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Antimicrobial Agents and Chemotherapy, February 2008, p. 427-434, Vol. 52, No. 2
0066-4804/08/$08.00+0     doi:10.1128/AAC.00375-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Replication of Colonic Crohn's Disease Mucosal Escherichia coli Isolates within Macrophages and Their Susceptibility to Antibiotics{triangledown}

Sreedhar Subramanian,1 Carol L. Roberts,1 C. Anthony Hart,2,{dagger} Helen M. Martin,1 Steve W. Edwards,3 Jonathan M. Rhodes,1 and Barry J. Campbell1*

Division of Gastroenterology, School of Clinical Science, University of Liverpool, Crown Street, Liverpool L69 3BX,1 School of Infection and Host Defence, University of Liverpool, Daulby Street, Liverpool L69 3GA,2 School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom3

Received 20 March 2007/ Returned for modification 22 May 2007/ Accepted 18 November 2007

There is increasing evidence that Escherichia coli organisms are important in Crohn's disease (CD) pathogenesis. In CD tissue they are found within macrophages, and the adherent-invasive CD ileal E. coli isolate LF82 can replicate inside macrophage phagolysosomes. This study investigates replication and antibiotic susceptibility of CD colonic E. coli isolates inside macrophages. Replication of CD colonic E. coli within J774-A1 murine macrophages and human monocyte-derived macrophages (HMDM) was assessed by culture and lysis after gentamicin killing of noninternalized bacteria and verified by electron microscopy (EM). All seven CD colonic isolates tested replicated within J774-A1 macrophages by 3 h (6.36-fold ± 0.7-fold increase; n = 7 isolates) to a similar extent to CD ileal E. coli LF82 (6.8-fold ± 0.8-fold) but significantly more than control patient isolates (5.2-fold ± 0.25-fold; n = 6; P = 0.006) and E. coli K-12 (1.0-fold ± 0.1-fold; P < 0.0001). Replication of CD E. coli HM605 within HMDM (3.9-fold ± 0.7-fold) exceeded that for K-12 (1.4-fold ± 0.2-fold; P = 0.03). EM showed replicating E. coli within macrophage vacuoles. Killing of HM605 within J774-A1 macrophages following a 3-h incubation with antibiotics at published peak serum concentrations (Cmax) was as follows: for ciprofloxacin, 99.5% ± 0.2%; rifampin, 85.1% ± 6.6%; tetracycline, 62.8% ± 6.1%; clarithromycin, 62.1% ± 5.6% (all P < 0.0001); sulfamethoxazole, 61.3% ± 7.0% (P = 0.0007); trimethoprim, 56.3% ± 3.4% (P < 0.0001); and azithromycin, 41.0% ± 10.5% (P = 0.03). Ampicillin was not effective against intracellular E. coli. Triple antibiotic combinations were assessed at 10% Cmax, with ciprofloxacin, tetracycline, and trimethoprim causing 97% ± 0.0% killing versus 86% ± 2.0% for ciprofloxacin alone. Colonic mucosa-associated E. coli, particularly CD isolates, replicate within macrophages. Clinical trials are indicated to assess the efficacy of a combination antibiotic therapy targeting intramacrophage E. coli.

* Corresponding author. Mailing address: Division of Gastroenterology, School of Clinical Science, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom. Phone: 44 151 7946829. Fax: 44 151 7946825. E-mail: bjcampbl{at}liv.ac.uk

{triangledown} Published ahead of print on 10 December 2007.

{dagger} Sadly, Tony Hart passed away after the original submission. He was an inspirational colleague who is greatly missed.

Antimicrobial Agents and Chemotherapy, February 2008, p. 427-434, Vol. 52, No. 2
0066-4804/08/$08.00+0     doi:10.1128/AAC.00375-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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