Comparative analysis of PIA and rSesC mixture, as vaccine candidate against the biolm forming Staphylococcus aureus.

Background: Staphylococcus aureus as a causative agent of hospital-acquired infections, has been considered as the primary concern in biomaterial-related infections (BAIs). Methods: Following the purication of polysaccharide intercellular adhesion (PIA) as an ecient macromolecule in biolm formation in the native condition, recombinant S. epidermidis surface exposed rSesC protein, with the most homology to clumping factor A (ClfA) in S. aureus was cloned and expressed in a prokaryotic host as well. Fourier transform infrared spectrometry (FTIR) and Western blotting procedure analyzed puried PIA and protein, respectively. Then, the immune response was evaluated by measuring total IgG titers. Moreover, the capacity of Anti-biolm forming activity of arisen antibodies to a biolm forming S. aureus strains was assessed by semi-quantitative micro-plate procedure. Results: Data showed that the total IgGs was boosted in mice immunized sera. By performing inhibition assay, biolm inhibitory effect of secreted antibodies to test strain was observed. Arisen antibodies against the mixture signicantly were more potent than PIA and rSesC, when comparing them in a biolm inhibition assay.


Background
Staphylococci, are opportunistic pathogens and determined as most common causes of infections related to implanted medical devices, infects both hospitalized patients and immunocompromised individuals [1].Considering diverse virulence factors, such as both capsule and cell wall-bound adhesion molecules, surface proteins, toxins, antibiotic resistance, and bio lm formation S. aureus could be make human and animal related infections. [2]. S. aureus is an etiological agent of the mild to severe related hospitalized patiense disorders including skin and tissue, bacteremia complicated by endocarditis, pneumonia, and metastatic infections. Finding reveled that, almost main part of adults are either permanently or transiently colonized by S. aureus due to the some discovered factors that makes the host susceptible for colonization.3] Up to 20-30% of humans, asymptomatically are colonized by S. aureus and 50-60% of those are intermittently colonized [4,5] Since the 1960s, rst (MRSA) strains were detected and then they have been spreading worldwide, becoming a global major challenge. Despite the importance of mentioned bacterium in human and animal infections, there are no available vaccines to prevent S. aureus relatedinfections yet [6]. Contamination of medical devices with S. aureus during insertion might be remarkably depended on the patient health care personal. Although there are some similarity in bio lm-associated infections with S. aureus and S. epidermidis, usually more intensive care is needed for the involvement of S. aureus In comparison with S. epidermidis bio lm-associated infections, not only S. aureus bio lm-associated infections are more di cult to be treated by antibiotic therapy, but also the devices need to get replaced more frequently [7]. The ica operon of staphylococcus spp, encode the production of polysaccharide intercellular adhesin (PIA), also known as poly-N-acetyl glucosamine (PNAG) [8]. The PIA-dependent mechanism is the best understood mechanism of bio lm formation.
Cerca et al. demonstrated that rabbit anti-PIA antibodies protect against infections with planktonic cells of PIA-positive S. aureus and S. epidermidis [9]. Based on a study by Maira-Litran et al., anti-dPIA antibodies mediated opsonic killing and protected against S. aureus infection [10]. Recent studies indicate other proteinaceous mechanisms of bio lm formation may exist. Shahrooei revealed that monoclonal antibodies against S. epidermidis surface-exposed Ses proteins can significantly reduce the accumulation phase [11]. Clumping factor A (ClfA) is a brinogen (Fg) -binding microbial surface molecule recognizing adhesive matrix molecules (MSCRAMM) of S. aureus; 65.1% similarity has been shown between SesC and a 341-aa fragment of ClfA [12].
Owing to the vital bio lm forming capacity role in the chronic staphylococcal disease development, evaluation of the bio lm inhibitory effect of PIA and recombinant SesC antibodies against a bio lm forming S. aureus, separatelyand in mixturewas targeted as the main goal of this study.

