Diversification of the P. aeruginosa populations in the CF lung, and the Staurosporine cost emergence of phenotypes such as mucoidy, are signs of adaptation leading to a chronic infection state. Diversification may also lead to enhanced antimicrobial resistance. Antibiotics that do not cause extensive diversification might be utilised
to prevent diversification, and possibly slow down the development of a chronic infection state. Therefore, being able to delay, control or possibly reduce diversification could be advantageous for the CF patient. This could also be achieved by using antibiotics that permeate the lung and the bacterial biofilms better to achieve inhibitory concentrations, but it could also be important to choose selleckchem Ricolinostat antibiotics that do not promote diversification. Hence a better understanding of the differential effects of various antibiotics on diversification of P. aeruginosa populations could provide valuable information to help clinicians choose the best antibiotics for CF patients. Methods ASM preparation and culture conditions The ASM was prepared following the protocol of Sriramulu et al. and Kirchner et al.. ASM contains mucin from porcine stomach (Sigma-Aldrich, Gillingham, UK), DNA (Sigma-Aldrich), the iron-chelator diethylene triamine pentaacetic acid (Sigma-Aldrich), NaCl (Sigma-Aldrich), KCl (Sigma-Aldrich), egg yolk emulsion (Sigma-Aldrich) and all essential
and non-essential amino acids (Fisher Scientific, Loughborough, UK and Sigma-Aldrich) at concentrations found in an average CF patient . A single colony of the genome-sequenced P. aeruginosa CF isolate LESB58  was used to inoculate LB broth and cultured for 18 h at 37°C and 200 rpm. The overnight culture was diluted in fresh LB to an A600nm of 0.05 (± 0.01) and Mannose-binding protein-associated serine protease 300 μl of this diluted LESB58 culture was added to 30 ml ASM. The ASM cultures were incubated at 37°C for 7 days at 50 rpm. Where appropriate, sub-inhibitory concentrations of either ceftazidime (0.125 μg/ml), colistin (1 μg/ml), meropenem (2 μg/ml), tobramycin (2 μg/ml) or azithromycin (0.25 μg/ml)
were added to the ASM. The minimum inhibitory concentrations were of ceftazidime 8 μg/ml, tobramycin 16 μg/ml, ciprofloxacin 168 μg/ml, colistin 8 μg/ml, meropenem 16 μg/ml, and azithromycin 16 μg/ml. Sub-inhibitory concentrations were determined by testing the growth of P. aeruginosa LESB58 exposed to a dilution series of these antibiotics in ASM. The antibiotics were then tested at 8, 16, 32, 64-fold below the minimum inhibitory concentration, and the antibiotic concentration used was the highest that did not affect the growth rate in ASM. Therefore, the sub-inhibitory concentration of each antibiotic was the highest concentration of antibiotic that still allowed culture absorbance readings similar to that of the negative control (LESB58 grown in the absence of antibiotics).