Detection and characterization of Helicobacter pylori from patients with gastroduodenal diseases

Detection and Characterization of Helicobacter pylori from Patients with Gastroduodenal Diseases Franco Busolo, Giuseppe Bertollo, Graziano Bordignon, Domenico Madia, and Davide Camposampiero

Polymerase chain reaction and cytotoxin assays were performed to identify as Helicobacter pylori type I (cagA1/ tox1) or type II (cagA2/tox2) 56 (59.6%) strains from 94 patients. Of these patients 64 were affected by nonulcer dyspepsia (NUD), 10 by gastric ulcer (GU), 19 by duodenal ulcer (DU), and 1 by both GU and DU. H. pylori strains were tested for cagA using two sets of primers; target sequences were detected in 40–42/56 (71.4–75%) depending on the set of primers used, while cytotoxin-producing strains (tox1) were 26/56 (46.4%). Tox1 strains were isolated in 13/32 (40.6%),

2/7 (28.6%), and 11/17 (64.7%) in NUD, GU, and DU patients, respectively. However, the different percentage between cagA1 strains from NUD patients (13/32; 40.6%) and patients with ulcerative diseases (13/23; 54.2%) is not statistically significant (p 5 0.462). Because the two sets of primers employed for amplification of cagA target sequences give different results, we concluded that cagA alone could not be taken as predictive factor for severity of gastroduodenal disease. It has been found that H. pylori type I is associated with duodenal ulcer disease. © 1998 Elsevier Science Inc.

INTRODUCTION

hol, blood group 0, male sex, and socioeconomical conditions) and bacterial virulence factors such as superoxide dismutase (Spigelhalder et al. 1993), urease (Smoot et al. 1990), adhesins (Evans et al. 1988), and flagella (Leying et al. 1992) seem to play an important role in the pathogenesis of gastroduodenopathic H. pylori–associated disease. In addition, two other bacterial factors that differ among H. pylori strains are the 128 to 140 kDa protein (probably needed for folding and export of the VacA protein) encoded by CagA (Covacci et al. 1993; Tummuru et al. 1993) and the approximately 90 kDa cytotoxin encoded by VacA (Cover and Blaser 1992; Cover et al. 1997; Papini et al. 1994; Schmitt and Haas 1994) that induce cellular vacuolation in different cell lines in vitro and gastric injury in animal models similar to that observed in humans (Tee et al. 1995). Four distinct populations of H. pylori for which cagA and tox1 in which markers have been correlated with the severity of disease in a mouse model (Ghiara et al. 1995) include: bacteria named type I (cagA1/tox1), bacteria named type II (cagA2/tox2),

Helicobacter pylori is a Gram-negative microaerophilic and spiral bacterium infecting the gastric mucosa of over 50% of people throughout the world (Blaser 1992). Most patients are asymptomatic or paucisymptomatic (Dooley et al. 1989). H. pylori is the causative agent of chronic and active antral gastritis (Lee et al. 1993) and it is considered a real lifetime risk (Sipponen et al. 1990) for the development of gastroduodenal ulcer (Nomura et al. 1994; Peterson 1991), gastric adenocarcinoma (Blaser et al. 1995; Parsonnet et al. 1991; Peek et al. 1997), or MALT lymphoma (Bayerdorffer et al. 1995) in about 10% of the infected patients. Both host factors (smoking, alcoFrom the Institute of Microbiology of Padua University, Faculty of Medicine, Padua (FB, DC), and the Gastroenterology and Microbiology Department of the Castelfranco Veneto Hospital, Treviso, Italy (DM). Address reprint requests to Dr. Franco Busolo, Institute of Microbiology, Padua University, Via A. Gabelli, 63, 35121 Padova, Italy. Received 31 October 1997; revised and accepted 28 March 1998.

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F. Busolo et al.

532 the intermediate strains carrying cagA but lacking of cytotoxin activity, and vice versa. The aim of the present study was to establish (i) the incidence of H. pylori strains and of vacuolating cytotoxin-producing strains from patients with nonulcer dyspepsia (NUD), gastric ulcer, (GU) and duodenal ulcer (DU); (ii) the correlation between type I and type II bacteria with NUD, GU, and DU; and (iii) the genetic variability of cagA using two sets of primers by polymerase chain reaction (PCR).

