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Journal of Clinical Microbiology, January 2004, p. 384-386, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.384-386.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Helicobacter Species in the Intestinal Mucosa of Patients with Ulcerative Colitis

Laboratory of Research in Bacteriology,¹ Departments of Internal Medicine,⁵ Microbiology,⁶ Pathology, Universidade Federal de Minas Gerais,⁷ Minas Gerais Institute of Gastrointestinal Procedures,³ Governador Israel Pinheiro Hospital, Belo Horizonte,⁴ Faculdade de Medicina do TriÂngulo Mineiro, Uberaba, Brazil²

Received 18 August 2003/ Returned for modification 18 September 2003/ Accepted 29 September 2003

	ABSTRACT

Top Abstract Introduction Nucleotide sequence accession... References

 
In a search for Helicobacter species in the intestinal mucosae of 42 patients with ulcerative colitis (UC) and 74 without UC, only H. pylori was found. Although the bacterium was detected in UC patients by culture (7.1%) and nested PCR (19.0%), its presence was not associated with the disease (P = 0.13).

	INTRODUCTION

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Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) of unknown etiology that is confined to the large bowel mucosa. It seems to be a multifactorial disorder involving both genetic and environmental components, particularly the bacterial gut microbiota (13). However, no specific pathogenic or commensal bacterium has been convincingly implicated as the etiologic agent (4). Numerous studies have demonstrated that indigenous Helicobacter species of the gut of mice—Helicobacter hepaticus and Helicobacter bilis—are able to induce a persistent inflammation of the colon and cecum in some immunocompromised mouse models (1, 5, 8, 15). Moreover, other Helicobacter species have been isolated from the gut of cotton-top tamarins (14) and rhesus monkeys (7) with IBD-like disease. We hypothesize that species of the genus Helicobacter may also be associated with the pathogenesis of UC in human beings. Therefore, we investigated the presence of Helicobacter species in the intestinal mucosa of patients with UC and controls, by culture and PCR.

We studied prospectively 42 patients with UC and 74 without IBD (controls) who were undergoing colonoscopy. Prior consent of all patients and approval of the institutional ethics committee were obtained. The UC diagnosis was established by standard clinic, radiologic, endoscopic, and histologic criteria. The extent of ulcerative colitis was determined by histology. The gastric H. pylori status was assessed by serology (Cobas-Core EIA; Roche Diagnostic Systems, Basel, Switzerland) (12) and ¹³C urea breath test (¹³C-UBT; NDIRIS; Wagner Analysen Technik, Bremen, Germany) (13). Patients' characteristics, including gastric H. pylori status, are shown in Table 1.

DNA from bacteria and mucosal fragments was extracted with a QIAamp DNA minikit (Qiagen GmbH, Hilden, Germany).

Primers C70 and B37 were used to amplify a product of 1.5 kb from the 16S rRNA gene of the isolated strains (10). The amplicons were then purified and directly sequenced as described by Queiroz et al. (10). The sequences were aligned and compared with those in the GenBank database.

For the detection of Helicobacter DNA in fragments of the intestinal mucosa, the 16S rRNA gene was amplified by nested PCR using an outer primer pair (C70 and C37) and two inner primer pairs for the genus Helicobacter (C97 and C98) and the species H. pylori (HP1 and HP2) (3, 6, 10). Another nested PCR for ureA specific to H. pylori was also used (16) (Table 2).

Gastric H. pylori infection was detected in 53.7% of the UC patients and in 52.1% of the controls, with no significant difference between the groups (P = 1.0). This result agrees with those obtained by Duggan et al. (2) and Piodi et al. (9).

Helicobacter strains were isolated from the intestinal mucosa throughout the colon in three (7.1%) patients with and one (1.4%) without UC, with no significant difference (P = 0.13). The isolates were gram negative and motile and had a slightly spiral appearance. All strains showed the same biochemical characteristics and were urease, catalase, oxidase, alkaline phosphatase, and -glutamyl transpeptidase positive but did not reduce nitrate or hydrolyze hippurate. They did not produce hydrogen sulfide and were resistant to nalidixic acid and sensitive to cephalothin. More than 96% of the complete 16S rRNA gene sequence was determined for all isolates. Comparison of consensus sequences showed >99% similarity to H. pylori.

Helicobacter DNA was detected by PCR in the intestinal mucosa of eight (19.0%) UC patients and seven (9.5%) controls. Although the sensitivity of the PCR assay was higher than that of the culture, no association was detected between the presence of Helicobacter in the intestine of controls and UC patients (P = 0.23). All DNA detected by specific 16S rRNA/ureA nested PCR belonged to H. pylori species.

All patients from whom Helicobacter strains were isolated or in whom Helicobacter DNA was detected in the intestinal mucosa were H. pylori positive, as assessed by ¹³C-UBT and serology.

Occurrence of diarrhea was not associated with Helicobacter isolation in UC patients (P = 1.0) or in controls (P = 1.0). Also, no association was found between H. pylori detection and activity (P = 0.19), extent (P = 0.32), or duration (P = 0.86) of UC, and treatment with sulfasalazine (P = 0.56) or corticosteroids (P = 0.29).

All Helicobacter strains isolated belonged to H. pylori, although the culture conditions adopted allow the isolation of most Helicobacter species, including those that have been recovered from the intestines of rodents and nonhuman primates with IBD. This result indicates that other cultivable Helicobacter did not colonize the lower gastrointestinal tracts of the patients and controls. Even considering that other Helicobacter species could be missed by culture, because they are fastidious, our hypothesis is reinforced by the concordant data obtained by a more sensitive method, the nested PCR assay, that is able to detect one copy of Helicobacter DNA fragment, equivalent to 10^-3 fg (16). Again, the only Helicobacter species detected by this method was H. pylori. However, neither the presence of viable H. pylori in the intestine, detected by culture, nor the presence of H. pylori DNA, detected by nested PCR, showed significant association with UC.

In conclusion, although we cultured H. pylori from the intestinal mucosae of a small number of UC patients, its presence was not associated with the disease. Since no other Helicobacter species was detected in the intestinal mucosa of the patients, we may speculate that the Helicobacter genus is not involved in the genesis of UC.

	Nucleotide sequence accession number.

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The sequences obtained in this study were deposited in GenBank (Table 3).

	ACKNOWLEDGMENTS

 
We thank the patients for participating in this study. This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Brazil.

	FOOTNOTES

 
* Corresponding author. Mailing address: Faculdade de Medicina, Av. Alfredo Balena, 190/4026, 30130-100, Belo Horizonte, MG, Brazil. Phone and fax: 55 31 3274 2767. E-mail: dqueiroz{at}medicina.ufmg.br.

	REFERENCES

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