Middlebrook 7H9 Medium Research Articles

Штаммы Mycobacterium tuberculosis с мутациями в gyrA различаются по уровню конкурентного фитнеса

As M. tuberculosis strains develop resistance to fluoroquinolones, pools of M. tuberculosis sensitive to drugs of this group and pools of M. tuberculosis with different resistance determinants can simultaneously coexist in the host organism. The goal of this research was to run an in vitro investigation of growth characteristics of M. tuberculosis strains which have different genetic determinants of resistance to fluoroquinolones, in the setting of competition for nutrients. The research used five clinical strains of multidrug-resistant M. tuberculosis differing in gyrA structure. Strains were cultured in pairs and individually under optimal conditions (Middlebrook 7H9 medium) and under conditions of multistress (50% Middlebrook 7H9 medium, 2 mM KNO2, 0.02% H2O2). The experiment took 21 days. The number of cells of each co-cultured strain was estimated from calibration curves. These curves showed the dependence of the threshold cycle of the polymerase chain reaction — respective to the channel targeted by the mutation — on the concentration of M. tuberculosis cells. The competitive fitness value and specific growth rate were calculated from the number of cells of each strain when co-cultured. M. tuberculosis strains with mutations in gyrA were found to be inferior in growth rate to the wild-type gyrA strain, which was particularly pronounced under multistress conditions. The strain with the most common gyrA_D94G mutation had the lowest growth rate of all strains examined. It has been hypothesised that the slow growth of M. tuberculosis with this mutation may lead to tolerance to anti-tuberculosis drugs, and as a result, the strain gains an advantage under chemotherapy conditions compared to other gyrA mutant variants.

Comparing minimum inhibitory concentrations of amikacin for pulmonary Mycobacterium avium complex disease: An analysis of culture media differences

Mycobacterium avium complex (MAC) is considered a paramount microbe, especially in East Asia, including Japan. The commonly used commercial Minimum Inhibitory Concentrations (MIC) assay using Middlebrook 7H9 (7H9) medium deviates from the latest Clinical and Laboratory Standards Institute (CLSI) guidelines. Alternatively, measurement with cation-adjusted Mueller-Hinton broth (CAMHB) that conforms to CLSI standards is not yet widely available. Following the approval and commercialization of amikacin liposome inhalation suspension (ALIS) in 2021, a more precise evaluation of amikacin (AMK) susceptibility in MAC is necessary for treatment decisions. In the present study, 33 sputum samples were extracted from 27 patients, and MICs of AMK were compared between the frequently used 7H9 and the recommended CAMHB of the isolated MAC strains. The history of exposure to aminoglycosides for each sample was also added as clinical information. The findings indicated that there was only an 18% concordance rate in MIC between the two media, with 19 samples (58%) indicating lower MICs in 7H9 relative to CAMHB. The 17 samples had a history of exposure to aminoglycosides for periods ranging from 1.5 to 28 months. Specifically, 10 samples were exposed to amikacin by inhalation and intravenous injection, and the remaining seven samples had a history of ALIS inhalation. Samples with a prior utilization of aminoglycosides were significantly predisposed to developing resistance to ALIS compared to those without such a history (P = 0.046). Physicians are encouraged to scrutinize the findings of susceptibility testing utilizing CLSI-endorsed MIC assay using CAMHB medium to ascertain the optimal therapeutic approach.

The effect of in vitro consecutive passages and culture medium on the genetic variations in BCG Pasteur 1173P2 vaccine.

Since the introduction of the Bacillus Calmette-Guérin (BCG) vaccine, the genomes of vaccine strains have undergone variations due to repeated passages in different laboratories and vaccine production facilities. Genetic variations have been considered as one of the effective factors in the BCG variable protective efficacy. Consecutive subcultures have been shown to play an essential role in causing genetic variations in several microorganisms, including Mycobacterium bovis BCG. Therefore, the world health organization (WHO) recommendation to limit the passages of master seed lot in the BCG vaccine production should be considered. Besides, the role of other external variables such as quality of the raw ingredients of the culture media, the type of the culture medium and the cultivation methods in the vaccine production has been poorly studied. Here, the effect of passages and culture medium on genetic variations in a BCG seed lot was investigated during a year. The findings of this study revealed a total of 19 variants compared to seed lot while the passages were more than the number recommended by WHO. The first culture of seed lot in the Sauton broth and Middlebrook 7H9 media, and the last subculture in Sauton broth had the least and the most variants, respectively. The observation of the higher number of variants in the last cultures on Sauton broth and Middlebrook 7H9 in comparison to the first and the middle cultures may indicate the effect of passages on the genetic variations in BCG. Additionally, more variants in BCG grown in the Sauton broth do not necessarily represent the greater ability of this medium to cause genetic mutations. For a better conclusion, it is required to examine the medium components as independent variables.

