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PWE-034 Evidence against intramacrophage replication of Crohn's disease E coli being dependent on an anaerobic environment within the phagolysosome: lack of efficacy for metronidazole
  1. P P Knight,
  2. B J Campbell,
  3. J M Rhodes
  1. Department of Gastroenterology, Clinical Sciences, University of Liverpool, Liverpool, UK

Abstract

Introduction Escherichia coli isolates from colonic Crohn's disease (CD) tissue have previously been demonstrated to replicate within J774-A1 macrophages, and to be sensitive to antibiotics such as ciprofloxacin, trimethoprim, tetracycline and rifampicin. It has been hypothesised that E coli internalised into macrophage vesicles may utilise anaerobic metabolism and become susceptible to the effect of metronidazole, an anaerobe specific antibiotic. Our aim was to evaluate efficacy of metronidazole used at Cmax (maximal plasma concentration achievable with standard dosing) on replication of CD colonic E coli isolate (HM605) within macrophages in-vitro, both as a single agent and in combination with ciprofloxacin

Methods Murine macrophages (J774-A1) in culture were infected with CD E coli isolate HM605 for 2 h, to allow bacterial internalisation, followed by removal of extracellular bacteria by gentamicin (20 μg/ml) treatment for 1 h. Macrophages were incubated for a further 3 h in either the absence of antibiotics or in the presence of metronidazole (20 μg/ml) orciprofloxacin (4 μg/ml), alone or in combination. Cells were then lysed and internalised bacteria enumerated by overnight growth on LB agar.

Results CD E coli HM605 survived and replicated 6.95-fold following gentamicin treatment. Following ciprofloxacin treatment alone, only 503±455 cfu were present within macrophages compared with 36 355±16 696 cfu in “no antibiotic” controls (p<0.001). Ciprofloxacin plus metronidazole resulted in low intramacrophage growth (240±196 cfu, p<0.001) unlike metronidazole alone (6.990±7.016, p=0.206), but with the combination not producing significantly enhanced killing when compared with ciprofloxacin alone (p=0.85).

Conclusion Metronidazole confers no additional effect to ciprofloxacin in intramacrophage killing of CD E coli HM605 in vitro.

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