Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection

Abstract

The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections.

INTRODUCTION

Staphylococcus aureus is one of the bacterial species most frequently associated with biofilm-mediated infections. It can be found as a commensal bacterium on the skin, nares, and mucosa, but in some situations, it can become the source of biofilm-related infections, where bacteria grow into multicellular communities attached to a surface and embedded in a self-produced extracellular matrix. S. aureus biofilms can occur on host tissues such as heart valves (endocarditis) and bone tissue (osteomyelitis), although they are more frequently related to medical devices (catheters, prostheses, and portacaths). Implanted medical devices are easily coated with plasma and extracellular matrix proteins such as fibrinogen and fibronectin (1). S. aureus has the ability to bind to these components via specific receptors, and thus, implants become colonized. After primary attachment to the polymeric surface, bacteria proliferate and accumulate in multilayered clusters surrounded by an extracellular matrix. The added level of bacterial resistance inside a biofilm makes these infections difficult to treat, and, as a consequence, in most situations, the device must be surgically removed and replaced (2). Bacteria from the biofilm can also propagate through detachment of small or large clumps of cells or by the release of individual cells, allowing bacteria to colonize other surfaces or tissues far from the original infection site. Bloodstream infections originating from device-associated infections account for 11% of all health care-associated infections. An estimation of 250,000 catheter-related bloodstream infections occur in the United States per year, resulting in significant morbidity, mortality, and costs for health care delivery (3–5). S. aureus is frequently associated with such infections, and therefore a great effort is being made to prevent and/or obtain effective treatments against this bacterium. Given the fact that bacteria living in a biofilm express a different set of genes than the same free-living bacteria (6–10), the process of antigen selection for the development of an efficient protection against S. aureus infections should also take into consideration the antigens expressed during biofilm growth. In this respect, a wide variety of extracellular compounds have been identified as mediators of staphylococcal biofilms, such as poly-N-acetylglucosamine exopolysaccharide (PNAG; also called polysaccharide intercellular adhesin [PIA]), (11–16), extracellular DNA (eDNA) (17, 18), and different surface-associated proteins, including the biofilm-associated protein (Bap), fibronectin-binding proteins (FnBPs), SasG, and protein A (19–23). Some of these biofilm mediators have already been proposed as vaccine antigens against S. aureus infections. Different studies have shown that administration of deacetylated PNAG conjugated with diphtheria toxin as a carrier protein induces an immunological response that protects against S. aureus infection (14, 24–26). Furthermore, a recent study by Cywes-Bentley et al. showed that PNAG or a structural variant of PNAG is a conserved surface polysaccharide produced by many pathogenic bacteria, fungi, and protozoal parasites and demonstrated that passive immunization with antibodies to PNAG protects mice against both local and systemic infections caused by many of these pathogens (27). Protein A and FnBPs have also been evaluated for vaccine development. These antigens generate an immune response that confers partial protection against S. aureus challenge using systemic infection models (28–30). However, no evidence of the efficiency of these molecules for protection against biofilm-based infections has been obtained.

In the last few years, several studies have demonstrated that biofilms harbor multiple cell types, resulting in heterogeneous populations that have followed different developmental pathways (31–33). In this regard, Brady et al. identified immunogenic cell wall proteins expressed during an S. aureus biofilm infection and demonstrated different expression patterns for each antigen (34, 35). These authors reasoned that immunization with a monovalent vaccine would likely mean that only a fraction of the biofilm would be targeted, and thus, the infection would persist (36, 37). Therefore, they used a quadrivalent vaccine, including four of the identified antigens (glucosaminidase, an ABC transporter lipoprotein, a conserved hypothetical protein, and a conserved lipoprotein) combined with antibiotic therapy and demonstrated a reduced S. aureus biofilm formation on infected tibias, using a chronic osteomyelitis model (37).

Taking into consideration that the biofilm matrix is the first primary interface between the host and bacteria during a biofilm-associated infection and the relevance of using a multivalent vaccine for the prevention of biofilm-type infections, in this study we aimed at investigating whether an extract containing all proteins secreted into the biofilm matrix might be a potential polyvalent vaccine candidate that protects against S. aureus biofilm-related infections. Thus, we first isolated and identified the exoproteins of both PNAG-dependent and -independent biofilm matrices produced by a methicillin-sensitive and a methicillin-resistant clinical strain. Notably, exoproteomes were uniform in that they contained a common set of proteins. Immunization with a biofilm matrix exoproteins extract effectively reduced biofilm formation in an in vivo model of mesh-associated biofilm infection, which significantly correlated with the production of immunoglobulins (IgG and IgM) with opsonic activity. Our results also suggested a role for interleukin 10 (IL-10) and IL-17 cytokines in biofilm matrix exoprotein-mediated protection. Finally, we showed that administration of this multicomponent protein extract reduces organ colonization by bacteria released via detachment from the biofilm.

MATERIALS AND METHODS

Ethics statement.

All animal studies were reviewed and approved by the Comité de Ética, Experimentación Animal y Bioseguridad, of the Universidad Pública de Navarra (approved protocol PI-019/12). Work was carried out at the Instituto de Agrobiotecnología building under the principles and guidelines described in European Directive 86/609/EEC for the protection of animals used for experimental purposes.

Bacterial strains and culture conditions.

Staphylococci were cultured on tryptic soy agar or broth at 37°C supplemented with glucose (0.25%) (TSB-Gluc) or NaCl (3%) (TSB-NaCl) when indicated. Strains used in this study are listed in Table 1. S. aureus 15981, 132, and 12313 were isolated at the Microbiology Department of the Clínica Universidad de Navarra (Pamplona, Spain) (23, 38). S. aureus V329 is a Bap-positive strain isolated from a case of bovine mastitis (19). S. aureus Newman_Bap is a Newman derivative strain containing a chromosomal copy of the bap gene (39). ISP479r is a derivative of ISP479 with a functional rsbU gene. As a biofilm-negative strain, we used S. aureus Newman (ATCC 25905).

TABLE 1.

