A total of 25 staphylococcal biofilm-forming clinical isolates were kindly provided by Hospital Universitario Donostia, Spain. In addition, they provided data on their antibiotic resistance phenotype, determined by diffusion discs on agar. The origin and antibiotic resistance phenotype of each isolate are shown in Table 1.

Swabs from the clinical isolates were plated on tryptic soy agar (TSA) and incubated at 37°C. Subsequently, a single colony of each isolate was grown in 10 ml of tryptic soy broth (TSB) supplemented with at least two antibiotics to which the strain was phenotypically resistant (see Table 1), at 37°C overnight. The culture was centrifuged at 8,000 × g for 10 min. Then, the pellet was resuspended in 2 ml of TSB medium containing 40% (v/v) glycerol and stored at −80°C.

For this study, strains were grown in TSB medium at 37°C and 200 rpm. TSB medium and TSA plates were supplemented, when required, with amoxicillin (8 μg/ml), cloxacillin [2 μg/ml for S. aureus and 0.5 μg/ml for coagulase negative staphylococci (CoNS)], erythromycin (4 μg/ml), mupirocin (520 μg/ml), tetracycline (8 μg/ml), gentamicin (20 μg/ml), rifampicin (2 μg/ml), or levofloxacin (2 μg/ml).

Plasmid DNA was extracted from the 25 clinical isolates with the ATP^TM Plasmid Midi kit (ATP biotech Inc., Taiwan), according to the manufacturer's instructions.

To detect small plasmids (molecular size < 20 kb), 1 μg of total extracted plasmid DNA was linearized by incubation with 30 U of Aspergillus oryzae nuclease S1 (Sigma, Spain) at 37°C for 45 min. Nuclease S1 cuts one strand of the DNA at the nick site and its activity results in linearized plasmids (Germond et al., 1974). Different enzyme concentrations were studied to optimize nuclease S1 digestion (data not shown). Linearized plasmids were visualized on 1% (w/v) agarose gels in 1 × TAE buffer.

Detection of large plasmids (molecular size > 20 kb) was carried out by Pulsed Field Gel Electrophoresis (PFGE) as described by Barton et al. (1995) with modifications. Bacteria were grown in 2 ml of TSB medium overnight at 37°C and 200 rpm. Cultures were diluted in PIV buffer [10 mM Tris-HCl (pH 8), 1 M NaCl] until OD₆₀₀ = 1. Then, 600 μl of diluted culture were centrifuged at 11,000 × g for 2 min. Subsequently, the pellet was washed with 500 μl of PIV buffer and centrifuged again. The pellet was resuspended in 300 μl of PIV buffer and incubated at 42°C for 10 min. Next, 150 μl of the sample were mixed with 150 μl of 2% (w/v) low-melting agarose (BioRad) which had been preincubated at 42°C. The mixture was transferred into the plugs, incubated at room temperature for 10 min and, subsequently, for 15 min at 4°C. Once solidified, gel plugs were incubated at 37°C for 5–6 h with shaking (600 rpm) in 1 ml of lysis buffer EC [6 mM Tris-HCl (pH 8), 1 M NaCl, 100 mM EDTA (pH 8), 0.2% (w/v) sodium deoxycholate, 0.5% (w/v) n-lauroylsarcosine, 100 μg/ml lysozyme, 50 μg/ml lysostaphin]. After cell lysis, gel plugs were transferred to new tubes containing 1 ml of EPS solution [1% (w/v) n-lauroylsarcosine, 0.5 M EDTA (pH 8), 100 μg/ml proteinase K] and then incubated at 56°C for 16–20 h. Next, five washes with 1 ml of TE buffer [10 mM Tris-HCl (pH 8), 1 mM EDTA (pH 8)] at 50°C for 30 min each were carried out. For nuclease S1 digestion, each gel plug was cut into two slices. Each slice was incubated twice in 100 μl of digestion solution [50 mM NaCl, 30 mM sodium acetate (pH 4.5), 5 mM ZnSO₄] at room temperature for 15 min. Then, slices were incubated at 37°C for 45 min with 1 U of A. oryzae nuclease S1 (Sigma) in 100 μl of digestion solution. The reaction was stopped by transferring the slices to 1 ml of TE buffer for 1 h. Digested slices were applied to wells in 1% (w/v) Pulsed Field Certified Agarose (BioRad) prepared in 0.5 × TBE buffer [45 mM Tris (pH 8), 45 mM boric acid, 1 mM EDTA] and run in CHEF-DR® III System (BioRad) at 6 V/cm, a field angle of 120°, and switch times of 5 to 35 s for 22 h. Lambda Ladder PFGE (New England Biolabs, Ispwich, U.S) was used as molecular size marker and pSK41 plasmid (46.4 kb) was used as positive control. Gels were stained with GelRed Nucleic Acid Stain (Biogen Científica, Madrid, Spain). Bands were visualized by ChemiDoc XRS System (BioRad). Images were analyzed by Quantity One 1-D Analysis Software (BioRad).

