JOURNAL OF CLINICAL MICROBIOLOGY, June 2005, p. 2635–2641 0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.6.2635–2641.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 43, No. 6
High Frequency of Gastric Colonization with Multiple Helicobacter pylori Strains in Venezuelan Subjects C. Ghose,1 G. I. Perez-Perez,1* L. J. van Doorn,2 M. G. Domı´nguez-Bello,3 and M. J. Blaser1,4 Departments of Microbiology and Medicine, New York University School of Medicine, New York, New York1; Delft Diagnostic Laboratories, Delft, The Netherlands2; Laboratory of Gastrointestinal Physiology, IVIC, Caracas, Venezuela3; and Department of Veterans Affairs Medical Center, New York, New York4 Received 29 November 2004/Returned for modification 22 December 2004/Accepted 17 February 2005
Multiple Helicobacter pylori strains may colonize an individual host. Using enzyme-linked immunosorbent assay and line probe assay (LiPA) techniques, we analyzed the prevalence of mixed H. pylori colonization in 127 subjects from Venezuela, a country of high H. pylori prevalence, from three regions representing different population groups: the Andes (Merida), where Caucasian mestizos predominate, a major city near the coast (Caracas), where Amerindian-Caucasian-African mestizos predominate, and an Amazonian community (Puerto Ayacucho), where Amerindians predominate and mestizos reflect Amerindian and Caucasian ancestry. Among 121 H. pylori-positive persons, the prevalence of cagA-positive strains varied from 50% (Merida) to 86% (Puerto Ayacucho) by LiPA. Rates of mixed colonization also varied, as assessed by LiPA of the vacA s (mean, 49%) and m (mean, 26%) regions. In total, 55% of the individuals had genotypic evidence of mixed colonization. vacA s1c, a marker of Amerindian (East Asian) origin, was present in all three populations, especially from Puerto Ayacucho (86%). These results demonstrate the high prevalence of mixed colonization and indicate that the H. pylori East Asian vacA genotype has survived in all three populations tested. strains produce little or none (9). Particular vacA genotypes vary in their geographic prevalence and serve as markers for the ancestry of the H. pylori isolates; for example, vacA s1c is a strong marker for East Asian ancestry (24, 29, 62, 68). H. pylori possesses an unusually high number of type II restriction-modification (R-M) systems, and each strain varies in its complement of R-M systems (58, 69). For the hpyI and the hpyIII R-M systems, the methyltransferase gene (either hpyIM or hpyIIIM) is present in all strains, but in some strains hpyIR has been replaced with another gene (hrgB) and/or hpyIIIR has been replaced by hrgA (5, 25). Each strain possesses either gene at the hpyIR locus and not both or neither; the same has been found for the hpyIIIR locus (4). Therefore, the detection of both alleles in a single gastric specimen implies that the host is colonized by multiple H. pylori strains. Because multiple H. pylori strains may colonize a single patient, intergenomic recombination occurs, and the H. pylori population structure indicates that such events have been relatively common (12, 38, 57). Colonization by multiple H. pylori strains appears more common in countries where H. pylori is highly prevalent (8, 36, 43, 45). Venezuela, a country of high H. pylori prevalence, was settled by persons of different ethnicities, and ethnic mixing continues to the present (22, 44). Nevertheless, in the South (Amazonas), Amerindians predominate and mestizos reflect Amerindian and Caucasian ancestry; in the Western Andes (Merida), Caucasian mestizos predominate; and in the NorthCentral region (Caracas), Amerindian-Caucasian-African mestizos predominate (18, 52). In this study, we analyzed the prevalence of mixed H. pylori colonization among persons in these three different locales in Venezuela. We hypothesized that we would find evidence for mixed colonization and that the circulating strains would reflect the ethnicities of the host population. Our analysis was
