Mycobacterium Tuberculosis and Avium Complex Investigation among Malaysian Free-Ranging Wild Boar and Wild Macaques at Wildlife-Livestock-Human Interface.

Simple SummaryThis study targeted a small epidemiological area of a selected wildlife-livestock-human interface in Selangor to detect important veterinary and public health mycobacteria in free-ranging wild boar (Sus scrofa) and wild macaques (Macaca fascicularis) using a combination of diagnostic methods, tuberculosis-like lesion (TBLL) detection and nucleic acids detection by conventional and molecular analyses. Conventional PCR on wild boar tissues showed that 75% (9/12) of the lymph node samples were positive for Mycobacterium bovis (95% CI: 46.8–91.1). For macaques, 33.3% (10/30) were positive for Mycobacterium avium (95% CI: 19.2–51.2).Wild animals are considered reservoirs, contributing to the transmission of emerging zoonotic diseases such as tuberculosis (TB). A cross-sectional study was conducted by opportunistic sampling from fresh carcasses of free-ranging wild boar (n = 30), and free-ranging wild macaques (n = 42). Stained smears from these tissues were tested for acid-fast bacilli (AFB) with Ziehl–Neelsen staining. Mycobacterial culture was conducted using Lowenstein–Jensen media and Middlebrook 7H11 agar media. Polymerase chain reaction (PCR) was performed through the detection of the 16S rRNA gene, with multiple sets of primers for the detection of Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium complex (MAC). In wild boars, 30% (9/30; 95% Confidence Interval: 16.7–47.9%) of examined samples showed gross tuberculosis-like lesions (TBLLs). Multiple nodular lesions that were necrotic/miliary with cavitation were found in the submandibular lymph nodes, tonsils, lungs, kidney and liver, while single nodular lesions were found in the mediastinal lymph nodes, spleen and mesenteric lymph nodes. Conventional PCR on the submandibular lymphoid tissues of wild boar (nine samples with TBLLs and three non-TBLL samples) showed that 75% (9/12) were positive for Mycobacterium bovis (95% CI: 46.8–91.1), and 91% (CI: 64.6–98.5) were positive for Mycobacterium avium. For macaques, 33.3% (10/30) were positive for M. avium (95% CI: 19.2–51.2) but negative for MTBC.

The 7H11 Agar Medium Supplemented with Calf Bovine Serum for Susceptibility Testing of Mycobacterium tuberculosis Isolates Against Pyrazinamide.

Despite its importance, pyrazinamide (PZA) is a blind spot in drug susceptibility testing in tuberculosis laboratories. The aim of this study was to set up a reliable agar-based proportion method for detection of PZA-resistant phenotypes using Middlebrook 7H11 agar supplemented with calf bovine serum (CBS) compared with albumin/dextrose/catalase (ADC) enrichment and pncA/rpsA sequencing results. The 7H11 agar medium supplemented with 10% ADC or 10% CBS (pH 6.2) and 100 μg/mL PZA was used to detect PZA resistance among 64 Mycobacterium tuberculosis isolates. Sanger sequencing and whole-genome sequencing were performed to track mutations in the pncA, rpsA, and their upstream regions. A total of 43 rifampicin/multidrug-resistant, 20 drug-susceptible, and 1 isoniazid mono-resistant M. tuberculosis isolates were investigated. The 7H11+ADC and 7H11+CBS could detect 22 and 23 PZA-resistant strains, respectively. With the same specificity, the sensitivity and accuracy of 7H11+CBS was found to be a little greater than 7H11+ADC in PZA resistance detection compared with sequencing results. Twenty-four mutant strains were found to have different mutations in pncA-upstream, pncA and rpsA genes, in which Gly97Asp was the most dominant mutation. The results obtained from 7H11+CBS were comparable to the results of 7H11+ADC. Therefore, the 7H11 agar proportion method would be a less-expensive test using CBS and produces reliable results.

Validation of a Novel Diagnostic Approach Combining the VersaTREK™ System for Recovery and Real-Time PCR for the Identification of Mycobacterium chimaera in Water Samples.