PCR and Southern blotting assays, specific for horizontal transfer and antibiotic resistance genes, were performed using the strains and plasmids indicated in Table 2 as reference DNA. Oligonucleotides used for gene detection are listed in Table 3. Each 25 μl PCR reaction mixture contained 1.25 U Taq polymerase (New England Biolabs, Ipswich, U.S.), 1 × PCR buffer, 0.5 μM of each primer, 0.2 mM deoxynucleoside triphosphates and 20 ng of template DNA (plasmid DNA). Amplifications were carried out in a C1000™ Thermal Cycler (BioRad). PCR temperature profiles are shown in Table 4. PCR products were separated by agarose gel electrophoresis, transferred to a membrane (Sambrook and Russel, 2001), and then hybridized with the corresponding specific DIG-labeled probe using the PCR DIG Probe Synthesis Kit (Roche, Mannheim, Germany). Detection of DNA sequences was performed with the DIG Luminescent Detection Kit (Roche) according to the manufacturer's instructions.

To test the 25 clinical isolates for biofilm formation, a quantitative adherence assay (Christensen et al., 1985) with some modifications was used. Briefly, 200 μl of TSB medium in 96-well flat-bottom polystyrene plates were inoculated with 10 μl overnight bacterial cultures and grown without shaking at 37°C for 24 h. Planktonic bacteria were removed from each well. Then, three washes with distilled water per well were carried out. Next, 125 μl of 0.1% (w/v) crystal violet solution were added to each well and incubated for 10 min at room temperature. Subsequently, three washes with distilled water were again performed. To solubilize the dye, 200 μl of 33% (v/v) glacial acetic acid solution were added to each stained well and incubated for 10 min at room temperature. TSB medium was used as negative control. The optical density of the attached bacteria was measured in a microplate reader at 570 nm (in triplicate for each strain). The ability to form biofilm was attributed as: OD₅₇₀ < 0.120, no biofilm-forming; 0.120 < OD₅₇₀ < 0.240, weak biofilm-forming; OD₅₇₀ > 0.240, strong biofilm-forming (Christensen et al., 1985; Di Rosa et al., 2006); and OD₅₇₀ > 1.5, very strong biofilm-forming. Dilutions were performed when absorbance values were higher than the limit of accurate detection. To classify the isolates into significant groups, statistical analysis was performed using SigmaPlot program and Student's t-test or Mann–Whitney U-test.

Plasmid DNA was extracted from the 25 clinical isolates and then analyzed by agarose gel electrophoresis and PFGE (Figures 1, 2). Since plasmid DNA samples are sometimes contaminated with chromosomal DNA, as suggested in Figure 1 for some of our isolates (i.e., 1, 3, 17, 21), after the extraction of plasmid DNA, we decided to test for such contamination. To this purpose, 16S rRNA from isolates 1, 3, 17, and 21 was amplified by PCR as explained in Broszat et al. (2014). The obtained amplicons were analyzed by 1% (w/v) agarose gel electrophoresis in 1 × TAE buffer. As observed in Supplementary Figure 1, some of our plasmid DNA samples appear to be contaminated with chromosomal DNA. Nonetheless, as reflected in Figures 1, 2, the majority of the extracted DNA corresponds to plasmid DNA.

As shown in Figures 1, 2 and Table 5, a total of 54 plasmids of sizes ranging from 2 to 200 kb were detected using both methods: 15 small plasmids (size < 20 kb) and 39 large plasmids (size > 20 kb; Shearer et al., 2011). All clinical isolates contained at least one plasmid, except isolate 3. The combination of agarose gel electrophoresis and PFGE is unable to detect plasmids between 13 and 45 kb. Then, a priori, our clinical isolates could harbor more plasmids than observed here. In particular, isolate 3 could harbor a plasmid between 13 and 45 kb, which could explain the apparent lack of plasmid observed for this isolate.

When agarose gel electrophoresis was used, it was observed that 44% of the clinical isolates contained at least one plasmid with a size <20 kb (Figure 1 and Table 5). In particular, nine of the isolates contained only one plasmid smaller than 20 kb. Isolate 12 harbored two plasmids smaller than 20 kb, while 4 plasmids of this size were identified in isolate 9.