Helicobacter pylori is a gram-negative microaerophilic bacterium that persistently colonizes the gastric mucosa of human hosts for decades or life (14). More than 50% of the world’s population carries H. pylori, with proportions as high as 80% in developing countries (27). Although most H. pylori-positive persons are asymptomatic, the presence of H. pylori is associated with increased risk for the development of peptic ulcer disease, gastric adenocarcinoma, and gastric lymphoma (6, 46, 50). Expression of disease is associated with particular host and bacterial factors (16, 17, 40, 47). Two H. pylori genes, vacA and cagA, have been especially associated with the differences in disease risk (19, 20, 61). In the Western world, cagA is present in ⬃60% of strains and is the marker for a 35- to 40-kb pathogenicity island that encodes a type IV secretion system responsible for the translocation of the CagA protein into host gastric epithelial cells (2, 48). Translocation of the CagA protein into host cells changes signal transduction pathways and induces proinflammatory cytokine production (31). In contrast, all H. pylori strains contain vacA, but its product is detectable in vitro in only about half the strains (20). The vacA genotypes for any given H. pylori strain are a mosaic of combinations of signal sequence and midregion genotypes or are chimerae (9). The vacA signal sequence (s sequence) has two major genotypes, s1 and s2, and there are three variations of s1: s1a, s1b, and s1c (62, 64). The midregion of vacA, about 700 bp, has two major genotypes, m1 and m2 (9, 10, 21). Strains of vacA s1/m1 and s1/m2 types produce high and moderate levels of vacuolating activity, respectively, whereas s2/m2
* Corresponding author. Mailing address: Infectious Diseases Laboratories, Department of Medicine, VAMC 6026 West, 423 East 23rd Street, New York, NY 10010. Phone: (212) 263-4105. Fax: (212) 2634108. E-mail:
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TABLE 1. Characteristics of the 127 subjects in the 3 studied populations in Venezuela % With clinical status Location (nb)
Merida (68) Caracas (29) Puerto Ayacucho (30) a b
Mean (⫾SD) age in yr (range)
42.2 ⫾ 17 (18–83) 35.5 ⫾ 15 (7–66) 33.9 ⫾ 13 (12–60)
Nonulcer dyspepsia
Othera
Unknown
75 86 100
3 14 0
22 0 0
Duodenal ulcer (n ⫽ 5), Barrett’s esophagus (n ⫽ 1). No. of subjects.
based on vacA s and m genotypes, using line probe assay (LiPA), and on cagA prevalence, using enzyme-linked immunosorbent assay (ELISA), and in one locale we used R-M alleles to define the extent of mixed colonization. Using biopsy-based methods instead of pure isolated strain-based methods and using relatively simple and widely available marker systems, we sought to determine the extent to which cocolonization with multiple H. pylori strains is present in the three Venezuelan populations studied. We also sought to determine whether the distribution of circulating vacA alleles is similar in the three locales, thus reflecting the origins of the H. pylori strains (whether East Asian or Western). MATERIALS AND METHODS Subjects. DNA was obtained from antrum biopsies from patients from Merida in the Andean state, from Caracas, the urban capital of Venezuela, and from Puerto Ayacucho, where medical care is provided to populations from surrounding Amazonian villages. The facilities at Puerto Ayacucho, Caracas, and Merida are parts of the public hospital system, where patients of lower socioeconomic status go for medical care. All of the studied patients were of lower socioeconomic status. The 30 persons studied from the Puerto Ayacucho area (mean age, 34 years), were of Amerindian ancestry (25 Piaroas and 5 Guajibos) and are representative of a rural poor community in the Amazonas state (Table 1). All 29 persons from Caracas (mean age 36, years) represent a poor urban community where Amerindian-Caucasian-African mestizos predominate. The 68 persons from Merida (mean age, 43 years) are representative of a poor urban community in the Andes where Caucasian mestizos predominate. From each patient two antrum biopsies were taken, 2 cm from the pylorus, one for culture and one for PCR. All patients were undergoing upper gastrointestinal endoscopy for dyspeptic symptoms and signed a consent form to participate in this study. The prevalence of peptic ulcer disease is low in all three populations, as has been reported previously (22). In our previous study, we analyzed the prevalence of the vacA s1c allele in populations from Caracas and Puerto Ayacucho by PCR followed by sequencing (29). We were able to subject to PCR and sequence 13 samples from Caracas and 16 samples from Puerto Ayacucho at the vacA s locus, of which 7 (54%) and 12 (75%), respectively, are included in our current study. Serum samples also were collected from the subjects in Caracas and Puerto Ayacucho but not from Merida. We were not able to establish ethnicity for individual patients. Specimens for LiPA analysis. DNA was extracted from biopsies using the QIAGEN DNeasy tissue kit (Valencia, CA). One hundred nanograms of total genomic DNA was used for PCR. PCR was performed using biotin-labeled primers specific for vacA s, m, and cagA, as described previously (64). Ten microliters of each biotin-labeled PCR product was denatured by the addition of 10 ml of 400 mM NaOH and 10 mM EDTA and used in the subsequent reverse hybridization LiPA assay. Reverse-hybridization LiPA analysis. PCR-LiPA was used to study the presence of cagA and the different vacA s and m genotypes in the patient specimens (64, 65). Briefly, allele-specific oligonucleotide probes for vacA s1a, s1b, and s1c, vacA m1 and m2, and cagA were tailed with poly(dT) and were immobilized as parallel lines on nitrocellulose strips, as described previously (65). The LiPA strips were incubated in prewarmed hybridization solution, and then denatured