Mycobacterium chimaera is an emerging pathogen associated with endocarditis and vasculitis following cardiac surgery. Although it can take up to 6–8 weeks to culture on selective solid media, culture-based detection remains the gold standard for diagnosis, so more rapid methods are urgently needed. For the present study, we processed environmental M. chimaera infected simulates at volumes defined in international guidelines. Each preparation underwent real-time PCR; inoculates were placed in a VersaTREK™ automated microbial detection system and onto selective Middlebrook 7H11 agar plates. The validation tests showed that real-time PCR detected DNA up to a concentration of 10 ng/µL. A comparison of the isolation tests showed that the PCR method detected DNA in a dilution of ×102 CFU/mL in the bacterial suspensions, whereas the limit of detection in the VersaTREK™ was <10 CFU/mL. Within less than 3 days, the VersaTREK™ detected an initial bacterial load of 100 CFU. The detection limit did not seem to be influenced by NaOH decontamination or the initial water sample volume; analytical sensitivity was 1.5 × 102 CFU/mL; positivity was determined in under 15 days. VersaTREK™ can expedite mycobacterial growth in a culture. When combined with PCR, it can increase the overall recovery of mycobacteria in environmental samples, making it potentially applicable for microbial control in the hospital setting and also in environments with low levels of contamination by viable mycobacteria.

In Vitro Activity of a Novel Quinolone, UB-8902, Against Ofloxacin-Resistant Mycobacterium tuberculosis Isolates.

The main objective of this study was to compare in vitro activities of a novel fluoroquinolone (FQ), UB-8902, with ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MOX) against Mycobacterium tuberculosis isolates. Eleven OFX-resistant and 11 drug-susceptible clinical isolates were studied. Individual minimum inhibitory concentrations of OFX, LFX, MOX, and UB-8902 were determined using Middlebrook 7H11 agar. The concentrations studied ranged from 0.125 to 128 μg/mL in twofold dilutions. UB-8902 was more active than LFX and similar to MOX for OFX-resistant M. tuberculosis isolates. In addition, UB-8902 and MOX showed equal activity against drug-susceptible isolates, both being more active than OFX and LFX. In conclusion, the new FQ, UB-8902, showed good activity against OFX-resistant isolates. Moreover, it showed better activity than OFX and LFX and was equivalent to MOX against FQ-susceptible clinical isolates. UB-8902 can be considered as a drug with potential antituberculous activity, similar to MOX.

The effect of sodium thiosulfate on the recovery of Mycobacterium chimaera from heater–cooler unit water samples

Heater-cooler units (HCUs) have been implicated in the recent global outbreak of invasive Mycobacterium chimaera infection among patients following cardiothoracic surgery. Because infected patients tend to remain asymptomatic for extended periods, detection of M.chimaera from HCUs in real time is essential to halting the ongoing M.chimaera HCU-associated outbreak. Sample collection protocols to evaluate the presence of M.chimaera offer conflicting recommendations regarding the addition of sodium thiosulfate (NaT) during the collection process. To study the effect of NaT on M.chimaera recovery and culture contamination. Seventy-six paired HCU water samples (with and without NaT) were collected, processed and cultured simultaneously into Lowenstein-Jensen slants, Middlebrook 7H10 agar plates, and mycobacterial growth indicator tubes (MGITs), and incubated at 37°C. A subset of 31 paired samples was additionally cultured on MGITs and incubated at 30°C. Of 76 samples incubated at 37°C in each of the three media, with and without NaT, M.chimaera was identified in at least one aliquot of 21 samples. The presence of NaT did not significantly increase the probability of recovering M.chimaera in a multi-variable conditional logistic model and culture contamination rates were similar between aliquots with and without NaT. In the subset of samples cultured on MGITs at both 30°C and 37°C, the presence of NaT again was not associated with M.chimaera recovery, but was significantly associated with reduced culture contamination.

BCG shortage: Reassessing the clinical viability of Bacillus Calmette-Guerin (BCG) after reconstitution.

534 Background: To address the current worldwide shortage of BCG, the AUA and SUO recommend reducing the dose to 1/3rd vial and enabling 3 patients to be treated in one setting. The manufacturer states that BCG must be used immediately after reconstitution, despite literature in the bacteriological world suggesting that M. Bovis might be viable for longer than a few hours. Herein we sought to study the viability of BCG after re-constitution at time points relevant to clinical practice. Methods: TICE BCG from separate lots was reconstituted per the manufacturer’s guidance and stored at 4 °C without light exposure. At predetermined time points, BCG was inoculated on Selective Middlebrook 7H11 agar in triplicate and incubated at 37°C with 5% CO2. M. smegmatis served as a positive control and un-inoculated media was incubated for 2 weeks as a negative control. CFUs were assessed between 3 and 4 weeks from plating. Acid-fast staining confirmed the presence of BCG. Data was analyzed as the mean of replicated experiments and compared to the reference (Time 0) using Student’s T-tests. Results: No significant difference in CFUs was observed for BCG between 0 and 8 hours after reconstitution (Table). Colony forming units significantly declined starting 24 hours after reconstitution, though the magnitude of this difference was less than 10 fold (which falls within the range of CFUs listed for the vial by manufacturer). Viability remained constant for both lots analyzed. Conclusions: Given the recurrent shortages of BCG, split dosing may become a clinical necessity. This often presents logistic quandaries since the manufacturer recommends each vial must be used within 2 hours. We have shown that the viability of TICE BCG is unaltered at least 8 hours after reconstitution and only begins to decline at 24 hours after reconstitution. This should allow pharmacists and physicians administering BCG more leeway in scheduling patients for split dose therapy. [Table: see text]

