25 0.25 2 2 0.5 0.5 Tigecycline 1 1 0.25 0.25 1 1 0.25

0.25 Meropenem 128 128 128 128 64 64 64 64 Imipenem MK-1775 mouse 32 32 32 32 64 64 64 64 Piperacillin 512 512 512 512 256 256 256 256 Oxacillin > 1024 >1024 > 1024 >1024 1024 1024 1024 1024 Ceftazidime 256 128 256 256 256 128 512 512 Erythromycin 512 512 512 512 512 512 512 512 Clindamycin 128 128 16 16 128 128 16 16 Trimethoprim 128 128 16 16 128 128 16 16 Gentamicin >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 Kanamycin >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 MIC (mg/L). Changes in MIC that are ≥ 4-fold are highlighted in bold. Although adeL and the adeFGH operon were expressed in DB and R2, albeit at a lower level that adeB and adeJ, inactivation of adeFGH in both

DB and R2 had minimal impact on the MDR phenotype of DB and R2 (Table  1). This is shown by the minimal change in antimicrobial susceptibility between the mutants that had only adeFGH inactivated (DBΔadeFGH and R2ΔadeFGH) and both adeFGH and adeIJK operons inactivated (DBΔadeFGHΔadeIJK and R2ΔadeFGHΔadeIJK) Selleck SN-38 (Table  1). The DBΔadeFGHΔadeIJK and R2ΔadeFGHΔadeIJK mutants had the same antimicrobial susceptibility as DBΔadeIJK and R2ΔadeIJK mutants, respectively (Table  1). Growth of pump deletion mutants The optical density at 600 nm measurements of liquid cultures of the parental strains and pump deletion mutants revealed no significant difference in growth kinetics (data not shown). Growth Mannose-binding protein-associated serine protease kinetics in the presence of sub-MIC concentrations of EIs were also carried out to simulate conditions in the H33342 accumulation assay (see below) and to ensure no inhibition of growth over a two-hour time period during the assay. These experiments Selleck Tideglusib showed that 30 mg/L CCCP and 50 mg/L PAβN did not restrict growth of R2 (data not shown). Viability of all strains was unaffected by H33342 concentrations of 2.5 μM, 5 μM and 10 μM

(data not shown). Accumulation of H33342 by efflux pump gene deletion mutants Compared with the parental isolate, R2, there was a significant 0.8 fold change in the level of H33342 accumulated at steady state in R2ΔadeFGH (Figure  5A). Compared with the parental isolate, accumulation of H33342 was significantly increased in R2ΔadeIJK and R2ΔadeFGHΔadeIJK, with a fold change of 1.18 and 1.16 respectively. The mutants created in isolate DB showed a different pattern of accumulation (Figure  5B). The level of H33342 accumulated at steady state was significantly higher in all three mutants, DBΔadeFGH, DBΔadeIJK and DBΔadeFGHΔadeIJK, compared with the parental strain, with fold-changes of 1.13, 1.26 and 1.22, respectively. Figure 5 Fold-change in fluorescence of H33342 at steady state levels of accumulation in efflux pump gene deletion mutants compared with the parental isolate. Three separate experiments showed consistent results and the average fold change is shown.