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Journal of Clinical Microbiology, January 1999, p. 245-247, Vol. 37, No. 1
Medical Department I, Technical University
Hospital, Dresden, Germany1;
Department
of Medicine, Veterans Affairs Medical Center, Baylor College of
Medicine, Houston, Texas2; and
University Hospital San Juan de Dios, Bogota,
Colombia3
Received 3 June 1998/Returned for modification 18 August
1998/Accepted 13 October 1998
In each of six gastric cancer patients, repetitive extragenic
palindromic PCR DNA fingerprints of 18 single colonies of
Helicobacter pylori from the gastric antrum, corpus, and
cardia were identical and matched that of the parental isolate. In
three additional gastric cancer patients, 17 of 18 single-colony DNA
fingerprints were identical to each other and to the DNA fingerprint of
the corresponding parental isolate.
The large degree of genetic
heterogeneity among Helicobacter pylori strains allows
individual clinical H. pylori isolates to be reliably
distinguishable from one another through the use of DNA fingerprinting
methods (1, 7). Repetitive extragenic palindromic (REP)
sequences are well-characterized repetitive DNA elements occurring in
various bacterial species, including H. pylori (4,
11). PCR amplification of genomic DNA located between these
repetitive sequences within the H. pylori genome (REP-PCR)
has been shown to be useful to generate stable and reproducible DNA
fingerprints despite the large degree of genetic heterogeneity among
H. pylori strains (4). cagA indicates
the presence of a so-called pathogenicity island which harbors other
potential virulence determinants (3).
From nine patients with intestinal-type gastric adenocarcinoma (four
men and five women; age range, 24 to 90 years; mean age, 59 years)
evaluated at the University Hospital San Juan de Dios, Bogota,
Colombia, gastric biopsy specimens from cardia, corpus, and antrum were
obtained. H. pylori isolates from all gastric biopsy
specimens were recovered under standard conditions and identified by
Gram-stain morphology and biochemical testing. Six single colonies from
each H. pylori isolate were obtained, giving a total of 18 single colonies per patient. Genomic DNA extraction and generation of
REP-PCR DNA fingerprints were performed as described previously
(4). cagA was detected by PCR amplification of a 297-bp region (nucleotides 1751 to 2048) and a ~1.4-kb DNA fragment further downstream in the cagA gene as described previously
(6).
REP-PCR DNA fingerprints were generated for a total of 162 single
colonies of H. pylori obtained from three different gastric sites and were compared with that of the parental isolate. Each patient
harbored an H. pylori strain with a unique DNA fingerprint. DNA fingerprints from the H. pylori parent isolate and the
corresponding 18 single colonies obtained from six of nine patients
demonstrated identical DNA band profiles (Fig.
1). In one patient, the H. pylori DNA fingerprints of 17 of the 18 single colonies matched
the parental isolate, and the remaining single-colony fingerprint was
highly similar (Fig. 2). In two patients,
two single-colony band patterns were highly similar but not identical
to the parental isolate DNA fingerprint, with one single colony being
very different from the parental isolate DNA fingerprint. Both the
297-bp and the ~1.4-kb PCR products for the cagA gene were
detected in all H. pylori isolates and the respective single
colonies, indicating the ubiquitous presence of the cagA
gene in this population (data not shown).
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
DNA Fingerprinting of Single Colonies of
Helicobacter pylori from Gastric Cancer Patients Suggests
Infection with a Single Predominant Strain
ABSTRACT
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FIG. 1.
DNA fingerprints of parental isolate and single colonies
from a single patient showing identical fingerprints from parental and
single-colony isolates. Lanes: 1 and 27, 1-kb molecular mass standards;
2 and 26, 100-bp ladder; 3, antral parental isolate; 4 to 9, single
colonies from antrum; 10, corpus parental isolate; 11 to 16, single
colonies from corpus; 17, cardia parental isolate; 18 to 23, single
colonies from cardia; 24, ATCC 49503 reference strain fingerprint; 25, negative control.
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FIG. 2.
DNA fingerprints of parental isolate and single colonies
from a single patient showing identical single-colony fingerprints
except one single colony from the corpus. Lanes: 1 and 24, 1-kb
molecular mass standards; 2, 100-bp ladder; 4, parental isolate from
antrum; 5 to 9, single colonies from antrum; 10, corpus parental
isolate; 11 to 16, single colonies from corpus; 17, cardia parental
isolate; 18 to 23, single colonies from cardia.
Our data suggest that gastric cancer patients from a country with a high prevalence of H. pylori infection appear to be infected with a genetically predominant strain throughout the stomach. Jorgensen et al. utilized randomly amplified polymorphic DNA (RAPD)-PCR and found that the majority of 106 patients were infected with a predominant strain, but multiple patients harbored more than one strain (5). However, this study was limited by the fact that no single colonies were investigated. Similarly, Taylor et al. detected identical DNA patterns produced by restriction enzyme analysis, RAPD-PCR, and PCR-restriction fragment length polymorphism, suggesting infection with a single strain in 56% of patients. However, this analysis was also not extended to single colonies (9).
With respect to H. pylori isolates obtained from different gastroduodenal sites, our data are in agreement with a study by Prewett et al. (8), who identified a single H. pylori strain in biopsy specimens from the antrum, corpus, and duodenum of 13 of 15 patients. Similarly, a study by Cellini et al. included antrum and corpus biopsy specimens from 32 H. pylori-infected patients and identified a single predominant strain infection in 27 patients (2).
The present study is unique in that H. pylori from the cardia region in addition to that from the gastric antrum and corpus was cultured, and in that six single colonies were subcultured from the parental isolate of each gastric site. Our finding of a homogeneous H. pylori population throughout the stomach may be explained by the following possibilities. (i) An individual patient may be exposed and colonized by one unique strain. (ii) A patient may have been infected with multiple strains, with one strain becoming dominant over time. Or (iii) the presence of a particular strain may inhibit or prevent colonization by different strains upon sequential exposure. In our study, all single colonies of H. pylori and the respective parental isolates possessed the cagA gene, which is considered a marker for the presence of the so-called pathogenicity island. The presence of two different regions of the cagA gene further supports the homogeneity in these isolates. This is in contrast to the observation by van der Ende et al. that three of eight family members from whom multiple single colonies were obtained contained both cagA-positive and cagA-negative H. pylori strains (10).
From our data, we conclude that REP-PCR can be used to generate reproducible and stable DNA fingerprints of all H. pylori strains we examined. Individual strains (single colonies) of the H. pylori population from individual patients almost always yield DNA fingerprints identical or highly similar to that of the parent isolate. H. pylori-positive individuals are infected with a single major genotype. If a single colony is selected from an individual patient for molecular analysis, it is possible but unlikely to select a single colony not representative of the H. pylori population infecting that individual host.
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ACKNOWLEDGMENTS |
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We thank Susan M. Small for her meticulous technical assistance in the culture and genetic characterization of these H. pylori isolates.
The research was supported by the NIH National Cancer Institute grant R01CA67469.
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FOOTNOTES |
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* Corresponding author. Mailing address: Veterans Affairs Medical Center (111 D), 2002 Holcombe Blvd., Houston, TX 77030. Phone: (713) 794-7231. Fax: (713) 790-1040.
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Journal of Clinical Microbiology, January 1999, p. 245-247, Vol. 37, No. 1
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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