Bacterial strains

S. aureus strain	Relevant characteristic(s)a	Reference
15981	MSSA clinical strain; biofilm positive; PNAG-dependent biofilm matrix	38
132	MRSA clinical strain; biofilm positive, able to alternate between a protein-dependent biofilm matrix (grown in TSB-Gluc) and a PNAG-dependent biofilm matrix (grown in TSB-NaCl)	23
ISP479c	MSSA clinical strain; biofilm positive; PNAG-dependent biofilm matrix	76
12313	MSSA clinical strain; biofilm positive; PNAG-dependent biofilm matrix	23
V329	Bovine subclinical mastitis isolate; biofilm positive; protein-dependent biofilm matrix	19
Newman	Strain used in systemic infection models	77

^aMSSA, methicillin-susceptible S. aureus; MRSA, methicillin-resistant S. aureus.

Biofilm formation and protein extract purification.

Biofilm formation under flow conditions was performed using 60-ml microfermentors (Pasteur Institute, Laboratory of Fermentation) with a continuous 40-ml h⁻¹ flow of medium and constant aeration with sterile compressed air (0.3 bar) (40). Submerged glass slides (spatulas) served as growth substrata. Approximately 10⁸ bacteria from an overnight culture of each strain grown in the appropriate medium (S. aureus 15981 was grown in TSB-Gluc and S. aureus 132 was grown in either TSB-Gluc or TSB-NaCl) were used to inoculate the microfermentors, which were then kept at 37°C for 24 h. The biofilm formed on the spatula was resuspended in 20 ml of PBS (phosphate-buffered saline) and vigorously homogenized by vortexing. The suspension was centrifuged at 4,800 × g for 30 min at 4°C. Then the supernatant was collected, centrifuged again at 4,800 × g for 30 min at 4°C, and filtered through a 0.45-μm filter (Sarstedt). Matrix proteins were extracted with 10% trichloroacetic acid. After precipitation, proteins were dissolved in PBS for quantification with the bicinchoninic acid protein assay kit (Sigma). The planktonic culture exoprotein extract (PLKE) was obtained as follows. An overnight culture of S. aureus 15981 was diluted 1:100 in an Erlenmeyer flask containing 50 ml of TSB-Gluc medium and was incubated overnight at 37°C with shaking. The culture was centrifuged at 4,800 × g. Supernatant was collected and filtered through a 0.45-μm filter (Sarstedt). Proteins secreted into the supernatant were precipitated by the addition of 10% trichloroacetic acid. Protein extracts were dissolved in PBS for quantification with the bicinchoninic acid protein assay kit (Sigma). Proteins were resolved using SDS-polyacrylamide gel electrophoresis and stained with Bio-Rad silver stain according to the manufacturer ′s recommendations. To obtain the bacterial heat extract, an S. aureus 15981 cell suspension containing 10⁸ CFU was heat inactivated at 80°C for 1 h (41).

Protein identification.

The extracellular protein extract was subjected to tryptic digestion and analyzed as previously described (22). Briefly, the tryptic peptide mixtures were injected onto a strong cationic exchange microprecolumn with a flow rate of 30 μl/min as a first-dimension separation. Peptides were eluted from the column as fractions by injecting salt of ammonium acetate at increasing concentrations. Ammonium salts were removed and peptides were analyzed in a continuous acetonitrile gradient on a C₁₈ reversed-phase self-packing nanocolumn. Peptides were eluted (at flow rate of 300 nl/min) from the reversed-phase nanocolumn to a PicoTip emitter nanospray needle (New Objective, Woburn, MA) for real-time ionization and peptide fragmentation on an Esquire HCT ion trap mass spectrometer (Bruker-Daltonics, Bremen, Germany). Every 1 s, the instrument cycled through acquisition of a full-scan mass spectrum and one tandem mass spectrometry (MS/MS) spectrum. A 4-Da window (precursor m/z ± 2), an MS/MS fragmentation amplitude of 0.80 V, and a dynamic exclusion time of 0.30 min were used for peptide fragmentation. Two-dimensional liquid chromatography (2D LC) was automatically performed on an advanced microcolumn-switching device (Switchos; LC Packings) coupled to an autosampler (Famos; LC Packings) and a nanogradient generator (Ultimate nano-high-pressure liquid chromatograph [HPLC]; LC Packings). The software Hystar 2.3 was used to control the whole analytical process. MS/MS spectra were batch processed by using DataAnalysis 5.1 SR1 and MS BioTools 2.0 software packages and searched against the S. aureus protein databases using Mascot software (Matrix Science, London, United Kingdom). The criteria for confirming highly confident protein identification were set as a MASCOT total protein score of ≥50 and at least one peptide E value of ≤0.05.

RNA extraction.

For planktonic growth conditions, an overnight culture of S. aureus 15981 was diluted 1:100 in an Erlenmeyer flask containing 50 ml of TSB-Gluc medium and was incubated to an optical density at 600 nm (OD₆₀₀) of 0.8 at 37°C with shaking. For biofilm growth conditions, microfermentors were inoculated as described above and incubated at 37°C for 6 h. Biofilm-grown and planktonically grown cells were harvested. Total RNA from bacterial pellets was extracted by using a TRIzol reagent method (42). Briefly, bacterial pellets were resuspended in 400 μl of solution A (10% glucose, 12.5 mM Tris [pH 7.6], 10 mM EDTA), mixed with 60 μl of 0.5 M EDTA, and transferred into lysing matrix B tubes containing 500 μl of acid phenol (Ambion). Cells were mechanically lysed by using the FastPrep apparatus (BIO101) at speed 6.0 for 45 s at 4°C. After centrifugation, the aqueous phase was transferred to 2-ml tubes containing 1 ml of TRIzol and 100 μl of chloroform. Tubes were centrifuged, and the aqueous phase was transferred into a 2-ml tube containing 200 μl of chloroform, mixed, and incubated for 5 min at room temperature. Tubes were centrifuged, and the aqueous phase containing the RNA was precipitated by addition of 500 μl of isopropanol and incubation for 15 min at room temperature. RNA concentrations were quantified, and RNA qualities were determined by using Agilent RNA Nano LabChips (Agilent Technologies). RNAs were stored at −80°C until needed.

cDNA labeling and DNA microarray hybridization.