Results
Puri cation of macromolecules: Basing on the cloning and expression procedure, 1399 bp encoding gene of 55KD recombinant protein representative of rSesC was cloned in pET11C vector and transformed to the BL21 (DE3) as a prokaryotic host. The expressed protein was puri ed taking advantage of a nity chromatography procedure as well.
Representative rSesC protein was con rmed basing on the size by SDS-PAGE procedure. Then presence of a C-terminal hexa-His-tagged residue in the protein was determined by a Western blot Following the Bradford assay procedure. Amount of the puri ed protein was assessed about 5mg. Fig.1.
Puri cation of PIA PIA as a main compartment of bio lm forming macromolecule in aggregation phase was puri ed basing on the native condition procedure using size exclusion chromatography procedure. To isolate PIA (100 KD), size-exclusion chromatography was performed by Sephacryl S-100. PIA was puri ed at a ow rate of 0.300 ml min−1 and fractions of 1.5 ml were collected every 5 min in a fraction collector.
Contaminated PIA by proteins and nucleic acid (RNA and DNA) was cleaned by enzymatic digestion too.

Endotoxin contents and general safety
According to the Limulus amebocyte lysate test to puri ed macromolecules, the content of endotoxin was determined as 4.5 EU ml−1. Pyrogenicity and toxicity were not observed following in vivo challenge.

Anti-antigens humoral response
Using antigen mediated ELISA in order to evaluate the total IgG antibody response against PIA in the mixture antigen, mice sera antibodies titers were determined (≤1: 200). Following the rst immunization by rSesC and PIA (P = 0.0057) low level IgGs production comparing to the control group was observed and this amount determined as not signi cant to rSesC immunized sera. An increase in IgG titres to PIA (P = 0.0004) and rSesC (P = 0.0025) was observed following the rst booster. The third group of mice received a mixture of PIA and rSesC and an increase in anti-PIA antibodies was observed following the second immunization (P<0.0001). Fig. 5.

Bio lm inhibition assay by microtiter plate
The bio lm inhibition assay was performed by comparing pre-post immune sera by semi-quantitative procedure. Experiments were observed with sera from mice that were boosted after speci c time spans (14, 28 and 42 days) and results were obtained by comparing the immunized and non-immunized sera, respectively. (Fig. 6). The data showed that those mice immune sera which immunized by PIA antigen provide signi cant (P>0.05) inhibition after rst booster dosage. The inhibitory effects of sera after rst boosted sera with rSesC (P>0.0314) after rst boosted the PIA/rSesC mixture, (P>0.0015) and after the second booster were also signi cant. Observed data showed that, vaccinated sera two weeks haven't signi cant bio lm inhibitory effect after the rst immunization (14 days) by PIA and rSesC antigens compared to the control group. However, this effect for sera which immunized by PIA/rSesC mixture (P = 0.0004) was signi cant when compared by the control sera. Detailed OD average listed in table 1.

Bacterial strains:
Puri cation of the PIA and related procedure were accomplished applying two bio lm-forming S. epidermidis strains, ATCC 14990 and 35984, also known as 1457 and RP62A, a transmutant strain, 1457-M10 and a wild type bio lm forming S. aureus strain, too. Moreover, Escherichia coli strain BL21 (DE3) was served as prokaryotic host to DNA manipulation and recombinant protein production.

