Genotype ntm dr

A total of 39 M. intracellulare strains, identified by GenoType NTM-DR test (Hain Lifescience), able to discriminate between M. intracellulare and Mycobacterium chimaera, were isolated between 2015 and 2019 from the same number of patients (23 males and 16 females; 70% older than 65 years) resident in Tuscany, Italy, with pulmonary infections at the Laboratory of Clinical Mycobacteriology of the University Hospital of Pisa, Italy.

PCR was carried out in the GTQ Cycler 96 using the protocol in the user guide (Hain Lifescience, Nehren, Germany). Speciation was carried out using GenoType®CM/AS (Hain Lifescience, Nehren, Germany) protocol on TwinCubator (Hain Lifescience, Nehren, Germany). Species-specific probes are mounted to the DNA strips to determine complementary strands from amplified DNA samples [28 (link), 29 (link)]. Table 1 below shows that MAC and Mycobacterium abscessus complex (MABC) that could not be speciated by this technique were further speciated using GenoType® NTM-DR (Hain Lifescience, Nehren, Germany) protocol [30 (link)]. DNA amplicon that could not be identified to species level the same day of amplification were stored at– 200C. On the other hand, isolates that GenoType® CM/AS assay could not GenoType® to species level are stored at– 800C for sequencing of 16S rRNA gene.

The M. abscessus strain 10 (Mab-10) used throughout this study was obtained from the European Centre for Disease Prevention and Control and identified as M. abscessus abscessus by the line probe assay GenoType NTM-DR (Hain Lifescience, Nehren, Germany). Mab-10 showed rough colonies on Middlebrook 7H10 agar (Difco, Detroit, MI, USA) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) (Becton Dickinson, Sparks, MD, USA). The strain was a clinical isolate from a lung infection.
The minimum inhibitory concentrations (MICs) of Mab-10 were determined by the broth microdilution method using the Sensititre RAPMYCOI plates (Thermo Fisher Scientific, Waltham, MA, USA). MICs were recorded after 5 days of incubation, with the exception of those of clarithromycin (CLR), which were recorded on days 5 and 14 in order to assess inducible CLR resistance [32 ]. The MICs of BDQ, RFB and CLF were performed with the same protocol of the RAPMYCOI assay. All MIC values are shown in the Supplementary Table S1. MIC interpretation was performed according to the Clinical and Laboratory Research Institute (CLSI) breakpoints [32 ] and showed that Mab-10 was susceptible only to CLR and AMK. These results were confirmed by the Whole Genome Sequence analysis of erm(41), rrl and rrs genes [5 (link),33 (link),34 (link)].