MATERIALS AND METHODS Patients and Collection of Specimens Ninety-four patients (62 males and 32 females with a mean age of 49.1 years, and ranging from 26 to 75 years of age) were scheduled for upper gastroduodenal endoscopy and enrolled by the Endoscopic Department of the Castelfranco Veneto Hospital (Treviso, Italy) in 1995 and 1996. Of these 64 were affected by NUD, 11 by GU, 20 by DU, and 1 had both GU and DU. The patients included in the present study did not receive antimicrobial agents within the preceding 3 weeks. Three biopsy specimens were collected from each patient: One was conserved in 10% buffered formalin for histologic examination, the second was frozen and stored at 280°C and the third, transferred to a vial containing 1 drop of sterile saline to avoid drying, was used for microbiologic examination within 2 h.

Culture and Identification of H. pylori Mucosal biopsy specimen was split in two parts: The first was squashed and placed between slides, dried, and fixed for Gram’s stain, and the second was finely minced and inoculated on the agar pylori medium (Becton Dickinson, Cockeysville, MD) plates. After incubation at 37°C under microaerophilic conditions (10% CO2, 5% O2, and 85% N2) for 8 to 10 days, the plates were examined for colonies that exhibited characteristic morphology. H. pylori was identified on the basis of morphology (Gram-negative spiral rod on Gram smear) and biochemical characteristics such as catalase, oxidase, and the rapid ability to hydrolyze urea (Balows et al. 1991).

H. pylori DNA Extraction and Amplification The reference strain of H. pylori, NCTC 11637, kindly provided by Dr. I. Luzzi (Istituto Superiore di Sanita`, Rome), was grown as described above and DNA was extracted using Chelex-resin 100 (Bio-Rad, Richmond, CA, USA). This method allows DNA extrac-

tion without phenol/chloroform and deproteinization steps. Briefly, after incubation in a boiling water bath for 8 min, a pellet from 1 mL of H. pylori suspension was incubated at 56°C for 30 min with an equal volume of 10% Chelex-resin. After further boiling for 5 min the sample was centrifuged (Biofuge A, Heraeus Sepatech, Germany) for 3 min at 12,000 3 g and the supernatant was processed for PCR. DNA extracted from the isolate was mixed with a solution of dATP, dTTP, dCTP, and dGTP (final concentration 0.4 mM each) in PCR buffer (10 mM tris-HCl, pH 8.3; 50 mM KCl; 3 mM MgCl2), containing 0.4 mM of each primer and 2 U of Taq polymerase (AmpliTaq; PerkinElmer, Saint-Quentin, France), in a final volume of 50 mL. Amplification was carried out for 40 cycles (1 min at 94°C, 1 min at 53°C, and 1 min at 72°C) in a DNA thermal cycler (Perkin-Elmer Cetus). Positive controls (DNA from the reference strain of H. pylori) and negative controls (H2O, human DNA and DNA from other bacteria such as Pseudomonas aeruginosa ATCC9027, Staphylococcus aureus ATCC 6538, and Mycoplasma pneumoniae strain-FH), were run in the same way. We used two sets of primers, the first was that named 93089 and 93261, used by Lage et al. (1995), and the second was selected from the published nucleotide sequence (Xiang et al. 1995) of the cytotoxinassociated antigen CagA gene (4826 bp) encoding for a 96- to 138-kDa protein that is associated with the production of vacuolating toxin (Covacci et al. 1993; Tummuru et al. 1993). The sequence of the primers from 59 to 39 are as follows: Set I 93089 (20 mers) 59 AAT ACA CCA ACG CCT CCA AG 39 93261 (20 mers) 59 TTG TTG CCG CTT TTG CTC TC 39 (2593–2973/400 bp) Set II cagA-up (21 mers) 59 ATC GTA TTG CTT TTG TTT CTA 39 cagA-lp (21 mers) 59 TCT TGG AGG CGT TGG TGT ATT 39 (2398–2593/216 bp) The numbers between parentheses indicate the sites of primer recognition and the size of the PCR product fragments.

Cytotoxin Assay Freshly isolated H. pylori strains were abundantly seeded in 15 mL of Brucella broth (Becton Dickinson) containing 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA) and 1% Isovitalex (Becton Dickinson). After 4 days of incubation at 37°C in microaerophilic atmosphere with continuous agitation (100 oscillations per min), the broth cultures were centrifuged at 30003 g and the supernatant was ster-

