Haplotype properties differ between different antibiotic exposures Diversification of P. aeruginosa LESB58 in ASM cultured with and without the various antibiotics was observed only with respect to colony morphology, pyocyanin production and antibiotic susceptibilities (Table 1). The culture of LESB58 in ASM with sub-inhibitory concentrations of ceftazidime

and colistin led to diversity in antimicrobial susceptibilities, changes in colony morphology and a loss of pyocyanin production (Table 1). LESB58 cultured in the presence of these antibiotics, generated more isolates that were outside the normal range of the antibiotic sensitivity profiles of LESB58 controls (Figure 3). In addition, exposure to azithromycin and tobramycin promoted increased cross-resistance CRT0066101 purchase to other antibiotics (Table 1, Figure 3). There

was no variation in the auxotrophic phenotype in the isolates analysed in all experimental and control groups (LESB58 has an auxotrophic phenotype). The H 89 populations exposed to meropenem exhibited no clear phenotypic diversification (Table 1 and Figure 2). Figure 3 Variations in zones of inhibition within LESB58 populations. The 120 LESB58 isolates obtained from the triplicate ASM cultures were assessed for susceptibility to six commonly used antibiotics (ceftazidime, ciprofloxacin, BV-6 colistin, meropenem, tazobactam/piperacillin and tobramycin). Boxplots showing the range in the diameter of the zones of inhibition to these antibiotics are presented. 1. LB (18 hours) 2. ASM 3. ASM with ceftazidime 4. ASM with colistin 5. ASM with meropenem 6. ASM with tobramycin 7. ASM with azithromycin 8. Normal range of LESB58 (Groups 1–8: n = 120). The red line represents the cut-off for Histone demethylase the sensitivity of P. aeruginosa to the antibiotics tested, in accordance with the guidelines of Andrews

and Howe [37]. The values above the red line denote a higher sensitivity to antibiotics and the values below the line denote a higher resistance. Table 1 Number of isolates in each group (total of 120) exhibiting each of the traits measured   Colony morphology Virulence Mutations Outside normal range of antimicrobials susceptibility Culture Green non-mucoid Straw non-mucoid Pyocyanin Hypermutability Ceftazidime Ciprofloxacin Tobramycin Meropenem Colistin Tazobactam/piperacillin ASM 120 0 117 0 3 0 19 0 2 8 ASM + CAZ 110 10 92 0 16 19 20 18 10 11 ASM + CT 113 7 84 0 17 37 29 15 7 9 ASM + AZT 120 0 120 0 0 16 34 0 4 4 ASM + MEM 120 0 118 0 1 8 4 0 0 1 ASM + TOBI 118 2 119 0 1 24 69 3 22 1 LB (18 hours) 120 0 120 1 0 0 0 0 0 0 Isolates that were characterized as being outside the normal range of antimicrobial susceptibility typically observed in LESB58, included isolates that had either an increased or reduced susceptibility to the antibiotic under test. ASM = Artificial Sputum Medium, LB = Luria Bertani, CAZ = Ceftazidime, CT = Colistin, AZT = Azithromycin, MEM = Meropenem and TOBI = Tobramycin.