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Objectives: To verify dissemination of daptomycin-non-susceptible Enterococcus faecium in a hospital where daptomycin was not in use and to understand the evolutionary pathways connecting daptomycin hypersusceptibility to non-susceptibility. Methods: Clonality of 26 E. faecium was assessed by PFGE and the STs of these isolates were determined. The most daptomycin-susceptible isolate was evolved in vitro by stepwise daptomycin selection, generating isolates for genome comparisons. Results: The spread of a high-risk daptomycin-non-susceptible VRE clone was detected, as was the occurrence of an unusual daptomycin-hypersusceptible strain (HBSJRP18). To determine the basis for daptomycin hypersusceptibility, we evolved HBSJRP18 in vitro and identified candidate genetic alterations potentially related to daptomycin susceptibility. Both lafB, encoding glycosyltransferase, which is putatively involved in lipoteichoic acid (LTA) biosynthesis, and dak, encoding a dihydroxyacetone kinase likely involved in fatty acid metabolism, were mutated in multiple independent experiments. Trans-complementation showed that the lafB polymorphism naturally occurring in HBSJRP18 caused its daptomycin hypersusceptibility. Fourier-transform infrared spectroscopy identified differences between the extracted LTA spectra from the hypersusceptible isolate and its revertant, as well as other non-susceptible variants, supporting a role for LafB in E. faecium LTA biosynthesis. Zeta potential difference was detected in one evolved dak mutant derivative. While much more susceptible to daptomycin, HBSJRP18 showed enhanced growth in the presence of piperacillin, suggesting that this, or another cell wall-targeting antibiotic, may have selected for the daptomycin-hypersusceptible phenotype. Conclusions: Our findings provide new information on the basis for daptomycin susceptibility in E. faecium, with implications for limiting the development and spread of daptomycin resistance.

Federal University of Saõ Carlos

Saõ Carlos Institute of Physics University of Saõ Paulo

Harvard Medical School

Massachusetts Eye and Ear Infirmary

Departamento de Doenca¸s Dermatológicas Infecciosas e Parasitárias Faculdade de Medicina de Saõ José Do Rio Preto-FAMERP

Instituto de Biociências Letras e Ciências Exatas (IBILCE) UNESP

Departments of Ophthalmology Microbiology and Immunobiology Harvard Medical School

Division of Infectious Diseases University of Pittsburgh School of Medicine, 3550 Terrace Street

Instituto de Biociências Letras e Ciências Exatas (IBILCE) UNESP