Heidarian, S.; Lohsen, S.; Guliaev, A.; Nicoloff, H.; Satola, S. W.; Andersson, D. I.; Hjort, K.

Heteroresistance (HR), the coexistence of a rare resistant subpopulation within a predominantly susceptible bacterial population, is a clinically relevant problem. The resistant subpopulation often escapes detection by standard susceptibility testing, which can lead to treatment failure. To investigate HR in Enterococcus faecalis, we performed population analysis profiling (PAP) on 40 clinical isolates against five clinically important antibiotics and whole-genome sequenced (WGS) the resistant subpopulations. HR was identified for daptomycin (20.0%), gentamicin (13.2%), and tigecycline (35.9%), but not for linezolid, and vancomycin. Genomic analysis revealed that daptomycin resistance was primarily caused by mutations affecting cell envelope integrity and stress-response pathways. Gentamicin resistance was linked to alterations in efflux regulation, ribosomal proteins synthesis, and transcriptional control. Tigecycline resistance involved deletions in the tet(M) leader peptide, resulting in a 25-fold increased tet(M) expression, as well as transposition of the transposon Tn916 carrying tet(M) to multiple chromosomal sites, causing an increased gene dosage of tet(M). These findings highlight the role of chromosomal mutations and mobile genetic elements in causing HR in E. faecalis Keywords: Enterococcus faecalis, antibiotic, heteroresistance, population analysis profile, point mutations, tet(M).