According to our PFGE data, 84% of the clinical isolates (all except isolates 1, 3, 4, and 16) contained at least one large plasmid (Figure 2 and Table 5): 32% of the isolates (6, 10, 15, 17, 18, 20, 21, and 25) harbored one large plasmid; 32% of the isolates (2, 5, 8, 13, 14, 19, 22, and 23) contained two large plasmids; and 20% of the isolates (7, 9, 11, 12, and 24) harbored three large plasmids.

Eight resistance genes commonly found in staphylococci were investigated by PCR and Southern blotting: genes encoding resistance to erythromycin (ermB, ermC, ermD, ermG), tetracycline (tetK, tetM), gentamicin (aac6-aph2a), and vancomycin (vanB). The presence of these genes was tested in our extracted DNA (i.e., putative plasmid DNA) because, initially, we were only interested in the risk of dissemination of antibiotic resistance from these clinical strains through bacterial conjugation.

Concerning erythromycin resistance, 15 of the strains had an erythromycin resistance phenotype (Table 1). Data at the genotype level for the different clinical isolates are shown in Table 5. ermC gene was observed in all the isolates (Figure 3), while ermD was not detected in any of the isolates (Supplementary Figure 2). Likewise, 72% of the isolates were ermB-positive (Supplementary Figure 3), whereas only 8% of the isolates (2 and 15) harbored the ermG gene (Supplementary Figure 4).

With respect to tetracycline, only two isolates (9 and 12) were observed to be tetracycline resistant at the phenotype level (Table 1). Regarding this antibiotic, 23 out of 25 isolates contained the tetK gene (Supplementary Figure 5), while only isolate 25 harbored the tetM gene (Supplementary Figure 6).

Similarly, the 25 isolates were analyzed for the occurrence of the gentamicin resistance aac6-aph2a gene. As shown in Supplementary Figure 7, this gene was detected in 88% of the isolates (all the isolates except 9, 11, and 21 showed a positive result for the aac6-aph2a gene). However, according to the phenotype (Table 1), only 44% of the isolates showed gentamicin resistance.

Finally, regarding vancomycin resistance, all the isolates were phenotypically sensitive to this antibiotic (Table 1). At the genotype level, only isolate 10 proved to be vanB-positive (Figure 4).

In order to find out whether the abovementioned antibiotic resistance genes were likely to be disseminated via conjugative transfer, we searched for horizontal transfer genes from two common staphylococcal plasmids: (i) conjugative pSK41 and (ii) mobilizable pT181 (Novick, 1989; Berg et al., 1998).

In relation to pSK41, the pre relaxase gene was found in all the isolates except isolate 14 (Supplementary Figure 8). In addition, isolates 1, 4, 7, 8, 11, 16, 17, and 18 contained the nes relaxase gene of pSK41 (Supplementary Figure 9). Five genes (traE, traG, traK, traL, and traM) from the transfer region of pSK41 were also analyzed: traE gene was present in 48% of the isolates (Supplementary Figure 10), traG gene was detected in 68% of the isolates (Supplementary Figure 11), traK gene was found in 56% of the isolates (Figure 5), and traL gene was detected in 88% of the isolates (Supplementary Figure 12). Finally, traM gene was found in only 36% of the isolates (Supplementary Figure 13).

In addition, we tested for the presence of the pre relaxase gene of the staphylococcal mobilizable plasmid pT181. As shown in Figure 6, this gene was detected in all the clinical isolates.

All the clinical strains were isolated from biofilms formed on medical devices such as catheters and prostheses, as well as from ulcer and articular fluids from patients with prostheses (Table 1). In order to confirm their biofilm forming capacity, we used the in vitro assay described above (Christensen et al., 1985; Di Rosa et al., 2006).

As shown in Figure 7, isolates were divided into four groups: (i) no biofilm-forming isolates: 1, 4, 14, 16, 18; (ii) weak biofilm-forming isolates: 2, 3, 11, 15; (iii) strong biofilm-forming isolates: 6, 7, 9, 10, 13, 17, 19, 20, 21, 23, 24; and (iv) very strong biofilm-forming isolates: 5, 8, 12, 22, 25. Additionally, our statistical analysis showed that the “strong biofilm-forming” group could be further divided into three different sub-groups with increasing biofilm forming capacity from “strong biofilm-forming (1)” to “strong biofilm-forming (3).” The distribution of isolates in these three sub-groups was as follows: “strong biofilm-forming (1)”: 7, 10, 17, 23; “strong biofilm-forming (2)”: 9, 19; and “strong biofilm-forming (3)”: 6, 13, 20, 21, 24 (Figure 7). Furthermore, the relationship between biofilm-forming capacity and Staphylococcus species was studied. S. epidermidis isolates had a significantly higher (p < 0.001) biofilm-forming capacity than S. aureus isolates.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer GDS and handling Editor declared their shared affiliation.