Bacterial cells are known to change the fatty acid (FA) composition of the phospholipids as a phenotypic response to environmental conditions and to the presence of toxic compounds such as antibiotics. FA ratios, and thus increased membrane fluidity. Furthermore, the phenotypic changes were transmitted to daughter cells grown in drug-free media. The fact that VBNC cells presented nearly the same FA composition as those obtained after cell growth in drug-free media, which could only be the result of growth of persister cells, suggest that VBNC and persister phenotypes share the same type of response to antibiotics at the lipid level. to concentrations of antibiotics several fold higher than the MIC to determine the phenotypic modifications occurring in the survivor cells, namely changes in the fatty acid (FA) composition of the phospholipids of the cellular membrane and of the cell surface properties. The method used, based on previously described techniques to produce persister samples (Keren et al., 2004; Hsu et al., 2011; Ca?as-Duarte et al., 2014), consists in promoting the lysis of normally growing cells and sedimenting the remaining cells by centrifugation. However, by using fluorescence microscopy to assess cell viability, it was found RBM45 that the survivor subpopulation contained more viable but non-culturable (VBNC) cells than persisters. Recent studies (Li et al., 2014; Ayrapetyan et al., 2015b) have defended that persister and VBNC cells coexist, are induced by the same conditions and may be part of a shared dormancy continuum. Nevertheless, all authors agree that the main distinction between persisters and VBNC cells is the ability of the former to resume normal growth when placed in fresh drug-free medium. Current isolation techniques do not allow the separation of persister and VBNC cells and most of the studies referring to persister cells must have been actually studying mainly VBNC cells (Orman and Brynildsen, 2013b). Since both types are in fact cells that have been able to maintain membrane integrity when their normal counterparts have suffered cell lysis, this indicates that if they are not the same subpopulation at least they must share similar adaptive mechanisms. Alterations of the FA composition in bacteria are known to be an adaptive mechanism during environmental challenges such as changes in pH, temperature and osmotic pressure, as well as in the presence of toxic compounds (Zhang and Rock, 2008; de Carvalho et al., 2009; de Carvalho, 2012). Phenotypic changes related to the membrane, including increased fluidity and increased positive surface charge associated with FA content and phospholipid composition, have been observed in exposed to daptomycin (Jones et al., 2008) and antimicrobial peptides (Nawrocki et al., 2014). Besides, the study of Mirani and Jamil (2013) showed that vancomycin has a significant impact on the phospholipid composition of vancomycin-resistant, -intermediate, and -susceptible strain used in this study also used modifications in the FA composition as an adaptation mechanism. To our knowledge, this is the first work studying lipid modifications occurring in cells challenged to very high concentrations of vancomycin and teicoplanin. The prospective role of membrane composition in the survival of these cells could help in the development of effective and specific therapies for persister-related infections. Materials and Methods Bacterial Strain Strain ATCC 25923 was used in this study. This strain is usually used as a vancomycin-susceptible control strain in studies comparing tolerance/resistance of isolates. Bacterial growth Xarelto was carried out in Mueller-Hinton broth (MHB; Fluka Analytical, Sigma-Aldrich) at 37C and 200 rpm in an Agitorb 200 incubator (Aralab), unless stated otherwise. Determination of the Minimum Inhibitory Concentration (MIC) The MICs for vancomycin (vancomycin hydrochloride from with a potency Xarelto 900 g/mg from Sigma-Aldrich, St. Louis, MO, USA) and teicoplanin (from with a purity 80% purchased from Sigma-Aldrich) were determined according to the Clinical and Laboratory Standards Institute Xarelto (CLSI) guidelines (CLSI, 2014). In summary, the antibiotics were serially diluted in twofold steps (from 100 to 0.037 mg/L) in 96-well microplates (Sarstedt, Inc., Newton, MA, USA) in MHB. To 150 T of medium comprising the antibiotic in each well, 50 T of an exponentially growing cell tradition diluted to 0.5 McFarland standard was added. The microplates comprising cells were incubated at 37C. The MIC was identified for each antibiotic, after 16 h of exposure, by visual inspection and by measuring the optical denseness.