PCR products from the patient specimens were added. After 30 min of incubation at room temperature, the strips were rinsed again, and 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate substrate were added. Positive hybridizations were visible as purple probe lines. Results were read at the moment of experiments, since positive bands fade over time. Moreover, results are qualitative and not quantitative; hence, no assumptions can be made as to which allele is the major/minor type present in one sample. DNAs from H. pylori strains with known genotypes were used as positive and negative controls. The LiPA has been previously used for detecting H. pylori in gastric biopsy samples, formalin-fixed samples, and paraffin-embedded samples and shows superior sensitivity compared with culture and histopathology (54, 55, 66). A sensitivity analysis was not performed for the LiPA, since it is a well-established technique with a detection capacity better than 1 in 20 (5%) to 1:10,000 (0.001%), as shown for hepatitis C virus and human papillomavirus detection (37, 67). Whole-cell and CagA ELISA. The presence of immunoglobulin G (antibodies to an H. pylori whole-cell (WC) antigen preparation or to CagA protein was assessed by enzyme-linked immunosorbent assays (ELISA), as described previously (28, 39). In brief, each antigen was dissolved in 0.5 M sodium bicarbonate (pH 9.6) and used to coat microtiter plates overnight at room temperature. Patient serum was added at 1:800 and 1:100 dilutions for WC and CagA antibodies, respectively, and incubated for 1 h at 37°C. Human anti-immunoglobulin G conjugated to horseradish peroxidase (Biosource International, Camarillo, CA) was used, and the mixture reincubated for 1 h. ABTS [2–2⬘-azino-bis(3ethylbenzthiazoline-6-sulfonic acid)] (Sigma Aldrich, St. Louis, MO) was added as a substrate, and plates were read at 405 nm. Each serum sample was tested in duplicate. Data were analyzed using the Revelation 2.0 program (Dynatech, Westwood, NJ). For WC, serum samples with ELISA units of ⱖ1.0 were considered seropositive, while a value of ⬍1 was considered negative. For CagA, serum samples with ELISA units of ⱖ0.35 were considered seropositive, while a value of ⬍0.35 was considered negative (39). Analysis of R-M systems. The presence of the restriction endonuclease or replacing gene in both the hpyI and hpyIII R-M loci was analyzed using PCR in 31 samples from Merida. Analysis was limited to a subset of Merida biopsies in which there was sufficient residual DNA for these PCR amplifications. Primers and PCR conditions for hpyIR, hrgB, hpyIIIR, and hrgA have been described previously (5, 25). The presence of both hpyIR and hrgB PCR products or hpyIIIR and hrgA PCR products in DNA from a single biopsy sample was considered evidence for the presence of multiple strains. Sensitivity analysis of R-M system allele PCR. A sensitivity analysis of the PCR for each R-M system gene used in the detection of mixed colonization was performed using genomic DNA from H. pylori strains. For the hpyIR allele, genomic DNA from strain 26695 (hpyIR⫹ hrgB⫺) was analyzed at concentrations from 100 ng per reaction to 0.001 ng per reaction as template DNA. For each reaction, genomic DNA from strain J99 (hrgB⫹ hpyIR⫺) was added at 100 ng per reaction as competing DNA. The converse concentrations were used for the sensitivity analysis of the hrgB PCR. For the hpyIIIR allele, dilutions were made with genomic DNA from 26695 (hpyIIIR⫹ hrgA⫺) as template DNA from 100 ng to 0.001 ng per reaction. Genomic DNA from strain 60190 (hrgA⫹ hpyIIIR⫺) was added at 100 ng per reaction as competing DNA. The converse concentrations were used for the sensitivity analysis of the hrgA allele. PCR conditions were as described previously (5, 25). Statistical analysis. A chi-square test was used to compare the frequencies of mixed or single colonizations in patients from the different locales. A P value of ⬍0.05 was considered to be significant.
