All animal experiments were performed in agreement with European and French guidelines (Directive 86/609/CEE and Decree 87-848 of 19 October 1987). The study received the approval by the Institut Pasteur Safety Committee (Protocol 11.245) and the ethical approval by the local ethical committee ‘Comité d’Ethique en Experimentation Animale N° 89 (CETEA)’ (CETEA 200037/ APAFiS #27688). Female, 7-week-old C57BL/6J mice were infected by aerosol route with Mtb H37Rv at a dose of 150–200 CFU per mouse with a homemade nebulizer as described (Sayes et al., 2012 (link)). 1 day prior to aerosol infection and 5 days a week thereafter, mice were given 100 mg/kg of FADS2 inhibitor SC-26196 by oral gavage. The lyophilized inhibitor was resuspended in 0.5% m/v methylcellulose – 0.025% Tween 20 as described (Obukowicz et al., 1999 (link)). At different time points, mice were sacrificed, and lungs and spleen were homogenized using a MM300 apparatus (QIAGEN) and 2.5 mm diameter glass beads. Serial dilutions in PBS were plated on 7H11 agar supplemented with 0.5% glycerol, 10% OADC, plus PANTA antibiotic mixture (BD Biosciences) for lung homogenates. CFUs were quantified after incubation at 37°C for 14–30 days.
Panta antibiotic mixture
Manufactured by BD
The PANTA™ Antibiotic Mixture is a laboratory reagent used for the selective isolation and cultivation of certain bacteria. It consists of a combination of antibiotics that inhibit the growth of unwanted microorganisms, allowing the target bacteria to be more easily identified and studied.
Automatically generated - may contain errors
Lab products found in correlation
7h11 plates, by BD (3 mentions)
Gentlemacs m tube, by Miltenyi Biotec (3 mentions)
Difco middlebrook 7h9, by BD (2 mentions)
Bbl middlebrook adc enrichment, by BD (2 mentions)
Mm300 apparatus, by Qiagen (1 mentions)
70 μm cell strainer, by BD (1 mentions)
70 m cell strainer, by BD (1 mentions)
Oleic acid, by BD (1 mentions)
Oadc enrichment, by BD (1 mentions)
Middlebrook 7h10 agar, by BD (1 mentions)
C57bl 6 mice, by Jackson ImmunoResearch (1 mentions)
Kanamycin, by BD (1 mentions)
Streptomycin, by BD (1 mentions)
Middlebrook 7h10 medium, by BD (1 mentions)
Albumin dextrose catalase, by BD (1 mentions)
Oleic acid albumin dextrose catalase, by BD (1 mentions)
Middlebrook 7h9 broth, by BD (1 mentions)
Ts100, by Nikon (1 mentions)
24 well plate, by Corning (1 mentions)
9 protocols using panta antibiotic mixture
1
Inhibition of FADS2 in Murine Tuberculosis
2
Aerosol Challenge of Mtb Erdman in Mice
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Ten weeks after the first immunization, mice were challenged with Mtb Erdman (ATCC 35801 / TMC107). Mtb Erdman was cultured in Difco™ Middlebrook 7H9 (BD) supplemented with 10% BBL ™ Middlebrook ADC Enrichment (BD) for two-three weeks using an orbital shaker (~110 rpm, 37°C). Bacteria were harvested in log phase and stored at −80°C until use. Bacterial stocks were thawed, sonicated for five minutes, resuspended with a 27G needle, and mixed with PBS to the desired inoculum dose. Using a Biaera exposure system controlled via AeroMP software, mice were challenged by the aerosol route with virulent Mtb Erdman in a dose equivalent to 50–100 CFUs. To determine vaccine efficacy, Mtb CFU were enumerated in lungs of aerosol infected mice. Left lung lobes were homogenized in 3 mL MilliQ water containing PANTA™ Antibiotic Mixture (BD, cat.no. #245114) using GentleMACS M-tubes (Miltenyi Biotec). Lymph nodes were forced through 70-μm cell strainers (BD Biosciences) in 1 mL PANTA solution. Tissue homogenates were serially diluted, plated onto 7H11 plates (BD), and grown for 14 days at 37°C and 5% CO2. CFU data were log-transformed before analyses.
