Enterohepatic Helicobacter in Ulcerative Colitis: Potential Pathogenic Entities?

Background Changes in bacterial populations termed “dysbiosis” are thought central to ulcerative colitis (UC) pathogenesis. In particular, the possibility that novel Helicobacter organisms play a role in human UC has been debated but not comprehensively investigated. The aim of this study was to develop a molecular approach to investigate the presence of Helicobacter organisms in adults with and without UC. Methodology/Principal Findings A dual molecular approach to detect Helicobacter was developed. Oligonucleotide probes against the genus Helicobacter were designed and optimised alongside a validation of published H. pylori probes. A comprehensive evaluation of Helicobacter genus and H. pylori PCR primers was also undertaken. The combined approach was then assessed in a range of gastrointestinal samples prior to assessment of a UC cohort. Archival colonic samples were available from 106 individuals for FISH analysis (57 with UC and 49 non-IBD controls). A further 118 individuals were collected prospectively for dual FISH and PCR analysis (86 UC and 32 non-IBD controls). An additional 27 non-IBD controls were available for PCR analysis. All Helicobacter PCR-positive samples were sequenced. The association between Helicobacter and each study group was statistically analysed using the Pearson Chi Squared 2 tailed test. Helicobacter genus PCR positivity was significantly higher in UC than controls (32 of 77 versus 11 of 59, p = 0.004). Sequence analysis indicated enterohepatic Helicobacter species prevalence was significantly higher in the UC group compared to the control group (30 of 77 versus 2 of 59, p<0.0001). PCR and FISH results were concordant in 74 (67.9%) of subjects. The majority of discordant results were attributable to a higher positivity rate with FISH than PCR. Conclusions/Significance Helicobacter organisms warrant consideration as potential pathogenic entities in UC. Isolation of these organisms from colonic tissue is needed to enable interrogation of pathogenicity against established criteria.

[1]  P. Malfertheiner,et al.  Identification of Enterohepatic Helicobacter Species in Patients Suffering from Inflammatory Bowel Disease , 2004, Journal of Clinical Microbiology.

[2]  F. Dewhirst,et al.  Helicobacter canadensis sp. nov. Isolated from Humans with Diarrhea as an Example of an Emerging Pathogen , 2000, Journal of Clinical Microbiology.

[3]  H. Flint,et al.  Degradation and utilization of xylans by the rumen anaerobe Prevotella bryantii (formerly P. ruminicola subsp. brevis) B(1)4. , 1997, Anaerobe.

[4]  M. Dave,et al.  Association between Helicobacter pylori infection and inflammatory bowel disease: A meta‐analysis and systematic review of the literature† , 2010, Inflammatory bowel diseases.

[5]  S. Goodison,et al.  16S ribosomal DNA amplification for phylogenetic study , 1991, Journal of bacteriology.

[6]  F. Dewhirst,et al.  Helicobacter pullorum sp. nov.-genotype and phenotype of a new species isolated from poultry and from human patients with gastroenteritis. , 1994, Microbiology.

[7]  P. Vandamme,et al.  Identification of Campylobacter cinaedi isolated from blood and feces of children and adult females , 1990, Journal of clinical microbiology.

[8]  J. Fry,et al.  PRIMROSE: a computer program for generating and estimating the phylogenetic range of 16S rRNA oligonucleotide probes and primers in conjunction with the RDP-II database. , 2002, Nucleic acids research.

[9]  J. Lennard-jones,et al.  Classification of inflammatory bowel disease. , 1989, Scandinavian journal of gastroenterology. Supplement.

[10]  R. Koch,et al.  Die Aetiologie der Tuberkulose , 1932, Klinische Wochenschrift.

[11]  J. Fox,et al.  Natural and experimental Helicobacter infections. , 2004, Comparative medicine.

[12]  F. Dewhirst,et al.  Helicobacter canis sp. nov., a new species from dogs: an integrated study of phenotype and genotype. , 1993, Journal of general microbiology.

[13]  A. Day,et al.  Nongastric Helicobacter Species Detected in the Intestinal Tract of Children , 2006, Journal of Clinical Microbiology.

[14]  P. Chipman,et al.  Helicobacter winghamensis sp. nov., a Novel Helicobacter sp. Isolated from Patients with Gastroenteritis , 2001, Journal of Clinical Microbiology.

[15]  Michael Wagner,et al.  probeBase—an online resource for rRNA-targeted oligonucleotide probes: new features 2007 , 2006, Nucleic Acids Res..