RESULTS Comparison of ELISA and LiPA methodologies for H. pylori detection and cagA prevalence in the studied populations. Of the 127 subjects studied, 121 (95%) had evidence by LiPA for H. pylori carriage (Table 2). The prevalence of cagA-positive strains in these 121 subjects, as determined by LiPA, varied in the three localities: Merida (50%), Caracas (77%), and Puerto Ayacucho (86%) (P ⬍ 0.0001). The results of the LiPA analysis of the strains and the ELISA analysis of the serum from the patients were compared with samples from Caracas and Puerto Ayacucho. For the specimens from Puerto Ayacucho, the two methodologies provide nearly identical results. For the specimens from Caracas, detection of carriage of cagA-positive H.
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TABLE 2. H. pylori and cagA status for 127 subjects from 3 locations in Venezuela as determined from analysis of gastric specimens by LiPA and serum IgG antibody responses by ELISA No. (%) of subjects in category
Patient status (H. pylori, cagA)
⫺, ⫹, ⫺, ⫹, a
Merida (n ⫽ 68)
⫺ ⫺ ⫹ ⫹
Caracas (n ⫽ 29)
Puerto Ayacucho (n ⫽ 30)
Total (n ⫽ 127)
LiPA
ELISA
LiPA
ELISA
LiPA
ELISA
LiPA
2 (4) 33 (48) 0 33 (48)
NDa ND ND ND
3 (10) 6 (21) 0 20 (69)
0 13 (45) 0 16 (55)
1 (3) 4 (13) 0 25 (83)
0 4 (13) 2 (7) 24 (80)
6 (5) 43 (34) 0 (0) 78 (61)
ND, not determined, because serum specimens were not available from patients in Merida.
pylori strains was observed in 77% and 61% of the individuals, using the LiPA and ELISA methodologies, respectively. Between the two populations, using the LiPA as the “gold standard,” the CagA ELISA had five false negatives (9%) and two false positives (14%). In total, these studies showed that H. pylori is highly prevalent in these populations, with a mixture of cagA-positive and cagA-negative strains. Prevalence of specific vacA s genotypes in the three studied populations of Venezuela. LiPA analysis of DNA extracted from biopsies can detect colonization by H. pylori strains that differ in vacA s and vacA m genotypes (26). Since H. pylori cells possess only a single copy of vacA, the presence of more than one vacA s or m genotype in a DNA sample indicates colonization by multiple strains (26). Of the 121 biopsies studied, a single vacA s genotype was observed in 65 (54%) of the samples, with the prevalence ranging from 31% (Caracas) to 62% (Puerto Ayacucho) (Table 3). Conversely, multiple genotypes were observed in 69%, 38%, and 41% of the specimens from Caracas, Puerto Ayacucho, and Merida, respectively. In the 56 biopsies (46%) from the three populations combined that showed multiple vacA s genotypes, 148 (mean, 2.57 ⫾ 0.53 per biopsy) alleles were observed, with frequencies varying between 2.48 ⫾ 0.50 (in Merida) and 2.72 ⫾ 0.65 (in Puerto Ayacucho). In Caracas, the most common circulating genotype was vacA s2, appearing in 21 (81%) of the subjects. In Merida, vacA s1b occurred in 51 (77%), and in Puerto Ayacucho, vacA s1c occurred in 25 (86%) of the subjects. The vacA s1a genotype never was detected as a single genotype in any patient, whereas vacA s1c was detected as a single genotype only in Puerto Ayacucho (48%). Twenty-five (86%) of the specimens from Puerto Ayacucho
