Abstract
BackgroundBiofilms are formed by a complex bacterial community encapsulated by a polymeric matrix, with strong adherent properties and persistent phenotype. Biofilms are considered one of the most challenging areas of modern medicine. Existing antibiotics have been developed against free-floating bacterial cells, and thus, many treatments of biofilm-related infection fail. In this study, we compared the effects of different media on biofilm growth of clinical reference strains of Staphylococci and Enterococci, including multi-drug resistant representatives. Further, we optimized the resazurin-based assay for determining the minimal biofilm inhibitory concentration (MBIC) of standard antibiotics, and evaluated its use for the determination of minimal biofilm eradication concentration (MBEC).ResultsWe showed that tryptic soy broth supplemented with 1% glucose was an optimal media for maximum biofilm growth of all strains tested, with an extended incubation time for Enterococci. A range of parameters were tested for the resazurin assay, including concentration, temperature and time of incubation. Using quality parameters to analyze the assay’s performance, the conditions for the resazurin assay were set as follows: 4 μg/mL and 8 μg/mL, with incubation at 25 °C for 20 min and 40 min for Staphylococci and Enterococci, respectively.ConclusionsIn summary, we defined conditions for optimal biofilm growth and for standardized resazurin assay for MBIC determination against six Gram-positive clinical reference strains. We also observed that MBEC determination by the resazurin-based assay is limited due to the poor detection limit of the assay. Complementary cell counting data is needed for precise determination of MBEC.
Highlights
Biofilms are formed by a complex bacterial community encapsulated by a polymeric matrix, with strong adherent properties and persistent phenotype
The broths used were Mueller hinton broth (MHB), Tryptic Soy (TS, BD), Tryptic Soy supplemented with 1% glucose (TSG, ICN Biomedicals, Irvine, CA, USA), or 2% glucose (TS2G), Brain Heart Infusion (BHI, Sigma-Aldrich, St Louis, MO, USA) and Brain Heart Infusion supplemented with 1% glucose (BHIG)
It was observed that supplementing Tryptic soy (TS) broth with glucose increased the biofilm production of S. aureus and E. faecium ATCC 35667 strains (Fig. 1a-c), a significant difference was only observed for the methicillin-resistant S. aureus (MRSA) strain (P < 0.05) (Fig. 1b)
Summary
Biofilms are formed by a complex bacterial community encapsulated by a polymeric matrix, with strong adherent properties and persistent phenotype. Existing antibiotics have been developed against free-floating bacterial cells, and many treatments of biofilm-related infection fail. Biofilms play a role in non-device-associated chronic bacterial infections [1, 6]. Treatment of these infections is not always successful due to bacterial tolerance to conventional antimicrobial agents, frequently leading to the surgical removal of the implanted device, involving risks and complications. In this context, biofilm-related infections caused by Gram-positive cocci are well established. Staphylococci is one of the leading causative bacteria of catheters and prosthetic related-infections [4], followed by Enterococci [6, 7]
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