Abstract
In vivo biofilms cause recalcitrant infections with extensive and unpredictable antibiotic tolerance. Here, we demonstrate increased tolerance of colistin by Pseudomonas aeruginosa when grown in medium that mimics cystic fibrosis (CF) sputum versus standard medium in in vitro biofilm assays, and drastically increased tolerance when grown in an ex vivo CF model versus the in vitro assay. We used colistin conjugated to the fluorescent dye BODIPY to assess the penetration of the antibiotic into ex vivo biofilms and showed that poor penetration partly explains the high doses of drug necessary to kill bacteria in these biofilms. The ability of antibiotics to penetrate the biofilm matrix is key to their clinical success, but hard to measure. Our results demonstrate both the importance of reduced entry into the matrix in in vivo-like biofilm, and the tractability of using a fluorescent tag and benchtop fluorimeter to assess antibiotic entry into biofilms. This method could be a relatively quick, cheap and useful addition to diagnostic and drug development pipelines, allowing the assessment of drug entry into biofilms, in in vivo-like conditions, prior to more detailed tests of biofilm killing.
Highlights
Biofilm infections of host tissues or indwelling medical devices impose a significant health and economic burden, due to the high tolerance of biofilm bacteria to host immune attack and to antibiotics
The inhibitory concentration of colistin varied depending on culture medium (SCFM usually >caMHB) and growth mode (MBEC >minimum inhibitory concentration (MIC) in all cases but one) (Fig. 1)
We selected cystic fibrosis (CF) isolates SED6, SED8, SED17 and SED19 for further work with the ex vivo porcine lung model (EVPL), as these had a range of MIC/minimum biofilm eradication concentration (MBEC) values (MIC 1–4 μg ml−1 in caMHB and 32–64 μg ml−1 in SCFM; MBEC 32–128 μg ml−1 in both media) and included two isolates for which MBEC=MIC in SCFM (SED6, SED8) and two for which MBEC >MIC in SCFM (SED17, SED19)
Summary
Biofilm infections of host tissues or indwelling medical devices impose a significant health and economic burden, due to the high tolerance of biofilm bacteria to host immune attack and to antibiotics. Biofilm antibiotic tolerance is a function of environmentally cued changes in bacterial physiology and gene expression, and reduced penetration of some antibiotic molecules through the biofilm matrix [1]. In the case of cystic fibrosis (CF) lung disease, plugging of small airways by aggregates of biofilm embedded in abnormal host mucus leads to reduced airflow and bronchiectasis [2, 3]. Bacterial lung infection was the strongest predictor of medication costs in CF, adding on average €3.6K per patient per year to direct healthcare costs [4, 5]. There is a narrow choice of antibiotics suitable for administration in CF, and a poor concordance between antibiotic susceptibility testing in diagnostic laboratories and patient outcome – even when standard in vitro biofilm platforms are employed for testing [7, 8]
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