contained vacA s1c, a significantly greater proportion than in Merida (26%; P ⬍ 0.0001) and in Caracas (42%; P ⫽ 0.001). Prevalence of specific vacA m genotypes in the three studied populations of Venezuela. Only two vacA m genotypes were distinguishable by LiPA (Table 4). Eleven percent of the specimens examined by the vacA m LiPA yielded untypable results, versus none for the vacA s genotype LiPA, reflecting the greater genetic complexity of the vacA m locus (10). In total, 24 (20%) of the 121 specimens analyzed showed evidence for both vacA m1 and m2 genotypes (Table 4). Mixed colonization was detected most often in Puerto Ayacucho (52%) and least in Merida (6%). The vacA m1 genotype was present in 90% of specimens from Puerto Ayacucho, ranging to 46% in Caracas. The vacA m2 genotype ranged from 62% in Puerto Ayacucho to 36% in Merida. In total, based on multiple genotypes in either the vacA s or m loci, the prevalence of mixed colonization detected in the specimens was 55% (Table 4). This methodology could permit detection of up to four vacA s genotypes and two vacA m genotypes. In Caracas, the majority of persons with mixed colonization carried three vacA s alleles (42%), whereas in Merida and Puerto Ayacucho, the majority were colonized with a single vacA s genotype (59% and 62%, respectively). In Puerto Ayacucho, there was evidence in one subject for all four vacA s genotypes (Table 3). Sensitivity analysis of R-M alleles as a tool for detection of mixed colonization. For the hpyIR and hpyIIIR alleles, the primers were able to amplify a product from 0.01 ng of template DNA when the majority of the DNA present in the PCR mix contained genomic DNA from strains that carried the heterologous allele (Fig. 1). For the hrgA and hrgB alleles, the
TABLE 3. Distribution of vacA s genotypes in gastric samples from 121 H. pylori-positive subjects in 3 studied populations in Venezuelaa
c
Location (n )
No. (%) of subjects with single genotype
s1a
s1b
s1c
s2
No. (%) of subjects with multiple genotypes
No. (%) of subjects with specified genotype
No. (%) of subjects with specified genotypeb s1a
s1b
No. (%) of subjects with the following no. vacA s genotypes:
s1c
s2
2
3
4
Merida (66) Caracas (26) Puerto Ayacucho (29)
39 (59) 8 (31) 18 (62)
0 0 0
25 (38) 4 (15) 3 (10)
0 0 14 (48)
14 (21) 4 (15) 1 (3)
27 (41) 18 (69) 11 (38)
1 (1) 26 (39) 4 (15) 15 (57) 1 (3) 9 (31)
17 (26) 11 (42) 11 (38)
23 (35) 17 (65) 9 (31)
14 (21) 7 (27) 4 (14)
13 (20) 11 (42) 6 (21)
0 0 1 (3)
Total (121)
65 (54)
0
32 (26)
14 (11)
19 (16)
56 (46)
6 (5)
39 (32)
53 (48)
25 (21)
30 (25)
1 (1)
a b c
Excludes the six persons whose biopsy did not yield an H. pylori signal. Percentages total ⬎100, since there were multiple genotypes per subject. No. of subjects.