RNAs (10 μg) were reverse transcribed using SuperScript II reverse transcriptase (Invitrogen Life Technologies). cDNA was digested by DNase I (Pierce) in 10× DNase I buffer (USB-Affymetrix), and the sizes of the digestion products were determined with an Agilent Bioanalyzer 2100 using RNA Nano LabChips to ensure that the fragmentation resulted in a majority of products in the range of 50 to 200 bp. The fragmented cDNA was then biotinylated using terminal deoxynucleotidyl transferase (Promega) and the GeneChip DNA labeling reagent (Affymetrix) following the manufacturer's recommendations. Biotinylated cDNA (5 μg per array) was hybridized to custom S. aureus tiling microarrays designed as described previously (43) (ArrayExpress accession no. A-AFFY-165) and incubated for 16 h according to the Affymetrix protocol in a total volume of 200 μl per hybridization chamber. Following incubation, the arrays were washed and stained in the Fluidics station 450 (Affymetrix) using the protocol no. FS450_0005. The arrays were then scanned using the GeneChip scanner 3000 (Affymetrix). The signal intensity of each probe cell was computed by the GeneChip operating software and stored in cell intensity files (.CEL extension) before preprocessing and analysis. Microarray data were analyzed using the LIMMA package (44).

Immunization studies.

CD1 mice were obtained from Charles River and maintained in the animal facility of the Instituto de Agrobiotecnología, Universidad Pública de Navarra. The biofilm matrix exoprotein (BME) extract used for immunization consisted of the exoproteins purified from the biofilm matrix produced by S. aureus 15981. Five-week-old female CD1 mice were injected intradermally with 10 μg of BME diluted in adjuvant (Sigma Adjuvant System). The control group was treated with PBS and adjuvant. Two weeks later, the vaccinated group received a booster dose of 5 μg of BME, while the control group received PBS and adjuvant. Mice were bled via the retroorbital venous plexus on day 0 (preimmune serum) and 21 days after the first vaccination (immune serum). Both serum samples were analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blotting for determination of antibody responses against the BME.

Detection of antibodies in the sera.

Serum IgG and IgM expression against BME were quantified by coating 96-well ELISA plates (Nunc Maxisorp; Millipore) with 100 μl/well of a 0.1-μg ml⁻¹ BME solution in carbonate buffer (0.5 M; pH 9.4). Plates were incubated at 4°C overnight. After incubation, wells were washed three times with PBS containing 0.1% Tween 20 (PBS-T; pH 7.4) and blocked with blocking buffer (5% nonfat dried milk powder in PBS-T) at room temperature for 1 h. After three washes with PBS-T, 100 μl of preimmune (negative-control) and immune serum diluted 1:100 in PBS was added to each well and incubated at 37°C for 2 h. After incubation, wells were washed three times with PBS-T, and 100 μl of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and IgM (Thermo Scientific) was added to each well. The plates were incubated for 1 h at 37°C and then washed three times. One hundred microliters of ABTS solution [diammonium 2,2′-azinobis(3-ethyl-2,3-dihydrobenzothiazole-6-sulfonate); Millipore] was added to each well, and the absorbance at 405 nm was determined on an ELISA reader. Results were reported as the OD₄₀₅ of immune serum/OD₄₀₅ of the control serum.

Immune response was also determined by Western blotting. For that, 5 μg of the BME or a planktonic culture exoprotein extract was resolved using SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and incubated with blocking buffer. Then the membrane was exposed to preimmune (negative-control) and immune serum at 4°C overnight. After five washes with washing buffer (0.1% PBS-T), the membrane was incubated with goat anti-mouse IgG and IgM (heavy plus light chain [H+L]) HRP-conjugated secondary antibody, and proteins were detected using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific).

Opsonophagocytic assays.

The opsonophagocytosis and killing assay was previously described (45). Briefly, 1 ml of a planktonic culture of strain S. aureus 132 grown overnight was pelleted for 5 min at 12,000 × g at 4°C, washed twice with PBS, and subsequently diluted to an OD₆₀₀ of 0.5. Bacteria were preincubated with 1% or 10% of immune serum, preimmune serum or PBS for 1 h at 4°C. The opsonophagocytosis assay was performed with fresh blood obtained from human healthy volunteers. Fresh whole blood from three volunteers was collected and mixed in tubes containing the anticoagulant heparin and then aliquoted into 1.5-ml microcentrifuge tubes (0.5 ml/tube). After preincubation, 10 μl of bacterial suspensions were added to the 1.5-ml microcentrifuge tubes containing 0.5 ml of fresh blood. Tubes were incubated at room temperature with gentle rocking, and after 30 min, samples were serially diluted and plated onto tryptic soy agar (TSA) plates to determine the number of surviving CFU. On the other hand, to analyze the opsonophagocytosis and killing of bacteria that are part of a biofilm, 0.5- by 0.5-cm polypropylene meshes (Prolene) were incubated with an 1:100 overnight dilution of a culture of the biofilm-forming strain S. aureus 132 for 2 h at 37°C with shaking. Meshes were then washed with PBS and preincubated with 1% or 10% immune serum, preimmune serum, or PBS for 1 h at 4°C. After preincubation, meshes containing bacteria inside a biofilm were added to the 1.5-ml microcentrifuge tubes containing 0.5 ml of fresh blood. Tubes were incubated at room temperature with gentle rocking, and after 30 min, meshes were removed, gently washed, placed in 1 ml of PBS, and vigorously vortexed. Samples were serially diluted and plated onto TSA plates for enumeration of viable staphylococci. Four independent samples of each treatment were tested. The amount of bacterial killing was calculated as follows: [1 − (number of CFU recovered from treated samples/number of CFU recovered from PBS control samples)] × 100.

Cytokine production by splenocytes.

Groups of 5 CD1 mice were immunized as described above in “Immunization studies.” One week after the second immunization, mice were sacrificed, and their spleens were collected under aseptic conditions. Cells suspensions were prepared by resuspending the spleens in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin and subsequent trituration and filtration through a 70-μm nylon mesh. Red blood cells were lysed using ACK lysing buffer. Splenocytes were counted and dispensed into 24-well plates at a concentration of 2 × 10⁵ cells/well. The cells were restimulated with either 1 μg of BME or with PBS during 24, 48, and 96 h. The supernatants were harvested and analyzed for IL-10, IL-2, IL-17A, and gamma interferon production using the appropriate ELISA kit (eBioscience) according to the manufacturer′s instructions.

Vaccination/challenge protocol using an in vivo model of mesh-associated biofilm infection.

