Induction of attachment-independent biofilm formation and repression of Hfq expression by low-fluid-shear culture of Staphylococcus aureus

Abstract

The opportunistic pathogen Staphylococcus aureus encounters a wide variety of fluid shear levels within the human host, and they may play a key role in dictating whether this organism adopts a commensal interaction with the host or transitions to cause disease. By using rotating-wall vessel bioreactors to create a physiologically relevant, low-fluid-shear environment, S. aureus was evaluated for cellular responses that could impact its colonization and virulence. S. aureus cells grown in a low-fluid-shear environment initiated a novel attachment-independent biofilm phenotype and were completely encased in extracellular polymeric substances. Compared to controls, low-shear-cultured cells displayed slower growth and repressed virulence characteristics, including decreased carotenoid production, increased susceptibility to oxidative stress, and reduced survival in whole blood. Transcriptional whole-genome microarray profiling suggested alterations in metabolic pathways. Further genetic expression analysis revealed downregulation of the RNA chaperone Hfq, which parallels low-fluid-shear responses of certain Gram-negative organisms. This is the first study to report an Hfq association with fluid shear in a Gram-positive organism, suggesting an evolutionarily conserved response to fluid shear among structurally diverse prokaryotes. Collectively, our results suggest S. aureus responds to a low-fluid-shear environment by initiating a biofilm/colonization phenotype with diminished virulence characteristics, which could lead to insight into key factors influencing the divergence between infection and colonization during the initial host-pathogen interaction.

Fig. 1.

Operation of the RWV bioreactor (Synthecon, Houston, TX). (A) Image of the NASA-designed RWV apparatus used to create the LSMMG environment for bacterial culture. (B) The altered positioning of the RWV that results in the two culture orientations, depicting the axis of rotation. The LSMMG environment is achieved by rotation of the RWV on an axis parallel to the ground, whereas the axis of rotation in the control orientation is perpendicular to the ground. (C) Depiction of the orbital path of a cell when cultured in the LSMMG orientation. The continued combination of the sedimentation effect, whereby gravity and lack of motility cause a cell to settle to the bottom of the vessel, and the clockwise solid body rotation of the medium results in the continuous suspension of the cell in an orbit.

Fig. 2.

Environmental scanning electron microscopy images of control and LSMMG-cultured S. aureus N315. Control-cultured S. aureus at 2,500× (A) and 10,000× (C) magnification revealed individual cells that were clearly visible. For low-fluid-shear-cultured S. aureus, at 2,500× (B) and 10,000× (D), the cells were much less visible and completely embedded in an EPS matrix.

Fig. 3.

Levels of antibiotic resistance for control and low-fluid-shear-cultured S. aureus N315. Low-shear-induced S. aureus aggregates were 1.72-fold more resistant to ciprofloxacin than bacteria cultured in the control-oriented RWV (*, P < 0.05).

Fig. 4.

Comparative growth curves of S. aureus N315 when cultured in either the LSMMG or control orientation. Culture in the LSMMG environment resulted in a 2.9-fold decrease in cell concentrations compared to control cultures.

Fig. 5.

Decreased carotenoid production of S. aureus N315 in response to LSMMG culture. (A) Centrifugation of the control and LSMMG cultures resulted in a visual difference in the pigmentation of the pellets. (B) To quantitate the differences in carotenoid production, the carotenoids were extracted and measured spectrometrically at 460 nm. There was a significant reduction in absorbance of the LSMMG-cultured bacteria compared to the control (*, P < 0.0001). S. aureus 8325, which does not produce carotenoids, was used as a negative control for comparison.

Fig. 6.

LSMMG-cultured S. aureus N315 is more susceptible to killing by oxidative stress. After 60 min (time zero), 50% of the LSMMG-cultured S. aureus had succumbed to the damage by H₂O₂, whereas the control cultures did not fall below 90% (*, P < 0.05). To test the duration of the LSMMG effect, samples of both the LSMMG and control bacteria were allowed to sit statically, out of the vessels, for a period of time (1, 1.5, and 2 h) and then subjected to H₂O₂. At 1.5 h there failed to be a significant (*, P < 0.05) difference in the survival rates of the LSMMG- and control-cultured bacteria after 60 min, and it was determined that the LSMMG effect had diminished.

Fig. 7.

Comparison of survival ability of LSMMG- and control-cultured S. aureus N315 when challenged with the immune components present in human whole blood. After 4 h, the LSMMG-cultured S. aureus displayed a significantly reduced ability to survive in whole blood compared to control cultures (*, P < 0.05).