50 (41)
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TABLE 4. Distribution of vacA m genotypes in gastric samples from 121 H. pylori-positive subjects in 3 studied populations in Venezuelaa m genotypes Location (nd)
No. (%) of subjects with single genotype
No. (%) with specified genotype m1
m2
No. (%) of subjects with both m1 and m2 genotypes
No. (%) of subjects with untypable samplesb
m and s genotypes: No. (%) of subjects with ⱖ2 genotypesc
Merida (66) Caracas (26) Puerto Ayacucho (29)
51 (77) 19 (73) 14 (48)
31 (47) 7 (27) 11 (38)
20 (30) 12 (46) 3 (10)
4 (6) 5 (19) 15 (52)
11 (16) 2 (8) 0
27 (41) 20 (77) 19 (66)
Total (121)
84 (69)
49 (40)
35 (29)
24 (20)
13 (11)
66 (55)
a
Excludes the six persons whose biopsy did not yield an H. pylori signal. Percentages total ⬎100, since there were multiple genotypes per subject. Including diversity of both vacA s-genotypes (see Table 3) and m-genotypes in the same individual. d No. of subjects. b c
primers were able to amplify a product from 0.001 ng of template DNA. Analysis of mixed colonization using R-M system genes. Of the 68 samples from Merida, 31 samples were further analyzed for the prevalence of mixed colonization based on the hpyIR and hpyIIIR loci (Fig. 2). Of the 31 samples analyzed, 8 (26%) showed the presence of mixed colonization, based on results involving either the hpyIII or hpyI locus (Table 5). For three (38%) of these eight samples, mixed colonization also had been detected by LiPA at the vacA s or m loci; thus, five new samples of mixed colonization were detected. DISCUSSION Studies comparing vacA sequences in isolates from different countries found geographic variation in the distribution of genotypes s1a, s1b, and s1c and differences in m1 and m2 distribution (10, 62). As elsewhere, the H. pylori alleles currently present in Venezuelans reflect the ancestry of their
progenitors (24, 29). That the population in Puerto Ayacucho, predominantly Amerindian, carries H. pylori with high prevalences of cagA and vacA s1c genotypes, as in East Asian communities, extends previous analyses suggesting a link between ancestral South American and East Asian populations (24, 29, 70). Although the s1c genotype was found in fewer than half of the study subjects in the two other regions, its presence is an indication of persistence despite the admixture of European and African strains, as competition and recombination continues (23, 24). The mixtures of strains found in the host populations reflect the mixing of the human ethnicities introduced into Latin America since Columbus. In the “less mestiza” Puerto Ayacucho, the ancestral marker (vacA s1c) was found to be much less diluted with European (vacA s1b or vacA s2) strains than in Merida and Caracas. No other study has previously reported the presence of H. pylori strains possessing the East Asian marker (vacA s1c) in major cities in Latin America (35, 70). The prevalence of vacA s1c in 86% of the specimens
FIG. 1. Sensitivity analysis of R-M system alleles for detecting mixed colonization using genomic DNA. For the positive control, the template DNA (T) is 100 ng of homologous genomic DNA. For one negative control, competing heterologous DNA (C) was used at 100 ng. For each reaction, template DNA at 100 ng, 10 ng, 1 ng, 0.1 ng, 0.01 ng, or 0.001 ng was added to 100 ng of competing DNA. Another negative control included water (W) only.
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FIG. 2. Agarose gel electrophoresis of PCR products involving the hpyI (locus 1) and hpyIII (locus 2) R-M loci in gastric biopsies from patients M20 and M67. Lanes indicate PCR products or their absence at each locus. Primers amplified hpyIR and/or hrgB at locus 1 and hpyIIIR and/or hrgA at locus 2. In both patients, there is evidence for at least two different strains in each gastric biopsy.
