4-fold higher than that of PAO1 (P = 0.0071). The mutation frequencies of both the 18A and PAO1 Venetoclax cell line biofilm communities were also quantified during biofilm development and dispersal (12 days). The number of morphotypic variants was enumerated to compare the mutation frequency with the frequency of morphotypic variants. The initial mutation frequency for 18A biofilm on day 0 was 3.17 × 10−8 ± 4.87 × 10−8 (Fig. 5a), which was also similar to the mutation frequency of the planktonic culture (3.10 × 10−8 ± 7.53 × 10−9). The mutation frequency decreased during the initial stages of biofilm development to 6.87 × 10−9 ± 7.4 × 10−9 by day 4. On day 8, the mutation frequency increased to 2.65 × 10−8 ± 3.68 × 10−8,

and by day 10, it was 6.11 × 10−8 ± 1.14 × 10−7, similar to MLN0128 concentration the mutation frequency observed at the start of biofilm development and the original planktonic culture. In contrast to PAO1, morphotypic variants appeared in the biofilm of 18A on day 4 and accounted for approximately 49% of the population. On day 10, when the mutation frequency was the highest for strain 18A, approximately 80% of the population consisted of morphotypic variants. Interestingly, by day 12, variants accounted for only 20% of the population at which time the mutation frequency also declined (4.11 × 10−8 ± 3.68 × 10−8). The mutation frequency for the PAO1 biofilm on day 0 was 1.26 × 10−8 ± 9.44 × 10−9 (Fig. 5a), which was similar to the mutation

frequency of the planktonic culture. During the course of biofilm development, it was observed that the mutation frequency decreased from day 0 to day 6 (2.71 × 10−9 ± 1.20 × 10−9

on day 6) and then increased to 5.76 × 10−9 ± 3.21 × 10−9 on day 8 and did not change significantly for the remaining 4 days of the experiment. Morphotypic variants were observed in the biofilms on day 8 and constituted approximately 2% of the total PAO1 biofilm population. The peak number of variants, 12%, was observed on day 10. It was observed that the biofilm of 18A developed more slowly than that of PAO1 (Fig. 5b), Farnesyltransferase which is in accordance with our observation that 18A has a lower growth rate than PAO1 (data not shown). Although the change in mutation frequency of the biofilm community was not statistically significant between the sampling days, there appears to be a positive correlation between the mutation frequency and the variant frequency. For strain 18A, both the mutation frequency and the percentage of variants increased from days 6 to 10 and decreased on day 12. In PAO1, the mutation frequency was observed to increase slightly between days 6–12, which coincided with the emergence of morphotypic variants. Pseudomonas aeruginosa has been shown to establish long-term colonisation of the lungs of CF sufferers. This process of chronic infection has been linked to the appearance of morphotypic variants (e.g. SCVs and mucoid colony types) as well as the selection of variants with reduced overt, or acute, virulence.