The vaccination protocol was performed as described above in “Immunization studies” using BME, PLKE, or 10⁸ heat-killed bacteria emulsified in adjuvant for immunization. Groups of 6 CD1 mice were used. One week after the second immunization, a model of mesh-associated biofilm infection was performed as previously described (46) with the following modifications. Prior to surgical procedure, 0.5- by 0.5-cm polypropylene meshes (Prolene) were incubated with 0.5 ml of a 1:100 overnight dilution of a culture of the biofilm-forming strain S. aureus 132 for 1 h and 15 min at 37°C with shaking. To calculate the initial inoculum, duplicate meshes were placed in 1 ml of PBS and vigorously vortexed. Samples were serially diluted and plated onto TSA plates for enumeration of viable staphylococci. Control and vaccinated CD1 mice were anesthetized by intraperitoneal injection of a ketamine/xylazine mixture. After abdominal epilation and antisepsis of the operative field, the animals were operated on. An incision of 1.5 cm in the skin was made, with displacement of the subcutaneous space and opening of the peritoneal cavity. Then a mesh coated with 10⁴ CFU of S. aureus strain 132 was fixed at the abdominal wall with one anchor point. Finally, the peritoneal cavity was closed by suture with 6/0 Monosyn. The animals were put in a warm environment, and when awake, they were put back in their cages. After 5 days, all animals were sacrificed. Mesh and surrounding tissue were extracted and placed in 1 ml of PBS and vigorously vortexed. Samples were serially diluted and plated onto TSA plates for enumeration of viable staphylococci.

To analyze the additional protection against the bacterial population that propagates through detachment from the biofilm, kidneys and livers from the animals that were operated on or from animals challenged by an intravenous injection of a bacterial suspension containing 10⁷ CFU of S. aureus Newman were extracted after 5 days. Viable counts were performed on the homogenates by plating the samples on TSA.

Microarray data accession number.

Raw data are available from ArrayExpress under accession no. E-MEXP-3924.

RESULTS

Identification of the S. aureus biofilm matrix exoproteome.

In order to isolate and identify the exoproteins present within the biofilm matrix, the biofilm formed by the clinical strain S. aureus 15981 grown in TSB-Gluc was isolated (38). This strain forms a PNAG-dependent biofilm when grown under the conditions tested. Exoproteins present within the PNAG-mediated biofilm matrix of 3 independent samples were purified as described in Materials and Methods. Proteins from these extracts were precipitated and then separated by 1D SDS-PAGE followed by trypsin digestion and identified by 2D LC-MS/MS. Only proteins identified in at least two of the three samples were considered for further analysis. Thus, a total of 33 extracellular proteins were detected with a MASCOT score higher than 50 (Table 2). Importantly, the proteins identified have been repeatedly detected in extracellular proteomes of various S. aureus isolates (34, 47–54). More notably, 28 out of the 33 proteins identified in our analysis have also been found in the biofilm exoproteome of S. aureus D30 strain, isolated from a persistent nasal carrier (Table 2) (50). These data reliably support the validity of the method used to identify exoproteins of the biofilm matrix.

TABLE 2.

Biofilm matrix exoproteomes

Category and NCBI GI no.	S. aureus N315 open reading frame ID	Putative protein	Presence in:			Theoretical pI	Theoretical Mw (10−3)	Total scorea	% coverage	Expression ratiob
			S. aureus 15981	S. aureus 132-PNAG	S. aureus 132-FnBPs
Exoproteins upregulated under biofilm conditions
15927581c	SA1813	Leukocidin	x			9.43	40.43	140.5	4.57	32.8
15926283c	SA0562	Alcohol dehydrogenase (Adh1)	x	x		5.34	36.05	113.51	10.12	16.6
15923805c	SA0746	Nuclease	x	x	x	9.27	25.12	1,306.41	25.44	14
15926008c	SA0295	Lipoprotein	x		x	9.49	33.35	321.51	17.15	7.8
15927994c	SA2204	Phosphoglyceromutase (GpmA)	x		x	5.23	26.68	191.13	7.02	7.7
15928224c	SA2431	Immunodominant antigen B (IsaB)	x		x	9.67	19.37	1,995,27	21.14	7.6
15925815c	SA0107	Protein A	x			5.54	56.44	289.57	13.78	6.9
15927579	SA1811	Truncated beta-hemolysin (Hlb)	x		x	7.68	31.26	463.98	6.57	5.9
15926570	SA0841	MAP hypothetical protein	x	x	x	9.28	15.84	2,018.96	18.06	5.7
15925596c	SA2399	Fructose-1,6-bisphosphate aldolase	x	x		4.88	33.04	376.91	10.14	4.1
15926551c	SA0823	Glucose-6-phosphate isomerase (Pgi)	x	x		4.83	49.82	69.43	8.8	3
15926634	SA0900	Cysteine protease precursor (SspB)	x		x	5.68	44.52	1,662.70	24.68	2.9
15926265	SA0544	Hypothetical protein		x	x	5.12	29.39	143.28	5.2	2.8
15927415c	SA1659	Foldase protein (PrsA)	x	x		9.01	38.64	105.90	3.44	2.8
15926291c	SA0570	Hypothetical protein	x		x	9.17	18.59	557.99	23.81	2.7
15927996c	SA2206	IgG-binding protein SBI	x		x	9.38	50.07	172.37	5.87	2.6
15927419c	SA1663	Hypothetical protein	x	x		4.33	13.31	227.81	34.21	2.6
15925985c	SA0272	Type VII secretion protein (EsaA)	x			6.24	114.78	99.62	1.19	2.5
15926635c	SA0901	Serine protease (SspA)	x			5.00	36.97	421.27	12.69	2.3
15926639c	SA0905	Autolysin (Atl)	x		x	9.60	136.75	3160.15	24.68	2
Non-differentially expressed exoproteins
15926452c	SA0730	Phosphoglycerate mutase (Pgm)	x			4.74	56.42	495.22	16.23	1.8
15926451c	SA0729	Triosephosphate isomerase (TpiA)	x	x	x	4.80	27.29	225.47	24.51	1.8
15926453c	SA0731	Enolase (Eno)	x	x		4.55	47.12	468.17	7.83	1.7
15923272c	SA0271	Hypothetical protein	x			4.61	11.04	2,443.79	74.23	1.6
15926190c	SA0471	Cystein synthase (CysK)		x		5.37	32.97	243.53	7.74	1.6
15926073c	SA0359	Putative secreted protease inhibitor		x		5.70	21.27	82.70	6.32	1.5
15928230c	SA2437	N-Acetylmuramoyl-l-alanine amidase	x			5.96	69.25	80.54	2.91	1.4
15928076c	SA2285	Cell wall surface protein (SasG)		x		5.35	178.53	73.21	1.93	1.4
15926396	SA0674	Sulfatase	x	x	x	9.04	74.4	1,308.21	4.64	1.3
15927054c	SA1305	DNA-binding protein II	x		x	9.52	9.63	676.57	52.22	1.2
15926091	SA0375	Inositol-monophosphate dehydrogenase		x		4.49	55.81	52.29	2.25	1.2
15927699c	SA1927	Fructose-bisphosphate aldolase (FbaA)	x	x		5.01	30.84	776.38	19.23	1.1
15926449c	SA0727	Glyceraldehyde-3-phosphate dehydrogenase	x	x	x	4.89	36.28	604.09	19.94	1.1
15926679c	SA0944	Pyruvate dehydrogenase E1 (PdhB)		x		4.65	35.24	74.69	10.46	1.1
15928148c	SA2356	Immunodominant antigen A (IsaA)	x	x	x	6.11	24.2	424.96	23.18	1
15927884	SA2097	Hypothetical protein			x	5.77	17.4	65.96	9.2	1
15925944c	SA0232	l-Lactate dehydrogenase (LctE)	x	x	x	4.95	29.45	98.7	11.04	−1
15926229	SA0509	Chaperone protein (HchA)		x		4.90	32.17	95.91	5.14	−1.1
15927133c	SA1382	Superoxide dismutase (SodA)	x	x	x	5.08	22.71	3,457.35	31.65	−1.1
15927879c	SA2093	Secretory antigen precursor (SsaA) homolog	x	x		8.96	29.33	324.85	22.85	−1.9
Exoproteins downregulated under biofilm conditions
15927670	SA1898	Similar to SceD precursor	x	x		5.52	24.07	57.82	7.79	−4.2