from Puerto Ayacucho, present either as a single or as a mixed colonization, is significantly higher than in either Merida or Caracas; in the latter cities, vacA s1c only was present in mixed colonization. We were able to identify the minor allele populations in Caracas and Merida because we used the LiPA assay. LiPA is a more-sensitive method for the detection of mixed infection than is sequencing, with capability of detecting minor populations at one part in 20 (5%) to up to one part in 10,000 (0.001%) (37, 54, 55). With PCR and sequencing, usually only the major allele is detected, and thus, minor populations were not well appreciated in previous work (29, 63, 66). The detection in a gastric specimen of multiple alleles at loci in which only one allele can be present per strain implies that the host is harboring multiple different isolates. The simplest interpretation is that the multiple isolates were present in the specimen at the time of sampling. An alternative explanation is that multiple strains once were present in that host and that recombination occurred before one (or more) of the strains was lost. Thus, the remaining strain(s) represents mixed populations that are largely similar but vary at the particular alleles. Recent data provide strong support that this phenomenon occurs as well (23). Regardless of whether the differences observed reflect recombination or the actual presence of two entirely distinct strains, detection of multiple alleles indicates the presence of multiple strains in a host, past or current. In this study, evidence of colonization with multiple H. pylori strains was frequently detected. Our results likely underestimate the extent of mixed colonization, particularly in places like Puerto Ayacucho, where the circulating strains are less diverse; mixed colonization with strains of the same vacA genotypes could not be detected in our analysis. Analysis of
TABLE 5. Distribution of vacA m and s genotypes and R-M system genes in gastric samples from 31 H. pylori-positive subjects in Merida, Venezuela No. (%) of subjects with: No. of subjects
ⱖ2 genotypes (m and s genotypes)
hpyI R-M locusa
hpyIII R-M locusb
Mixed colonization (vacA and R-M loci)
31
7 (23)
4 (13)
4 (13)
3 (10)
a b
hpyIR and hrgB. hpyIIIR and hrgA.
2 signals
additional multiallelic genes would allow the identification of a greater extent of mixed infection. Analysis of biallelic loci in two R-M systems provided independent confirmation of the general phenomenon of colonization by multiple H. pylori strains. For example, in Merida, of 31 samples analyzed for R-M system alleles, 5 samples that were previously believed to show a single colonization based on vacA s and m LiPA were identified as having mixed colonization based on R-M system alleles. Moreover, analysis of more than one biopsy per patient from more than one location (antrum and corpus) would increase the power of detection of mixed colonization, as has been reported previously (30). Our major conclusions are that colonization by multiple different H. pylori strains is more common than previously reported and that the greater the number of polymorphic markers used, the greater the extent of the intrahost diversity appreciated. Previous studies have not reported or have underreported mixed colonization due to the techniques that have been used, chiefly culture-based methods followed by sequencing. The use of high-resolution techniques such as LiPA and the analysis of multiple loci have led to the understanding that mixed colonization is highly prevalent. van Doorn et al. found an 11% rate of multiple strains in hosts from northern South America, respectively (62). Other reports of colonization with multiple strains from different geographic locales have included Korea (60%), Brazil (15%), Mexico (65%), Chile (32%), and Portugal (30%) (8, 26, 36, 45, 63). In a study of 400 biopsy samples from 20 H. pylori-positive subjects in Mexico, there was evidence for colonization with multiple strains in 17 (85%) of the 20 subjects (45). Population genetics studies of H. pylori indicate a history of substantial recombination; the high rates of mixed colonization that we and others have detected provide the substrate for this extensive recombination (1, 7, 23, 57). H. pylori has been disappearing with socioeconomic development (11, 49, 51, 53). As hygienic conditions have improved, leading to diminishing H. pylori transmission, the mean number of strains per colonized person likely has declined, a phenomenon implying that the overall extinction of H. pylori may be greater than previously appreciated (13). If loss of recombination possibilities affects the health of the H. pylori population in an individual host, the likelihood of persistence, and
