Pathogenic Nocardia cyriacigeorgica and Nocardia nova evolve to resist trimethoprim-sulfamethoxazole by both expected and unexpected pathways [PublishAheadOfPrint]

Nocardia spp. are Gram positive opportunistic pathogens that affect largely immunocompromised patients leading to serious pulmonary or systemic infections. Combination therapy using the folate biosynthesis pathway inhibitors, trimethoprim and sulfamethoxazole (TMP-SMX) is commonly used as an antimicrobial therapy. Not surprisingly, as antibiotic therapies for nocardiosis can extend for many months, resistance to TMP-SMX has emerged. Using experimental evolution, we surveyed the genetic basis of adaptation to TMP-SMX across 8 strains of Nocardia nova and 2 strains of Nocardia cyriacigeorgica. Employing both continuous experimental evolution to provide longitudinal information on the order of changes and characterization of resistant end-point isolates, we observe changes that are consistent with modification of two enzymes of the folate biosynthesis pathway: dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS/FolP) with the mutations often clustered near the active site of the enzymes. While changes to DHFR and DHPS might be expected, we also noted that mutations in a previously undescribed homolog of DHPS (DHPS2/FolP2) that was annotated as "non-functional" were also sufficient to generate TMP-SMX resistance which serves as a cautionary tale for the use of automated annotation by investigators and for future drug discovery against this genus. Additionally, folP2 overlapped with glucosyl-3-phosphoglycerate synthase. Remarkably, an adaptive frameshift mutation within the overlapping region resulted in a new in-frame fusion to the downstream gene to produce a potentially new bifunctional enzyme. How a single potentially bifunctional DHPS2 might confer resistance is unclear. However, it highlights the unexpected ways in which adaptive evolution finds novel solutions to selection.

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