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533

ilized by filtration through a 0.22-mm-pore-size filter and stored at 280°C until examination on cell cultures. The HeLa 229 cell monolayers, which appear to be the most sensitive cells for the detection of vacuolating cytotoxin (Leunk et al. 1988; Tee et al. 1995), were used as the target cells for the cytotoxin assay. The day before assay the cells were trypsinized and resuspended in Earle (EMEM) 1 10% FBS at the concentration of 105 cells/mL and 200 mL were seeded in each well of a 96-well flat-bottom plate (Costar, Cambridge, MA, USA) to produce a semiconfluent monolayer after 24 h of incubation at 37°C. The culture medium was removed from each well and the assays were performed in duplicate on 24-h semiconfluent monolayers. A 200-mL aliquot of an equal volume of the filtered broth culture and of EMEM 1 2% FBS was added to the cell monolayers. After incubation at 37°C in a 5% CO2 atmosphere cells were examined for cytotoxic effect by reading at 2-h intervals within the first 12 hours, 24 hours, and 48 hours after exposure. The broth culture of the reference H. pylori NCTC 11637 strain was used as positive cytotoxin control and uninoculated broth was used as a negative control. At the present time the technique for detection of vacuolating cytotoxin is not yet standardized and this makes it difficult to define the threshold of detectable cytotoxin. H. pylori was considered as cytotoxic (tox1) when vacuolating effect was seen in more than 50% of the cells within 48 hours of incubation. Statistical analysis of comparison of percentages was performed by the chisquared method.

RESULTS Ninety-four patients were examined. Of them, 64 were affected by NUD, 11 by GU, and 20 by DU. In the last group a patient with both GU and DU has been included. Fifty-six (59.5%) H. pylori strains were isolated; 32 (50%) from patients with NUD, 7 (63.6%) from patients with GU, and 17 (85%) from patients with DU (Table 1). These differences in percentage are statistically significant (p 5 0.02). TABLE 1 Presence of H. pylori in Gastroduodenal Diseases H. pylori Clinical Disease (no. of patients)

No.

%

NUD (64) GU (11) DU (20) total patients (94)a

32 7 17 56

50 63.6 85 59.5

a

One H. pylori–positive patient affected by gastric and duodenal ulcers has been included in the DU group (Chi-squared 5 7.827 (d.f. 5 2); p 5 0.02).

FIGURE 1 Vacuolating effect of a filtrated supernatant of a cytotoxic H. pylori strain on 24-h HeLa 229 semiconfluent cell monolayer.

Using the method specified above, we tested the 56 wild strains for the ability to produce the vacuolating cytotoxin. The cytopatic effects on the HeLa cell monolayers (Figure 1) were an evident intracellular vacuolization similar to that previously described by other authors (Cover and Blaser 1992; Ghiara et al. 1995; Xiang et al. 1995). Out of the 56 strains of H. pylori tested, 26 (46.4%) produced a vacuolating cytotoxin and were defined as tox1 strains: 13 (40.6%) strains were from NUD patients and 11 (64.7%) were from DU patients. Only in two cases (Table 2) were tox1 H. pylori strains isolated from patients affected by GU (p 5 0.164). The presence of the gene coding for cagA protein was investigated in all wild strains using two different sets of primers: set I and set II. Analysis of our clinical isolates indicate that when using both sets the cagA target sequence was detected with a high frequency, ranging from 71.4% (40/56 strains) to 75% (42/56 strains) using set I and set II, respectively. In Figure 2 are reported only data obtained with set I. When tested with the first set of primers H. pylori were cagA1 in 22/32 (68.7%) strains of NUD patients, 3/7 (42.8%) of GU patients, and 15/17 (88.2%) of patients affected by DU (Table 3). According to the definition of H. pylori type I (cagA1/tox1) and H. pylori type II (cagA2/tox2) we

TABLE 2 H. pylori Producing Vacuolating Cytotoxin and Gastroduodenal Diseases Clinical Disease

H. pylori tox1/H. pylori

%

NUD GU DU

13/32 2/7 11/17 26/56

40.6 28.6 64.7 46.4

Chi-squared 5 3.614 (d.f. 5 2); p 5 0.164.

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FIGURE 2 Typical electrophoresis of PCR products of 4 H. pylori strains Cag A1 with both set I and set II of primers (set I: lanes 2, 3, 4, and 7; set II: lanes 10, 11, 12, and 15). One H. pylori strain Cag A2 with both set I and set II of primers (lanes 6 and 14). One H. pylori strain CagA2 with set I and CagA1 with set II (lanes 5 and 13). DNA extracted from the reference H. pylori NCTC 11637 strain was used as template (lanes 1 and 9). Molecular weight markers: 557 bp, 374 bp, 308, and 216 bp (lane 8).

found type I in 23/56 (41%) and type II in 13/56 (23%) of the total strains isolated. H. pylori type I was found in 15/38 males (39.4%) with lower frequency than in female patients (8/18; 44.4%). Using caution in interpreting data on small patients groups, the more virulent H. pylori type I was found significantly more often (10/17; 58.8%) in patients affected by DU than in patients with GU (only in one patient).

