Mgit tube

Samples for mycobacteria identification were digested, decontaminated by the NALC-NaOH method [19 , 20 ], and inoculated in both Löwenstein-Jensen medium and MGIT tubes (Becton-Dickinson, Sparks, MA), according to the manufacturer’s specifications. Samples labelled as biopsies were additionally inoculated in Stonebrink culture medium. All positive cultures were further identified by DNA probe (Accuprobe, San Diego, CA). During the study period, all M. tuberculosis complex isolates were identified to the species level. We defined M. bovis as a positive culture with dysgonic growth and biochemical tests results (niacin production, nitrate reduction, thiophen-2-carboxylic acid anhydride susceptibility, and pyrazinamidase deamidation) typical of this species, and M. tuberculosis as a positive culture with eugonic growth and compatible biochemical test results (niacin production, nitrate reductase positive test and heat-stable catalase activity) [21 ]. Additionally, both M. bovis and M. tuberculosis identification was confirmed by spoligotyping from 2000 to 2013 and by the GenoType MTBC (Hain Lifescience GmbH, Nehren, Germany) test from 2013 to 2015 [22 (link), 23 (link)]. Susceptibility testing for rifampicin, streptomycin, isoniazid, and ethambutol was performed using the radiometric BACTEC 460 or the BACTEC 960 culture system (Becton-Dickinson, Sparks, MA) [24 (link)].

Further analysis of M. tuberculosis-containing samples required transfer to laboratories operating without biosafety level so that the inactivation of H37Rv by gamma irradiation had to be established. To this end, either lung tissue biopsy specimens or pulmonary cryosections frozen on dry ice were put into the irradiation chamber of a Biobeam 8000 (Gamma-Service Medical GmbH, Leipzig, Germany). The instrument is equipped with a ¹³⁷Cs gamma ray source (gamma energy = 0.662 MeV) with a half-life of 30.17 years and an activity of 81.21 TBq. Radiation doses were measured using alanine dosimetry. Elimination of M. tuberculosis was assessed by incubating the irradiated samples for at least 6 weeks in MGIT tubes (Becton, Dickinson GmbH, Heidelberg, Germany) at the National Reference Center for Mycobacteria at the Research Center Borstel. The MGIT contain 7 mL of modified Middlebrook 7H9 broth base for the cultivation of almost all mycobacteria, including M. tuberculosis. The complete medium contains OADC enrichment to optimize the growth of mycobacteria. Any positive sample was examined for AFB and cord formation.

Deep samples obtained by surgical procedures, i.e., joint fluids, crushed tissue or bone biopsies, were inoculated on 5 % sheep blood, chocolate, Mueller-Hinton, trypticase soy and MacConkey agar plates (BioMérieux, France) and incubated at 37 °C in a 5 % CO2 atmosphere and in an anaerobic atmosphere for 10 days. For mycobacterial culture we inoculated the samples in the MGIT tubes (Becton Dickinson, Pont-De-Claix, France) or on a home-made 5 % sheep blood agar (BioMérieux, La Balme-les-Grottes, France) for 2–45 days at 32 °C or 37 °C as previously described [11 (link), 12 (link)]. Pure bacterial cultures, obtained by picking isolated colonies, were identified with semi-automated Gram staining (Aerospray Wiescor, Elitech), catalase and oxidase activity tests, and the Vitek 2 system (BioMérieux, Marcy l’Etoile, France). The antibiotic susceptibility of P. multocida isolates were determined and interpreted according to the recommendations of the French Society for Microbiology (http://www.sfm-microbiologie.org/UserFiles/files/casfm/CASFM_EUCAST_V1_0_2014.pdf).

All of the specimens for AFB smears and cultures were pre-treated by decontamination with 4% (w/v) NaOH and centrifugation at 3000 × g for 20 min. The AFB smears were examined after auramine-rhodamine staining. Culture was considered positive if M. tuberculosis was cultured in liquid broth media. For the cultures, sediment was incubated in MGIT tubes (Becton, Dickinson and Co., Sparks, MD, USA) for 6 weeks. Bronchoscopy was performed in suspected pulmonary TB patients who could not expectorate sputum or in those with negative AFB staining of sputum if needed. For PCR of bronchial washing or fluid from bronchoalveolar lavage, the Amplicor Mycobacterium tuberculosis test (Roche Molecular Systems, Branchburg, NJ, USA) or Xpert MTB/RIF assay (Cepheid, Inc., Sunnyvale, CA, USA) was used.