However, careful examination of the agar plates for pinpoint colonies after 3 days of incubation would help find sVISA. Even stored culture of
VISA strains may contain the original sVISA strain as a minor subpopulation. In fact, we identified a few pinpoint colonies on the drug-free agar plates streaked with the glycerol stocks of 3 of the 33 VISA clinical strains listed in Table 1. The slow-growing strains established from these pinpoint colonies expressed high and unstable vancomycin resistance as well as a colony morphology similar to those observed with Mu3-6R-P (Table 3). All of the strains lost the VISA phenotype and returned to the large colony morphology within a week’s passage. This indicated that generation of sVISA is not infrequent. They are generated during vancomycin therapy but escaped FG-4592 manufacturer recognition since they easily reverted to hVISA or became stabilised as VISA during propagation in the clinical laboratory. Generation of sVISA during vancomycin Paclitaxel therapy may well explain the discrepancy between the frequency of vancomycin therapeutic failure and the frequency of VISA isolation from patient samples. Whole-genome sequencing of Mu3-6R-P revealed only one single nucleotide polymorphism relative to the Mu3 genome.
The mutation was identified in the rpoB gene, changing the arginine-512 to proline. Sequence determination of the rpoB gene identified non-synonymous rpoB mutations in 7 (21%) of 28 sVISA strains; they Sorafenib were rpoB(G744R), rpoB(S746F) (2 strains), rpoB(H929T) (2 strains) and rpoB(G977V). Unlike the rpoB mutations in extant VISA strains, these mutations were all identified outside of the RRDR and, except for rpoB(R512P), were located in the C-terminus half of the RpoB protein ( Fig. 5). Three Mu3-derived sVISA strains Mu3-6R-P, 17–6 d and 21–4 d carrying rpoB(R512P), rpoB(S746F) and rpoB(H929T), respectively, were cultivated in drug-free medium, and large-colony derivative strains were established to determine their rpoB gene sequences. The rpoB(H929T) mutation was back-mutated to wild-type
and the rpoB(R512P) mutation was replaced by alternate mutations such as rpoB(R512L), rpoB(R512H) or rpoB(R512S). Only the rpoB(S746F) mutation was not changed in three independently isolated large-colony strains. All of the large-colony strains reverted to hVISA phenotype with comparable levels of vancomycin resistance and DTs to that of Mu3. Therefore, it is likely that certain rpoB mutation does serve as an on-and-off switch for the sVISA phenotype. A single mutation in the rpoB gene can make the cell survive otherwise growth-inhibitory concentration of vancomycin. After the vancomycin selective pressure is lifted, the bacteria can start to diverge themselves beyond the constraints imposed by the rpoB regulatory mutation.