Staphylococcus epidermidis surfactant peptides promote biofilm maturation and dissemination of biofilm-associated infection in mice

Biofilms are surface-attached agglomerations of microorganisms embedded in an extracellular matrix. Biofilm-associated infections are difficult to eradicate and represent a significant reservoir for disseminating and recurring serious infections. Infections involving biofilms frequently develop on indwelling medical devices in hospitalized patients, and Staphylococcus epidermidis is the leading cause of infection in this setting. However, the molecular determinants of biofilm dissemination are unknown. Here we have demonstrated that specific secreted, surfactant-like S. epidermidis peptides — the β subclass of phenol-soluble modulins (PSMs) — promote S. epidermidis biofilm structuring and detachment in vitro and dissemination from colonized catheters in a mouse model of device-related infection. Our study establishes in vivo significance of biofilm detachment mechanisms for the systemic spread of biofilm-associated infection and identifies the effectors of biofilm maturation and detachment in a premier biofilm-forming pathogen. Furthermore, by demonstrating that antibodies against PSMβ peptides inhibited bacterial spread from indwelling medical devices, we have provided proof of principle that interfering with biofilm detachment mechanisms may prevent dissemination of biofilm-associated infection.

Surface-attached cellular agglomerations of microorganisms called biofilms are an important virulence determinant in bacteria (1), mainly because biofilm formation significantly increases resistance to antibiotics and host defenses (2). Many biofilm-associated infections occur in the hospital setting by contamination of indwelling medical devices from the epithelial flora of the patient or health care personnel. Staphylococcus epidermidis, a normal inhabitant of human skin, is the predominant causative agent of these infections (3). Importantly, bacterial dissemination from biofilm-infected catheters represents the most frequent source of severe S. epidermidis infections such as sepsis (4). Especially neonates often develop sepsis from S. epidermidis catheter infection, and S. epidermidis infection is a significant global source of morbidity and death, particularly in very low birth weight infants (5).

The formation of a biofilm begins with the attachment of bacteria to a surface and is followed by proliferation and maturation, which ultimately leads to the characteristic 3D biofilm structure with mushroom-shaped bacterial agglomerations surrounded by fluid-filled channels (6). Later, cells may detach from the biofilm in a process believed to be of crucial importance for the dissemination of a biofilm-associated infection. The molecular basis of biofilm maturation and detachment is poorly understood, but presumably involves mechanisms to disrupt cell-cell adhesion. In vitro evidence obtained in Pseudomonas aeruginosa and Bacillus subtilis indicates cell-cell disruption may be accomplished by surfactants (7–10), while enzymatic digestion of biofilm matrix molecules appears to promote biofilm detachment in Actinobacillus actinomycetemcomitans (11). These mechanisms are commonly under control of cell density (“quorum sensing”) and are likely to ascertain a tightly regulated degree of biofilm expansion (12, 13). In staphylococci, the molecular effectors of cell-cell disruption during biofilm development are not known. Furthermore, whether biofilm detachment mechanisms contribute to the in vivo dissemination of biofilm-associated infection has not been investigated in any bacterium.

We and others have identified a family of short staphylococcal peptides, the phenol-soluble modulins (PSMs) (14, 15), which are strictly regulated by the quorum-sensing system agr (16–18) and whose amphiphilic α-helical structure suggests surfactant-like properties. This prompted us to analyze the role of PSMs in biofilm development. In the present study, we demonstrate that the β-type PSMs represent key effectors of S. epidermidis biofilm maturation and detachment. Furthermore, we show that these peptides facilitate the dissemination of biofilm-associated infection, providing evidence for in vivo significance of biofilm detachment.