Construction and puri cation of His-tagged rSesC protein
Based on the previously reported procedure, [12] a 1,359-bp fragment of SesC encoding a 459aa extracellular part of SesC containing a six-His-tag at the C-terminus was ampli ed. In short, following the cloning and expression of the representative sequence in a prokaryotic host, 55KD rSesC protein was determined by SDS-PAGE and Coomassie Brilliant Blue staining. Protein was expressed in Eshershia coli BL21 DE3 following the induction by 1mM imidazole in optical density 0.7-0.9 in a shaker incubator (150 rpm in 37 °C for 3 hours). Purifying the recombinant protein was done using the commercial a nity chromatography Kit (GE health care, Sweden) according to the manufacture's recommendation followed by extraction of soluble protein from induced host using sonication (4 times for 30 s on ice) with an additional centrifugation (12 000 r.p.m. for 20 min at 4 °C) to the clari cation of the targeted protein. The purity of the recombinant protein was determined by Coomassie blue staining of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and then analyzed by western blotting. The expression of the protein was done as follows: E. coli BL21 (DE3) cells were transformed with the vector. Cells were grown with shaking at 250 rpm at 37 °C in Luria-Bertani broth with 100 g/ml ampicillin to an optical density at 600 nm of 0.6 to 0.8. Isopropyl-Dthiogalactopyranoside (IPTG) in 1 mM as a nal concentration was added on media to induction of protein expression. Following cooling on ice, cells were collected by centrifugation at 8,000 rpm for 5 min at 4 °C, resuspended in imidazole buffer, and frozen at -20 °C. Harvested pellets was sonicated three times for 30 s on ice. The expressed protein was determined by SDS-PAGE and coomassie brilliant blue staining (Fig. 1).
Extraction and puri cation of native PIA PIA was puri ed as previously described [13,14]. Brie y, the cells were harvested from incubated (37 °C for 24 h with shaking at 40-50 rpm/min) 2 liters of trypticase soy broth (TSB) by centrifugation (4,500 rpm for 20 min at 4 °C) and were re-suspended in 20 ml of 50 mM sodium phosphate buffer (pH 7.5).
After that, the suspended colonies were sonicated four times for 30 s on ice. The extracts were cleaned up by centrifugation at 12,000 rpm for 15 min at 4 °C. The supernatant was dialyzed overnight in a 12 KD dialysis bag against the same buffer and concentrated using Centriprep 10 (Amicon, Witten, Germany). The soluble proteins were eliminated by proteinase-K, and then sample was directly loaded onto an equilibrated 1.6 x 100 cm Sephacryl S-100 column (Pharmacia LKB GmbH, Freiburg, Germany) with 50 mM sodium phosphate. At the end, puri ed PIA was stored at -20 °C in Amicon cell (Fig. 2). The concentration of the puri ed PIA was assessed via the amount of hexosamine, taking advantage of the 3methyl-2-benzothiazolone hydra-zine hydrochloride method with N-acetyl glucosamine as standard (see Fig. S1, which is available in the online version of this article) (18).

Biochemical analysis
The amount of protein content in the puri ed rSesC was assessed by the Bradford assay (17). The constitution and structure of the puri ed native PIA was analysed using Fourier transform infrared spectroscopy (FTIR) and colorimetric assays (Fig. 3, 4) as well.
Pyrogenicity test and general safety Taking advantage of the previously procedure, the pyrogenicity and toxicity of the antigens were checked [15]. The amount of endotoxin in the prepared antigens was measured by a commercial Limulus amebocyte lysate kit (Thermo Scienti c, Waltham, MA, USA) according to the manufacturer's recommendations.
Immunization of mice 6-8-week-old female 18±2 g BALB/c inbred mice (purchased from the Research Institution of Pasteur Karaj, IR Iran) were divided into four groups of six mice each for the vaccination experiments. The mice were housed in standard stainless cages at 23-25 ° C and 60-70 % humidity, with a 12 h light/dark cycle, for a week before the experience. The mice were given free access to a standard diet and water. Each mouse in the speci c group was immunized thrice subcutaneously with the respective lyophilized antigens (PIA, rSesC, PIA/ rSesC, and PBS) in 1 % alum (Brentag, Denmark) dissolved in PBS ( ltered at 0.22 nm pore diameter). The quantity of candidate vaccines were adjusted to a concentration of PIA and protein in the mixture then an immunogenicity of candidate vaccines was compared to control. After two weeks of each immunization (immunization days were 0, 7, 14 and 28) 500 µl of peripheral blood was collected from the tail vein of ve mice in each group. Collected sera were stored at −20 °C followed by centrifugation (3000 r.p.m. for 5 min) of peripheral blood [16].
The experimental groups were as follows: Using the halogenated ether procedure as an inhalant anesthetic, mice were euthanized as well.

