A brief exploration of EPS composition in biofilms of Staphylococcus spp ATCC reference strains

Abstract

Antibiotic resistance is expected to cause 10 million deaths per year worldwide by 2050. One of the mechanisms for the resilient nature of bacteria toward antibiotics is through the formation of biofilm. Bacterial biofilms are sessile communities of microorganisms, which exist in a matrix of proteins, carbohydrates, eDNA and other various components – collectively known as extracellular polymeric substances. Biofilms slow the penetration of drugs, and also contribute to the development of a resistant phenotype known as persisters. Thus, understanding biofilm composition might contribute to the development of anti-biofilm strategies. The aim of this study was to explore biofilm formed by five Staphylococcus spp ATCC strains, commonly used in research as references: S. aureus 25923, S. aureus 29213, S. aureus 43300 (methicillin-resistant), S. aureus 6538 and S. epidermidis 12228. Biofilm mass and its components were analysed after 24h and 72h of biofilm growth. Bacterial biofilm was prepared in 96-well microtiter plates, in Trypticase Soy Broth supplemented with 1% glucose. After incubation at 37°C, absorbance measurements and crystal violet staining were performed and the specific biofilm formation determined for each strain. Extracellular polymeric substances were extracted using a combination of physical and chemical methods; including centrifugation, vortexing and the use of 1.5M NaCl. In these assays, biofilms were grown in polystyrene tubes containing 10 ml of same media mentioned above. The concentration of protein, carbohydrate and eDNA was determined using the Bicinchoninic acid assay, phenol-sulfuric acid method and DNeasy^® Blood and Tissue Kit, respectively, followed by spectroscopy. Our data demonstrated heterogeneity between the biofilm-forming capabilities and EPS components within staphylococcal strains and species. Strains 25923 and 6538 had the highest value for biofilm formation at both time points. Interestingly, strain 43300 was the only one to show a significant increase in biofilm after 72h. Contradictory to previous findings, S. epidermidis 12228 was found to be a good biofilm producer. At both time points studied, strains demonstrated considerably higher concentrations of protein (varying from 172 µg/mL – 345 µg/mL) and carbohydrate (56 µg/mL - 372µg/mL) in EPS compared to eDNA (2.74 µg/mL – 8.12 µg/mL). On average, strains 43300 and 12228 had the highest concentration of protein, and the latter also had the highest carbohydrate and eDNa amounts at 72h. Strains 25923 and 6538 had a significant decrease in eDNA concentration over time. Based on this brief study, the relative quantities of EPS components investigated is similar to that of other studies with protein being the most plentiful component followed by carbohydrate and then considerably lower amounts of eDNA. Differences in specific biofilm formation did not directly reflect variations observed in abundance of a particular constituent in the matrix of EPS. This study also showed that S. epidermidis 12228, usually classified as a weak or non-biofilm former, was able to grow a relatively substantial biofilm under the conditions tested here.