Methods: A DAP-susceptible E. faecium (HOU503; MIC 3 mg/ml) harboring the above LiaSR substitutions, was subjected to simulated human doses of 6, 8, and 10 mg/kg/d in the model for 14 days using a starting inoculum of 109 CFU/ml. Sixteen DAP-R isolates were recovered from the SEV model: 5 isolates from 6 mg/kg (D6 isolates from day 2 – 14); 5 isolates from 8 mg/kg (D8 isolates from day 2 – 8); and 6 isolates from 10 mg/kg (D10 isolates from day 1 – 14) which were subjected to whole genome sequencing. Reads from each sequenced isolate were mapped against the HOU503 genome for SNP analyses. Variant calling was done with GATK, SamTools and the low frequency variant detector from CLC Genomics Workbench 8.5. Variants detected by the three callers were selected and annotated with SnpEff; then compared among the different groups of isolates accordingly to the DAP doses that were exposed.
Results: We detected a total of 16 proteins exhibiting substitutions consistently in all the DAP-R sequenced isolates; including mobile genetic elements (9), hypothetical proteins (2), a bacteriocin, a cysteine desulfurase, a N-acetylglucosamine-specific PTS system, a N-acetylmannosamine-6-phosphate 2-epimerase and a MurR/RpiR, which is a transcriptional regulator that represses the operon MurPQ involved in the uptake and degradation of N-acetylmuramic acid. Notably, mutations in cardiolipin synthase were present only in isolates recovered under D8 dose. The LiaRS substitutions remained in all isolates.
Conclusion: Using a humanized SEV PK/PD model and SNP based analyses we were able to uncover possible novel genetic pathways associated with the development DAP-R via the LiaFSR system in enterococci.
T. T. Tran,
R. Rios, None
A. Dinh, None
S. Jahanbakhsh, None
R. Kebriaei, None
M. J. Rybak, Allergen: Scientific Advisor , Consulting fee
C. Arias, None