Enzyme-linked immunosorbent assay (ELISA)
Anti-PIA antibodies were introduced into the immunized mice sera by applying a commercial enzymelinked immunosorbent assay after each immunization. Brie y, 96-well plates (Extra gene, USA) were coated overnight with 100 µl of PIA (1 µg/well) in PBS at 4 °C. Then, the plates were washed three times with washing buffer [0.05 % (v/v) Tween 20 in PBS], followed by blocking with PBS/Tween 20 containing 5% bovine serum albumin (BSA) for 2 h at 37 °C. Fling blocking and washing, the mouse sera were (1: 2 to 1: 1024) in blocking buffer and 100 µl of the samples was added to the wells in duplicate. The plates were incubated for 2 h at 37 °C, washed three times and incubated with HRP-conjugated anti-mouse IgG (Sigma, USA) diluted to 1 : 10 000 (as a secondary antibody) at 37 °C for 2 h. The plates were washed as described above; enzymatic activity was measured by adding 100 µl of tetramethylbenzidine (TMB) substrate.
In vitro bio lm inhibition assay Bio lm inhibitory effect ofpre-and post-immune IgGs against injected antigens on in vitro biofilm formation was analysed using a semi-quantitative microtiter plate method17]. (Fig. 6). In short, 5×10 5 c.f.u. ml −1 of an overnight culture of S. aureus grown in BHI for the initial attachment in fresh sterile trypticase soy supplemented by 1 % glucose (TSBg) broth was prepared.
A while later, in a polystyrene microtiter plate (Corning Joined Life Sciences, Lowell, MA, USA), a mixture of 200 μl of diluted bacterial cultures and 50 μl of twofold diluted post and pre-immunized mouse sera were inoculated into three parallel wells. After incubation at 37 °C for 20 h, respectively, the attached cells and biofilm were stained by crystal violet as previously described [18]. The OD at 595 (OD595) of the dissolved stain in 160 µl of 30 % (vol/vol) acetic acid was measured in a multipurpose UV/VIS plate reader. A negative control of sterile TSBg without bacteria was included and the assay was repeated independently three times. A previously described in vitro bio lm formation assay previously described was also performed [18].