Screening for Anti Mycobacterium tuberculosis Activity of Streptomyces sp. from Lapindo Mud in Sidoarjo, Indonesia

BACKGROUND: Streptomyces sp. from Indonesian soil have not been explored and isolated to find new strains as a source of antibiotics for the treatment of tuberculosis (TB) disease. AIM: In this study, the effect of Streptomyces spp. from Lapindo mud in Sidoarjo, Indonesia be observed, to find out whether Streptomyces spp. has anti-TB activity. METHODS: The primers Strep F; 5-AGAGTTTGAT CCTGKGTCAG-3 and Strep R; 5-AAGGGAG GTGATCCAKKGKGA-3 were used in polymerase chain reaction amplification of the 16S rRNA gene against Streptomyces strains. The anti-TB activity of Streptomyces sp. was determined by broth dilution method using Middlebrook 7H9 media. RESULTS: The results showed that new types of Streptomyces spp., namely, Streptomyces A, Streptomyces D, Streptomyces Ea, Streptomyces Ep, Streptomyces I, Streptomyces F, and Streptomyces G from garbage dump soils. This result also showed that the activity of Streptomyces I, Streptomyces F, and Streptomyces G could inhibit the Mycobacterium TB growth by with inhibitory zones, respectively, 2 ± 0.3; 8 ± 0.7 and 15 ± 0.9mm, while Streptomyces A, Streptomyces D, Streptomyces Ea, and Streptomyces Ep did not inhibit M. TB. CONCLUSION: Thus, from the results obtained, it can be concluded that Streptomyces extract mainly Streptomyces G has promising anti-TB activity by preliminary in vitro techniques. Therefore, it has the definite potential as a source of compounds that may be developed further into antimycobacterial drugs.

Pigmented hypopyon – An approach to management

A 60-year-old Indian male presented with diminution of vision in the right eye for 3 months duration. Subsequent evaluation showed signs of granulomatous anterior uveitis, scleral thinning and brown colored pigmented hypopyon in the right eye. Patient was thoroughly evaluated for all possible associations of pigmented hypopyon. He was found to have positive mantoux test, QTB Gold test, positive culture for Mycobacterium tuberculosis on Middlebrook 7H9 medium and positive MPT64 antigen test confirming the diagnosis of intraocular tuberculosis. He was treated with anti-tubercular treatment and oral steroids and subsequently with dexamethasone intravitreal implant. He had complete resolution of pigmented hypopyon with improvement in visual acuity. This case highlights an approach to management in cases of pigmented hypopyon and also highlights that an aggressive management in such cases is pertinent to saving the eye and vision.

Proteome characterization of the culture supernatant of Mycobacterium bovis in different growth stages

This study aimed to identify proteins secreted by Mycobacterium bovis into culture medium at different stages of bacterial growth. A field strain of M. bovis was grown in Middlebrook 7H9 media and culture supernatant was collected at three-time points representing three different phases of growth (early exponential, late exponential, and stationary phases). Supernatants were double filtered, digested by trypsin and analyzed by LC-MS/MS. The study found 15, 21, and 16 proteins in early, mid and late growth phases, respectively. In total, 22 proteins were identified, 18 of which were reported or predicted to have a cell wall or extracellular localization. To our knowledge, this is the first study to identify proteins secreted into the culture medium by a field strain of M. bovis in three different stages of growth. The dataset generated here provides candidate proteins with the potential for the development of serological diagnostic reagents or vaccine for bovine tuberculosis. Data are available via ProteomeXchange with identifier PXD017817.

