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

Abstract

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 (C max ) 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% C max , with ciprofloxacin, tetracycline, and trimethoprim causing 97% ؎ 0.0% killing versus 86% ؎ 2.0% for ciprofloxacin alone. Colonic mucosaassociated 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.

Crohn’s disease as an immunodeficiency

“…A few CD-associated microbial species have been described (notably AIEC and Mycobacterium species) that appear able to either bypass an intact intestinal barrier or evade or suppress macrophage function [24,[67][68][69]. These species might therefore be able to initiate a chronic inflammatory response even in 'innate immunocompetent' hosts.…”

“…Although it is impossible to establish causality with this approach, focused metagenomic studies represent a powerful tool for further study of this aspect of the intestinal environment in health and disease and should be applied across populations. Bifidobacterium species and Faecalibacterium prausnitzii appear to have a clinically relevant association with CD, but it remains to be determined whether 'restoration' or alteration of the balance of microbial species in the bowel is of therapeutic benefit [65,66] (although again, this would not necessarily demonstrate causality).A few CD-associated microbial species have been described (notably AIEC and Mycobacterium species) that appear able to either bypass an intact intestinal barrier or evade or suppress macrophage function [24,[67][68][69]. These species might therefore be able to initiate a chronic inflammatory response even in 'innate immunocompetent' hosts.…”

Activity of Species-specific Antibiotics Against Crohnʼs Disease–Associated Adherent-invasive Escherichia coli

“…Antimicrobial treatments were colicin E1, colicin E9, and colicin E9-H575A, a variant protein that lacks antibacterial activity because of a mutation in the active site of the cytotoxic DNase domain, or ciprofloxacin, which has previously been shown to be highly active against intramacrophagic AIEC. 45 For antibiotic-free controls, the number of bacteria present in RAW264.7 macrophages at 4 hours and 24 hours was approximately 1.6 · 10 4 and 800 CFUs per well, respectively (see Fig., Supplemental Digital Content 2, http://links.lww.com/IBD/A972). Both colicin E9-and colicin E1-treated macrophages showed reduced intracellular survival of LF82 within macrophages at both 4 and 24 hours, but to a lesser extent than ciprofloxacin (Fig.…”

The Role of Bacteria in the Pathogenesis of Inflammatory Bowel Disease

“…Studies in our laboratory concluded that azithromycin, ciprofloxacin, rifampicin, sulfamethoxazole, tetracycline and trimethoprim were all effective against E. coli within macrophages. 56 Use of single antibiotics is likely to be unsuccessful in Crohn's disease because of the development of bacterial resistance. Thus a controlled trial showed that clarithromycin as a single agent was apparently effective for one month but then lost efficacy by the principal endpoint at 3 months.…”

“…16,56 They can be seen within double membrane vesicles, implying that they are able to survive within autophagosomes. Further research is needed to understand how they evade killing by the macrophages.…”