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Comparison of biofilm formation between major clonal lineages of methicillin resistant Staphylococcus aureus.

Vanhommerig E, Moons P, Pirici D, Lammens C, Hernalsteens JP, De Greve H, Kumar-Singh S, Goossens H, Malhotra-Kumar S - PLoS ONE (2014)

Bottom Line: Only 16 strains successfully formed biofilms under both conditions, of which 13 harboured SCCmec IV and included all tested USA300 strains (n = 3).However, USA300 demonstrated remarkably lower percentages of cell-occupied space (6.6%) compared to the other clones (EMRSA-15 = 19.0%) under dynamic conditions.USA300 demonstrated abundant biofilm formation under both conditions, which probably confers a competitive advantage, contributing to its remarkable success as a pathogen.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, University of Antwerp, Antwerp, Belgium; Vaccine & Infectious Disease Institute, University of Antwerp, Antwerp, Belgium.

ABSTRACT

Objectives: Epidemic methicillin-resistant S. aureus (MRSA) clones cause infections in both hospital and community settings. As a biofilm phenotype further facilitates evasion of the host immune system and antibiotics, we compared the biofilm-forming capacities of various MRSA clones.

Methods: Seventy-six MRSA classified into 13 clones (USA300, EMRSA-15, Hungarian/Brazilian etc.), and isolated from infections or from carriers were studied for biofilm formation under static and dynamic conditions. Static biofilms in microtitre plates were quantified colorimetrically. Dynamic biofilms (Bioflux 200, Fluxion, USA) were studied by confocal laser-scanning and time-lapse microscopy, and the total volume occupied by live/dead bacteria quantified by Volocity 5.4.1 (Improvision, UK).

Results: MRSA harbouring SCCmec IV produced significantly more biomass under static conditions than SCCmec I-III (P = 0.003), and those harbouring SCCmec II significantly less than those harbouring SCCmec I or III (P<0.001). In the dynamic model, SCCmec I-III harbouring MRSA were significantly better biofilm formers than SCCmec IV (P = 0.036). Only 16 strains successfully formed biofilms under both conditions, of which 13 harboured SCCmec IV and included all tested USA300 strains (n = 3). However, USA300 demonstrated remarkably lower percentages of cell-occupied space (6.6%) compared to the other clones (EMRSA-15 = 19.0%) under dynamic conditions. Time-lapse microscopy of dynamic biofilms demonstrated that USA300 formed long viscoelastic tethers that stretched far from the point of attachment, while EMRSA-15 consisted of micro-colonies attached densely to the surface.

Conclusions: MRSA harbouring SCCmec types IV and I-III demonstrate distinct biofilm forming capacities, possibly owing to their adaptation to the community and hospital settings, respectively. USA300 demonstrated abundant biofilm formation under both conditions, which probably confers a competitive advantage, contributing to its remarkable success as a pathogen.

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Space occupied by live (green) or dead (red) or total (grey) cells by different epidemic MRSA clones in a biofilm.Error bars denote standard deviations were calculated using 1 (USA300, EMRSA-15), 2 (USA600, Hungarian/Brazilian, Iberian) or 3 (USA500) independent z-stacks.
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pone-0104561-g004: Space occupied by live (green) or dead (red) or total (grey) cells by different epidemic MRSA clones in a biofilm.Error bars denote standard deviations were calculated using 1 (USA300, EMRSA-15), 2 (USA600, Hungarian/Brazilian, Iberian) or 3 (USA500) independent z-stacks.