^aMASCOT score obtained by 2D LC-MS/MS analysis.

^bRatio of gene expression levels between biofilm and planktonic growth conditions.

^cAlso found in the S. aureus D30 biofilm exoproteome (50).

Specifically, exoproteome analyses revealed the presence in the extracellular biofilm matrix of many proteins involved in pathogenesis, such as toxins (leukocidin, EsaA, and truncated beta-hemolysin) and immunomodulatory proteins (lipoprotein, immunodominant antigen B, immunodominant antigen A, protein A, IgG-binding protein, secretory antigen precursor SsaA, and SceD). The biofilm matrix also contained a markedly large number of proteins involved in carbohydrate metabolism, namely, phosphoglycerate mutase, triosephosphate isomerase, enolase, glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate isomerase, alcohol dehydrogenase, l-lactate dehydrogenase, and fructose bisphosphate aldolase. Finally, albeit to a lesser extent, enzymes involved in cell wall peptidoglycan synthesis (autolysin and N-acetylmuramoyl-l-alanine amidase) and DNA metabolism and stress proteins (foldase protein, DNA binding protein II, nuclease, and superoxide dismutase) were also encompassed in the biofilm exoproteome.

With the aim of extending the biofilm matrix exoproteome analysis to other S. aureus strains, we used strain 132, which is a methicillin-resistant S. aureus (MRSA) clinical isolate able to alternate between a PNAG-independent biofilm matrix mediated by the fibronectin-binding proteins (FnBPs) and an exopolysaccharidic PNAG-mediated biofilm depending on whether it is cultured under TSB-Gluc or TSB-NaCl growing conditions, respectively (23). S. aureus 132 was incubated in microfermentors under these different conditions, which allowed the formation of the two biofilm matrices, and subsequently, these were isolated for matrix exoprotein identification. Analysis of the PNAG-mediated biofilm matrix revealed the presence of 24 proteins, 17 of which (71%) had been previously identified in the S. aureus 15981 exoproteome. On the other hand, analysis of the FnBP-mediated biofilm matrix led to the identification of 19 proteins, nearly half of which were also present in the exoproteome of the PNAG-mediated matrix and the other half of which were found in the S. aureus 15981 exoproteome. When we considered the biofilm matrices formed by S. aureus 132 under these two experimental conditions as a unit, results showed that almost 80% of the matrix exoproteins were included in the biofilm matrix exoproteome of S. aureus 15981.

In conclusion, we identified the PNAG-dependent and FnBP-dependent biofilm matrix exoproteomes of a methicillin-resistant S. aureus isolate and also the PNAG-dependent exoproteome of a methicillin sensitive clinical strain. The results indicated that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. The biofilm matrix exoprotein extract used for the rest of the study consisted of the 33 exoproteins identified in the biofilm matrix produced by S. aureus 15981 and was referred to as BME.

Transcriptional analysis of genes coding for biofilm matrix exoproteins.

Previous studies with several bacteria have shown that gene expression and protein production differ when bacteria are grown under biofilm conditions in comparison with planktonic growth (6–9). Therefore, we proceeded to investigate whether genes coding for the biofilm matrix exoproteins identified in the proteomic analysis were differentially expressed under biofilm conditions and planktonic growth conditions. Transcriptome analyses revealed that S. aureus 15981 cells grown under biofilm conditions expressed a markedly different repertoire of genes in comparison to their planktonic counterparts. In total, we observed that 626 genes were differentially expressed under biofilm conditions. From these, 276 genes were expressed in larger amounts in biofilm cells, while 350 genes were downregulated under biofilm conditions. Then we focused on expression levels of the genes coding for the BME previously identified and found that expression of more than half of the identified proteins (58%) was upregulated under biofilm conditions (Table 2). Importantly, genes encoding 39% of matrix exoproteins were not differentially expressed under biofilm conditions, indicating that the S. aureus biofilm matrix encompasses not only proteins that are specific to the biofilm mode of growth but also a set of proteins that S. aureus expresses at the same level during planktonic growth.

Biofilm extracellular proteins induce a humoral immune response in mice.

In order to investigate whether this multivalent extract might be able to induce a protective immune response against S. aureus, we firstly evaluated the antibody response in mice immunized with BME. For that, groups of 8 mice were immunized with BME. Blood and serum samples were obtained at days 0 and 21 postimmunization, and serum IgG and IgM levels were determined by ELISA. Results showed that immunoglobulin levels were significantly higher in sera from mice immunized with BME than in sera from control mice (Fig. 1A).