Performance of Different Solid and Liquid Culture Media for the Improvement of Tuberculin Production

Tuberculosis (TB) is one of the most important zoonotic bacterial diseases. A huge economic loss which could be direct or indirect are associated with the disease. Currently, the primary methods used for detection of TB in humans and cattle include the measurement of a delayed type hypersensitivity to purified protein derivative (PPD). So, the need for preparation of purified PPD with adequate properties and increasing the final PPD yield with decreasing the time of tuberculin production has stimulated the interest in the development of its preparation. Our study was performed to compare between the standard and modified media for improving tuberculin production. Middle brook 7H10 agar medium was used as a modified basic medium for mycobacterial growth, followed by cultivation of mycobacteria on Middle brook 7H9 broth medium. For the production, strains were inoculated onto the culture medium (Dorest Henly synthetic medium). Other steps for tuberculin production was done according to standard Weighbridge protocol. The results demonstrated that the using of both Middle brook 7H10 agar and Middle brook 7H9 broth instead of Lowenstein-Jensen (LJ) and glycerin broth media which used in currently produced tuberculin, have better physical and chemical properties. In addition, reducing the time required for production by accelerating the time of microbial growth. Also, it was found that the tuberculin produced using modified media was slightly more potent or the same as currently tuberculin produced. So, both Middle brook 7H10 agar and Middle brook 7H9 broth media are recommended for production of tuberculin saving time and increasing potency of the product but more investigation was recommended for estimation types of protein present in both locally prepared and modified tuberculin.

Detection of airborne bacteria from patient spaces in tuberculosis hospital.

The spread of nosocomial bacterial infection greatly threatens public health and the impact of nosocomial infection worsens if highly pathogenic bacteria, Mycobacterium tuberculosis as an instance, involves. In this study, we have investigated the presence of airborne M. tuberculosis in a specialized tuberculosis hospital. The study sites selected were waiting room I, II, and ward VI patient lounge, Masan National Tuberculosis Hospital, where the modern ventilation system is on the operation for opportunistic infection prevention. The air samples were collected from the different sites three times for 1 day, and after air collection, air sampled disposable filter membrane was incubated for 4 weeks on nine Middlebrook 7H11 agar plates. Our data showed that out of nine incubated 7H11 plate agars, four plates showed bacterial growth and these grown bacterial colonies were isolated and identified. Among bacterial species identified, there was a colony of Mycobacterium mageritense, one of nontuberculous Mycobacteria. Although there was no M. tuberculosis, the cause of tuberculous disease and transmitted through the nosocomial infection, all pathogens detected were known to be associated with nosocomial infection. Hospitals dealing with infectious diseases should always be wary that ventilation system does not guarantee safety from airborne pathogen exposure hence should continuously monitor the presence of other hospital-associated infection causing pathogenic microorganisms.

Evaluation of a novel rapidly-growing mycobacteria medium for isolation of Mycobacterium abscessus complex from respiratory specimens from patients with bronchiectasis

This single center study assessed the performance of a novel solid rapidly-growing mycobacteria (RGM) medium for the recovery of nontuberculous mycobacteria (NTM), especially Mycobacterium abscessus complex, in patients with underlying bronchiectasis. A total of 297 mycobacterial sputa from 116 patients were plated directly on RGM medium and, following decontamination, onto an agar biplate [Middlebrook 7H11 and Mitchison (selective) agar] and into broth media (VersaTrek). The recovery of M. abscessus complex was increased by approximately 12% by implementation of the RGM medium. Contamination was reduced to 2% from 48% and 95% on routine solid media and broth cultures respectively. Our study corroborated previous studies in that recovery of M. abscessus complex was enhanced and contamination was virtually eliminated without the need for specimen decontamination when utilizing RGM medium.

RpoB Mutations Causing Discordant Rifampicin Susceptibility in Mycobacterium tuberculosis: Retrospective Analysis of Prevalence, Phenotypic, Genotypic, and Treatment Outcomes.

