Transcription of clumping factor A in attached and unattached Staphylococcus aureus in vitro and during device-related infection

Abstract

Staphylococcus aureus is one of the pathogens most frequently isolated in device-related infections. S. aureus is equipped with surface-associated proteins promoting specific binding to matrix molecules. Clumping factor A (ClfA, encoded by clfA) mediates adhesion to fibrinogen. Whereas the contribution of ClfA to pathogenicity is well documented, the influence of different growth and host parameters on gene activity is unclear. To elucidate this question, we investigated clfA transcript levels in an animal model of device-related infection and in planktonic and sessile bacteria grown in vitro. Specific mRNA from the S. aureus strains Newman, Reynolds, and RN6390 was quantified by LightCycler reverse transcription-PCR. In vitro, clfA transcript levels were low in the early logarithmic growth phase, but a clear increase was observed after the late logarithmic phase. Quantities of clfA transcripts were four to six times higher in the planktonic than in the sessile bacterial subpopulations grown to the stationary phase. During infection, in strains Newman and Reynolds levels of clfA transcripts in exudates accumulating in the infected devices were lower than those in the bacteria grown in vitro to stationary phase. clfA mRNA levels in the exudates increased during the initial phase of infection and remained constant after 96 h postinoculation. In contrast to the in vitro results, quantities of clfA transcripts in the unattached bacteria of the exudates never exceeded the level of clfA transcripts in the sessile bacteria attached to glass beads. However, a clear increase in clfA quantities in the sessile bacteria was observed late in infection after 144 h. In conclusion, maximal clfA transcript levels are reached late during growth in vitro and in vivo.

FIG. 1.

Quantitative transcript analysis of S. aureus strains Newman (A), Reynolds (B), and RN6390 (C) by LightCycler RT-PCR in vitro. Levels of clfA transcripts 8, 12, 24, and 144 h after inoculation were quantified in reference to the levels of gyrB transcripts (left y axis). Levels of clfA transcripts were determined in planktonic bacteria (black bars) and in sessile bacteria (hatched bars). At each time point, bacterial densities (right y axis) in planktonic (black diamonds; CFU per milliliter) and in sessile bacteria (white squares; CFU per bead) were determined.

FIG. 2.

Slot blot hybridization for the detection of clfA mRNA in planktonic and sessile bacteria in vitro. Equal amounts of total RNA were serially diluted (1:1, 1:5, 1:25) and hybridized with a digoxigenin-labeled gene probe specific for clfA.

FIG. 3.

Quantitative transcript analysis of S. aureus strains Newman (A), Reynolds (B), and RN6390 (C) by LightCycler RT-PCR during device-related infection. Levels of clfA transcripts in bacteria of the exudates (black bars) and in sessile bacteria attached to glass beads (hatched bars) were quantified in reference to the levels of gyrB transcripts (left y axis). Bacterial densities (right y axis) in the exudates (black diamonds; CFU per milliliter) and on glass beads (white squares; CFU per bead) were determined at each time point.