Statistical analysis
Utilizing multiple-group analysis of variance (ANOVA), statistical analysis of results was accomplished and a P-value<0.05 was considered signi cant.
Abbreviations: Not applicable Discussion S. aureus as a main human primary pathogen because of the ability to bio lm formation and antibiotic resistance pattern has been assigned as an essential concern in worldwide healthcare system [19]. Staphyloccocus species specially S. aureus and S. epidermidis despite being a part of human ora,wellknown ability to attach to surfaces of medical devices and develop into recalcitrant community multilayered structures, referred to as "bio lm", makes them problematic [20]. Bio lm forming ability is a surveillance factor in Staphyloccocus spp., mediating the adherence of bacterial cells to biomaterials and helps organisms to avoid the host immune defense [21]. Staphylococcalability to form bio lm, considered the most important factor involved in the pathogenesis, and its colonization on medical devices, makes it increasingly resistant not only to multiple antibiotics but also to host defenses. There is an essential need to replace the medical devices after S. epidermidis bio lm infection, and practical studies on bio lm-preventing vaccines is necessary as well [22]. The role of PIA and proteins in bio lm formation has been clearly demonstrated. Targeting of the SesC protein and PIA macromolecules involved in attachment and accumulation bio lm forming phase's and antibodies that arise in response to them may be suitable options for antibodies-dependent treatment of biofilms [21]. In the current study, the evaluation of arisen antibodies against PIA and rSesC protein, e cacy of antibodies to bio lm inhibition process and putative vaccine candidate activity against the mentioned antigens of S. aureus have been listed as the main purposes. Taking advantage of cloning, rSesC protein was generated and con rmed with precision by Western blotting procedure. The DNA sequence of the truncated protein representative of the anchor site on the SesC protein was inserted into cloning vector and transformed into the prokaryotic host. 55KD protein representative of rSesC previously puri ed [12,23]. Our results were con rmed when compared by mentioned studies. Less than 70% similarity described to homologous proteins. Although, the speci c function of the SesC proteins hadn't describe, but the closest homologous protein to SesC, is a 341 aa fragment nominated Clumping factor A in S. aureus with speci c function(Identity 26.6% and Homology 65.11%) [12].
Reported data by previously published demonstrate that the arisen antibody to immunized animals with conjugated a deacetylated PNAG (≤15% acetyl) to diphtheria toxoid (DT) as a carrier protein shown a killing activity against three strains of S. aureus and a PIA dependent bio lm forming S. epidermidis M187 [14].
Because of this homology, bio lm inhibitory effect of the arisen antibodies to rSesC was targeted to bio lm forming S. aureus. Based on the results, rSesC protein has been determined as the suitable bio lm inhibitory candidate vaccine following the injection of 25 mg/ml of the mentioned antigens in mice. This protein not only caused to arise the speci c antibodies after the rst booster, but also bio lm inhibitory effect of raised antibodies was con rmed. Despite that raised mentioned antibodies were not signi cant when compared to the rst immunization (14/28 days) but the amount of arisen antibodies when compared to the second booster was signi cantly increased. Raised antibodies against rSesC protein, as a S. aureus clumping factor A protein homolog, successfully decreased bio lm formation in a bio lm forming wild type S. aureus comparing the collected sera after injection times. Our results showed that rSesC, due to the bio lm inhibitory effect, separately could be a vaccine candidate in bio lm forming S. aureus, too. Taking advantage of reported ndings [25], although rSesC and PIA were described as two effective vaccine candidates in bio lm forming S. epidermidis, the conjugation of PIA to rSesC will enhance the opsonic activity of secreted antibodies, too. In this study, a mixture of rSesC and PIA was prepared and arisen antibodies of mixture against bio lm forming wild type S. aureus were assessed. Findings support the hypothesis that rSesC could be hopefully considered as a suitable candidate vaccine against bio lm forming S. aureus, too.
PIA as the main component of bacterial accumulation during bio lm formation, was extracted from wildtype strain 1457 basing on the native puri cation process [13]. The composition of puri ed PIA con rmed by FTIR and it was observed that our data and other published data are similar [13,21]. We tried to evaluate the PIA and rSesC e cacies as immunoprophylaxis and immunotherapy against bio lmforming S. aureus. According the previous studies, the purity of native puri ed PIA can elicit an antibody response against the mentioned antigen [20,[24][25][26]. Although the antigenicity of most polysaccharides is poor, increased antibodies to PIA have shown a bio lm inhibitory effect. Corresponding antigens had previously been analysed as a useful candidate vaccine against bio lm-forming S. epidermidis, but the opsonic rate of the rSesC protein was lower in puri ed IgGs [21].
At the present research, 6-8 weeks female mice were immunized by PIA, rSesC and mix of them in four categorized. Control group received PBS as basic solvent of antigens. All mice were boosted by each speci c antigens two weeks after rst immunization. Immunized sera were collected in 14, 28 and 42 days after the rst immunization. According to the obtained results in ELISA, to evaluation of the total IgGs by checker board method, diluted sera in 1: 200 determined as titers for all of the antigens. PIA and rSesC Immunized sera were not statistically signi cant when compared by the control 14 days after the rst immunization. After the rst booster dosage, increasing of the PIA and rSesC antibodies titers were observed, while titers of arisen antibodies in 28 (P = 0.0025) and 42 (P = 0.0001) collected immune sera were statistically signi cant. Arisen antibodies to mixture of PIA and rSesC in all immunized sera were statistically signi cant. Similar results were observed in previously published study based on the antigenicity of mentioned macromolecules [21].
Regarding the results reported in previous studies, the presence of amine groups in the PIA structure arisen titers in the rst and second boosters is justi able. The alum as an adjuvant could be stimulated the secreted antibodies and induction of Th2 immunity as well.
Since one of the most important characteristics of vaccine candidates is the in vitro and in vivo effects of them, in this study, the features of polyclonal secreted antibodies analysed on bio lm formation under laboratory conditions. Data showed that, immune sera after 14 days had bio lm inhibitory effect, however after the rst and second reminders, the effect of bio lm inhibition in the mixture group and other groups showed a signi cant difference compared with the control group in the last reminder. E cacy of arisen antibodies in immunized sera showed that, at the rst immunization for PIA (P = 0.3466), rSesC (P = 0.371), bio lm inhibitory effect were not statistically signi cant when compared by the control group. Immunized sera by mix of PIA and rSesC in two week after the rst immunization showed the bio lm inhibitory effect was statistically signi cant (P = 0.0004). Bio lm inhibitory effect of antigens comparing the different immunization era (14/28 days) showed that the immunized sera by PIA, rSesC (P = 0.0314) and Mix of them (P = 0.0003) were statistically signi cant as well.
Previous researches reported that the mixture of mentioned macromolecules had a good bio lm inhibitory effect. Our published study [25] reported that using a mixture of PIA and rSesC, the opsonic activity of arisen antibodies enhanced against bio lm forming S. epidermidis [25]. In this study, bio lm inhibition capability to PIA/rSesC mixture antibodies against bio lm forming S. aureus was evaluated. Although opsonic activity and in vivo challenge for survival not cheeked at the current research but bio lm inhibition assay demonstrated that mixture of PIA and rSesC, because of suitable decreasing of the bio lm against a wild type bio lm forming S. aureus, could be considered as a good candidate vaccine regarding inhibition of bio lm formation in both S. epidermidis an S. aureus.

