Microbiological Procedures
Sample preparation and mycobacterial cultures were performed as
described elsewhere (Ghielmetti et al.,
2018). Briefly, BBL MGIT liquid media tubes supplemented with Bactec
MGIT 960 growth supplement, BBL MGIT PANTA (Polymyxin B, Amphotericin B,
Nalidixic acid, Trimethoprim, Azlocillin) antibiotic mixture (Becton,
Dickinson, BD, Allschwil, Switzerland) and 50 μg/ml sodium-pyruvate were
each inoculated with 0.5 ml of decontaminated and homogenized specimen.
In addition, one Löwenstein-Jensen and one Stonebrink agar slants (BD)
were inoculated with the same inoculum and incubated up to eight weeks
at 37°C. In order to obtain pure mycobacterial cultures, subcultures on
7H10 agar-plates and on Stonebrink agar slants (BD) were performed at
intervals of three to ten days. Simultaneously, 400 μl of culture
inoculum was suspended in 100 μl ATL buffer (Qiagen, Hilden, Germany)
and transferred onto a Lysing Matrix E tubes (MP Biomedicals, Eschwege,
Germany). Genomic DNA was extracted through mechanical cell lysis using
a TissueLyser II (Qiagen, Hilden, Germany) and enzymatic digestion with
Proteinase K (Qiagen) overnight. Automated DNA purification was
performed using the QIAcube instrument in accordance with the QIAamp
cador Pathogen Mini Kit protocol (Qiagen). DNA concentration in the
final eluate was measured by reading the absorbance at 260 nm using a
NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific, Reinach,
Switzerland), diluted to a maximal concentration of 100 ng/µl and stored
at -20°C until use. Purified DNA was used for direct MTBC detection
using the qPCR assay targeting insertion sequence IS6110 as
described by Reed et al. with slight modifications
(Reed et al., 2016). Briefly, the MTB
IS6110 probe was double-quenched with iQ500 and BHQ1 instead of ZEN and
3IABkFQ, respectively. Moreover, the qPCR assay internal control was
substituted by eGFP as described by Hoffmann and colleagues and
performed on a 7500 Fast real-time PCR system (Applied Biosystems, Zug,
Switzerland) (Hoffmann et al., 2006).
DNA from cultured mycobacteria was extracted by inoculating a loop-full
of cell materials into 200 μl of chelating ion-exchange resin (InstaGene
Matrix, Cressier, Switzerland) and centrifuged at 13000 g for 10
minutes. The supernatant was used in downstream reactions.
Pure cultures that presented acid-fast bacilli (AFBs) by Ziehl-Neelsen
(ZN) staining and negative MTBC qPCR results were classified as NTM and
further characterized by sequence analysis of a combination of
housekeeping genes and matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF MS). Sanger sequencing of
16S rRNA (Scherrer et al., 2018),rpoB (Adékambi et al., 2003) andhsp65 (Telenti et al., 1993)
housekeeping genes was performed in duplicates followed by gene homology
analyses. For isolates identified as members of the MAC, the completehsp65 gene was sequenced as proposed by Turenne et al.using primers MAChsp65F and MAChsp65R
(Turenne et al., 2006). DNA sequencing
was performed at Microsynth (Balgach, Switzerland). Resulting sequences
were assembled using CLC Genomics Workbench 7.5.1 (Qiagen) and BLAST
similarity searching for multiple sequence alignment was performed
(https://blast.ncbi.nlm.nih.gov/Blast.cgi). Control strains included
were M. avium subsp. avium ATCC 25291, M. aviumsubsp. hominissuis ATCC 700898, M. peregrinum ATCC 700686.
For samples resulted positive by direct MTBC real-time PCR, mycobacteria
could not be cultivated from any of the described culture media, even
after 12 months of incubation. Molecular characterization by
mycobacterial interspersed repetitive unit and variable number tandem
repeats (MIRU-VNTR) and species determination using spoligotyping were
therefore performed using DNA extracts of lymph nodes as previously
described (Ghielmetti et al., 2017).