In fact, MccJ25 was able to inhibit both intracellular targets in the resistant Salmonella strains carrying E. coli fhuA[9]. Based on these results it was postulated that the

outer membrane is the principal barrier that MccJ25 has to overcome to reach its targets. Recently, we demonstrated that the membrane permeabilizing peptide, (KFF)3K, allows the MccJ25 uptake independently of FhuA and SbmA receptors thus turning microcin naturally resistant strains into susceptible ones [10]. Moreover, the same effect of (KFF)3K on S. Typhimurium susceptibility to MccJ25 was observed in bacteria replicating buy Fer-1 within eukaryotic cells. Furthermore, an interesting observation was that MccJ25 itself was able to inhibit 30% of the S. Typhimurium intracellular replication [10]. The goal of the present study was to address the mechanism causing this phenomenon. Our data demonstrate that the low pH affects the bacterial membrane permeability in vitro, indicating that this mechanism could be also responsible for the S. Typhimurium sensitization

once it is phagocytized and transferred to vacuoles. Results and buy PKC412 discussion Effect of macrophages internal environment on S. Typhimurium sensitivity to MccJ25 We previously showed that, ARRY-162 mouse although S. Typhimurium is a MccJ25-resistant strain in vitro, its intracellular replication was moderately inhibited by the antibiotic (about 30%, 6 h after bacteria internalization) [10]. In the present work we observed that the number of surviving S. Typhimurium cells within macrophages

decreased 60% after 8 h of MccJ25 exposition compared with the control (without MccJ25). This effect was strongly increased with time, reaching between an 80-90% of intracellular replication inhibition after 18 h of MccJ25 treatment (Figure 1). A potential ioxilan explanation for this effect is an unspecific MccJ25 uptake produced when the pathogen grows within macrophages. In order to prove this hypothesis we determined, in vitro, the MccJ25 sensitivity of S. Typhimurium cells grown for 8 h within macrophages in the absence of the antibiotic (see Methods). We observed a 58% viability decrease when bacteria directly harvested from macrophages (fraction from lysed macrophages) were incubated with MccJ25 for 6 h, compared with the control (bacteria incubated without antibiotic) (Figure 2, macrophage). Additionally, we determined the MccJ25 sensitivity of bacteria grown on LB medium and resuspended in Triton X-100 (solution used to harvest intracellular bacteria) and no MccJ25 effect on bacterial viability was observed (Figure 2, LB medium). Figure 1 Effect of MccJ25 on S. Typhimurium intracellular replication. RAW 264.7 macrophages were seeded in 24-well plates and grown for 24 h before bacterial infection with an overnight culture of S. Typhimurium 14028s strain. The infected macrophages were treated with MccJ25 (117.