[16]  J. Fox,et al.  Helicobacter mustelae-associated gastritis in ferrets. An animal model of Helicobacter pylori gastritis in humans. , 1990, Gastroenterology.

[17]  H. Flint,et al.  Assessment of microbial diversity in human colonic samples by 16S rDNA sequence analysis. , 2002, FEMS microbiology ecology.

[18]  M. Kamm,et al.  Evaluation of Helicobacter species in inflammatory bowel disease , 2003, Alimentary pharmacology & therapeutics.

[19]  B. N. Anderson,et al.  Atypical Campylobacters Associated with Gastroenteritis , 1988, Journal of clinical microbiology.

[20]  H. Neu,et al.  Oligonucleotide probe for detection and identification of Campylobacter pylori , 1989, Journal of clinical microbiology.

[21]  F. Mégraud,et al.  Association between entero‐hepatic Helicobacter species and Crohn’s disease: a prospective cross‐sectional study , 2009, Alimentary pharmacology & therapeutics.

[22]  M. Ferrús,et al.  Use of fluorescent in situ hybridization to evidence the presence of Helicobacter pylori in water. , 2003, Water research.

[23]  H. Mitchell,et al.  Detection of Helicobacter Colonization of the Murine Lower Bowel by Genus-Specific PCR-Denaturing Gradient Gel Electrophoresis , 2002, Applied and Environmental Microbiology.

[24]  M. Blaut,et al.  Quantification of Different Eubacteriumspp. in Human Fecal Samples with Species-Specific 16S rRNA-Targeted Oligonucleotide Probes , 2000, Applied and Environmental Microbiology.

[25]  R. Schell,et al.  Case report of an unclassified microaerophilic bacterium associated with gastroenteritis , 1988, Journal of clinical microbiology.

[26]  J M Ward,et al.  Helicobacter hepaticus Triggers Colitis in Specific-Pathogen-Free Interleukin-10 (IL-10)-Deficient Mice through an IL-12- and Gamma Interferon-Dependent Mechanism , 1998, Infection and Immunity.

[27]  Z. Shen,et al.  Hepatic Helicobacter species identified in bile and gallbladder tissue from Chileans with chronic cholecystitis. , 1998, Gastroenterology.

[28]  E. El-Omar,et al.  Could Helicobacter organisms cause inflammatory bowel disease? , 2011, FEMS immunology and medical microbiology.

[29]  J. Heesemann,et al.  Rapid and specific detection of Helicobacter pylori macrolide resistance in gastric tissue by fluorescent in situ hybridisation , 2000, Gut.

[30]  K. Schleifer,et al.  The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. , 1999, Systematic and applied microbiology.

[31]  J. Fox,et al.  Helicobacter mustelae-associated gastritis in ferrets , 1990 .

[32]  S. Bereswill,et al.  Isolation of Helicobacter pullorum from patients with enteritis. , 1997, Scandinavian journal of infectious diseases.

[33]  F. Powrie,et al.  Inflammatory bowel disease: an immunity-mediated condition triggered by bacterial infection with Helicobacter hepaticus , 1997, Infection and immunity.

[34]  A. Lastovica,et al.  Efficient Isolation of Campylobacteria from Stools , 2000, Journal of Clinical Microbiology.

[35]  M. Nei,et al.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 2007, Molecular biology and evolution.

[36]  J. Satsangi,et al.  Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. , 2005, Canadian journal of gastroenterology = Journal canadien de gastroenterologie.

[37]  R. Amann,et al.  Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations , 1990, Applied and environmental microbiology.

[38]  M. Weizenegger,et al.  Acute appendicitis is characterised by local invasion with Fusobacterium nucleatum/necrophorum , 2009, Gut.

[39]  J. Nicolet,et al.  Gastroenteritis associated with Helicobacter pullorum , 1994, The Lancet.

[40]  U. Schaad,et al.  Novel Campylobacter-like organism resembling Helicobacter fennelliae isolated from a boy with gastroenteritis and from dogs , 1993, Journal of clinical microbiology.

[41]  M. Wannemuehler,et al.  Helicobacter bilis triggers persistent immune reactivity to antigens derived from the commensal bacteria in gnotobiotic C3H/HeN mice , 2006, Gut.

[42]  G. Daskalopoulos,et al.  Absence of Mucosa-Associated Colonic Helicobacters in an Australian Urban Population , 2004, Journal of Clinical Microbiology.