In vitro activity of bedaquiline against Mycobacterium avium complex.

Introduction. Nontuberculous mycobacteria (NTM) are widespread in the environment and can cause various diseases in humans, especially immunocompromised patients.Hypothesis. Treatment of diseases caused by NTM is a complicated issue, mainly due to the resistance of the pathogen to most antimicrobial agents. Bedaquiline (Bdq) is now widely used for the treatment of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB).Aim. The main goal of our study was to evaluate the activity of Bdq against Mycobacterium avium complex (MAC), the most common species among NTM.Methodology. A total of 166 MAC cultures (124 Mycobacterium avium and 42 Mycobacterium intracellulare) were studied. The minimum inhibitory concentrations (MICs) of Bdq for M. avium and M. intracellulare were obtained by twofold serial dilutions in the Middlebrook 7H9 medium. MIC ranges were determined and the MIC50, MIC90 and ECOFF values were obtained.Results. The MICs in respect of M. avium ranged from 0.003 to 1.0 µg ml-1; those for M. intracellulare ranged from 0.003 to 0.5 µg ml-1. The Bdq MIC50 and MIC90 values were found to be 0.015 and 0.12 µg ml-1 , respectively, for M. avium and 0.007 and 0.06 µg ml-1, respectively, for M. intracellulare. The tentative ECOFF values for M. avium and M. intracellulare were 0.12 and 0.06 µg ml-1, respectively.Conclusion. The main bedaquiline susceptibility parameters for MAC strains isolated in the Moscow region were determined.

Mycobacterial Populations Partly Change the Proportions of the Cells Undergoing Asymmetric/Symmetric Divisions in Response to Glycerol Levels in Growth Medium.

Twenty to thirty percent of the septating mycobacterial cells of the mid-log phase population showed highly deviated asymmetric constriction during division (ACD), while the remaining underwent symmetric constriction during division (SCD). The ACD produced short-sized cells (SCs) and normal/long-sized cells (NCs) as the sister–daughter cells, but with significant differential susceptibility to antibiotic/oxidative/nitrite stress. Here we report that, at 0.2% glycerol, formulated in the Middlebrook 7H9 medium, a significantly high proportion of the cells were divided by SCD. When the glycerol concentration decreased to 0.1% due to cell-growth/division, the ACD proportion gradually increased until the ACD:SCD ratio reached ~50:50. With further decrease in the glycerol levels, the SCD proportion increased with concomitant decrease in the ACD proportion. Maintenance of glycerol at 0.1%, through replenishment, held the ACD:SCD proportion at ~50:50. Transfer of the cells from one culture with a specific glycerol level to the supernatant from another culture, with a different glycerol level, made the cells change the ACD:SCD proportion to that of the culture from which the supernatant was taken. RT-qPCR data showed the possibility of diadenosine tetraphosphate phosphorylase (MSMEG_2932), phosphatidylinositol synthase (MSMEG_2933), and a Nudix family hydrolase (MSMEG_2936) involved in the ACD:SCD proportion-change in response to glycerol levels. We also discussed its physiological significance.

Multicentre testing of the EUCAST broth microdilution reference method for MIC determination on Mycobacterium tuberculosis

ObjectivesThe first objective of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee for antimycobacterial susceptibility testing (AMST), launched in 2016, was to set a reference method for determining the MICs of antituberculous agents, since many protocols are used worldwide and a consensus one is needed for the determination of microbiological breakpoints. MethodsDuring 2017 and 2018, MIC determination protocols were evaluated prospectively in a multicentre study within the four AMST laboratories. MIC results were obtained for isoniazid, levofloxacin and amikacin on the reference strain Mycobacterium tuberculosis H37Rv ATCC 27294. Broth microdilution (BMD) in Middlebrook 7H9 and solid medium dilution (SMD) in Middlebrook 7H10 were performed using two inoculum concentrations. MICs were interpreted with regard to visual and 99% inhibition after 7, 14 or 21 days of incubation for BMD and 21 days for SMD. ResultsFollowing the EUCAST reference protocol, intra- and inter-assay agreements were within ±1 MIC dilution for >95% of the observations for the three drugs in both methods. MIC values, presented as MIC mode (range) for BMD and SMD respectively, were: 0.03 (0.015–0.06) mg/L and 0.12 (0.06–0.25) mg/L for isoniazid, 0.25 mg/L (0.25–0.5) and 0.5 mg/L (0.12–0.5) for levofloxacin, and 0.5 mg/L (0.5–1.0) and 0.5 mg/L (0.5–1.0) for amikacin. ConclusionsBoth SMD and BMD were reproducible and eligible as a reference method for MIC determination of the Mycobacterium tuberculosis complex (MTBC). BMD was finally selected as the EUCAST reference method. From now on it will be used to set epidemiological cut-off values and clinical breakpoints of new and old antituberculous agents.