BackgroundDiscordant genotypic/phenotypic rifampicin susceptibility testing in Mycobacterium tuberculosis is a significant challenge, yet there are limited data on its prevalence and how best to manage such patients. Whether to treat isolates with rpoB mutations not conferring phenotypic resistance as susceptible or multidrug-resistant tuberculosis (MDR-TB) is unknown. We describe phenotypic and genotypic characteristics of discordant isolates and clinical characteristics and treatment outcomes of affected patients in KwaZulu-Natal, South Africa.MethodsWe analyzed clinical isolates showing rifampicin resistance on GenoType MTBDRplus while susceptible on 1% agar proportion method. We measured rifampicin minimum inhibitory concentrations (MICs) using Middlebrook 7H10 agar dilution and BACTEC MGIT 960. Sensititre MYCOTB plates were used for drug-susceptibility testing, and rpoB gene sequencing was performed on all isolates. Local MDR-TB program data were reviewed for clinical information and patient outcomes.ResultsDiscordant isolates constituted 4.6% (60) of 1302 rifampicin-resistant cases over the study period. Of these, 62% remained susceptible to isoniazid and 98% remained susceptible to rifabutin. Rifampicin MICs were close to the critical concentration of 1 µg/mL (0.5–2 µg/mL) for 83% of isolates. The most frequent rpoB mutations were Q513P (25.3%), D516V (19.2%), and D516Y (13.3%). Whereas 70% were human immunodeficiency virus infected, the mean CD4 count was 289 cells/mm3 and 87% were receiving antiretroviral therapy. Standard therapy for MDR-TB was used and 53% achieved successful treatment outcomes.ConclusionsRifampicin-discordant TB is not uncommon and sequencing is required to confirm results. The high susceptibility to rifabutin and isoniazid and poor treatment outcomes with the current regimen suggest a potential utility for rifabutin-based therapy.

Validation of bedaquiline drug-susceptibility testing by BACTEC MGIT 960 system for Mycobacterium tuberculosis.

Bedaquiline (BDQ) is a new antituberculosis (TB) drug effectively used for the treatment of multidrug-resistant and extensively drug-resistant TB. However, the reports on drug-susceptibility testing (DST) for BDQ are scarce. The study aimed to validate and standardize BDQ DST by BACTEC MGIT 960 system for Mycobacterium tuberculosis. A panel of ten M. tuberculosis isolates comprising 8 BDQ sensitive and 2 BDQ resistant strains were used to test accuracy, repeatability, and reproducibility of BDQDST by MGIT 960. BDQ DST by Middlebrook 7H11 agar method using polystyrene tubes was used as a standard method to calculate the accuracy of the validation. DST by MGIT for BDQ showed 100% accuracy, repeatability, and reproducibility, although variations were observed in the growth units of the "test" MGIT tubes between technologist and drug stocks while testing for reproducibility. BDQ DST by MGIT 960 system is accurate, repeatable, and reproducible and hence can be implemented in certified laboratories routinely performing DST by MGIT 960 system.

Isolation and analysis of the molecular epidemiology and zoonotic significance of Mycobacterium tuberculosis in domestic and wildlife ruminants from three states in India.

The majority of tuberculosis cases in ruminants are caused by Mycobacterium bovis (MB). However, in this study, the authors reported the isolation of Mycobacterium tuberculosis (MT) from bovine milk, nasal swabs and post-mortem tissue samples (n = 841) collected from cattle and buffaloes in the states of Telangana, Maharashtra and Gujarat in India in the period from 2010 to 2015. The isolates (n = 7) were confirmed as Mycobacterium due to their growth characteristics and colony morphology in a commercial liquid medium Mycobacterial Growth Indicator Tube (MGIT)™ employing the BD BACTEC™ MGIT™ 960 system and the Löwenstein-Jensen (LJ) medium supplemented with glycerol but not with sodium pyruvate, and BD-DIFCO™ Middlebrook 7H10 agar containing oleic albumin dextrose catalase (OADC). These isolates were initially identified as members of the M. tuberculosis complex (MTC) using a commercial nested polymerase chain reaction (PCR) kit based on the IS6110 MTC specific nucleotide sequence. The isolates were confirmed as MT using three commercial line probe assay kits, were further genotyped, and the spoligotypes identified were of East African Indian (EAI) 3_IND, EAI5, Central-Asian (CAS) 1_DELHI, U and T1 lineages. Two MT isolates from one antelope (Antilope cervipara) andone gazelle (Gazella bennettii) from Gujarat, which were identified previously, were spoligotyped during this study and identified as belonging to EAI3_IND and EAI5 lineages, respectively. The epidemiological significance and zoonotic implications of regional presence and documentation of the same or two differents poligotypes in different species within the family Bovidae as well as humans is discussed.

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.

Deletion of PimE mannosyltransferase results in increased copper sensitivity in Mycobacterium smegmatis.