FIG 1.

Immunogenicity of BME extract in mice. Mice (n = 8) were immunized twice at an interval of 2 weeks with 10 and 5 μg of the BME extract (immune serum) or with the adjuvant alone (control serum). Sera were collected at 0 and 1 week after the last immunization. (A) IgG titers in response to mouse immunization were determined by ELISA. Results were reported as the OD₄₀₅ of immune serum (treated)/OD₄₀₅ of the control serum (control) (T/C). The BME extract (B) and PLKE from the supernatant of a planktonic culture (C) were separated on an SDS gel and silver stained. Proteins were transferred to a nitrocellulose membrane by Western blotting, probed with immune or control serum, and detected with goat anti-mouse IgG and IgM (H+L) HRP-conjugated secondary antibody. (D) Western blot analysis of matrix exoproteins extracts of biofilms formed by different S. aureus strains, probed with immune and control sera. ISPr, S. aureus ISP479r; Nw-Bap, S. aureus Newman_Bap.

Next, BME were separated in an SDS-PAGE gel (Fig. 1B) and interrogated with a pool of sera obtained either from immunized or control mice. Results showed that the majority of the BMEs were recognized by sera from immunized mice, while only a slight cross-reaction, probably caused by the presence of protein A, was observed when a serum pool from control mice was used (Fig. 1B). Also, because BME contains a group of proteins that are expressed equivalently under biofilm and planktonic growth conditions, we tested immune and control sera against an extract containing extracellular proteins secreted by S. aureus cells grown planktonically (PLKE). As expected, immune serum recognized part of the proteins present in the planktonic extract (Fig. 1C).

Finally, with the aim of analyzing if antibodies raised against the BME extract recognized the biofilm formed by different S. aureus strains, we isolated biofilm matrix exoproteins from biofilms formed by several S. aureus strains, and these were interrogated with immune and control sera. In particular, we tested S. aureus 132 (PNAG- and FnBP-mediated biofilms), V329 and Newman_Bap (Bap-dependent biofilms), and ISP479 and 12313 (PNAG-mediated biofilms). As shown in Fig. 1D, immune sera against the BME extract recognized many proteins present in all extracts analyzed.

Taken together, these data showed that BME was able to induce a humoral immune response and that many of the proteins present in the extract contributed to this immunogenicity. Also, antibodies generated against BME were capable of targeting a broad range of biofilm matrices, suggesting that this multivalent extract might be effective against a large number of relevant biofilm-producing strains.

Antibodies against BME induce opsonophagocytic killing of S. aureus.

The presence of IgG and IgM in the immune serum can be correlated with high opsonic activity (55). Thus, our next objective was to evaluate whether hyperimmune serum obtained against BME promoted opsonophagocytic killing of S. aureus. S. aureus 132 grown under planktonic or biofilm conditions was preincubated with preimmune serum, 1% or 10% BME-specific sera, or PBS as a control. After incubation, bacteria were mixed with whole blood for 30 min (45). Staphylococcal killing was monitored by spreading sample aliquots on TSA agar medium followed by colony formation and enumeration. Results showed that antibodies against BME significantly induced opsonophagocytic killing of both planktonic and sessile S. aureus cells (Fig. 2). Additionally, data showed that killing of biofilm S. aureus cells was slightly higher than killing of planktonic cells.

FIG 2.

Opsonization with immune serum against BME enhances killing of S. aureus. Bacteria grown in planktonic form (white) or attached to polypropylene meshes (gray) were tested for their ability to survive in human blood after preincubation with sterile PBS, preimmune serum, or 1% or 10% immune serum. Surviving bacteria were measured by viable counting. Results are expressed as percent killing, calculated as follows: [1 − (number of CFU recovered from treated samples/number of CFU recovered from PBS control samples)] × 100. Multiple comparisons were performed by one-way analysis of variance combined with the Bonferroni multiple comparison test (GraphPad Instat, version 5).

BME induces the production of IL-10 and IL-17 in ex vivo-stimulated splenocytes.

We next sought to characterize the cellular response stimulated by BME. For that, cytokine production was examined after ex vivo splenocyte stimulation with BME as described in Materials and Methods. Supernatants of stimulated cells were analyzed for the production of gamma interferon (IFN-γ) and IL-2 (prototype Th1 cytokines), IL-10 (prototype Th2 cytokines), and the Th17-associated cytokine IL-17. When production of IL-17 was analyzed over time, a 10-fold increase was observed at the early time of 24 h poststimulation, when supernatants of splenocytes from mice immunized with the BME extract were compared to supernatants of control mouse splenocytes. This difference increased to 50-fold at 96 h poststimulation (Fig. 3). It is important to note that levels of IL-17 over time were barely detectable in supernatants of control mice splenocytes (Fig. 3). With respect to cytokine IL-10, an approximately 2.5-fold increase was observed at 24 h poststimulation that was maintained over time (P < 0.05) (Fig. 3). Lastly, mouse immunization with BME led to neither stimulation of cytokine IL-2 production nor induction of IFN-γ (Fig. 3). Taken together, these results showed that immunization with BME induced a cellular response characterized by production of the cytokines IL-17 and IL-10.

FIG 3.

BME-induced production of cytokines in splenocytes. Mice were immunized twice at an interval of 2 weeks with 10 and 5 μg of the BME extract (treated) or with adjuvant alone (control). Three weeks postimmunization, splenocytes were harvested and restimulated for 24 h, 48 h, and 96 h with 1 μg of the BME extract. Cell supernatants were harvested and analyzed for IL-2, IL-10, IL-17, and IFN-γ production using respective ELISA kits. Results are representative of three independent samples. Statistical analysis was carried out using the unpaired Student t test.

Immunization with BME reduced biofilm formation in a mesh biofilm model.