Optimisation of Mycobacterium bovis BCG Fermentation and Storage Survival.

Mycobacterium bovis Bacillus Calmette–Guérin (M. bovis BCG) was generated over a century ago for protection against Mycobacterium tuberculosis (Mtb) and is one the oldest vaccines still in use. The BCG vaccine is currently produced using a pellicle growth method, which is a complex and lengthy process that has been challenging to standardise. Fermentation for BCG vaccine production would reduce the complexity associated with pellicle growth and increase batch to batch reproducibility. This more standardised growth lends itself to quantification of the total number of bacilli in the BCG vaccine by alternative approaches, such as flow cytometry, which can also provide information about the metabolic status of the bacterial population. The aim of the work reported here was to determine which batch fermentation conditions and storage conditions give the most favourable outcomes in terms of the yield and stability of live M. bovis BCG Danish bacilli. We compared different media and assessed growth over time in culture, using total viable counts, total bacterial counts, and turbidity throughout culture. We applied fluorescent viability dyes and flow cytometry to measure real-time within-culture viability. Culture samples were stored in different cryoprotectants at different temperatures to assess the effect of these combined conditions on bacterial titres. Roisin’s minimal medium and Middlebrook 7H9 medium gave comparable, high titres in fermenters. Flow cytometry proved to be a useful tool for enumeration of total bacterial counts and in the assessment of within-culture cell viability and cell death. Of the cryoprotectants evaluated, 5% (v/v) DMSO showed the most significant positive effect on survival and reduced the negative effects of low temperature storage on M. bovis BCG Danish viability. In conclusion, we have shown a reproducible, more standardised approach for the production, evaluation, and storage of high titre, viable, BCG vaccine.

Aktivitas Anti-Mycobacterium tuberculosis Kombinasi (-)-Epigallocatechin-Gallate (EGCG) dan Obat Antituberkulosis Lini Pertama

Tuberculosis is a global health problem, and there is even an increase in cases of multidrug-resistant tuberculosis in the world. Therefore, research is needed that can find new anti-tuberculosis drugs (OAT) that are more effective for tuberculosis treatment. In this study, the effect of (-)-epigallocatechin-gallate (EGCG) of tea leaves (Camellia sinensis) combined with the first-line OAT will be observed, in order to find out whether EGCG has anti-tuberculosis activity and can increase the potential of first-line OAT in-vitro. The anti-tuberculosis activity of EGCG was determined by broth dilution method using Middlebrook 7H9 media at concentration of 50, 100, 150, dan 200 ppm, then the potential of first-line OAT before and after combined with the EGCG was observed. The results showed that the activity of EGCG at concentration 50 ppm and 100 ppm could inhibit the Mycobacterium tuberculosis growth by 80%, at concentration 150 ppm by 90%, and at concentration 200 ppm by 100%. First-line OAT activity before combined with EGCG was ≥ 90% at 5 ppm rifampicin, 0.5 ppm isoniazid, 50 ppm pyrazinamide, and 5 ppm ethambutol. Whereas after combined with EGCG, the potential of each drug increased, marked by anti-tuberculosis activity achieved ≥ 90% at lower concentrations, i.e. rifampicin 0.5 ppm, isoniazid 0.25 ppm, pyrazinamide 20 ppm, and ethambutol 2 ppm. These results indicated that the potential of each first-line OAT increases after being combined with EGCG, and EGCG has potentiation effect when combined with those drugs. In conclusion, EGCG can increase the first-line OAT activity

