William Chamberlin

Crohn’s disease and the mycobacterioses: A quarter century later. Causation or simple association?

It has been more than 25 years since Mycobacterium paratuberculosis was first proposed as an etiologic agent in Crohn’s disease based on the isolation of this organism from several patients. Since that time, a great deal of information has been accumulated that clearly establishes an association between M. paratuberculosis and Crohn’s disease. However, data are conflicting and difficult to interpret and the field has become divided into committed advocates and confirmed skeptics. This review is an attempt to provide a thorough and objective summary of current knowledge from both basic and clinical research from the views and interpretations of both the antagonists and proponents. The reader is left to draw his or her own conclusions related to the validity of the issues and claims made by the opposing views and data interpretations. Whether M. paratuberculosis is a causative agent in some cases or simply represents an incidental association remains a controversial topic, but current evidence suggests that the notion should not be so readily dismissed. Remaining questions that need to be addressed in defining the role of M. paratuberculosis in Crohn’s disease and future implications are discussed.

Microbial Population Differentials between Mucosal and Submucosal Intestinal Tissues in Advanced Crohn's Disease of the Ileum.

Since Crohns disease is a transmural disease, we hypothesized that examination of deep submucosal tissues directly involved in the inflammatory disease process may provide unique insights into bacterial populations transgressing intestinal barriers and bacterial populations more representative of the causes and agents of the disease. We performed deep 16s microbiota sequencing on isolated ilea mucosal and submucosal tissues on 20 patients with Crohns disease and 15 non-inflammatory bowel disease controls with a depth of coverage averaging 81,500 sequences in each of the 70 DNA samples yielding an overall resolution down to 0.0001% of the bacterial population. Of the 4,802,328 total sequences generated, 98.9% or 4,749,183 sequences aligned with the Kingdom Bacteria that clustered into 8545 unique sequences with <3% divergence or operational taxonomic units enabling the identification of 401 genera and 698 tentative bacterial species. There were significant differences in all taxonomic levels between the submucosal microbiota in Crohns disease compared to controls, including organisms of the Order Desulfovibrionales that were present within the submucosal tissues of most Crohns disease patients but absent in the control group. A variety of organisms of the Phylum Firmicutes were increased in the subjacent submucosa as compared to the parallel mucosal tissue including Ruminococcus spp., Oscillospira spp., Pseudobutyrivibrio spp., and Tumebacillus spp. In addition, Propionibacterium spp. and Cloacibacterium spp. were increased as well as large increases in Proteobacteria including Parasutterella spp. and Methylobacterium spp. This is the first study to examine the microbial populations within submucosal tissues of patients with Crohns disease and to compare microbial communities found deep within the submucosal tissues with those present on mucosal surfaces. Our data demonstrate the existence of a distinct submucosal microbiome and ecosystem that is not well reflected in the mucosa and/or downstream fecal material.

Primary treatment of Crohn’s disease: combined antibiotics taking center stage

Although controversial, the use of properly chosen antibiotics in Crohn’s disease appears beneficial. Evidence supporting the use of targeted antibiotic therapy comes in two forms: statistical evidence derived from meta-analyses of multiple formal studies and the documented clinical and endoscopic responses in patients treated with antibiotic combinations outside of formal clinical studies. This article reviews evidence from both categories that support the use of properly chosen antibiotic regimens in treating Crohn’s disease, comments on the advantages and disadvantages of antibiotic therapy, and attempts to present a unifying hypothesis related to the role of enteric bacteria, mucosal immunity and antibiotic therapy. Relevant studies identified through a Medline search from 1976 to 2011 were assessed for inclusion by two independent observers who resolved any disagreements by consensus. References from all identified articles and recent review articles were cross-checked to ensure a thorough search. Papers were selected based on scientific merit as to which presented original contributions to the results.

Mycobacterium avium ss paratuberculosis-associated Diseases Piecing the Crohn's Puzzle Together

The relation of Mycobacterium avium ss paratuberculosis (MAP) to Crohn’s Disease (CD) and other MAP-associated conditions remains controversial. New data, coupled with the analogous Helicobacter pylori (H. pylori) story, has permitted us to piece together the MAP puzzle and move forward with a more scientific way of treating inflammatory bowel disease, particularly CD. As infection moves centre stage in inflammatory bowel disease, the dated “aberrant reaction” etiology has lost scientific credibility. Now, our growing understanding of MAP-associated diseases demands review and articulation. We focus here on (1) the concept of MAP-associated diseases; (2) causality, Johne Disease, the “aberrant reaction” hypothesis; and (3) responses to published misconceptions questioning MAP as a pathogen in CD.

Resolution of Crohn's disease and complex regional pain syndrome following treatment of paratuberculosis

A cohort of family members with various chronic diseases including Crohns disease, asthma, complex regional pain syndrome, hypothyroidism, type 1 diabetes mellitus, and lymphangiomatosis and/or evidence of infection by Mycobacterium avium subsp. paratuberculosis (MAP) are described in this series of case reports. MAP was cultured from the blood of three members affected by the first five diseases and there was accompanying elevated anti-MAP IgG in two members. The patient affected by the sixth disease has a markedly elevated anti-MAP titer. The two patients affected by the first four diseases have been treated with a combination of anti-MAP antibiotics and ultraviolet blood irradiation therapy with resolution of the disease symptomatology and inability to culture MAP in post treatment blood samples. These case reports of patients with MAP infections provide supportive evidence of a pathogenic role of MAP in humans.