DISCUSSION In a previous study Xiang et al. (1995) proposed that most of the clinical isolates can be divided into two broad groups: type I bacteria with cagA and producing vacuolating cytotoxin (cagA1, tox1), and type II bacteria that do not have cagA and do not produce the vacuolating cytoxin (cagA2, tox2). Statistical analysis of our results indicate that H. pylori is significantly more present in GU and DU patients when compared with NUD patients (p 5 0.02). H. pylori tox1 strains were isolated from all the groups exam-

ined, but no statistically significant difference (p 5 0.164) was observed. According to the above mentioned definition, we found that the comparison of the frequency of H. pylori type I among the groups of NUD, GU, and DU patients did not reveal any statistically significant difference. Nevertheless, in the last group of patients, the association between H. pylori type I and DU (10/17; 58.8%) as well as the low rate of H. pylori type II in the same group (1/17; 5.9%), appear very suggestive, in agreement with data presented by Cover et al. (1995). How can we explain the high rate of H. pylori type I (12/32; 37.5%) in NUD patients? Some hypotheses can be proposed: (i) NUD patients may be at the initial phase of the ulcerative disease and follow-up will allow us to verify this assumption; (ii) Some NUD patients are more resistant to the virulence factors, or differ in cytokines secretion ability (Phadnis et al. 1994). H. pylori type I induces significantly higher IL-8 secretion in gastric epithelial cell lines than type II and this may contribute to the development of gastroduodenal ulcerative diseases (Crabtree et al. 1994); and (iii) tox1 strains are influenced in the secretion and/or the release of the vacA product (Logan and Berg 1996; Phadnis et al. 1994). On the other hand it has been demonstrated that, in vivo, H. pylori can become type I by recombination between genomes (Garner and Cover 1995) and that tox1 and tox2 strains differ substantially within the middle region of vacA (Cover et al. 1994). This region exhibits a great diversity (Atherton et al. 1995; Jiang et al. 1996) and various vacA genotypes of H. pylori are correlated with a different level of in vitro cytotoxin activity (Atherton et al. 1995). Comparison between percentages of cagA1, tox2 strains in NUD patients (10/32; 31.2%) and patients with ulcerative diseases (7/24; 29.2%) did not show significant difference. This might probably indicate that both factors, cagA gene and cytotoxin production, are needed for the complete expression of pathogenicity of the strain. The presence of cagA gene, encoding for a protein probably needed for folding and export of the vacA protein (Tee et al. 1995; Xiang et al. 1995), and the ability of H. pylori to produce vacA cytotoxin have been

TABLE 3 Genetic and Phenotypic Characteristics of 58 Isolates of H. pylori from Patients with Gastroduodenal Diseases Disease (no. of isolates)

cagA1, tox1

cagA2, tox2

cagA1, tox2

cagA2, tox1

NUD (32) GU (7) DU (17) Total strains (56)

12 1 10 23

9 3 1 13

10 2 5 17

1 1 1 3

CagA1 was detected using set I of primers. H. pylori (cagA1, tox1) type I vs H. pylori not type I: chi-squared 5 4.457 (d.f. 5 2); p 5 0.108.

Helicobacter pylori in Gastroduodenal Diseases correlated with severity of the clinical disease (Tee et al. 1995; Telford et al. 1994). Our data on amplification of cagA target sequence in 56 isolates using two different sets of primers seem to be in accordance with the existence of variable sequences. The fact that only two cases with cagA were detected by set II where set I failed indicates a variability within the gene. Therefore, not only vacA, but also cagA contains variable regions. This is probably due to small deletions within cagA and it could be compatible with the variable size, 128 to 140 kDa, of the cagA protein. Furthermore, the fact that cagA may be detected by using a specific set of primers and not with other, suggests that cagA alone in H. pylori could not be used as a single reliable predictor for higher risk of development of disease. We found that patients with DU were more fre-

535 quently infected with H. pylori (cagA1) strains (88.8%) than GU patients (42.8%; that becomes 57% with the second set of primers); these data are substantially in agreement with those of Cover et al. (1995) who found cagA1 in 81% and 68% of patients with DU and GU, respectively. Nevertheless, because in the present study the percentage of the cagA-containing strains depends on the set of the primers used, and no significant association was found between the presence of cagA and ulcerative (18/24; 75%) or nonulcer dyspepsia (22/ 32; 68.7%) diseases, at the present time the detection of cagA gene alone in H. pylori cannot be used as a predictive index for a patient’s developing ulcer disease. According to our data, the characterization of H. pylori for its possible role in human disease must be based on the demonstration of both virulence factors.

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