Several major findings emerge from this investigation of the impact of sub-inhibitory concentrations of anti-staphylococcal drugs on S. aureus adhesion and invasion phenotypes. S. aureus binding to human fibronectin and the transcriptional levels of the fnbA/B genes encoding the fibronectin-binding proteins were differentially modulated by antimicrobial agents. Oxacillin, moxifloxacin and linezolid treatment led to the development of a hyper-adhesive phenotype, along with an increase in fnbA/B mRNA levels relative to the gyrB internal standard. The same hyper-adhesive phenotype was induced by clindamycin treatment, although no significant change in fnbA/B mRNA levels was observed. Rifampin was the only antimicrobial agent among those tested that significantly inhibited S. aureus binding to fibronectin without affecting relative fnbA/B transcription profiles. Vancomycin and gentamicin induced no change in either the adhesion phenotype or the fnbA/B transcription. S. aureus adhesion to and invasion of live eukaryotic cells was also assessed after oxacillin, linezolid or rifampin treatment in an ex vivo infection model of cultured human osteoblasts. Oxacillin treatment significantly increased S. aureus adhesion but not invasion, while no significant change in adhesion or invasion levels was observed after linezolid or rifampin treatment.
Several recent studies have focused on the influences of sub-inhibitory concentrations of antimicrobial agents on the expression of various virulence factors produced by S. aureus and on the various regulation mechanisms involved in this modulation [6, 8, 17]. Similarly, the expression and surface display of FnBPs are regulated by a complex network of global regulators and stress response pathways that can be triggered by antimicrobial agents in a drug-specific fashion. Fluoroquinolones, by activating the SOS system (a global response system to DNA damage), have been shown to induce fnbB up-regulation and fibronectin binding in S. aureus through a LexA-RecA-dependant pathway . Moreover, in a rabbit S. aureus infection model, moxifloxacin treatment inhibited the expression of agr global regulator , which acts as a repressor of surface protein expression, including fnbA/B, and as an activator of exotoxin expression . Beta-lactams, besides inducing the SOS response system , have also been reported to up-regulate virulence factor expression, including fnbB, through the two-component system SaeRS .
Clindamycin and linezolid are protein synthesis inhibitory agents known to repress exotoxin secretion by S. aureus [6–8]. Thus, their positive effect on fibronectin binding in S. aureus makes it tempting to speculate that their impact on protein expression involves selective inhibition of agr. We recently showed that sub-inhibitory concentrations of linezolid repress early agr expression in S. aureus . Furthermore, sub-inhibitory concentrations of clindamycin have been shown to decrease saeRS expression , thus possibly interfering with fnbB expression. An alternative explanation for the effects of clindamycin has been reported by Blickwede et al., who observed that fnbB mRNA levels were selectively increased after clindamycin treatment and that this increase was due to mRNA stabilisation in the presence of clindamycin . Whether linezolid also affects fnbA/B mRNA levels through mRNA stabilisation remains unknown, and this question merits further investigations.
With respect to sub-inhibitory rifampin treatment, the decrease in fibronectin binding observed here was not accompanied by a transcriptional decrease of fnbA/B relative to the internal control gyrB, suggesting that fibronectin binding inhibition takes place at the post-transcriptional level. Mechanisms underlying the effects of rifampin in this context are still to be elucidated. We speculate that these mechanisms could involve either a decrease of sortase activity, which is responsible for cell wall anchorage of several MSCRAMMs including FnBPs [26, 27], or an increase of protease activity, which has been shown to dramatically influence fibronectin-binding in S. aureus .
Interestingly, fibronectin-binding modulation by oxacillin, linezolid or rifampin only partially correlated with host cell adhesion and invasion under our experimental conditions. Although oxacillin-treated S. aureus displayed significantly increased binding to cultured osteoblasts, its invasiveness did not differ significantly from that of the untreated control. Beta-lactams interfere with cell division and induce dramatic changes in staphylococcal morphology even at sub-inhibitory concentrations . Of note, the inhibition of cell separation ultimately leads to the formation of so-called pseudomulticellular staphylococci . These aberrant forms were present following oxacillin treatment under our experimental conditions, whereas bacterial size and morphology were unchanged in bacteria either untreated or treated with rifampin or linezolid, as objectivated by microscopic examination after fluorescence staining of the cell wall (data not shown). It is likely that the larger size of pseudomulticellular staphylococci hampers their internalization by osteoblasts, which could negatively compensate the increase in adhesiveness induced by oxacillin. In the same way, we failed to identify a change in adhesion and invasion phenotypes after linezolid or rifampin treatment. A putative explanation for these discrepancies between phenotypes observed under both controlled in vitro conditions and more complex ex vivo infection assays is adhesin redundancy. Although FnBPs play a major role in S. aureus-host cell interactions, whole cell adhesion involves several other MSCRAMMs , which are also likely regulated by antibiotics and thus could hamper or cancel the effects of FnBPs modulation. This outcome is illustrated by our finding that strain DU5883 lacking fnbA/B still adhered significantly to cultured osteoblasts. The same is probably true with respect to S. aureus invasiveness, although a more limited number of factors are involved along with FnBPs in the cell invasion process. FnBPs are required and sufficient for host cell invasion , as confirmed in our model by the observation that invasiveness was abolished in strain DU5883. However, the multifunctional protein eap, which also binds fibronectin, acts additively with FnBPs to mediate host cell invasion in eap-positive strains such as 8325-4  and can partially compensate for loss of FnBP functions . Additional studies are warranted to determine whether compensatory mechanisms occur to sustain host cell invasion, despite rifampin-mediated FnBP expression decrease.