Introduction Mucosa-associated adherent, invasive E.coli (AIEC), found in increased number in Crohn disease (CD) ileal and colonic mucosa, can replicate within host mucosal macrophages^.1,2 Other pathogens, known to survive within macrophages (e.g. Mycobacteria and Salmonella), possess stress-response inducible systems that allow them to tolerate environmental stress. Here we aimed to establish whether CD AIEC are better able than non-AIEC E.coli to tolerate low pH, high nitrosative and oxidative stress conditions, that mimic the environment within the macrophage phagolysosome.

Methods CD AIEC isolates possessing ability to replicate intracellularly in J774-A1 macrophages (ileal isolate LF82 and 3 colonic isolates HM427, HM605 and HM615; range 4.5 to 9-fold replication) were compared to E.coli strains killed by macrophages (XL1 and EPI300). Bacterial cultures were grown at 37^oC in Luria-Bertani (LB) medium to OD_600nm 0.1 and diluted in 10-fold steps in 0.9%w/v saline. Samples were spotted onto LB agar containing one of the following stress agents: 100mM 4-Morpholine ethanesulfonic acid (MES) pH5, 100mM MES pH5 + 1mM NaNO₂ (high nitrosative stress), 1mM H₂O₂ pH7 (high oxidative stress) and 1mM methyl viologen (MV) pH7 (super oxidative stress). Plates were incubated overnight at 37^oC.

Results All four CD AIEC showed tolerance to low pH, high nitrosative and oxidative stress mimicking the intra-phagolysosome environment. CD AIEC demonstrated greater tolerance to growth at pH5. Compared to growth seen on LB agar pH7 (100%), LF82 showed 99.3 ± 22.2% growth at pH5 [mean±SD]. Likewise, growth of HM427 (104.4 ± 15.2%), HM605 (81.1 ± 15.5%) and HM615 (94.2 ± 9.4%) was also seen at pH5. Conversely,% growth seen for laboratory strains was only 64.2 ± 9.9% for XL-1 and 61.3 ± 8.7% for EPI300; N=4 expts, n = 3 replicates (P < 0.01; ANOVA). Most remarkable was tolerance on super oxidative LB agar containing 1mM MV, with LF82 showing growth at 95.9 ± 11.5%, HM427 81.3 ± 17.9%, HM615 102.4 ± 14.3% and HM605 86.1 ± 13.5% vs. that seen on standard LB agar (100%). Non-AIEC strains showed little tolerance to all stress conditions tested (in 1mM MV; XL-1, no growth; EPI300, 1.8 ± 1.2% growth vs. LB agar alone, 100%); (P < 0.0001). The four CD AIEC were also observed to grow better after 8h in a low nutrient M9 medium supplemented 0.1% casamino acids (pH4) than the laboratory E.coli tested and 3 additional E.coli isolates from healthy controls (ECOR1, ECOR35, ECOR51); N = 1, n = 3.

Conclusion Crohn’s AIEC, unlike non-AIEC E. coli, tolerate low nutrient, low pH and high chemical stress conditions that mimic the macrophage phagolysosome environment.

References

1. Bringer MA, et al. Cell. Microbiol 2006;8:471–84.

2. Subramanian S, et al. Antimicrob. Agents Chemother 2008;52:427–34.

Disclosure of Interest A. Tawfik: None Declared, J. Rhodes Grant/research support from: Provexis plc., Consultant for: advisory boards for Atlantic, Procter and Gamble and Falk, Speaker bureau with: honoraria from Abbott, Falk, Ferring, Glaxo Smith Kline, Procter and Gamble and Schering Plough, Conflict with: the University of Liverpool and Provexis plc, holds a patent for use of a soluble fibre preparation as maintenance therapy for Crohn’s disease, B. Campbell Grant/research support from: Provexis plc, The Bo and Vera Axe:son Johnson Foundation for Nature Medicine Ltd and Arcis Biotechnology, Speaker bureau with: honorarium from Amgen Inc.