In the present study, we show that biofilm maturation and detachment in S. epidermidis
are mediated by a specific class of surfactant peptides, the β-type PSMs. Notably, these findings provide a mechanistic basis for crucial steps in staphylococcal biofilm development that were not previously understood on a molecular level. Furthermore, using a premier biofilm-forming pathogen, our results lend support to the evolving concept that biofilm maturation in bacteria is accomplished by the use of surfactants (7
). However, it is important to note that the various bacterial systems appear to share only the surfactant physicochemical properties of the biofilm maturation effectors and quorum-sensing control of their production. In contrast, the chemical nature of the surfactants and genetic basis of their biosynthesis are completely unrelated.
Detailed understanding of the molecular determinants governing biofilm development is of utmost importance for potential therapeutic interference with biofilm-associated infection. In biofilm-forming pathogens, biofilm detachment processes have exceptionally high significance, as they are believed to lead to the systemic dissemination of infection. However, molecular effectors of biofilm maturation and detachment have not been analyzed in vivo in any biofilm-forming pathogen. It is therefore important to stress that our study establishes in vivo relevance for biofilm detachment by demonstrating a key role of surfactant-like peptides in promoting the dissemination of S. epidermidis biofilm-associated infection.
Biofilm maturation and detachment are commonly under quorum-sensing regulation to ascertain a well-controlled degree of channel formation and biofilm expansion (12
). This is also the case for staphylococci (26
). Importantly, our findings indicate that the PSMβ peptides are the main determinants that exert the impact of quorum-sensing control on these mechanisms of biofilm development in S. epidermidis
on the molecular level. This is in good agreement with our previous observation in S. aureus
showing that the mechanism of psm
control by agr
is different from, and likely evolved before, agr
control of other staphylococcal virulence determinants (16
). Our finding that PSMs have a key role in biofilm development as a basic phenotype of staphylococcal physiology during both commensal life and infection provides a plausible explanation for the stringency and early evolution of that regulation. Interestingly, the relatively higher concentration of PSMβ peptides compared with other PSM peptides in biofilm versus planktonic culture indicates that the psm
β operon is regulated by a yet unknown regulator in addition to the psm
master regulator agr
. Furthermore, we have previously shown that Agr-dysfunctional and thus, PSM-negative strains are more frequently found among strains isolated from biofilm-associated infection (26
). Our present results suggest that such strains have lost the capacity to detach and owing to the lack of PSMβ production may form more compact and extensive biofilms.
Blocking the agr
quorum–sensing system has been frequently proposed as a potential basis for novel therapeutics interfering with staphylococcal virulence, as many toxins are under positive control of agr
). On the other hand, it has been noted that this may lead to increased biofilm formation, owing to negative agr
control of biofilm expansion (33
), which is in accordance with results achieved in the present study. However, our results also indicate that such treatment may inhibit bacterial dissemination from biofilms, showing that interference with agr
might potentially be beneficial also during biofilm-associated infection. In addition, we provide proof of principle that the specific inhibition of biofilm detachment surfactants may prevent dissemination, a strategy we believe should be further evaluated for potential therapeutic use.
Surfactants are not the only molecules that have been proposed to contribute to biofilm maturation and detachment in staphylococci. According to an alternative hypothesis, these processes may also be accomplished by enzymatic degradation of biofilm matrix molecules. In that regard, recent research indicated that staphylococcal biofilm formation is accomplished by protein and/or exopolysaccharide biofilm matrix components (24
). Hypothetically, enzymatic degradation of these polymers may thus contribute to biofilm maturation; and a proteolytic detachment mechanism has been proposed for protein-dependent biofilms of S. aureus
). However, direct evidence for such a mechanism is scarce and no evidence has been produced for clinical strains or in vivo. Furthermore, it has been shown that such enzymes specifically degrade only those staphylococcal biofilms that are based on proteins, or exopolysaccharides, respectively (35
). Moreover, it has become clear that staphylococci do not produce an enzyme to degrade the PIA/PNAG exopolysaccharide, the most well-established staphylococcal biofilm matrix component (36
). It is therefore important to stress that, in contrast, our results indicate that the PSM-based surfactant detachment mechanism is largely independent of the biofilm matrix chemical composition. Finally, presence of PSMs and PSM-like molecules in many staphylococci including S. aureus
) indicates that PSMs may also contribute to biofilm structuring in other staphylococcal species, which remains to be investigated.
In summary, our study presents a mechanism of S. epidermidis biofilm maturation and detachment that is likely used in a similar form by other staphylococcal biofilm–forming strains. Furthermore, using S. epidermidis as an example, our study provides evidence for the importance of biofilm detachment molecules in the dissemination of biofilm-associated infections, thus identifying a potential target for therapeutics aimed at preventing complication and spread of such infections.