PSMβ peptides are the main PSM type produced in S. epidermidis biofilm culture. To investigate the role of PSMs in biofilms, we first determined production of PSM peptides in planktonic versus biofilm culture. We found that PSM production was overall lower in biofilm culture, while relative production of β-type PSMs was significantly increased (Figure 1). Of note, β-type PSMs were virtually the only PSM type produced in the biofilm mode of growth. We detected similar production patterns in all S. epidermidis strains in our collection, with the exception of 24 strains that completely lacked production of PSMs, including the agr-encoded δ-toxin. Thus, they are most likely naturally occurring mutants in the agr quorum–sensing system, whose deletion or lack of functionality leads to absence of PSM production (16–18). Of note, all those 24 strains were shown to contain psmβ genes by analytical PCR (data not shown).

Figure 1

PSM production under biofilm and planktonic modes of growth. PSM production under planktonic (shaken 125 ml flasks containing 50 ml TSB/0.5% glucose, 18 hour growth, 37°C) or biofilm (24 hour static culture in microtiter plates, 37°C) modes of growth was assayed by RP-HPLC/ESI-MS. PSMs were determined in supernatants of planktonic or biofilm cultures after centrifugation. Absolute amounts are shown at the top, relative composition at the bottom. S. epid. 1457, S. epidermidis 1457.

The psmβ locus forms a transcriptional unit. β-type PSMs are distinguished from α-type PSMs and δ-toxin (~20–25 amino acids) by their larger size (~45 amino acids) and absence of cytolytic activity at physiological concentrations (18). As PSM production patterns suggested that especially the β-type PSMs may play a role in biofilm development, we first analyzed organization and transcription of the psmβ genes (Figure 2A). The psmβ operon is composed of 4 genes in strains S. epidermidis ATCC12228 and RP62A (19, 20), whose genome sequences are available, while the clinical isolate 1457 (21) used in our study has only 3 psmβ genes. This is due to an exact duplication of the psmβ1 gene in strains ATCC12228 and RP62A, while we determined by DNA sequencing that this duplication is missing from strain 1457. The remaining sequence of the psmβ locus of strain 1457 was exactly the same as in ATCC12228. In the shotgun-sequenced strain M23864, the psmβ operon appears to consist of 5 genes with slightly different peptide products. In keeping with the psmβ1 gene duplication, strains RP62A and ATCC12228 secreted more PSMβ1 than PSMβ2, but combined production of the PSMβ peptides was not higher than in strain 1457 (data not shown). Furthermore, we never detected a peptide in culture filtrates of any S. epidermidis strain corresponding to the gene product of the psmβ3 gene, indicating that the hypothetical PSMβ3 peptide is not secreted. To analyze whether the psmβ genes form an operon, and thus are transcribed together, we performed Northern blot analysis (Figure 2B). We detected 1 predominant band, whose size corresponded exactly to the postulated size of a transcript comprising all 3 psmβ genes of strain 1457, which therefore form an operon.

Figure 2

The psmβ operon. (A) Organization of the psmβ locus in genome-sequenced S. epidermidis strains and the 1457 strain used in this study. Construction of the psmβ mutant allelic replacement strain is shown at the bottom. (B) Northern blot analysis of S. epidermidis 1457 RNA from different growth phases using a psmβ probe. The psmβ signal is marked by an arrow.

PSM peptides influence S. epidermidis biofilm formation in vitro. To investigate whether PSMβ peptides impact biofilm development, we first assayed in vitro biofilm formation with synthetic PSMβ peptides. The peptides were added to growing biofilm cultures of an agr mutant of strain 1457 (22), which owing to strict regulation of PSMs by agr (16–18), is devoid of PSM production. The PSMβ peptides promoted biofilm formation in a medium concentration range, while they inhibited biofilm formation at higher concentrations (Figure 3A). Of note, this phenotype is in accordance with a putative function of PSMβ peptides in cell-cell disruption on the molecular level (3, 23): at lower concentrations, this process is thought to facilitate the formation of channels, which increases biofilm formation on the macroscopic level, whereas higher concentrations promote detachment and thus a reduction in biofilm mass (Figure 3B).

