Molecular characterization and improved diagnostics of strains isolated over the last two decades at a German tertiary care center.

Nocardiosis is a rare but life-threatening infection caused by aerobic of the genus particularly affecting immunocompromised hosts. The identification of and antibiotic susceptibility testing by standard microbiological methods are incomplete and molecular techniques may improve diagnostics. We studied 39 strains isolated from 33 patients between 2000 and 2018. Twenty-four patients (72.7 %) were immunocompromised. Whole genome sequencing (WGS) revealed a broad taxonomic range of those isolates spanning 13 different species, including four strains that belonged to three novel species based on average nucleotide identity (ANI < 95 % with currently available genome sequences). 16S rRNA gene analyses mirrored WGS results. Conventional MALDI-TOF analysis correctly identified 29 isolates at the species level (74.4 %). Our advanced protocol with formic acid and acetonitrile treatment increased identification to 35 isolates (89.7 %). Antibiotic resistance was tested using both a microdilution method and MIC strip testing. Results were in good concordance with an overall trimethoprim-sulfamethoxazole (SXT) resistance rate of 13.5 % WGS of a SXT resistant isolate showed a deletion of several amino acids in a homolog of dihydropteroate synthase (FolP2) that was not seen in sensitive members of this species. Diversity of isolates was high and involved many different species, suggesting that this taxon has broadly distributed mechanisms for infecting individuals. Widely applicable diagnostic methods including MALDI-TOF and 16S rRNA gene analyses correctly identified most strains. WGS additionally revealed molecular insights into SXT resistance mechanisms of clinical isolates highlighting the potential application of (meta)genomic-based diagnostics in the future.