ASSESSMENT OF THE MICROSCOPIC– OBSERVATION- DRUG - SUSCEPTIBLITY ASSAY (MODS) AND GENEXPERT ASSAY FOR DIAGNOSIS OF PULMONARY TUBERCULOSIS

Background: Tuberculosis (TB) is considered the most common and highly contagious disease worldwide. Early and accurate diagnosis of TB with detection of multidrug-resistant Mycobacterium tuberculosis (M.TB) is of primary importance for both patient management and infection control. Objectives: To evaluate the performance of Microscopic-Observation Drug-Susceptibility (MODS) assay and GeneXpert MTB/RIF( GeneXpert ) assay in diagnosis of TB and detection of MDR-TB directly from sputum specimens in comparison to the convential diagnostic methods (direct Ziehl-Neelsen "Z-N" stained smear and culture on Lowenstein-Jenson media "LJ"). Patients and Methods: After approval of institutional ethical committee a total of 120 suspected pulmonary TB patients admitted to chest hospitals and dispensaries in Egypt (Giza, Cairo, Alexandria, Dakahlia, Suez, Beheira, Monufia, Qena). Their age was more than 16 years; all of them did not receive any anti tubercular drugs. Sputum samples were collected from each patient and subjected to direct ZN smear, culture and sensitivity of M.TB on LJ and Middlebrook 7H9 medium for MODS assay. Finally Genexpert M.TB/RIF assay was done directly for all sputum samples. Results: Infection rate among males was higher than female (81.7% & 18.7%). Also, it was observed that 56.7% of sputum samples gave positive direct Z-N smear, while 64.2% were positive for both LJ culture &MODS assay. On the other hand, by Genexpert, 68% of cases were positive. The sensitivity and specificity of the various diagnostic methods were studied in relation to LJ culture (gold standard). It was found that MODS assay was rapid that results were obtained in a median of 8 days ranged from (7– 21 days), while median time of LJ was 21dayes ranged from (21– 60). MODS had 100% sensitivity and specificity. Direct smear was less sensitive (88.3%), and GeneXpert was less specific (88.4%), but more rapid results were obtained in 2 hours. Finally, detection of MDR-TB using various methods for 77 isolated M.TB strains ranged from 16.9-19.5% to isoniazid (INH) and 9.8%-18.2% to rifampicin (RMP) by various diagnostic methods with statistically insignificant difference between them. Conclusion: MODS was an optimal alternative rapid diagnostic technique for TB and detection of M.TB and MDR-TB in resource limited settings, results were obtained in a median of 8 days. On the other hand, Gene Xpert was a novel integrated diagnostic PCR analysis in a single hand free step for rapid diagnosis of TB and detection of RMP resistance in clinical specimens in 2 hours but it was expensive.

ASSESSMENT OF THE MICROSCOPIC– OBSERVATION- DRUG - SUSCEPTIBLITY ASSAY (MODS) AND GENEXPERT ASSAY FOR DIAGNOSIS OF PULMONARY TUBERCULOSIS