3
Quantifying Mycobacterium tuberculosis Burden
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Ten weeks after the first immunization, mice were challenged with Mtb Erdman (ATCC 35801/TMC107). Mtb Erdman was cultured in Difco ™ Middlebrook 7H9 (BD) supplemented with 10% BBL ™ Middlebrook ADC Enrichment (BD) for two-three weeks using an orbital shaker (~110 rpm, 37 °C). Bacteria were harvested in log phase and stored at −80 °C until use. On the day of the experiment, the bacterial stock was thawed, sonicated for five minutes, thoroughly suspended with a 27G needle, and mixed with PBS to the desired inoculum dose. Using a Biaera exposure system controlled via AeroMP software, mice were challenged by the aerosol route with virulent Mtb Erdman in a dose equivalent to 50–100 CFUs.
In order to determine vaccine efficacy, Mtb CFUs were enumerated in lungs and spleens. Left lung lobes or spleens were homogenized in 3 mL MilliQ water containing PANTA™ Antibiotic Mixture (BD, cat.no. #245114) using GentleMACS M-tubes (Miltenyi Biotec). Tissue homogenates were serially diluted, plated onto 7H11 plates (BD), and grown for ~14 days at 37 °C and 5% CO2. CFU data were log-transformed before analyses.
In order to determine vaccine efficacy, Mtb CFUs were enumerated in lungs and spleens. Left lung lobes or spleens were homogenized in 3 mL MilliQ water containing PANTA™ Antibiotic Mixture (BD, cat.no. #245114) using GentleMACS M-tubes (Miltenyi Biotec). Tissue homogenates were serially diluted, plated onto 7H11 plates (BD), and grown for ~14 days at 37 °C and 5% CO2. CFU data were log-transformed before analyses.
4
Enumerating BCG and Mtb in Tissues
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In order to determine vaccine efficacy or cross-reactivity, BCG and Mtb CFU were enumerated in lungs, spleens, and lymph nodes. Left lung lobes or spleens were homogenized in 3 mL MilliQ water containing PANTA™ Antibiotic Mixture (BD, cat.no. #245114) using GentleMACS M-tubes (Miltenyi Biotec). Lymph nodes were forced through 70-µm cell strainers (BD Biosciences) in 1 ml PANTA solution. Tissue homogenates were serially diluted, plated onto 7H11 plates (BD), and grown for approximately 14 days at 37 °C and 5% CO2. CFU data were log-transformed before analyses.
5
Mtb Culture Filtrate Production Protocol
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The laboratory strain of Mtb, H37Rv, was used to produce culture filtrate (CF), as described previously (Loraine et al., 2016 (link)). Briefly, Mtb cultures were grown in Middlebrook 7H9 medium containing 0.05% tween and supplemented with 10% OADC (oleic acid, albumin, dextrose, and catalase; BD Biosciences) in vented flasks and kept stationary at 37°C until mid-exponential phase was reached (OD600 nm 0.6–1.0). Cultures were centrifuged at 4,000 × g for 15 min and supernatants were sterilized by double filtration using sterile, disposable Durapore Membrane Filters PVDF (0.22 μm pore size). CF was supplemented with fresh 7H9 media (at a 1:1 ratio) and polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA™ antibiotic mixture, BD Biosciences) were added to increase subsequent selectivity for Mtb growth. A 450 μL aliquot of the CFSM was added to a 48-well cell culture plate (Nunc, Thermo Scientific). Sterility of the CF was verified by including neat aliquots in control wells of the cell culture plate.
6
Detecting BCG in Infected Mouse Tissues
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The ability of the assay to identify BCG directly from tissue was determined using infected mice. Three C57BL/6 mice (Jackson Laboratories, Bar Harbor, MA, USA) were intravenously infected with 2.3 × 108 CFU BCG Russia. After 3 weeks, the mice were sacrificed, and spleen, liver, and lung samples were collected. The organs were homogenized, and infection was confirmed by serial dilution and plating onto Middlebrook 7H10 agar (Becton, Dickinson), OADC enrichment (Becton, Dickinson), 0.5% glycerol (Sigma-Aldrich), and PANTA antibiotic mixture (Becton, Dickinson). DNA was extracted from organ homogenates as previously described (23 (link)). DNA was then quantified and analyzed by the two-step real-time PCR assay.