Figure 3

S. epidermidis in vitro biofilm formation under influence of PSMβ peptides. (A) Biofilm formation in microtiter plates (24 hours, 37°C). PSMβ peptides at different concentrations were added at the time of inoculation with S. epidermidis agr (devoid of PSMs). Biofilms were made visible using safranin staining (see example for PSMβ1 at the bottom), and biofilm formation was measured using an ELISA reader. Error bars depict mean ± SEM. (B) Schematic presentation of biofilm cell-cell disruptive processes leading to channel formation and detachment.

Biofilm formation in staphylococci has been shown to be exopolysaccharide dependent or independent (24, 25). To analyze whether PSMβ peptides mediate biofilm detachment in both exopolysaccharide-dependent and –independent S. epidermidis biofilms, we first determined presence of the ica genes responsible for production of the main biofilm exopolysaccharide polysaccharide intercellular adhesin (PIA, or poly-N-acetylglucosamine [PNAG]) in the 24 agr-negative S. epidermidis strains. Then we determined in vitro biofilm formation by these strains. Interestingly, biofilm-forming capacity was significantly higher among the strains that contained ica genes than among the ica-negative strains (Figure 4A), confirming the importance of PIA/PNAG for efficient biofilm formation in S. epidermidis (24). We selected the 2 ica-negative strains with the highest biofilm-forming capacity and 1 ica-positive strain, in addition to strain 1457, to confirm the universal importance of PSMβ peptides for biofilm detachment in vitro. In all strains, PSMβ1 had a biofilm detachment effect very similar to that observed with strain 1457 (Figure 4D), demonstrating that the impact of PSMβ peptides on biofilm detachment in vitro is not dependent on whether biofilm formation is exopolysaccharide dependent or independent.

Figure 4

Specific impact of PSMβ1 on in vitro biofilm detachment. (A) Biofilm formation by functionally agr-negative (without PSM production) ica-positive versus ica-negative strains. Biofilms were grown in microtiter plates for 24 hours and stained with safranin. ***P < 0.0001, t test. Colored symbols represent the strains used for the analyses shown in D. Error bars depict mean ± SEM. (B) α-Helical wheel presentation and (C) sequences of PSMβ1 and the mutated PSMβ1* peptide. The α-helical part (positions 17–44) is shaded in yellow. In the sequence, amino acids changed in PSMβ1* in comparison with PSMβ1 are shown in red; in the wheel presentation, exchanges are shown using red arrows. (D) Impact of PSMβ1 versus PSMβ1* on in-vitro biofilm development by 2 ica-positive and 2 ica-negative S. epidermidis strains. Conditions are the same as those used for the experiment shown in Figure 3A. *P < 0.05; **P < 0.01, t tests comparing values for the PSMβ1- versus PSMβ1*-treated samples at corresponding concentrations of added peptide.

To confirm the specificity of PSMβ detachment activity and analyze the importance of the PSMβ amphipathic α-helix in the observed detachment process, we synthesized a PSMβ1 derivative, PSMβ1*, in which 5 amino acids were altered to prevent the formation of an amphipathic α-helix (Figure 4, B and C). As the formation of an amphipathic α-helix is the basis of surfactant properties, these experiments were also conducted to gain insight into whether the detachment mechanism is dependent on the surfactant properties of PSMβ1. According to analysis by HeliQuest ( http://heliquest.ipmc.cnrs.fr), the amphipathic α-helical part of PSMβ1 extends from amino acids 17 to 44 (C terminus). This was predicted by the hydrophobic face that is characteristic of an amphipathic α-helix and was found for 18–amino acid stretches comprising those, but not amino acids N-terminal of position 17 (stretches 1–18 to 16–43). In the C-terminal part of PSMβ1, 2 lysine residues (positive charge) were changed to proline residues (K22P, K40P), and 3 uncharged amino acids were altered to negatively charged amino acids (W20E, I27E, V35E) to abolish α-helicity and amphipathy in PSMβ1* (Figure 4, B and C). With PSMβ1*, the detachment effect was absent (in strains 1457, BM26, and BM33) or strongly decreased (strain BM32) compared with PSMβ1 (Figure 4D), indicating that the impact of PSMβ1 on biofilm detachment is specific and dependent on the PSMβ amphipathic α-helix. Furthermore, these results strongly suggest that the mechanism by which PSMβ peptides contribute to biofilm maturation and detachment is based on their surfactant properties.