Conclusions
We found that a PIA/rSesC mixture vaccine could inhibit bio lm-formation process in bio lm producer S. aureus by eliciting high titer anti-PIA antibodies. It seems that this mixture could be employed for patients from the colonization and bio lm formation of mentioned bacterial. According to our ndings, the immunization of high-risk patients with mixture of vaccine candidate or treatment of them using monoclonal antibodies such as IgG2a could help to eradicate bacterial bio lms. The preparation and puri cation of speci c anti-PIA IgG2a are a possible means to inhibit medical device infections caused by S. epidermidis and S. aureus.

Declarations
Ethics approval and consent to participate: Availability of data and material: All the results of this study have been classi ed and maintained by the dissertation in the Pasteur Institute of Iran. We have indeed provided all raw data on which our study is based.    Puri cation of PIA. Representative Fast protein liquid chromatography (FPLC) chromatogram for native PIA. Sample in 1ml nal volume was injected after 36 minutes column equilibration. PIA was eluted near the void volume following the 110 minutes after sample injection by using 0.3ml/min owrate. Polysaccharide was identi ed at a wavelength of 206 nm and protein was identi ed at 280 nm.

Figure 3
Standard curve to hexosamine assay. According to the procedure, three concentration (µg/ml) of Nacetylglucosamine as standard were used and optical density of these concentrations evaluated in 650 nm.
Page 17/19 Figure 4 IR spectra of Puri ed PIA in the 4000-500 cm−1 range and the result of this deconvolution. Infrared spectroscopy of puri ed polysaccharide was investigated using the regularized method of deconvolution.
According to the composition of the PIA molecules C=O groups in 1739.327 of the native polysaccharide in the FTIR pattern, was detected.

Figure 5
Total arisen antibodies to antigens. The booster effect of the assessed antigens at different times. ELISA was performed by coating the native PIA and rSesC. The titers of the antibodies was assessed for immunized sera compared to the controls. Signi cant effects were observed from use of the mixture and conjugate booster. Six weeks after the rst injection, the titres of antibodies had increased. Antibody titration was assessed in 1: 200 titer, the error bar is representative of the mean±sd (n=3).