Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against <em>Pseudomonas aeruginosa</em> in Conditions More Relevant to the Cystic Fibrosis Lung

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 antibiotic 1 . However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic 2 . 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 tests 3 .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 divergence 4,5,6 . 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 methods 3 . Several in vitro models have been used previously to study P. aeruginosa biofilms 7,8 . However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung 9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa 2 and affect antibiotic susceptibility 10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility. Video LinkThe video component of this article can be found at https://www.jove.com/video/3857/ Protocol Preparation of Artificial Sputum Medium (ASM)1. Add 4 g DNA from fish sperm to 250 ml sterile water very slowly over a period of several hours. The DNA tak...

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