Crohn's Disease May Be Differentiated Into 2 Distinct Biotypes Based on the Detection of Bacterial Genomic Sequences and Virulence Genes Within Submucosal Tissues

Objective: To determine whether bacterial pathogens can be detected within the diseased submucosal tissues of patients with Crohn’s disease by molecular techniques independent of cultural methods. Design: We designed a quantitative polymerase chain reaction to detect 32 virulence genes and transposons within submucosal tissues of patients with Crohn’s disease and controls and compared the microbiome of the submucosa with mucosal bacterial populations. Results: Within submucosal tissues, the bacterial invasion/adherence genes eaeA and invA were detected in 43% of patients (P=0.01 and 0.008 vs. mucosa and controls, respectively) and the Mycobacterium-specific IS900 and 251F genes detected in 50% of patients (P=0.03 vs. mucosa and controls). These findings were mutually exclusive: invasion/adhesion genes and Mycobacterium-associated transposons were not detected in the same patient. Metagenomic sequencing and quantitative polymerase chain reaction results confirmed effective separation of the submucosal and mucosal microbiome and the existence of a submucosal bacterial population within diseased tissues. Conclusions: This study is the first to examine the microbial populations of submucosal tissues during intestinal disease and provide evidence of a distinct submucosal microbiome and biotypes within Crohn’s disease. These data suggests that Crohn’s disease may not be a single disease, but a spectrum that can be divided into distinct biotypes based on the presence of invasion/adherence genes or Mycobacterium-associated transposons. If corroborated by larger population studies, these findings could revolutionize the diagnosis, management, and treatment of Crohn’s disease by the identification of patient biotypes and the application of targeted chemotherapeutic treatments that go beyond supportive in nature.

The Consensus from the Mycobacterium avium ssp. paratuberculosis (MAP) Conference 2017

On March 24 and 25, 2017 researchers and clinicians from around the world met at Temple University in Philadelphia to discuss the current knowledge of Mycobacterium avium ssp. paratuberculosis (MAP) and its relationship to human disease. The conference was held because of shared concern that MAP is a zoonotic bacterium that poses a threat not only to animal health but also human health. In order to further study this problem, the conferees discussed ways to improve MAP diagnostic tests and discussed potential future anti-MAP clinical trials. The conference proceedings may be viewed on the www.Humanpara.org website. A summary of the salient work in this field is followed by recommendations from a majority of the conferees.

What IsMycobacterium aviumsubsp.paratuberculosis?Authors' Reply

We read with interest the article by Beumer et al. (1) on the detection of Mycobacterium avium subsp. paratuberculosis in 81% of drinking water and biofilm samples by PCR. Although these findings could have a significant influence on our understanding and the potential public impact of M. avium subsp. paratuberculosis as an agent of disease, much of the information on M. avium subsp. paratuberculosis based on genomic sequence detection and the environment tends to raise more questions than answers and fuels an ever-increasing mass of conflicting data. n nIt must first be questioned whether the detection of a specific sequence in bulk-extracted DNA actually represents a microbe, an in situ microbe, and a member of the microbial community without establishing a direct link between the molecular sequence and an actual in situ active microbe. It may be a leap of faith to assume that it does. Perhaps it would be more accurate to express such studies as detection of a genomic sequence rather than detection of an actual microbial species? n nAlthough IS900 has proved to be species specific and reliable for the detection and confirmation of M. avium subsp. paratuberculosis infection in animals with Johnes disease (ruminant paratuberculosis) (2, 4), it may again be a leap of faith to extrapolate that specificity to other biological systems such as the environment and human tissues. In todays era of gene sequencing and genomic databases, we tend to assign sequence specificity based on information contained within those databases without appreciating that they reflect only a small fraction of known bacteria and even a smaller percentage of the genomic diversity of wild-type strains. Within the human gut microbiome, for example, only 20% of ribosomal gene sequences align with known cultivable species of bacteria, suggesting that 80% of all bacteria within the human gut are unknown and/or uncultivable (7). We should expect similar findings in other ecosystems and microbial communities. n nBeumer et al. (1) have recognized and shown that other mycobacterial species that contain IS900 and/or IS900-like sequences have been identified (although not in genomic databases) and that some strains of M. avium subsp. paratuberculosis may not contain IS900. We agree with the authors premise that bona fide strains of M. avium subsp. paratuberculosis are IS900 and 251F positive, as documented in their specificity studies (1), those of others (8), and our own. But what do we make of those strains that are IS900 negative but 251F positive and those that are IS900 positive but 251F negative? Are they or are they not M. avium subsp. paratuberculosis? n nHistorically, the identification of M. avium subsp. paratuberculosis has not been stringent and has been based solely on acid fastness, slow growth, and mycobactin dependency (4). With the advent of liquid culture systems and molecular biological methods, identification has been diminished further to simply acid fastness and IS900 positivity (2). Combined with the knowledge that “species-specific” insertion sequences have been shown to cross species barriers (5), the sole reliance on IS900 as a means of identifying M. avium subsp. paratuberculosis would also seem to be a misguided leap of faith when applied to remote ecosystems. n nAccepting that the detection of IS900 is prima facie evidence of M. avium subsp. paratuberculosis and that M. avium subsp. paratuberculosis is widely distributed in the environment, the data still fail to hold water. If M. avium subsp. paratuberculosis was as widespread as suggested by Beumer et al. and others (6) and the primary biomass of M. avium subsp. paratuberculosis was the environment, why are not all domestic and wild ruminants infected? Was it not these precise inconsistencies that led to the realization that M. avium subsp. avium (the causative agent of avian tuberculosis) was not an environmental organism and to the designation of M. avium subsp. hominissuis as the only bona fide environmental M. avium strain (9)? Is the overreliance on IS900 as the sole basis for the identification of M. avium subsp. paratuberculosis going to result in a state of confusion similar to that created by strain 18 (3)?