Mm300 apparatus

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.

Animal studies were carried out in strict accordance with the Amsterdam protocol on animal protection and welfare, the Directive 2010/63/EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes and the French Decree 2013-118. The protocol was approved by the Minister of Higher Education and Research after favorable opinion of the Ethics Committee (CEEA Nord-Pas de Calais/INSERMU1019 n° 00579.01 from 23/07/2014). All efforts were made to minimize suffering of the animals. Six-weeks-old female BALB/c mice (Janvier) were challenged with mycobacterial strains via the intranasal route with 10 μl/nostril. Challenge suspensions were adjusted in order to obtain an inhaled dose of approximately 1,000 CFU/lungs. At day 0, 28, and 49 post-infection, lungs from euthanized mice were collected and homogenized by using an MM300 apparatus (Qiagen) and 2.5-mm diameter glass beads. Ten-fold serial dilutions of lung homogenates were plated on 7H11/OADC agar medium and CFUs enumerated, as previously described (Palucci et al., 2016 (link)).

Virulence studies were performed in SCID mice. Briefly, groups of 6 week old C.B-17/Icr SCID mice (Charles River) were intravenously infected with a bacterial suspension containing 1–2 × 10⁵ CFU/mouse. One day and 28 days after infection, mice were euthanized, and spleen and lungs were homogenized in 2 ml tubes containing 500 µl Sauton medium and 2.5 mm diameter glass beads using an MM300 apparatus (Qiagen). CFU numbers in target organs were determined by plating 5- or 10-fold serial dilutions of organ homogenates on solid medium and incubation at 37 °C. These studies were approved by the Institut Pasteur Safety Committee (Protocol 11.245; experimentation authorization number 75–1469), in accordance with European and French guidelines (Directive 86/609/CEE and Decree 87–848 of 19 October 1987), and implicating approval from local ethical committees (CETEA 2013–0036 and CETEA dab180023).