Influence of biofilm growth age, media and antibiotics exposure time on Staphylococcus aureus and Pseudomonas aeruginosa biofilm removal in vitro

Abstract

Biofilm is known to be tolerant towards antibiotics and difficult to eradicate. Numerous studies have reported Minimum Biofilm Eradication Concentration (MBEC) values of antibiotics for many known biofilm pathogens. However, the experimental parameters applied in these studies differ considerably, and often the rationale behind the experimental design are not well described. This makes it difficult to compare the findings. To demonstrate the importance of experimental parameters, we investigated the influence of biofilm growth age, antibiotic treatment duration and growth media on biofilm eradication in this study. The commonly used biofilm model Calgary biofilm device was used to grow 24 h and 72 h biofilms of Staphylococcus aureus and Pseudomonas aeruginosa , which were treated with time-dependent vancomycin and concentration-dependent tobramycin, respectively. Two common bacteriological growth media Tryptic Soy Broth (TSB) and Cation-adjusted Mueller Hinton Broth (CaMHB) were tested. We found for both species that biofilms were more difficult to kill in TSB than in CaMHB. Furthermore, young biofilms (24 h) were easier to eradicate than old biofilms (72 h). In agreement with vancomycin being time-dependent, extension of the vancomycin exposure increased killing of S. aureus biofilms. Tobramycin treatment of 24 h P. aeruginosa biofilms was found concentration-dependent and time-independent, however, increasing killing was indicated for 72 h P. aeruginosa biofilms. This study demonstrated biofilm removal efficacy was influenced by media, biofilm age and antibiotics treatment duration. It is therefore necessary to taking these parameters into consideration when designing experiments.

Dynamics of a Staphylococcus aureus infective endocarditis simulation model

“…With this current work, we wanted to evaluate if biofilm development in our IEV model requires full 24 h, which is the duration of several other studies to observe the mature biofilms [27,28]. We could, however, demonstrate that biofilms could already develop after 6 h under the chosen conditions, which is in line with similar observations made in P. aeruginosa biofilm development [29].…”

“…This correlates with the generally known phenomenon that the minimal biofilm inhibitory concentration (MBIC) and the minimal biofilm eradication concentration (MBEC) are often 2-1000-fold higher compared with the MIC of the bacteria [24][25][26]. With this current work, we wanted to evaluate if biofilm development in our IEV model requires full 24 h, which is the duration of several other studies to observe the mature biofilms [27,28]. We could, however, demonstrate that biofilms could already develop after 6 h under the chosen conditions, which is in line with similar observations made in P. aeruginosa biofilm development [29].…”