Nongastric Helicobacter Species Detected in the Intestinal Tract of Children

Abstract

The presence of members of the family Helicobacteraceae in the colon of children was investigated using PCR, denaturing gradient gel electrophoresis, and fluorescent in situ hybridization. The rate of detection of species belonging to the Helicobacteraceae family in children with inflammatory bowel disease (92%) and irritable bowel syndrome (100%) was found to be significantly higher than that in healthy children (25%) (P < 0.05).Helicobacter species are gram-negative curved to spiralshaped bacteria that naturally colonize the mucus layer of the gastrointestinal tract of a range of mammalian hosts (15). Based on their preferential site of colonization, species of the Helicobacter genus have been divided into gastric and enterohepatic subgroups. Helicobacters in the gastric group, represented by Helicobacter pylori, have been associated with gastritis and other gastric disorders (15, 16). Enterohepatic Helicobacter species, which colonize the intestine and hepatobiliary system, have been linked to chronic hepatic and intestinal diseases (9, 16) as well as inflammatory bowel disease (IBD) in immunodeficient rodents (10, 13). The role of Helicobacter species in human IBD is currently controversial (4,5,19).In this study, we assessed the presence and spatial distribution of members of the Helicobacteraceae family (genera Helicobacter and Wolinella) in the colon of 21 children (16/21 male, aged 3 to 15 years) undergoing diagnostic colonoscopy and determined their association with colonic inflammation. The H. pylori status of all children, as assessed by histological examination and rapid urease testing of gastric biopsies, showed that 20/21 children were negative for H. pylori.Three colonic biopsies were collected from each child at colonoscopy. One biopsy was fixed in 10% neutral formalin for routine histological examination, the second biopsy was fixed in Carnoy's fixative for fluorescent rRNA in situ hybridization (FISH) analysis and Alcian blue-periodic acid-Schiff (PAS) staining, and the third biopsy was placed directly into 600 l of lysis buffer (Gentra Systems, Minneapolis, Minn.) for DNA extraction.DNA was extracted from freshly collected biopsy tissues using the PUREGENE DNA extraction kit (Gentra Systems, Minneapolis, Minn.). The16S rRNA gene of members of the family Helicobacteraceae was amplified by PCR using primers H276f and H676r with modified thermal cycle conditions (18). Hot-start PCRs were performed in a 25-l volume containing 10 pmol of each primer, 1ϫ PCR buffer (Fisher Biotech), 200 nM concentrations of each nucleotide triphosphate, 1.5 mM MgCl 2 , and 200 to 250 ng of extracted DNA. Thermal cycling conditions were 94°C for 5 min and 35 cycles of 94°C for 5 s, 57°C for 5 s, and 72°C for 30 s. Twenty-microliter PCR products were examined on a 1.5% agarose gel. PCR-denaturing gradient gel electrophoresis (DGGE) analysis was conducted according to a method described previously by Grehan et al. (11). Individual DGGE bands were excised and sequenced.Slides prepared from biopsies fixed in Carnoy's fi...

Isolation and characterization of a novel Helicobacter species, Helicobacter jaachi sp. nov., from common marmosets (Callithrix jaachus)

“…However, this study did not perform colonic biopsies to ascertain if the H. cinaedi-positive monkeys had histological evidence of chronic colitis, a common finding in rhesus monkeys (Fernandez et al, 2002). Isolation of other novel helicobacters from inflamed colons of rhesus monkeys and cotton-top tamarins is also consistent with the increasing recognition of enteric helicobacters in children with gastroenteritis who reside in developing countries (Fox et al, 2001b;Lastovica & le Roux, 2000;Saunders et al, 1999) and the identity of enteric helicobacters in the lower bowel of children with inflammatory bowel disease (Thomson et al, 2011;Zhang et al, 2006). Our findings in this study are consistent with previous NHP studies, in that we identified Helicobacter jaachi sp.…”

Isolation and Characterization of a Novel Helicobacter Species, “ Helicobacter macacae ,” from Rhesus Monkeys with and without Chronic Idiopathic Colitis

“…In selected cases, some of these species are also cultured from the blood or extraintestinal sites in patients with and without immune defects (4,13). Enteric helicobacters have also been identified in the stools of patients with inflammatory bowel disease (14). Children with diarrhea were transiently positive for H. pylori by an ELISA of antigens in their stool samples; they not only had H. pylori DNA amplified by PCR and identified by sequence, but in addition, a significant percentage of their antigen-positive stool samples had enteric Helicobacter species identified, including, for the first time, MIT 99-5504, a strain of H. macacae (8).…”

Role of Helicobacter Species in Chinese Patients with Inflammatory Bowel Disease

“…In this study, which consisted of the largest number of IBD patients included in a study to date, we employed for the first time two pairs of highly sensitive and specific primers to detect Helicobacter species in Chinese IBD patients and controls (14,21). In our study, no significant difference in the prevalence of Helicobacter species DNA in the intestinal mucosa was found between patients with IBD and controls.…”

“…Currently, the data on whether the presence of Helicobacter species is increased in patients with IBD are inconclusive. While some studies have found an increased prevalence rate (3,10,12,14,18,21), other studies have found otherwise (1, 2, 5, 7, 13). Discrepancies among studies may be explained by geographical and genetic differences among countries and populations and/or differences in disease stage, the length of time since initial diagnosis, and previous antibiotic or other treatments received.…”

“…DNA samples were extracted from intestinal biopsy specimens (about 5 mg per biopsy sample) by using the QIAamp DNeasy tissue kit (Qiagen, Hilden, Germany). Two different Helicobacter genus-specific primer sets, pair 1 (H276f and H676r) and pair 2 (C97 and C98) (14,21), were used to amplify a fragment of approximately 400 bp within the 16S rRNA gene that is conserved among members of the Helicobacter genus. Positive (H. pylori DNA) and negative (water) controls were included in all PCR runs.…”