Biofilm-Forming Clinical Staphylococcus Isolates Harbor Horizontal Transfer and Antibiotic Resistance Genes

Abstract

Infections caused by staphylococci represent a medical concern, especially when related to biofilms located in implanted medical devices, such as prostheses and catheters. Unfortunately, their frequent resistance to high doses of antibiotics makes the treatment of these infections a difficult task. Moreover, biofilms represent a hot spot for horizontal gene transfer (HGT) by bacterial conjugation. In this work, 25 biofilm-forming clinical staphylococcal isolates were studied. We found that Staphylococcus epidermidis isolates showed a higher biofilm-forming capacity than Staphylococcus aureus isolates. Additionally, horizontal transfer and relaxase genes of two common staphylococcal plasmids, pSK41 and pT181, were detected in all isolates. In terms of antibiotic resistance genes, aac6-aph2a, ermC, and tetK genes, which confer resistance to gentamicin, erythromycin, and tetracycline, respectively, were the most prevalent. The horizontal transfer and antibiotic resistance genes harbored on these staphylococcal clinical strains isolated from biofilms located in implanted medical devices points to the potential risk of the development and dissemination of multiresistant bacteria.

Keywords: Staphylococci; antibiotic resistance; biofilm; nosocomial infections; relaxases.

Figure 1

Detection of plasmids from 25 staphylococcal clinical isolates by agarose gel electrophoresis after digestion with nuclease S1. One microgram of plasmid DNA from each isolate was digested with 30 U of nuclease S1 at 37°C for 45 min. After digestion, the plasmids were analyzed by 1% (w/v) agarose gel electrophoresis in 1 × TAE buffer. Lanes 1–25: digested plasmid DNA from each strain (lane numbers correspond to the number of the isolate). Lanes M: DNA molecular weight marker 1 kb Plus DNA Ladder. Bands corresponding to plasmids are indicated with arrows.

Figure 2

Detection of plasmids in 25 staphylococcal clinical isolates by PFGE. Large plasmids (>30 kb) were analyzed by PFGE after digestion with nuclease S1 at 37°C for 45 min. Lanes 1–25: clinical isolates; lane number corresponds to the number of the isolate. Lane C: positive control, plasmid pSK41 (46.4 kb) extracted from SK5428 strain. Lane M: Lambda Ladder PFGE molecular size marker. Arrows point to detected plasmids.

Figure 3

Detection of erythromycin resistance gene ermC in 25 clinical isolates by PCR (A) and Southern blotting (B). Amplicons of ermC (477 bp) were visualized on 1% (w/v) agarose gels. Lanes 1–25: clinical isolates. Lanes +: positive control. Lanes –: negative control. Lanes M1: DNA molecular weight marker 1 kb Plus DNA Ladder. Lanes M2: DNA molecular weight marker VI DIG-labeled.

Figure 4

Detection of vancomycin resistance gene vanB in 25 clinical isolates by PCR (A) and Southern blotting (B). Amplicons of vanB (539 bp) were visualized on 1% (w/v) agarose gels. Lanes 1–25: clinical isolates. Lanes +: positive control. Lanes −: negative control. Lanes M1: DNA molecular weight marker 1 kb Plus DNA Ladder. Lanes M2: DNA molecular weight marker VI DIG-labeled.

Figure 5

Detection of traK gene in 25 clinical isolates by PCR (A) and Southern blotting (B). Amplicons of traK gene (573 bp) were visualized on 1% (w/v) agarose gels. Lanes 1–25: clinical isolates. Lanes +: positive control. Lanes −: negative control. Lanes M1: DNA molecular weight marker 1 kb Plus DNA Ladder. Lanes M2: DNA molecular weight marker VI DIG-labeled.

Figure 6

Detection of pre_pT181 gene in 25 clinical isolates by PCR (A) and Southern blotting (B). Amplicons of pre_pT181 gene (397 bp) were visualized on 1% (w/v) agarose gels. Lanes 1–25: clinical isolates. Lanes +: positive control. Lanes −: negative control. Lanes M1: DNA molecular weight marker 1 kb Plus DNA Ladder. Lanes M2: DNA molecular weight marker VI DIG-labeled.

Figure 7

Biofilm-forming capacity of staphylococcal clinical isolates. 96-well flat-bottom polystyrene plates were incubated for 24 h at 37°C without shaking. Cells attached to the wells were stained with 0.1% (w/v) crystal violet. Absorption at 570 nm was measured to quantify biofilm formation. Results are the mean ± SEM of at least 4 independent biological experiments performed in triplicate. To classify the isolates into significant groups, statistical analysis was performed using Student's t-test or Mann–Whitney U-test (^*p < 0.05, ^***p < 0.001).