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Intestinal bacteria have been implicated in the initiation and amplification of inflammatory bowel disease (IBD). The dysbiosis theory, reviewed by Tamboli et al (Gut 2004;53:1), is that an imbalance between putative “harmful” versus “protective” bacterial species may promote chronic intestinal inflammation. Although several studies published so far support this hypothesis, the most vexing question posed by Tamboli et al remains “what is the origin of dysbiosis?”. Bacteriophages outnumber bacteria by a factor of 10 in many natural ecosystems, exert a strong influence on bacterial diversity and population structure, and are probably involved in dysbiosis by destabilising bacterial communities.1 They could be involved indirectly through gene transfer and genome reorganisation within the bacterial population or directly as immunomodulating agents2 or by steric competition for microbe-associated molecular patterns on bacterial surfaces. However, bacteriophages are a neglected component of the gut microbiota. The first viral metagenomic study demonstrated a wide diversity (1200 genotypes) of uncultured bacteriophage species.3 The present study aimed at measuring the total viral community associated with the gut mucosa and comparing viral abundance between healthy individuals and patients with Crohn’s disease (CD), and also between the ulcerated and non-ulcerated mucosa of these patients.
Fourteen healthy individuals and 19 CD patients were recruited and gave their informed consent. Biopsies were obtained during colonoscopy and, for CD patients, from non-ulcerated and ulcerated tissues. Biopsies were disrupted by ultrasonication, filtered through 0.22 μm membranes and fixed in glutaraldehyde. For epifluorescence microscopy, biopsy supernatants were filtered through 0.02 μm membranes. Virus-like particles (VLPs) were stained with SYBR Green and counted on triplicate subsamples. VLPs were detected in every sample, and no contaminating bacteria were observed (fig 1A). Strikingly high numbers of VLPs were observed (fig 1B), with an average of 1.2×109 VLPs/biopsy (4.4×107–1.7×1010). Transmission electron microscopy demonstrated that viral particles corresponded to bacteriophages, with morphotypes consistent with Siphoviridae, Myoviridae and Podoviridae being the dominant families (fig 2). Each individual seemed to be colonised by one dominant phage family. This result was confirmed by pulse-field gel electrophoresis of viral genomic DNA (data not shown). At the mucosal level and with specific reference to the potential role of bacteriophages in dysbiosis, CD patients harboured significantly more VLPs than healthy individuals (2.9×109 vs 1.2×108 VLPs/biopsy; Wilcoxon test p = 0.024). Moreover, decreased amounts of VLPs were detected in CD ulcerated mucosa, with an average of 2.1×109 VLPs/biopsy compared with 4.1×109 VLPs/biopsy from non-ulcerated mucosa (fig 1). It can be hypothesised either that more viruses are produced or that they can survive longer in non-ulcerated areas. Whether bacterial composition and abundance differ when comparing non-ulcerated with ulcerated mucosa is still controversial although differing bacteriophage life cycles (lysis, lysogeny) could be responsible for some of these discrepancies.
This study shows for the first time a dense bacteriophage community specifically associated with the gut mucosa, reaching 1010/mm3 of tissue. Significantly more bacteriophages were detected in the mucosa from CD patients than from healthy individuals. Tamboli et al discussed risk factors known to affect the gut microbial composition (host genetic background, method of birth delivery, early bacterial colonisation of neonates, diet and environment) which could be responsible for dysbiosis. Based on our results, we postulate that bacteriophages might also play a key role in the dysregulated immune response of IBD patients to the mucosal-associated bacterial population. More detailed studies that relate phage populations to disturbances in bacterial populations and the dysregulated host immune response on larger cohorts would help in establishing their role in the pathogenesis of IBD.
Competing interests: None.
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