7
Culturing and Isolating Mycobacterial Strains
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All mycobacterial strains were grown in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) with 0.2% glycerol, 0.1% Tween 80, and 10% albumin-dextrose-catalase (Becton, Dickinson and Co., Sparks, MD) with rotation at 37 °C. For colony isolation, the bacteria were plated on Middlebrook 7H10 medium supplemented with 10% oleic acid-albumin-dextrose-catalase (Becton, Dickinson and Co.). To grow MAP, 0.1% mycobactin J (Allied Monitor, Fayette MO) was added to liquid and solid media. The antibiotics kanamycin (50 µg/mL), streptomycin (50 µg/mL), or PANTA antibiotic mixture (Becton, Dickinson and Co.) were included in the media when required.
8
Lyophilized MODS Media Cultivation
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The lyophilized media was prepared as the standard MODS culture medium. After autoclaving and adding the sterile OADC solution as was prepared for the standard MODS media, fresh Middlebrook 7H9 culture media was distributed in 25 mL aliquots and frozen at -70°C for 24 hours. Lyophilization was performed at -46°C with a pressure between 50 x 10 -3 to 70 x 10 -3 MBAR in the lyophilizer LYPHLOCK (Labconco). The room was UV irradiated for 15 min before lyophilization.
Four culture variations of the LM were evaluated based on the addition of PANTA antibiotic mixture (Becton Dickinson), and gamma irradiation delivered at 5 KGy (Figure 2).
Four culture variations of the LM were evaluated based on the addition of PANTA antibiotic mixture (Becton Dickinson), and gamma irradiation delivered at 5 KGy (Figure 2).
9
Rapid Tuberculosis Diagnosis from Sputum
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Clinical sputum samples were collected between January to February during 2020 in the Regional Tuberculosis Reference Laboratory, Callao, Peru (Ethical approval number 345-15-19) . A total of 31 samples with positive acid-fast bacilli smear (AFB)
were selected for the study. Sputum samples were decontaminated according to the standardized protocol (Coronel et al. 2008) . Briefly, two milliliters of sample were mixed with two milliliters of 2% NaLc-NaOH, vortexed roughly, and incubated at room temperature for 15 min. Next, the mix was neutralized by adding 10 mL of phosphate buffer and centrifuged at 3000 x g for 15 min at 17°C. Finally, the pellet was resuspended with 2.5 mL phosphate saline buffer pH 6.8.
From the decontaminated samples, 300 µL were added to two milliliters of each selected culture medium variant and dispensed in two wells of a 24-well plate (Corning). The 24-well plate was placed in a resealable bag (Ziploc) and incubated at 37°C in for up to 30 days. The standard MODS medium (Coronel et al. 2008) with PANTA antibiotic mixture (Becton Dickinson) was used as 100% growth reference.
Bacterial growth was evaluated since day three using 40X magnification in an inverted microscope (TS100-Nikon) and reported as growth percentage. Culture results were registered the first day that bacterial growth was observed.
were selected for the study. Sputum samples were decontaminated according to the standardized protocol (Coronel et al. 2008) . Briefly, two milliliters of sample were mixed with two milliliters of 2% NaLc-NaOH, vortexed roughly, and incubated at room temperature for 15 min. Next, the mix was neutralized by adding 10 mL of phosphate buffer and centrifuged at 3000 x g for 15 min at 17°C. Finally, the pellet was resuspended with 2.5 mL phosphate saline buffer pH 6.8.
From the decontaminated samples, 300 µL were added to two milliliters of each selected culture medium variant and dispensed in two wells of a 24-well plate (Corning). The 24-well plate was placed in a resealable bag (Ziploc) and incubated at 37°C in for up to 30 days. The standard MODS medium (Coronel et al. 2008) with PANTA antibiotic mixture (Becton Dickinson) was used as 100% growth reference.
Bacterial growth was evaluated since day three using 40X magnification in an inverted microscope (TS100-Nikon) and reported as growth percentage. Culture results were registered the first day that bacterial growth was observed.
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