Use of artificial sputum medium to test antibiotic efficacy against pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung

Kirchner, S; Fothergill, J; Wright, E; James, C; Mowat, E; Winstanley, C

Abstract

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic
fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used
to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa
populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between
bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free
DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population
divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on
growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with
antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This
assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under
aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some
evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found
with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a >128 fold, towards tobramycin in the ASM
system when compared to assays carried out in aerobic conditions.
The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3.
Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms,
whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown
to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a
more realistic environment in which to study antimicrobial susceptibility.

Citation

Kirchner, S., Fothergill, J., Wright, E., James, C., Mowat, E., & Winstanley, C. (2012). Use of artificial sputum medium to test antibiotic efficacy against pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung. Journal of Visualized Experiments, 64, 1-8. https://doi.org/10.3791/3857

• Journal Article Type: Article
• Publication Date: Jun 1, 2012
• Deposit Date: Mar 21, 2013
• Journal: Journal of Visualised Experiments
• Publisher: Journal of Visualized Experiments
• Peer Reviewed: Peer Reviewed
• Volume: 64
• Pages: 1-8
• DOI: https://doi.org/10.3791/3857
• Publisher URL: http://dx.doi.org/10.3791/3857
• Related Public URLs: http://www.jove.com/
• Additional Information: Funders : The NIHR School for Social Care Research (SSCR);The Dr Hadwen Trust;The Wellcome Trust;Novartis Pharmaceuticals