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

Abstract

Following a pressure treatment of a clonal Staphylococcus 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 typical S. 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. These S. aureus variants showed phenotypic similarities to previously isolated Listeria monocytogenes piezotolerant mutants that contained mutations in ctsR. Because of these similarities, possible alterations in the ctsR hypermutable regions of the S. aureus variants were investigated through amplified fragment length polymorphism analysis. No mutations were identified, and subsequently we sequenced the ctsR and hrcA genes of three representative variants, finding no mutations. This work demonstrates that S. aureus probably possesses a strategy resulting in an abundance of multiple-stressresistant variants within clonal populations. This strategy, however, seems to involve genes and regulatory mechanisms different from those previously reported for L. monocytogenes. We are in the process of identifying these mechanisms.

Population Diversity of Listeria monocytogenes LO28: Phenotypic and Genotypic Characterization of Variants Resistant to High Hydrostatic Pressure

“…All LO28 variants were therefore tested for mutations in this specific repressor gene (hrcA), its promoter region, and binding site, but no mutations were found (data not shown). A similar observation was also made in the study with HHP-resistant S. aureus isolates (16).…”

“…SCVs have also been described for HHP-resistant staphylococci. These staphylococcal SCVs resulted from a deficiency in aerobic electron transport chain activity, resulting in lower ATPgenerating capacity and consequently reduced growth yields under aerobic conditions (16). On the contrary, for the LO28 CtsR SCVs the maximum growth rates under aerobic conditions were similar to that of the wild type at 7°C and similar or only slightly lower at 30°C.…”

“…Recent characterizations of HHP-resistant S. aureus variants and pressure-tolerant L. monocytogenes strains revealed in both studies no mutations in ctsR, suggesting that also differences in other genomic regions are responsible for their phenotype (3,16). An obvious candidate may be the HrcA repressor, which controls expression of class I stress response genes encoding chaperones (29).…”

“…The other Scott A stable variants have unknown mutations (14). Stable HHP-resistant variants of other food-borne pathogens, including Staphylococcus aureus (16) and Escherichia coli (8,24), have also been isolated. A few of their phenotypic characteristics have been described, and the studies revealed only diversity in heat resistance among the resistant variants.…”

Isolation of Highly Heat-Resistant Listeria monocytogenes Variants by Use of a Kinetic Modeling-Based Sampling Scheme

“…Those isolates were individually subcultured five times during five consecutive days using 0.5% (vol/vol) inocula in fresh BHI medium (equivalent to approximately 40 generations) and on day 5 retested for resistance to HHP. In the present study, variants were searched by an optimized series of repetitive stress challenge cycles as described previously for the isolation of resistant variants of Escherichia coli and Staphylococcus aureus (15,21). To increase the probability of isolating resistant variants, three challenge treatments were used, and this was shown to be effective.…”