Background: Tuberculosis (TB) is considered the most common and highly contagious disease worldwide. Early and accurate diagnosis of TB with detection of multidrug-resistant Mycobacterium tuberculosis (M.TB) is of primary importance for both patient management and infection control. Objectives: To evaluate the performance of Microscopic-Observation Drug-Susceptibility (MODS) assay and GeneXpert MTB/RIF( GeneXpert ) assay in diagnosis of TB and detection of MDR-TB directly from sputum specimens in comparison to the convential diagnostic methods (direct Ziehl-Neelsen stained smear and culture on Lowenstein-Jenson media LJ). Patients and Methods: After approval of institutional ethical committee a total of 120 suspected pulmonary TB patients admitted to chest hospitals and dispensaries in Egypt (Giza, Cairo, Alexandria, Dakahlia, Suez, Beheira, Monufia, Qena). Their age was more than 16 years; all of them did not receive any anti tubercular drugs. Sputum samples were collected from each patient and subjected to direct ZN smear, culture and sensitivity of M.TB on LJ and Middlebrook 7H9 medium for MODS assay. Finally Genexpert M.TB/RIF assay was done directly for all sputum samples. Results: Infection rate among males was higher than female (81.7% & 18.7%). Also, it was observed that 56.7% of sputum samples gave positive direct Z-N smear, while 64.2% were positive for both LJ culture &MODS assay. On the other hand, by Genexpert, 68% of cases were positive. The sensitivity and specificity of the various diagnostic methods were studied in relation to LJ culture (gold standard). It was found that MODS assay was rapid that results were obtained in a median of 8 days ranged from (7– 21 days), while median time of LJ was 21dayes ranged from (21– 60). MODS had 100% sensitivity and specificity. Direct smear was less sensitive (88.3%), and GeneXpert was less specific (88.4%), but more rapid results were obtained in 2 hours. Finally, detection of MDR-TB using various methods for 77 isolated M.TB strains ranged from 16.9-19.5% to isoniazid (INH) and 9.8%-18.2% to rifampicin (RMP) by various diagnostic methods with statistically insignificant difference between them. Conclusion: MODS was an optimal alternative rapid diagnostic technique for TB and detection of M.TB and MDR-TB in resource limited settings, results were obtained in a median of 8 days. On the other hand, Gene Xpert was a novel integrated diagnostic PCR analysis in a single hand free step for rapid diagnosis of TB and detection of RMP resistance in clinical specimens in 2 hours but it was expensive.

Plasma concentrations of second-line antituberculosis drugs in relation to minimum inhibitory concentrations in multidrug-resistant tuberculosis patients in China: a study protocol of a prospective observational cohort study

IntroductionIndividualised treatment through therapeutic drug monitoring (TDM) may improve tuberculosis (TB) treatment outcomes but is not routinely implemented. Prospective clinical studies of drug exposure and minimum inhibitory concentrations (MICs) in...

The stability of antimycobacterial drugs in media used for drug susceptibility testing

The emergence of drug-resistant tuberculosis and disease caused by nontuberculous mycobacteria has increased the need for accurate drug susceptibility testing of mycobacteria. The stability of the tested drugs in relevant test media have been understudied.We assessed the stability of isoniazid, rifampicin, clarithromycin, linezolid and amikacin in Middlebrook 7H9 medium and that of clarithromycin, amikacin and cefoxitin in the cation-adjusted Mueller Hinton broth. We used ultra-performance liquid chromatography (UPLC) methods for rifampicin and isoniazid and a microbiological assay for rifampicin, clarithromycin, amikacin, cefoxitin and linezolid.Rifampicin and isoniazid concentrations in Middlebrook 7H9 medium had decreased by 92% and 54% after 7 days. The microbiological assay revealed decreases in drug concentration of ≥75% (rifampicin, clarithromycin, cefoxitin) and 60% (linezolid) after 14 days.With the exception of amikacin, all antimycobacterial drugs were unstable during 14 days of incubation in the preferred media for DST. Drug stability may influence minimum inhibitory concentration measurements.

Phenotypic and Genotypic Characterization of Mycobacteria Isolates from Buruli Ulcer Suspected Patients Reveals the Involvement of Several Mycobacteria in Chronic Skin Lesions