Mentions: Of the 76 strains tested here, 39 (51%) strains successfully formed biofilms under shear flow. Successful biofilm formation was defined as the ability of cells to adhere to the surface in 2 of 2 or 3 independent experiments and was considered negative when no adherent cells could be shown in 2 of 2 or in 3 of 4 such experiments. Of the strains that were found positive for biofilm formation, 22 (66%) harboured SCCmec types I-III (n = 33) and 17 (40%) SCCmec type IV (n = 43) (P = 0.036, χ2). All USA600 (n = 3, CC45-SCCmec II) and USA300 (n = 3, CC8-SCCmec IV), but none of the South-West Pacific (n = 5, CC30-SCCmec IV) and Paediatric (n = 5, CC5-SCCmec IV) clones formed biofilms in the shear flow assay (Figure 2). The remaining 33 strains positive for biofilm formation under shear flow belonged to the following clones: Hungarian/Brazilian (8/12), EMRSA-15 (5/7), Berlin (5/11), EMRSA-16 (3/4), New York/Japan (3/4), Southern Germany (3/6), USA500 (3/8), Iberian (2/4) and European (1/4). On confocal microscopy, differences in the biofilms between various clones were evident, with the Iberian and USA600 clones forming thicker, more confluent and densely packed biofilms in comparison to those formed by USA500, USA300 and the Hungarian/Brazilian clones that showed gaps making the biofilms less confluent (USA500) or formed a less densely packed biomass (USA300 and Hungarian/Brazilian) (Figure 3). Since live/dead staining only stains bacterial cells and not the matrix, differences in cell densities within the biofilm could be captured through 3-dimensional quantification of the space occupied by micro-organisms. Both the USA300 and Hungarian/Brazilian clones demonstrated remarkably lower percentages of cell-occupied space (6.6% and 4.0%, respectively) compared to the other clones (EMRSA-15 = 19.0%, USA500 = 25.2%, USA600 = 15.7% and Iberian  = 9.4%) (Figure 3 and 4). Time-lapse microscopy demonstrated the strength of an MRSA biofilm under shear flow conditions (movie S1 and movie S2). Even when flow was 4× higher than normal growth conditions (2.0 dyne/cm2), the USA300 biofilm remained attached to the surface. Long elastic matrix strands kept the cells together. The USA300 (CC8-MRSA-IV-PVL-ACME) strain showed individual cells attached more to each other than to the surface resulting in the formation of long streamers/tethers, while attachment to the surface was more evident for the EMRSA-15 (CC22-MRSA-IV) strain (movie S1 and movie S2).


Comparison of biofilm formation between major clonal lineages of methicillin resistant Staphylococcus aureus.

Vanhommerig E, Moons P, Pirici D, Lammens C, Hernalsteens JP, De Greve H, Kumar-Singh S, Goossens H, Malhotra-Kumar S - PLoS ONE (2014)

Space occupied by live (green) or dead (red) or total (grey) cells by different epidemic MRSA clones in a biofilm.Error bars denote standard deviations were calculated using 1 (USA300, EMRSA-15), 2 (USA600, Hungarian/Brazilian, Iberian) or 3 (USA500) independent z-stacks.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4126748&req=5

