Molecular identification of Helicobacter DNA present in human colorectal adenocarcinomas by 16S rDNA PCR amplification and pyrosequencing analysis

Abstract

Seroepidemiological studies have indicated that Helicobacter pylori infection might be a possible risk factor for colorectal adenocarcinoma (CRC) development. However, limited information is available as to whether or not Helicobacter species are present in CRC tissues. In this study the presence of Helicobacter DNA in 77 CRC biopsies was investigated by means of a Helicobacter species-specific 16S rDNA PCR assay and real-time DNA pyrosequencing of the 16S rDNA variable V3 region. Pyrosequencing revealed the presence of Helicobacter DNA sequences in 21 of 77 biopsy specimens (27 %). 16S rDNA sequences corresponding to H. pylori 26695 and H. pylori J99 were most commonly found. Intriguingly, one sequence belonged to Helicobacter mustelae, previously identified in ferrets. No significant correlations were found in the prevalence of Helicobacter DNA between colon and rectum tumour biopsies (P ¼ 0 . 815), nor between Dukes' classes A/B and C/D (P ¼ 0 . 262). 16S rDNA PCR amplification combined with pyrosequencing analysis of 16S rDNA variable V3 regions provides a powerful molecular tool to identify Helicobacter species in human biopsy specimens. INTRODUCTIONHelicobacter pylori is a microaerophilic Gram-negative spiral-shaped bacterium (Marshall & Warren, 1983) associated with chronic gastritis, peptic ulcer and gastric adenocarcinoma development (Parsonnet et al., 1991;Cover & Blaser, 1992;Logan, 1994). It has been shown that there is a significant geographical relationship between gastric cancer mortality rates and prevalence of H. pylori infection. Subtypes of H. pylori might differ in pathogenicity (Xiang et al., 1995), and a number of studies have shown that the prevalence of H. pylori expressing the cytotoxin-associated gene A (cagA) in gastric cancer patients is much higher than in age-and gender-matched controls (Parsonnet et al., 1997; Konturek et al., 2003;Semino-Mora et al., 2003). Recent reports imply that H. pylori may be an association factor involved in colorectal cancer (CRC) development in patients infected with H. pylori strains (Meucci et al., 1997;Shmuely et al., 2001). However, it is far from clear whether H. pylori is present in CRC tissues and whether or not H. pylori plays a similar role in colorectal carcinogenesis as has been proposed for gastric cancer development.16S rDNA sequence analysis has demonstrated considerable genomic diversity among H. pylori clinical isolates, and numerous sequence-specific PCR assays, combined with 16S rDNA sequencing, have been developed to identify Helicobacter species (Thoreson et al., 1995;Blom et al., 2002). We recently described a method for the rapid simultaneous molecular identification and subtyping of H. pylori by pyrosequencing analysis of the 16S rDNA variable V1 and V3 regions (Monstein et al., 2001).In this report we describe the molecular identification of Helicobacter DNA in CRC biopsies by means of a 16S rDNA PCR amplification assay combined with pyrosequencing analysis. METHODSTissue collection and DNA isolation. A total of 77 patients (45 w...

Seroprevalence of Helicobacter pylori in patients with colon adenomas in a brazilian university hospital

“…Some studies were able to identify the genetic material of the bacteria in colonic neoplasias (3,8,15) . The effect of the intestinal flora as a carcinogenic agent has been evaluated and the possibility exists that bacteria such as H. pylori circulating in the intestines could directly activate carcinogenesis (33) .…”

Helicobacter pylori Infection and the Risk of Colorectal Adenoma and Adenocarcinoma: an Updated Meta-analysis of Different Testing Methods

“…Like serologic studies, studies using PCR methods have been equivocal. Two studies found that detection of H. pylori via PCR was significantly higher in colorectal adenocarcinoma tissues compared with normal colorectal tissues (Grahn et al, 2005;Jones et al, 2007), but a third study found that only 1.2% of malignant colorectal tissue samples were positive for H. pylori, compared with 6% of normal tissues (Bulajic et al, 2007). The 13C-urea breath test has 97% sensitivity and specificity in H. pylori detection (Chen et al, 2003); however, as in PCR-based studies, the evidence is not convincing.…”

Helicobacter pylori in Colorectal Carcinoma Tissue

“…Recent reports suggest that H. pylori may be an association factor involved in the development of colorectal cancer in patients infected with certain strains (9,10). The Swedish group of Grahn et al (11) was the first who previously described the potential molecular identification of Helicobacter DNA in colorectal cancer biopsies by means of a 16S rDNA PCR amplification assay combined with pyrosequencing analysis. Furthermore, Maggio-Price et al (12) proved that infection of SMAD3 À/À mice with Helicobacter triggers colon cancer in 50% to 66% of those animals.…”