is a cutaneous mycobacterial disease that occurs in tropical countries in sub-Saharan Africa, South-East Asia, Australia and America. The responsible pathogen is Mycobacterium ulcerans. Cote d'Ivoire is the most affected country, with more than 30 endemic health districts reporting a large number of chronic skin lesions. The clinical forms and the severity of ulcers vary from one patient to another. Samples from suspected patients were analyzed by PCR at Pasteur Institute of Cote d'Ivoire, as recommended by WHO. IS2404 sequence was detected in 61% of cases, incriminating M. ulcerans in chronic cutaneous lesions. For the other cases the etiology was not identified, thus raising several questions. Are all reported Buruli ulcer cases really caused by M. ulcerans? Would other mycobacteria be involved in the occurrence of chronic skin lesions considered as Buruli ulcer? BU suspected patients were enrolled in endemic areas of Cote d’Ivoire. Samples were collected from cutaneous lesions and transported to the lab at +4°C in 2 ml of Middlebrook 7H9 medium supplemented by Cetylpiridium chloride. The centrifugation pellet was taken with saline buffer to perform microscopic examination and mycobacteria isolation on Lowenstein-Jensen medium. Biochemical characteristics were described by the nicotinic acid detection according to Konno protocol, by the Nitrates reduction test, by the catalase activity detection at 22° and 68°C and the Wayne's Tween 80 hydrolysis test. Genotypic characteristics were determined by PCR with 1 ml of bacterial suspension targeting the insertion sequences (IS6110, IS2404, and IS2606), the plasmid virulence genes and Miru-VNTR loci (Miru-1, VNTR 6, VNTR 19, ST-1). A total of 47 mycobacterial strains were isolated with 3 different types of colonies whose microscopic examination showed Acid-Alcohol-Resistant Bacilli. 65.9% of isolates expressed biochemical characters in favor of M. ulcerans strains and 6.4% in favor of M. marinum strains. For 29.8% of isolates, the characteristics were related to atypical mycobacterial species. The genotyping targeting the IS6110, IS2606 and IS2404 insertion sequences allowed simultaneous amplification in 53.2% of isolates. IS2404 was amplified in 93.6% of isolates; IS6110 was amplified in 74.5% of isolates and IS2606 was amplified in 70.2% of isolates. Five genotypes were identified corresponding to various species of mycobacteria: genotypes 1 and 2 accounted for 63.8% with all the 3 insertion sequences and biochemical characteristics in favor of M. ulcerans strains; genotype 4 accounted for 6.4% of isolates with insertion sequences and biochemical characteristics in favor of M. marinum strains; The strains of genotypes 3 and 5 expressed molecular and biochemical characters relating to various non-M. ulcerans mycobacteria. Virulence genes were found in 72.3% of isolates corresponding to 90% of M. ulcerans strains and 60.7% of non-M. ulcerans mycobacteria. This study confirmed the involvement of several genotypes of M. ulcerans and other mycobacteria in chronic cutaneous lesions suspected as cases of in Cote d’Ivoire.

Rapid culture-based detection of living mycobacteria using microchannel electrical impedance spectroscopy (m-EIS)

BackgroundMultiple techniques exist for detecting Mycobacteria, each having its own advantages and drawbacks. Among them, automated culture-based systems like the BACTEC-MGIT™ are popular because they are inexpensive, reliable and highly accurate. However, they have a relatively long “time-to-detection” (TTD). Hence, a method that retains the reliability and low-cost of the MGIT system, while reducing TTD would be highly desirable.MethodsLiving bacterial cells possess a membrane potential, on account of which they store charge when subjected to an AC-field. This charge storage (bulk capacitance) can be estimated using impedance measurements at multiple frequencies. An increase in the number of living cells during culture is reflected in an increase in bulk capacitance, and this forms the basis of our detection. M. bovis BCG and M. smegmatis suspensions with differing initial loads are cultured in MGIT media supplemented with OADC and Middlebrook 7H9 media respectively, electrical “scans” taken at regular intervals and the bulk capacitance estimated from the scans. Bulk capacitance estimates at later time-points are statistically compared to the suspension’s baseline value. A statistically significant increase is assumed to indicate the presence of proliferating mycobacteria.ResultsOur TTDs were 60 and 36 h for M. bovis BCG and 20 and 9 h for M. smegmatis with initial loads of 1000 CFU/ml and 100,000 CFU/ml respectively. The corresponding TTDs for the commercial BACTEC MGIT 960 system were 131 and 84.6 h for M. bovis BCG and 41.7 and 12 h for M smegmatis, respectively.ConclusionOur culture-based detection method using multi-frequency impedance measurements is capable of detecting mycobacteria faster than current commercial systems.

In vitro activity of sitafloxacin against Mycobacterium tuberculosis with gyrA/B mutations isolated in Japan.

