In vitro serial passage of Staphylococcus aureus: changes in physiology, virulence factor production, and agr nucleotide sequence.
Recently, we observed that Staphylococcus aureus strains newly isolated from patients had twofold-higher aconitase activity than a strain passaged extensively in vitro, leading us to hypothesize that aconitase specific activity decreases over time during in vitro passage. To test this hypothesis, a strain recovered from a patient with toxic shock syndrome was serially passaged for 6 weeks, and the aconitase activity was measured. Aconitase specific activity decreased 38% (P < 0.001) by the sixth week in culture. During serial passage, S. aureus existed as a heterogeneous population with two colony types that had pronounced (wild type) or negligible zones of beta-hemolytic activity. The cell density-sensing accessory gene regulatory (agr) system regulates beta-hemolytic activity. Surprisingly, the percentage of colonies with a wild-type beta-hemolytic phenotype correlated strongly with aconitase specific activity (rho = 0.96), suggesting a common cause of the decreased aconitase specific activity and the variation in percentage of beta-hemolytic colonies. The loss of the beta-hemolytic phenotype also coincided with the occurrence of mutations in the agrC coding region or the intergenic region between agrC and agrA in the derivative strains. Our results demonstrate that in vitro growth is sufficient to result in mutations within the agr operon. Additionally, our results demonstrate that S. aureus undergoes significant phenotypic and genotypic changes during serial passage and suggest that vigilance should be used when extrapolating data obtained from the study of high-passage strains.
Somerville
Greg A
GA
Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases/NIH, 903 South 4th Street, Hamilton, Montana 59840, USA.
Beres
Stephen B
SB
Fitzgerald
J Ross
JR
DeLeo
Frank R
FR
Cole
Robert L
RL
Hoff
Jessica S
JS
Musser
James M
JM
D000154:Aconitate Hydratase / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000378:metabolism; D001483:Base Sequence; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial; D006460:Hemolysin Proteins / Q000378:metabolism; D006461:Hemolysis; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D009876:Operon / Q000235:genetics; D012692:Serial Passage; D012772:Shock, Septic / Q000382:microbiology; D013203:Staphylococcal Infections / Q000382:microbiology; D013205:Staphylococcal Protein A / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity / Q000502:physiology; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics*; D014774:Virulence
C095698:Agr protein, Staphylococcus aureus; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003470:Culture Media; D006460:Hemolysin Proteins; D013205:Staphylococcal Protein A; D015534:Trans-Activators; C021091:staphylococcal alpha-toxin; D000154:Aconitate Hydratase