Piezotolerant Small-Colony Variants with Increased Thermotolerance, Antibiotic Susceptibility, and Low Invasiveness in a Clonal Staphylococcus aureus Population

Following a pressure treatment of a clonalStaphylococcus aureus culture with 400 MPa for 30 min, piezotolerant variants were isolated. Among 21 randomly selected survivors, 9 were piezotolerant and all formed small colonies on several agar media. The majority of the isolates showed increased thermotolerance, impaired growth, and reduced antibiotic resistance compared to the wild type. However, several nonpiezotolerant isolates also demonstrated impaired growth and the small-colony phenotype. In agglutination tests for the detection of protein A and fibrinogen, the piezotolerant variants showed weaker agglutination reactions than the wild type and the other isolates. All variants also showed defective production of the typicalS. aureus golden color, a characteristic which has previously been linked with virulence. They were also less able to invade intestinal epithelial cells than the wild type. TheseS. aureus variants showed phenotypic similarities to previously isolatedListeria monocytogenes piezotolerant mutants that contained mutations inctsR . Because of these similarities, possible alterations in thectsR hypermutable regions of theS. aureus variants were investigated through amplified fragment length polymorphism analysis. No mutations were identified, and subsequently we sequenced thectsR andhrcA genes of three representative variants, finding no mutations. This work demonstrates thatS. aureus probably possesses a strategy resulting in an abundance of multiple-stress-resistant variants within clonal populations. This strategy, however, seems to involve genes and regulatory mechanisms different from those previously reported forL. monocytogenes . We are in the process of identifying these mechanisms.