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Glycopeptides are the basis of the treatment of infections caused by MRSA (Methicillin-Resistant Staphylococcus aureus). Previously, it was demonstrated that antibiotic tolerant phenotypes are formed during selection of resistance under the influence of high concentrations of antibiotics. The present study uses a similar in vitro selection model with vancomycin. Clinical isolates of MRSA belonging to genetic lines ST8 and ST239, as well as the MSSA (ATCC29213) strain, were included in the experiment. Test isolates were incubated for five hours in a medium with a high concentration of vancomycin (50 μg/ml). Test cultures were grown on the medium without antibiotic for 18 hours after each exposure. A total of ten exposure cycles were performed. Vancomycin was characterized by bacteriostatic action; the proportion of surviving cells after exposure was 70–100%. After selection, there was a slight increase in the MIC to vancomycin (MIC 2 μg/ml), teicoplanin (MIC 1.5–3 μg/ml) and daptomycin (MIC 0.25–2 μg/ml). According to the results of PAP analysis, all strains showed an increase in the area under curve depending on the concentration of vancomycin after selection, while a heteroresistant phenotype (with PAP/AUC 0.9) was detected in three isolates. All isolates showed walK mutations (T188S, D235N, E261V, V380I, and G223D). Exposure to short-term shock concentrations of vancomycin promotes the formation of heteroresistance in both MRSA and MSSA. Formation of VISA phenotypes is possible during therapy with vancomycin.

Daptomycin is an antibiotic used to treat infections caused by multidrug-resistant gram-positive pathogens. With increasing resistance to other antibiotics, an increase in daptomycin consumption is expected, which poses a potential threat of emergence of antibiotic resistance. These facts explain the need to study possible mechanisms for resistance emergence. In vitro selection of resistance to daptomycin in methicillin-resistant Staphylococcus aureus SA0420 of the ST239 genetic line was carried out. After 40 passages, the derivative strain was characterized by an increase in daptomycin minimum inhibitory concentration (MIC) from 1 to 64 µg/mL and a decrease in the doubling time and growth rate, while the induced autolytic activity did not change. During the selection and assessment of population diversity by the whole genome sequencing, both homo-mutations (nucleotide substitutions in 96–100% of reads after the alignment with the control genome at the particular position) and hetero-mutations (5‒95% of alternative reads) were revealed. After the fifth passage, the mutation in the S295L codon of the mprF gene was identified. The 40th passage revealed an additional mutation in the R50C codon; this mutation was not previously described. A new variant of the A214T mutation was detected in the Cls2 protein. The hetero-mutation in the Q493K codon of the walK regulatory gene associated with the cell wall was identified during the fifth passage and was not detected during further passaging. Hetero-mutations were also found in the ktrA and ntpA genes encoding the potassium uptake protein and phosphate transport protein, respectively. By the 40th passage of the selection, stop codons in the hypothetical gene yhfP (SACOL1927) were identified. Therefore, daptomycin resistance arises through the development of hetero-populations. After the 5th, 20th, and 40th passages, homo- and hetero-mutations in the genes with the proved role in the emergence of daptomycin resistance (mprF, cls2, and walK), as well as in the potential candidates (ktrA, ntpA, and yhfP), were revealed.

Mutations in genes of omnipotent nonsense suppressors SUP35 and SUP45 in yeast Saccharomyces cerevisiae encoding translation termination factors eRF3 and eRF1, respectively, and prionization of the eRF3 protein may lead to the suppression of some frameshift mutations (CPC mutations). Partial inactivation of the translation termination factor eRF3 was studied in strains with unstable genetically modified prions and also in transgenic yeast S. cerevisiae strains with the substitution of the indigenous SUP35 gene for its homolog from Pichia methanolica or for a recombinant S. cerevisiae SUP35gene. It was shown that this partial inactivation leads not only to nonsense suppression, but also to suppression of the frameshift lys2-90 mutation. Possible reasons for the correlation between nonsense suppression and suppression of the CPC lys2-90 mutation and mechanisms responsible for the suppression of CPC mutations during inactivation of translation termination factors are discussed.

Site-directed mutagenesis and nucleotide sequence analysis were used to study the roles of the global and local contexts in suppression of the lys2-90 frameshift (FS) mutation inSaccharomyces cerevisiae. Global context features established for the LYS2 mRNA region containing the extra T (lys2-90) were similar to those characteristic of regions involved in translational frameshifting. These were a potential ability of the region to form a pseudoknot and the presence of heptanucleotide CUU UGA C with the “hungry” UGA nonsense codon in the pseudoknot. Some local context features proved to be essential for the phenotypic expression of FS suppression as a result of translational frameshifting. Two amino acid substitutions determined by the nucleotide sequence between the extra U and the UGA nonsense codon lacked expression. A dependence was observed between the efficiency of FS suppression and the type of the nonsense codon located at a particular position downstream of the extra nucleotide (UGA > UAG > UAA). When translation termination was inactivated, nonsense suppression and FS suppression correlated with each other. These results suggest that translational frameshifting, which underlies suppression in the case of inactivation of translation termination, most likely takes place on the nonsense codon arising as a result of insertion of an extra nucleotide.