Biofilm structuring and detachment by PSMβ peptides is likely accomplished during biofilm development, while mature S. epidermidis biofilms that are stabilized by an exopolysaccharide network may be largely resistant to macroscopic decomposition by externally added PSMβ peptides. To test this hypothesis, we added PSMβ1 peptide at 1 mg/ml to a 24-hour biofilm of the PIA/PNAG-producing strain 1457. We observed only a very minor impact on the biofilm (data not shown). In agreement with a surfactant-based mechanism of PSMβ detachment, these results suggest that PSMβ peptides must be present during biofilm development to promote biofilm structuring and detachment, while the presence of an established matrix network prevents the large-scale detachment of biofilm clusters by PSMβ peptides.

PSMβ peptides promote biofilm structuring and detachment in vitro. To validate the hypothesis that PSMβ peptides promote biofilm detachment, we analyzed biofilm development under flow using confocal laser scanning microscopy (CLSM). To measure expression of the psmβ operon, we constructed a psmβ promoter-egfp transcriptional fusion. The egfp gene was synthetically assembled using codon usage optimized for staphylococci (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI42520DS1) to increase fluorescence intensity. Expression of psmβ was limited to the outer layers of the biofilm (Figure 5A) in accordance with a function in detachment and control by agr, for which we have previously shown a similar spatial expression pattern in S. epidermidis biofilms (26). Importantly, we observed that strong psmβ expression was followed by the appearance of void spaces (Figure 5, B and C). Furthermore, there were significantly more psmβ-expressing cells in the effluent compared with the biofilm cells (Figure 5D). These results confirmed our hypothesis that psmβ expression leads to biofilm cluster detachment during biofilm development.

Figure 5

Role of PSMβ peptides in S. epidermidis biofilm development: cell detachment. A psmβ-promoter egfp fusion construct (see Supplemental Figure 1 for the synthetic egfp gene with optimized Staphylococcus codon usage) was produced to monitor psmβ expression in dynamic S. epidermidis biofilms using flow cells with CLSM (green). Entire biofilms were stained with propidium iodide (red). (A) Mature (24 hours) biofilm showing expression of psmβ at the outer biofilm layer. (B and C) Biofilm cluster detachment observed during biofilm development. Lower (A) and higher (B) temporal and spatial resolution is shown. (D) Comparative analysis of the relative amount of cells expressing psmβ (green fluorescence) in effluent and biofilm. The ratio of fluorescent versus nonfluorescent cells was determined using IMARIS software in effluent and biofilm samples. **P < 0.01, t test. Error bars depict mean ± SEM.

To further investigate the role of PSMβ peptides in S. epidermidis biofilm development, we constructed a deletion mutant of the entire psmβ operon in S. epidermidis 1457 (Figure 2A). CLSM images revealed that static biofilms of the psmβ operon deletion mutant in comparison with the isogenic WT strain lacked channel formation and were thicker (Figure 6, A and C). Genetic complementation restored the phenotype observed in the WT (Figure 6A). Image analysis of the biofilms using IMARIS software showed a significant increase in the total and average biovolumes, indicative of an increase in total biofilm formation and decrease in the degree of channel formation, respectively, comparing the mutant to the WT, while complementation restored WT levels (Figure 6B). These findings showed that PSMβ peptides promote channel formation in S. epidermidis biofilms. In addition, they further substantiated that PSMβ peptides facilitate detachment, as the lack of detachment leads to biofilm expansion.