The unique cell envelope structure of Mycobacterium tuberculosis is fundamental to its pathogenesis. Phosphatidylinositol (PI)-anchored glycolipids, such as phosphatidylinositol mannosides (PIMs), lipomannan and lipoarabinomannan, are essential components of the cell envelope widely conserved among mycobacteria, but their roles in the cell envelope integrity are not fully understood. We previously identified PimE in Mycobacterium smegmatis, a nonpathogenic model organism, as a mannosyltransferase that catalyzes the fifth mannose transfer for the biosynthesis of hexamannosyl PIMs. Our analyses, reported here, further demonstrate that the growth of the pimE deletion mutant (ΔpimE) is defective in the presence of copper. We first found that the small colony phenotype of ΔpimE on a solid Middlebrook 7H10 agar surface was alleviated when grown on M63 agar. Comparative analysis of the two media led us to identify copper in Middlebrook 7H10 as the cause of growth retardation seen in ΔpimE. We further demonstrated that ΔpimE is sensitized to several antibiotics, but the increased sensitivities were independent of the presence of copper. We conclude that the deletion of the pimE gene does not cause growth defects under optimal growth conditions, but makes the cell envelope vulnerable to toxic compounds such as copper and antibiotics.

Evaluation of the BACTEC MGIT 960 SL DST Kit and the GenoType MTBDRsl Test for Detecting Extensively Drug-resistant Tuberculosis Cases.

The present study aimed to evaluate the performances of the BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl test for detecting second-line antituberculosis drug resistance in Multidrug-resistant TB (MDR-TB) cases. Forty-six MDR-TB strains were studied. Second-line antituberculosis drug resistances were detected using the BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl test. The Middlebrook 7H10 agar proportion method was used as the reference test. The sensitivity and specificity values for the BACTEC MGIT 960 SL DST kit were both 100% for amikacin, kanamycin, capreomycin (4 µg/mL), and ofloxacin; 100% and 95.3%, respectively, for capreomycin (10 µg/mL); and 85.7% and 100%, respectively, for moxifloxacin (0.5 µg/mL). The sensitivity and specificity values for the GenoType MTBDRsl test to detect fluoroquinolone and aminoglycoside/cyclic peptide resistance were 88.9% and 100%, respectively, for ofloxacin and 85.7% and 94.9%, respectively, for moxifloxacin (0.5 µg/mL). The accuracy of the GenoType MTBDRsl assay for kanamycin, capreomycin, ofloxacin, and moxifloxacin was lower than that of the BACTEC MGIT 960 SL DST. The BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl were successful in detecting second-line antituberculosis drug resistance. Preliminary results of the GenoType MTBDRsl are very valuable for early treatment decisions, but we still recommend additional BACTEC MGIT 960 SL DST kit usage in the routine evaluation of drug-resistant tuberculosis.

Mycobacterium stephanolepidis sp. nov., a rapidly growing species related to Mycobacterium chelonae, isolated from marine teleost fish, Stephanolepis cirrhifer.

A previously undescribed rapidly growing, non-pigmented mycobacterium was identified based on biochemical and nucleic acid analyses, as well as growth characteristics. Seven isolates were cultured from samples collected from five thread-sail filefish (Stephanolepis cirrhifer) and two farmed black scraper (Thamnaconus modestus). Bacterial growth occurred at 15-35 °C on Middlebrook 7H11 agar. The bacteria were positive for catalase activity at 68 °C and urease activity, intermediate for iron uptake, and negative for Tween 80 hydrolysis, nitrate reduction, semi-quantitative catalase activity and arylsulfatase activity at day 3. No growth was observed on Middlebrook 7H11 agar supplemented with picric acid, and very little growth was observed in the presence of 5 % NaCl. α- and α'-mycolates were identified in the cell walls, and a unique profile of the fatty acid methyl esters and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiles of the protein and cell-wall lipids were acquired. Sequence analysis revealed that the seven isolates shared identical sequences for the 16S rRNA, rpoB, hsp65, recA and sodA genes. Phylogenetic analysis of the five gene sequences confirmed that the isolates were unique, but closely related to Mycobacterium chelonae. Antibiotic susceptibility testing revealed the minimum inhibitory concentration (MIC) of clarithromycin against this novel species was <0.25 µg ml-1, which was lower than that for Mycobacterium salmoniphilum. The hsp65 PCR restriction enzyme analysis pattern differed from those of M. chelonae and M. salmoniphilum. Based on these findings, the name Mycobacterium stephanolepidis sp. nov. is proposed for this novel species, with the type strain being NJB0901T (=JCM 31611T=KCTC 39843T).