2.1. Bacterial Strains and Growth Conditions

A clinical isolate of E. faecalis (1390) was tested for penicillin, gentamicin, and streptomycin resistance as well as high-level streptomycin resistance (HLSR) and high-level gentamicin resistance (HLGR) according to Clinical and Laboratory Standards Institute (CLSI) guidelines [11]. This strain was selected from a susceptibility profile of 90 clinical strains by the Instituto Nacional de Cardiología in Mexico City and based on a plasmid analysis performed according to a previously published report [12] as well as the phenotype and genotype of HLAR and prg B (Supporting Information Table S1). The strain was subjected to selective pressure with ampicillin (Sigma-Aldrich, Saint Louis, Missouri, USA) continuously at subinhibitory concentrations (0.25×, 0.5× and 0.75×) as previously described [13]. The recovered clones were thereafter stimulated continuously with higher concentrations of ampicillin minimum inhibitory concentration (MIC) (1×, 2×, 4×, 8×, 16×, 32×, 64×, and 128×); afterward, the resistant clones were analyzed based on a plasmid profile. The resistant clone with plasmid was named 1390R and considered a donor clone and able to express aggregation substances (AS). The 1390R strain was subjected to treatment with ascorbic acid (2, 5, 10, and 15 mM) to induce plasmid loss as previously reported for Staphylococcus [14]. The plasmid profile of the resulting clones was tested, and a resistance analysis was conducted for ampicillin as well as HLGR and HLSR. The recovered clone that was plasmid-free of plasmids and sensitive to the tested antibiotics, was named C29 and considered a receptor and pheromone producer. Susceptibility was determined by an analysis of viability. Briefly, the strains were grown for 6 h at 37 °C, and the pellet was recovered by centrifugation and adjusted to 0.5 McFarland standard (1.4 × 10⁸ bacterial/mL). The MIC of ampicillin of each strain was tested following the CLSI criteria. The tubes were grown for 4 h as previously described, and the bacterial suspension was diluted to 10,000 bacteria/mL in PBS 1× (pH 7.4) and EDTA 0.05 M. Rhodamine 123 (Sigma-Aldrich) was added to a concentration of 2 µg mL⁻¹, and propidium iodide (Sigma-Aldrich ) was added to a concentration of 10 µg mL⁻¹. Each dye was added to the bacterial suspensions. To establish the kinetics of survival in each strain, the strains were analyzed by flow cytometry in a FACSCalibur device with CellQuest software (Beckton Dickinson, Becton Drive Franklin Lakes, NJ, USA).