Figure 6

Role of PSMβ peptides and agr in S. epidermidis biofilm development: channel formation and biofilm expansion. (A) CLSM pictures of S. epidermidis 1457 WT, isogenic psmβ mutant, and psmβ-complemented strains. The WT and psmβ mutant strains were transformed with the control plasmid pT181mcs to ensure comparability. Static biofilms were grown for 24 hours, stained with propidium iodide, and imaged using CLSM. View is from the top. (B) Analyses of total and average biovolumes, which are measures of total biofilm and degree of channel formation, respectively, using IMARIS software of the biofilm samples shown in part A. Note that increased average biofilm volume corresponds to decreased channel formation. **P < 0.01; ***P < 0.001, 1-way ANOVA with Bonferroni’s post tests. Error bars depict mean ± SEM. (C) CLSM pictures of S. epidermidis 1457 WT, isogenic agr, and psmβ operon deletion mutant biofilms. Growth conditions are as in A.

Furthermore, we compared the phenotype of the psmβ deletion mutant with that of an isogenic agr mutant. The biofilm phenotypes of these strains were very similar (Figure 6C), indicating that PSMβ peptides represent the main effector molecules of quorum-sensing regulated biofilm maturation in S. epidermidis.

PSMβ peptides promote dissemination from in vivo biofilms. To analyze the role of β-type PSMs in biofilm detachment in vivo, we performed a murine model of device-related infection (Figure 7 and Table 1). The development of human sepsis is a rare event compared with the frequency of catheter infection and possibly due to yet unknown roles of host factors; it is thus difficult to mimic in an animal model. We therefore focused on analyzing bacterial spread from the catheters to the lymph and organs. Furthermore, we used immune-compromised Nu/Nu mice, as we showed previously that those mice develop more pronounced and longer lasting catheter-related infection (27).

Figure 7

Role of PSMβ peptides in the dissemination of biofilm-associated infection. Catheter pieces with equal cell numbers of preformed biofilms of either the S. epidermidis 1457 WT or isogenic psmβ mutant strains were inserted under the skin of mice at the dorsum. Each mouse received 2 pieces, left and right, 1 of which was coated with WT and 1 with mutant biofilms. At days 2 and 4 after insertion (this timing found in a pilot experiment to be optimal), catheter pieces were removed and dissemination was analyzed. In a first experiment, body fluids (from the i.p. cavity) and organs were analyzed. Almost all disseminated bacteria detected were of the WT, and significantly more WT than mutant bacteria were detected in the body fluids and liver. Number of mice: n = 9 (day 2); n = 6 (day 4). In a second experiment, lymph nodes at day 2 after infection were analyzed and showed predominantly WT bacteria, except for in the nodes adjacent to the catheter infected with psmβ mutant bacteria. Numbers represent percentages of WT among total bacteria from all tested mice (n = 7) in the respective lymph nodes. Blue, WT; green, psmβ mutant strains. See Table 1 for details.

Table 1

Catheter-related animal infection model

In this model, 2 catheters, precolonized with equal numbers of either the psmβ mutant or WT strain, were inserted s.c. on the right and left dorsum, respectively. In a first experiment, we measured systemic dissemination into body fluids as assessed by taking body fluid samples from the peritoneal cavity. Bacteria harvested from body fluids were overwhelmingly and significantly more of the WT than psmβ mutant strain (Table 1), indicating dissemination is favored in the WT over the mutant strain. Bacteria were rarely found in organs, but those that were found were almost exclusively of the WT. Owing to low and strongly varying numbers, differences in the organs only reached statistical significance in the liver sample at day 4 (Table 1). Finally, bacteria on the WT infected catheter were only of the WT strain, while on the psmβ mutant–infected catheter, rare infiltration of WT bacteria was detected, in accordance with dissemination occurring only for WT bacteria.

In a second experiment, we focused on determining bacterial dissemination into the lymph nodes, as those would be first encountered by bacteria after dissemination. Bacteria in all lymph nodes were almost exclusively of the WT strain (Figure 7 and Table 1). Only bacteria in the right brachial and axillary lymph nodes were mainly of the mutant strain, most likely due to involvement of adjacent tissue during surgical insertion of the catheters. In contrast, no bacteria were found in the blood. This suggests that bacteria found in the body fluids from the peritoneal cavity in the first experiment originated from the lymph, while host defenses had cleared bacteria in the blood. Together, these data demonstrated that systemic dissemination from the indwelling device was strongly and significantly favored in the WT compared with the psmβ deletion strain and thus, PSMβ peptides play a key role in the dissemination of biofilm-associated infection. Finally, results from a bacteremia control experiment indicated that PSMβ peptides do not have an impact on the development of disease that is unrelated to biofilm formation, as no significant difference was detected when monitoring death of animals injected with WT or psmβ mutant bacteria, respectively (Supplemental Figure 2).

