Bbl mgit panta antibiotic mixture

Following strains were used for the study at different colony forming units (CFU)/ml (10⁴–10⁷), which were cultivated in Löwenstein–Jensen medium (LJ): M. abscessus 9547/00 (type strain, =AB1), M. abscessus 8562/11 (clinical isolate, =AB2), M. avium 3725/07 (strain 104, =AV2), M. avium 3439/10 (clinical isolate, =AV1), M. tuberculosis 9679/00 (type strain H37Rv, =TB2), and M. tuberculosis 1616/12 (clinical isolate from a German patient, =TB1). In order to precipitate mycobacterial clumps, suspensions were centrifuged at low speed (100 × g) for 5 min. BBL™ MGIT™ PANTA™ antibiotic mixture (BD diagnostics, USA) was added to the suspensions to prevent other bacterial growths. The concentrations of viable mycobacteria (CFU/ml) in the stock suspensions were controlled three times during the study. Basically, the stock solutions were serially diluted (1:10 each) until 10⁰ CFU/ml. From 10⁰ to 10³ CFU/ml 0.3 ml were cultured in petri dishes containing LJ medium. Cultivation time for M. abscessus was 1–2 weeks and for M. avium, as well as M. tuberculosis 4–6 weeks, respectively. The mycobacterial colonies were counted visually and the CFU/ml were determined. For infection of the lung tissue specimens, 2 ml of suspension from each strain were used.

The assessment of initial bacterial burden was performed 3 days postinfection by growing viable bacteria from whole-lung homogenates. For the other time points, lung and spleen single-cell suspensions were incubated with 0.1% saponin (Sigma-Aldrich) for 10 min to release intracellular bacteria. The CFU was determined by plating 10-fold serial dilutions of saponin-treated cell suspensions on Middlebrook 7H11 agar plates supplemented as described in “Bacterial growth and stocks.” BBL MGIT PANTA antibiotic mixture (BD Bioscience) was added to prevent the contamination of lung samples. Viable M. tuberculosis colonies were counted after 21 to 28 days of incubation at 37°C.

A 5-mL aliquot of the BAL fluid was homogenized by shaking and centrifuged. The pellet was suspended (0.5 mL), inoculated in conventional fungal medium [23 (link)], and incubated at 37 °C ± 1 for 14 days. Another aliquot was used for Gram and fungifluor staining, and immediate inoculation onto Columbia blood agar, MacConkey agar, Columbia colistin-nalidixic acid (C. N. A.) agar, chocolate agar, and brain–heart broth. The solid media and brain–heart broth were incubated in a CO₂-enriched atmosphere for 4 days at 37 °C ± 1. The identification of the C. jeikeium and R. dentocariosa isolates was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Maldi Biotyper 2.0, Bruker Daltonics, Bremen, Germany) according to the manufacturer’s instructions. Acid-fast bacilli cultures were processed by using the BBL MGIT PANTA Antibiotic Mixture (Becton Dickinson), in addition to Stonebrink and Coletsos media (incubated for 14 weeks at 36.5 °C). The identification of acid-fast bacilli and fungal pathogens was carried out by PCR and sequencing as described previously [24 (link),25 (link)]. All antibiotic susceptibility testing (AST) was performed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (edited in 2014; Available online: http://www.eucast.org/).