Purpose. Sitafloxacin (SFX) is a new fluoroquinolone (FQ) that has shown a strong bactericidal effect against Mycobacterium tuberculosis (Mtb) in vitro. However, data on SFX efficacy against Mtb with gyrA/B mutations and its epidemiological cut-off (ECOFF) value remain limited. Therefore, we evaluated and compared the in vitro activity of SFX against gyrA/B-mutant Mtb to that of moxifloxacin (MFX), levofloxacin (LFX) and ciprofloxacin (CFX), and determined the ECOFF for SFX.Methodology. A total of 109 clinical Mtb isolates, including 73 multidrug-resistant (MDR) isolates, were subjected to minimum inhibitory concentration (MIC) analysis in oleic-albumin-dextrose-catalase (OADC)-supplemented Middlebrook 7H9 medium. Our results showed that SFX had lower cumulative MIC than MFX, LFX and CFX. Furthermore, we performed direct DNA sequencing of the quinolone-resistance-determining regions (QRDRs).Results. We identified the following mutations: D94G, D94A, A90V, D94H, D94N and G88A in gyrA; and A543V, A543T, E540D, R485C, D500A, I552S and D577A in gyrB. Based on our results, an ECOFF of 0.125 µg ml-1 was proposed for SFX. With this ECOFF, 15 % of LFX-resistant isolates with MIC ≥2 µg ml-1 were susceptible to SFX.Conclusion. SFX had the lowest cumulative MIC and a relatively low ECOFF value against Mtb, indicating that SFX was not only more effective against gyrA-mutant isolates, but also MDR isolates in Japan.

Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection

Johne's disease is a chronic granulomatous enteritis of ruminants caused by the intracellular bacterium Mycobacterium avium subsp. paratuberculosis (Map). We previously demonstrated that Map isolates from sheep persisted within host macrophages in lower CFUs than cattle isolates after 7 days of infection. In the current study, we hypothesize that these phenotypic differences between Map isolates may be driven be the fatty acids (FAs) present on the phosphadidyl-1-myo-inositol mannosides of the Map cell wall that mediate recognition by the mannose receptors of host macrophages. FAs modifications may influence Map's envelope fluidity ultimately affecting pathogenicity. To test this hypothesis, we investigated the responses of two Map isolates from cattle (K10 isolate) and sheep (2349/06-1) to the bovine and ovine macrophage environment by measuring the FAs content of extracellular and intracellular bacteria. For this purpose, macrophages cell lines of bovine (BOMAC) and ovine (MOCL-4) origin were infected with the two isolates of Map for 4 days at 37°C. The relative FAs composition of the two isolates recovered from infected BOMAC and MOCL-4 cells was determined by gas chromatography and compared with that of extracellular bacteria and that of bacteria grown in Middlebrook 7H9 medium. Using this approach, we demonstrated that the FAs composition of extracellular and 7H9-grown bacteria was highly conserved within each Map isolate, and statistically different from that of intracellular bacteria. Analysis of FAs composition from extracellular bacteria enabled the distinction of the two Map strains based on the presence of the tuberculostearic acid (18:0 10Me) exclusively in the K10 strain of Map. In addition, significant differences in the content of Palmitic acid and cis-7 Palmitoleic acid between both isolates harvested from the extracellular environment were observed. Once the infection established itself in BOMAC and MOCL-4 cells, the FAs profiles of both Map isolates appeared conserved. Our results suggest that the FAs composition of Map might influence its recognition by macrophages and influence the survival of the bacillus within host macrophages.

Analysis of uracil phosphoribosyltransferase expression in Mycobacterium tuberculosis and evaluation of upp knockout strain in infected mice.

The upp (Rv3309c)-encoded uracil phosphoribosyltransferase from Mycobacterium tuberculosis (MtUPRT) converts uracil and 5-phosphoribosyl-α-1-pyrophosphate into pyrophosphate and uridine 5΄-monophosphate, the precursor of all pyrimidine nucleotides. A M. tuberculosis knockout strain for upp gene was generated by allelic replacement. Knockout and complemented strains were validated by a functional assay of uracil incorporation. A basal level of MtUPRT expression is shown to be independent of either growth medium used, addition of bases, or oxygen presence/absence. The upp disruption does not affect M. tuberculosis growth in Middlebrook 7H9 medium, and it is not required for M. tuberculosis virulence in a mouse model of infection. Thus, MtUPRT is unlikely to be a good target for drugs against M. tuberculosis.