We next hypothesized whether immunization with BME might reduce the number of bacterial cells inside a biofilm formed in vivo. To analyze this hypothesis, we compared the efficiency of BME in a mesh biofilm model with the protective effect of an extract containing the secreted proteins of S. aureus 15981 grown planktonically (PLKE) and also that of a heat extract obtained from S. aureus 15981 (HE). Mice were immunized at an interval of 2 weeks with 10 μg and 5 μg of BME or PLKE, with 10⁸ heat-killed bacteria (HE), or with adjuvant alone. After immunization, sera from immunized mice were extracted and were interrogated against the BMEs. Results showed that sera from mice immunized with PLKE and HE recognized fewer proteins of the BME extract than sera from BME-immunized mice (Fig. 4B).

FIG 4.

BME extract protects against a biofilm-related infection. (A) Mice were immunized twice at an interval of 2 weeks with 10 and 5 μg of the BME or PLKE, with 10⁸ heat-killed bacteria (HE), or with adjuvant alone (control). Polypropylene meshes coated with 10⁴ CFU of S. aureus strain 132 were fixated at the abdominal wall. After 5 days, animals were sacrificed, and meshes were extracted and placed in 1 ml of PBS. Samples were serially diluted and plated onto TSA plates for enumeration of viable staphylococci. Results are representative of six independent mice. Multiple comparisons were performed by one-way analysis of variance combined with the Bonferroni multiple comparison test. (GraphPad Instat, version 5). (B) BMEs were transferred to a nitrocellulose membrane by Western blotting and probed with sera purified from mice immunized with BME, PLKE, or HE. (C) Biofilm-infected meshes after 5 days of infection. (D) Vaccination with the BME extract also reduced colonization by bacteria that are released from the biofilm. Livers, kidneys, and mesh-surrounding tissue from vaccinated and control mice were extracted after 5 days of insertion of contaminated meshes. Viable staphylococci in the organs and tissue were enumerated by plate counting.

Seven days after the second immunization, polypropylene meshes coated with 10⁴ CFU of the biofilm-forming strain S. aureus 132 were implanted in the intraperitoneal cavity of immunized and control mice. After 5 days, all animals were sacrificed, and meshes were extracted. When the abdominal cavities of mice were opened, abdominal wall adhesions were observed in all animals. Meshes removed from nonvaccinated mice (controls) were more difficult to extract from the abdominal cavity than meshes from vaccinated mice. Also, as shown in Fig. 4C, meshes from control mice were surrounded by purulent and necrotic tissue, while a healthier and a more vascularized tissue surrounded the meshes from immunized mice. When the number of bacteria on meshes was determined, results showed that immunization with BME significantly reduced the number of bacteria attached to the polypropylene meshes (P ≤ 0.05) (Fig. 4A). In contrast, immunization with PLKE or HE showed a slight but not statistically significant reduction of the number of bacteria in the mesh biofilm model (Fig. 4A).

Finally, we decided to investigate whether BME-vaccinated mice were additionally protected against bacterial population that propagates via detachment from the biofilm. To do so, mesh-surrounding tissue, kidneys, and livers from BME-immunized mice were extracted, and bacterial colonization was determined. In contrast to the nonvaccinated group (control), mice immunized with BME presented a significantly reduced number of bacteria in liver and mesh-surrounding tissue (P ≤ 0.05) (Fig. 4D). Although there was also a slight reduction in kidney colonization in immunized mice, differences between control and vaccinated mice were not statistically significant (P = 0.06) (Fig. 4D).

Reduction of organ colonization in immunized mice might be the consequence of not only reduction of the biofilm formation capacity inside the animal, and thus a reduction in the number of bacteria released from the biofilm, but also the efficacy of the immune response against organ colonization by released bacteria. In order to analyze this possibility, we tested whether vaccination with the exoprotein extract might protect against a systemic infection and subsequent organ colonization caused by S. aureus. For this, mice were immunized as described above and were challenged with a retroorbital injection containing 10⁷ CFU/mice of S. aureus. Five days after the infection, animals were killed and kidneys and livers were removed. No bacteria were found in the livers of either vaccinated or control mice. In contrast, visual examination of kidneys from nonvaccinated mice showed the presence of abscesses all around the surface of the organs. Many fewer abscesses were detected on kidneys from immunized mice (Fig. 5). Enumeration of S. aureus cells from the organs showed that kidneys of immunized mice were significantly less colonized than kidneys of control mice (P < 0.01) (Fig. 5).

FIG 5.

Immunization with the BME extract generates a significant protective immunity against S. aureus infection. Vaccinated and control mice were infected with a retroorbital injection containing 10⁷ CFU of S. aureus Newman. (Top) Viable counts were performed on kidney homogenates by plating the samples on TSA. (Bottom) Abscesses (black arrows) formed in kidneys from control and vaccinated mice.

From all these results we inferred that immunization with BME significantly reduced biofilm formation in an in vivo model of mesh-associated biofilm infection and also moderated organ colonization conducted by bacteria that were released via detachment from the biofilm.

DISCUSSION

In recent years, S. aureus has emerged as one of the most critical nosocomial pathogens. Success of S. aureus as a pathogen is the result of different abilities, such as the capacity to invade a wide variety of cell types, to secrete a diversity of proteins and toxins, and to persist in the host, remaining resistant to clearance by the immune system or antibiotics through a biofilm mode of growth. Numerous approaches have been adopted in order to identify staphylococcal surface- and cell wall-associated proteins as antigenic candidates for a vaccine against S. aureus infections (34, 49, 51, 53, 56–60). However, few works have been focused on the selection of antigens that could also protect against biofilm-associated bacteria (14, 24–26). This is particularly important because S. aureus biofilms play a major role in persistent infections formed on the surface of implanted medical devices and in deep tissues. In this study, we have demonstrated that a multicomponent extract containing biofilm matrix exoproteins is able to elicit a protective immune response against S. aureus biofilm-mediated infections.

According to Harro et al. (36), the selection of appropriate antigens effective in preventing the establishment of a biofilm-related infection should meet the following criteria: (i) they must be expressed in vivo throughout the infection cycle in a large number of genetically unrelated strains, (ii) they must target the entire microbial population of the biofilm, and (iii) they must also induce a protective immune response against planktonic bacteria.

