New insights in Staphylococcus pseudintermedius pathogenicity: antibiotic-resistant biofilm formed by a human wound-associated strain.

Objectives: Staphylococcus pseudintermedius is recognized as the leading cause of skin and ear infections in dogs and cats. However, cases of human infection have been recently reported. The main aim of this work was to evaluate, for the first time, the ability of human S. pseudintermedius to form biofilm, both under conditions relevant at wound site (serum, pH), and in the presence of antibiotics. Methods: S. pseudintermedius Sp32 strain, we recently isolated from an abdominal wound in a patient underwent to an allogeneic bone marrow transplant for chronic lymphoblastic leukemia, was tested. The effect of pH (5.5, 7.1, and 8.7) and serum (diluted at 1:2, 1:10, and 1:100; either used as free or substratum-adsorbed) on biofilm formation was simultaneously assessed by crystal violet staining. Biofilm amount was normalized on bacterial growth by calculating the specific biofilm formation (SBF). The in vitro activity of several antibiotics (chloramphenicol, gentamycin, cefoxitin, linezolid, rifampicin, vancomycin, tetracycline, and tigecycline) against biofilm formation and mature (48h-old) biofilms (MBEC50 and MBEC90, minimum concentration eradicating 50% and 90% of biofilm viability, respectively) was assessed by crystal violet staining or viable cell counting, respectively. Confocal laser and scanning electron microscopy were performed to evaluate kinetic of biofilm formation and biofilm architecture. Results: In the absence of serum, biofilm formation at pH 7.1 was significantly higher than at pH 5.5 or 8.7 (SBF: 5.3±1.0 vs 2.8±1.2 and 3.1±1.0, respectively; p<0.05). In the presence of free serum, biofilm forming ability resulted to be significantly affected, regardless of pH value tested (% reduction vs control: 45.3-87.5%, 74.3-95.5%, and 21.9-96.5% at pH of 5.5, 7.1, and 8.7, respectively). Polystyrene pre-treatment with serum significantly reduced biofilm formation under acid and neutral pH (% reduction vs control: 59.4-78.5%, and 82.7-88.3% at pH 5.5 and 7.1, respectively). Notably, 1% serum under acid environment significantly stimulated biofilm formation (120% vs control). Antibiotics at sub-inhibitory concentrations reduced biofilm formation in a dose-dependent manner, although at different extent. Cefoxitin was the most active, causing a significant reduction already at 1/8xMIC (% reduction vs control: 87.5±4.9). To the contrary, vancomycin exhibited the worst activity, being able to affect biofilm formation at 1/2xMIC only. Rifampicin was the most active antibiotic against preformed biofilms (MBEC50 and MBEC90: 1x and 2xMIC, respectively), while gentamicin was the worst one (MBEC50 and MBEC90: 64xMIC), even significantly stimulating biofilm formation at 1x and 2xMIC, compared to controls. Microscopic observation showed that S. pseudintermedius rapidly adhered to polystyrene (30 min), formed small microcolonies (4h), then a mature biofilm (24-72h) showing a complex “mushroom-like” architecture consisting of microcolonies embedded in a fibrillar extracellular matrix. Conclusion: S. pseudintermedius is able to form biofilm ultrastructurally complex and inherently resistant to antibiotics, thus confirming its pathogenic potential to humans.