Antibodies to PSMβ peptides block dissemination of biofilm-associated infection. To further substantiate the involvement of PSMβ peptides in the dissemination of biofilm-associated infection, we investigated whether antibodies against PSMβ peptides prevent dissemination. To that end, we first produced antibodies to PSMβ1 and PSMβ2 in mice. PSMβ peptides proved immunogenic and elicited a strong IgG response (Supplemental Figure 3). Antisera were treated using extracts from the psmβ deletion mutant, and the resulting blocked antisera were highly specific for PSMβ1 and PSMβ2, respectively (Supplemental Figure 3). We pooled the PSMβ1 and PSMβ2 antisera and analyzed the efficacy of the obtained serum in reducing the dissemination of biofilm-associated infection using the same device-related infection model. In contrast to animals treated with PBS or control serum, no bacteria were found in the livers, spleens, or kidneys of animals treated with the anti-PSMβ antibodies, and bacterial load in the lymph nodes was significantly reduced (Figure 8). Reduced bacterial counts were most likely not due to an opsonophagocytosis-enhancing effect of the anti-PSMβ antibodies, as in an in vitro control experiment, we did not observe enhanced bacterial killing with anti-PSMβ compared with control serum (Supplemental Figure 4). Thus, these results confirmed that PSMβ peptides are essential for the dissemination of biofilm-associated infection.

Figure 8

Blocking dissemination of biofilm-associated infection using anti-PSMβ serum. Specific antisera against PSMβ1 and PSMβ2 were mixed 1:1 and used to protect mice in the biofilm infection/dissemination model. *P < 0.05, versus PBS and control serum for the lymph nodes, versus PBS for the kidney results; 1-way ANOVA with Bonferroni’s post-tests. For spleen and liver, differences were not statistically significant owing to the fact that dissemination into organs only occurred in a limited number of mice and therefore SEM values were high. However, dissemination into the organs of mice that received anti-PSMβ serum was never observed (average CFU/100 μl ≤ 1). Number of mice: controls, n = 9; anti-PSMβ, n = 7. Control serum was from animals that received injections containing CFA and IFA. Error bars depict mean ± SEM.

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 (7–10). 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, 26, 28, 29). This is also the case for staphylococci (26, 28–31). 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 psmβ 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 (32–34). 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, 25). 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 (28). 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 (18, 37, 38) 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.

View Supplemental data

This study was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID), NIH. The authors thank Frank DeLeo for critically reading the manuscript, Saravanan Periasamy, NIAID, for help with confocal microscopy, and Daniel C. S. Tan, Nanyang Polytechnic, Singapore, for technical assistance.

Address correspondence to: Michael Otto, National Institute of Allergy and Infectious Diseases, NIH, 9000 Rockville Pike, Building 33 1W10, Bethesda, Maryland 20892, USA. Phone: 301.443.5209; Fax: 301.480.3633; E-mail: motto@niaid.nih.gov.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2011;121(1):238–248. doi:10.1172/JCI42520.

Rong Wang’s present address is: Meat Safety and Quality Research Unit, U.S. Meat Animal Research Center, Clay Center, Nebraska, USA.

Shu Y. Queck’s present address is: Nanyang Polytechnic, Singapore.

Kok-Fai Kong’s present address is: Department of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.

Max Jameson-Lee’s present address is: Myles Thaler Center, University of Virginia, Charlottesville, Virginia, USA.

Burhan A. Khan’s present address is: Southcentral Foundation, Anchorage, Alaska, USA.

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