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thus transmission, may have been diminishing, further accelerating H. pylori’s disappearance. Evidence in animal models suggests that colonization with one particular “founding” strain may diminish subsequent colonization by another strain (32, 33, 41, 42, 56, 59, 60). The likelihood of establishing new colonization may depend upon whether the new strain has a selective advantage (15). The presence of coexisting strains of Helicobacter pylori within a single patient may be explained by limited competition for the same mucosal surface by competing strains (34). The evidence of mixed colonization can be utilized in mathematical models to better define parameter values; however, whether particular H. pylori genotypes confer survival advantages may be both host and context specific (3, 15). ACKNOWLEDGMENTS This work was supported by the National Institutes of Health grant R01GM63270, the Medical Research Service of the Department of Veterans Affairs, the Institute for Urban and Global Health (NYU), the Instituto Venezolano de Investigaciones Cientificas, and the Filomena D’Agostino Foundation. We acknowledge the collaboration of the Gastroenterology Service at Hospital General del Oeste in Caracas, Euclides Gonzalez from the Hospital Central de Puerto Ayacucho, Hospital Jose Gregorio Hernandez in Puerto Ayacucho, and Imad Kansau at the Universidad de Los Andes in Merida. REFERENCES 1. Achtman, M., T. Azuma, D. E. Berg, Y. Ito, G. Morelli, Z. J. Pan, S. Suerbaum, S. A. Thompson, A. van der Ende, and L. J. van Doorn. 1999. Recombination and clonal groupings within Helicobacter pylori from different geographical regions. Mol. Microbiol. 32:459–470. 2. Akopyants, N. S., S. W. Clifton, D. Kersulyte, J. E. Crabtree, B. E. Youree, C. A. Reece, N. O. Bukanov, E. S. Drazek, B. A. Roe, and D. E. Berg. 1998. Analyses of the cag pathogenicity island of Helicobacter pylori. Mol. Microbiol. 28:37–53. 3. Akopyants, N. S., K. A. Eaton, and D. E. Berg. 1995. Adaptive mutation and cocolonization during Helicobacter pylori infection of gnotobiotic piglets. Infect. Immun. 63:116–121. 4. Ando, T., R. A. Aras, K. Kusugami, M. J. Blaser, and T. M. Wassenaar. 2003. Evolutionary history of hrgA, which replaces the restriction gene hpyIIIR in the hpyIII locus of Helicobacter pylori. J. Bacteriol. 185:295–301. 5. Ando, T., T. M. Wassenaar, R. M. Peek, Jr., R. A. Aras, A. I. Tschumi, L. J. van Doorn, K. Kusugami, and M. J. Blaser. 2002. A Helicobacter pylori restriction endonuclease-replacing gene, hrgA, is associated with gastric cancer in Asian strains. Cancer Res. 62:2385–2389. 6. Anonymous. 1994. Infection with Helicobacter pylori. IARC Monogr. Eval. Carcinog. Risks Hum. 61:117–240. 7. Aras, R. A., J. Kang, A. I. Tschumi, Y. Harasaki, and M. J. Blaser. 2003. Extensive repetitive DNA facilitates prokaryotic genome plasticity. Proc. Natl. Acad. Sci. USA 100:13579–13584. 8. Ashour, A. A., P. P. Magalhaes, E. N. Mendes, G. B. Collares, V. R. de Gusmao, D. M. Queiroz, A. M. Nogueira, G. A. Rocha, and C. A. de Oliveira. 2002. Distribution of vacA genotypes in Helicobacter pylori strains isolated from Brazilian adult patients with gastritis, duodenal ulcer or gastric carcinoma. FEMS Immunol. Med. Microbiol. 33:173–178. 9. Atherton, J. C., P. Cao, R. M. Peek, Jr., M. K. Tummuru, M. J. Blaser, and T. L. Cover. 1995. Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with cytotoxin production and peptic ulceration. J. Biol. Chem. 270:17771–17777. 10. Atherton, J. C., P. M. Sharp, T. L. Cover, G. Gonzalez-Valencia, R. M. Peek, Jr., S. A. Thompson, C. J. Hawkey, and M. J. Blaser. 1999. Vacuolating cytotoxin (vacA) alleles of Helicobacter pylori comprise two geographically widespread types, m1 and m2, and have evolved through limited recombination. Curr. Microbiol. 39:211–218. 11. Banatvala, N., K. Mayo, F. Megraud, R. Jennings, J. J. Deeks, and R. A. Feldman. 1993. The cohort effect and Helicobacter pylori. J. Infect. Dis. 168:219–221. 12. Beji, A., P. Vincent, I. Darchis, M. O. Husson, A. Cortot, and H. Leclerc. 1989. Evidence of gastritis with several Helicobacter pylori strains. Lancet 2:1402–1403. 13. Blaser, M. J. 1998. Helicobacters are indigenous to the human stomach: duodenal ulceration is due to changes in gastric microecology in the modern era. Gut 43:721–727.
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