pone-0104561-g004: Space occupied by live (green) or dead (red) or total (grey) cells by different epidemic MRSA clones in a biofilm.Error bars denote standard deviations were calculated using 1 (USA300, EMRSA-15), 2 (USA600, Hungarian/Brazilian, Iberian) or 3 (USA500) independent z-stacks.
Mentions: Of the 76 strains tested here, 39 (51%) strains successfully formed biofilms under shear flow. Successful biofilm formation was defined as the ability of cells to adhere to the surface in 2 of 2 or 3 independent experiments and was considered negative when no adherent cells could be shown in 2 of 2 or in 3 of 4 such experiments. Of the strains that were found positive for biofilm formation, 22 (66%) harboured SCCmec types I-III (n = 33) and 17 (40%) SCCmec type IV (n = 43) (P = 0.036, χ2). All USA600 (n = 3, CC45-SCCmec II) and USA300 (n = 3, CC8-SCCmec IV), but none of the South-West Pacific (n = 5, CC30-SCCmec IV) and Paediatric (n = 5, CC5-SCCmec IV) clones formed biofilms in the shear flow assay (Figure 2). The remaining 33 strains positive for biofilm formation under shear flow belonged to the following clones: Hungarian/Brazilian (8/12), EMRSA-15 (5/7), Berlin (5/11), EMRSA-16 (3/4), New York/Japan (3/4), Southern Germany (3/6), USA500 (3/8), Iberian (2/4) and European (1/4). On confocal microscopy, differences in the biofilms between various clones were evident, with the Iberian and USA600 clones forming thicker, more confluent and densely packed biofilms in comparison to those formed by USA500, USA300 and the Hungarian/Brazilian clones that showed gaps making the biofilms less confluent (USA500) or formed a less densely packed biomass (USA300 and Hungarian/Brazilian) (Figure 3). Since live/dead staining only stains bacterial cells and not the matrix, differences in cell densities within the biofilm could be captured through 3-dimensional quantification of the space occupied by micro-organisms. Both the USA300 and Hungarian/Brazilian clones demonstrated remarkably lower percentages of cell-occupied space (6.6% and 4.0%, respectively) compared to the other clones (EMRSA-15 = 19.0%, USA500 = 25.2%, USA600 = 15.7% and Iberian  = 9.4%) (Figure 3 and 4). Time-lapse microscopy demonstrated the strength of an MRSA biofilm under shear flow conditions (movie S1 and movie S2). Even when flow was 4× higher than normal growth conditions (2.0 dyne/cm2), the USA300 biofilm remained attached to the surface. Long elastic matrix strands kept the cells together. The USA300 (CC8-MRSA-IV-PVL-ACME) strain showed individual cells attached more to each other than to the surface resulting in the formation of long streamers/tethers, while attachment to the surface was more evident for the EMRSA-15 (CC22-MRSA-IV) strain (movie S1 and movie S2).

Bottom Line: Only 16 strains successfully formed biofilms under both conditions, of which 13 harboured SCCmec IV and included all tested USA300 strains (n = 3).However, USA300 demonstrated remarkably lower percentages of cell-occupied space (6.6%) compared to the other clones (EMRSA-15 = 19.0%) under dynamic conditions.USA300 demonstrated abundant biofilm formation under both conditions, which probably confers a competitive advantage, contributing to its remarkable success as a pathogen.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, University of Antwerp, Antwerp, Belgium; Vaccine & Infectious Disease Institute, University of Antwerp, Antwerp, Belgium.

ABSTRACT

Objectives: Epidemic methicillin-resistant S. aureus (MRSA) clones cause infections in both hospital and community settings. As a biofilm phenotype further facilitates evasion of the host immune system and antibiotics, we compared the biofilm-forming capacities of various MRSA clones.

Methods: Seventy-six MRSA classified into 13 clones (USA300, EMRSA-15, Hungarian/Brazilian etc.), and isolated from infections or from carriers were studied for biofilm formation under static and dynamic conditions. Static biofilms in microtitre plates were quantified colorimetrically. Dynamic biofilms (Bioflux 200, Fluxion, USA) were studied by confocal laser-scanning and time-lapse microscopy, and the total volume occupied by live/dead bacteria quantified by Volocity 5.4.1 (Improvision, UK).

Results: MRSA harbouring SCCmec IV produced significantly more biomass under static conditions than SCCmec I-III (P = 0.003), and those harbouring SCCmec II significantly less than those harbouring SCCmec I or III (P<0.001). In the dynamic model, SCCmec I-III harbouring MRSA were significantly better biofilm formers than SCCmec IV (P = 0.036). Only 16 strains successfully formed biofilms under both conditions, of which 13 harboured SCCmec IV and included all tested USA300 strains (n = 3). However, USA300 demonstrated remarkably lower percentages of cell-occupied space (6.6%) compared to the other clones (EMRSA-15 = 19.0%) under dynamic conditions. Time-lapse microscopy of dynamic biofilms demonstrated that USA300 formed long viscoelastic tethers that stretched far from the point of attachment, while EMRSA-15 consisted of micro-colonies attached densely to the surface.

Conclusions: MRSA harbouring SCCmec types IV and I-III demonstrate distinct biofilm forming capacities, possibly owing to their adaptation to the community and hospital settings, respectively. USA300 demonstrated abundant biofilm formation under both conditions, which probably confers a competitive advantage, contributing to its remarkable success as a pathogen.

Show MeSH
Related in: MedlinePlus