Numerous evidence have demonstrated that S. aureus is able to produce polysaccharidic and proteinaceous biofilm matrices (11–16). Therefore, potential antigens against S. aureus biofilm infections should be expressed by strains that form either type of biofilm matrix. Our results showed that BME from exopolysaccharidic matrices of two unrelated clinical strains (S. aureus 15981 and 132) comprised a high number of proteins common to both strains. Moreover, all proteins except one present in the BME isolated from a proteinaceous matrix produced by S. aureus 132 were also contained in PNAG-dependent matrices (Table 2). Also, it is important to note that 85% of exoproteins encompassed in the BME of S. aureus 15981 are identical to those in the first S. aureus biofilm exoproteome identified, which was produced by the nasal carrier strain S. aureus D30 (50). Accordingly, here we showed that antibodies raised against an extract from a PNAG-dependent biofilm formed by strain 15981 recognized many proteins from biofilms of different characters produced by different S. aureus strains (Fig. 1D). These data might explain why immunization with a BME extract obtained from strain 15981 was effective in protecting against challenges with the clinically relevant MRSA strain 132 (Fig. 4) and with S. aureus Newman (Fig. 5).

Because individual cells within biofilms can display different protein expression patterns depending on nutrient availability, respiratory conditions, or environmental stresses, Harro et al. (36) proposed that vaccines that aim at only one specific antigen would likely eliminate the section of the biofilm in which the antigen is expressed, whereas other biofilm areas that do not express the vaccinated antigen would probably persist. Hence, BME extract comprising most exoproteins of the biofilm matrix may ensure that not only different areas of the biofilm but also various cell types present within the biofilm are targeted. It is important to note that vaccinations with other multicomponent extracts, such as a heat-killed or a PLKE, which have been shown to provide protection against S. aureus infections (41, 61, 62), were less efficient than BME in reducing the number of bacteria inside a biofilm, using a mesh-associated biofilm infection model. The reason behind the low efficiency of heat-killed and PLKEs might be that they probably do not include biofilm-specific antigens like the BME extract (Fig. 4). Additional experimentation will be required to arrive at a detailed picture of the localization of BMEs in the biofilm structure.

Biofilm formation is a dynamic process that occurs through sequential steps in which the initial attachment of planktonic bacteria to a surface is followed by their subsequent proliferation and accumulation in multilayer cell clusters, where bacteria are enclosed in a self-produced polymeric matrix. As the biofilm ages, bacterial cells escape from the matrix and return to a planktonic existence, enabling them to reach other locations in the host. This step represents a potentially important mechanism for the dissemination of bacteria during infection. Our proteomic, transcriptomic, and immunological analysis showed that BME extract contains antigens that S. aureus produces under both planktonic and biofilm conditions (Table 2). As a consequence, sera from BME-immunized mice recognized several proteins in the exoproteome extract of planktonic bacteria (Fig. 1C). Accordingly, mice immunized with this extract showed not only a reduction in the number of bacteria inside an S. aureus biofilm but also a moderated tissue and organ colonization by bacteria that were released through detachment from the biofilm. Nevertheless, clearance of the infection would likely require an added antimicrobial treatment, as proposed by Brady et al. (37).

With respect to the immune response mounted after mice immunization with BME, results showed an increase in the production of total immunoglobulins. The primary antibody function in protection against S. aureus infections is neutralization and opsonization of bacteria for phagocytosis. Although reduction in the number of biofilm bacteria on polypropylene meshes in the opsonophagocytic experiment could be due to both neutralization and the opsonic activity of antibodies, we did not observe a significant direct effect of BME antibodies on S. aureus biofilms in vitro, in the absence of immune system components (see Fig. S1 in the supplemental material). Hence, BME antibodies seem to protect against S. aureus infections, likely through an increase in opsonization. Importantly, these opsonic antibodies may help in the phagocytosis of bacteria inside a biofilm that otherwise would be inaccessible due to the extracellular matrix coating. Although antibodies unquestionably play an important role in the protection against S. aureus infections, they may not be decisive for vaccine protective efficacy, since animals and humans have enough circulating antibodies to S. aureus (56, 63, 64). Certain indications show a partial role of these antibodies in protecting humans against staphylococcal infections (65). However, patients with defects in humoral immunity are not particularly prone to S. aureus infections (66). In this respect, a cellular response mediated by IL-17 is considered critical for immunity against this pathogen. It has been shown that vaccination with heat-killed S. aureus provides protection in systemic infection via staphylococcal lipoproteins that stimulate Th17/IL-17 (67). Also, IL-17 induction has been shown to be a determinant in the clearance of IsdB-immunized mice (68). In biofilm-related infections, IL-17 cytokine production increases during the development of the infection, indicating that infected mice mount a robust Th17 response (69, 70). Bacteria in biofilm are embedded in an extracellular matrix and are largely protected from phagocytosis by neutrophils and macrophages. The release of inflammatory cytokines by Th17 cells provokes the recruitment and activation of neutrophils and might aid to devitalize the biofilm surface, thus helping bacterial clearance. In the case of BME extract immunization, it not only induced a humoral response but also stimulated the production of IL-17, which might help to clear bacteria in the biofilm. In order to elucidate the role of the induction of IL-17 by BME administration in the efficiency of this multicomponent extract, we performed a preliminary experiment in which IL-17 cytokine was neutralized by administration of an antibody against IL-17. BME-immunized mice that had been administered the neutralizing antibody to IL-17 showed a nonsignificant reduction in the number of bacteria recovered from biofilm-infected meshes compared with control BME-immunized mice (see Fig. S2 in the supplemental material). These preliminary results suggest a putative role of IL-17 cytokine in the immune response against an S. aureus biofilm-related infection. BME-immunized mice also exhibited significantly higher levels of IL-10 than nonvaccinated mice. IL-10 has been shown to protect the host from staphylococcal enterotoxin, endotoxin, and septic shock (71–73). Furthermore, administration of an anti-IL-10 monoclonal antibody to mice inhibits the clearance of S. aureus, suggesting that IL-10 might play a beneficial role in host resistance to S. aureus systemic infections (74, 75). Further studies are needed to explore the role of IL-10 induction by BME administration in the clearance of S. aureus biofilm-related infections.

In summary, the work presented here shows that an extract containing biofilm matrix exoproteins induces a protective immune response against an S. aureus biofilm-related infection and thus reduces colonization and persistence. This is likely because this multicomponent vaccine ties together cell-mediated immunity and a humoral response where opsonic antibodies play a supportive role to eradicate the biofilm infection. In future work, it would be interesting to determine the contribution of each antigen present in the BME extract to its immunogenicity in order to define a particular antigen combination that provides efficient protection against S. aureus biofilm infections.