PTU-062 Crohn’s Disease-associated Escherichia Coli Can Tolerate Low Nutrient, Low Ph And High Chemical Stress Environments Encountered Within The Phagolysosome Of Mucosal Macrophages
Authors: A Tawfik, JM Rhodes, BJ Campbell
Journal: Gut (2014)

<h3>Introduction</h3> Mucosa-associated adherent, invasive <i>E.coli</i> (AIEC), found in increased number in Crohn disease (CD) ileal and colonic mucosa, can replicate within host mucosal macrophages<sup>.1,2</sup> Other pathogens, known to survive within macrophages (e.g. Mycobacteria and <i>Salmonella</i>), 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 <i>E.coli</i> to tolerate low pH, high nitrosative and oxidative stress conditions, that mimic the environment within the macrophage phagolysosome. <h3>Methods</h3> 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 <i>E.coli</i> strains killed by macrophages (XL1 and EPI300). Bacterial cultures were grown at 37<sup>o</sup>C in Luria-Bertani (LB) medium to OD<sub>600nm</sub> 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 NaNO<sub>2</sub> (high nitrosative stress), 1mM H<sub>2</sub>O<sub>2</sub> pH7 (high oxidative stress) and 1mM methyl viologen (MV) pH7 (super oxidative stress). Plates were incubated overnight at 37<sup>o</sup>C. <h3>Results</h3> 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 &lt; 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 &lt; 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 <i>E.coli</i> tested and 3 additional<i> E.coli</i> isolates from healthy controls (ECOR1, ECOR35, ECOR51); N = 1, n = 3. <h3>Conclusion</h3> Crohn’s AIEC, unlike non-AIEC <i>E. coli</i>, tolerate low nutrient, low pH and high chemical stress conditions that mimic the macrophage phagolysosome environment. <h3>References</h3> Bringer MA, <i>et al</i>. <i>Cell. Microbiol </i>2006;8:471–84. Subramanian S, <i>et al</i>. <i>Antimicrob. Agents Chemother </i>2008;52:427–34. <h3>Disclosure of Interest</h3> 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.