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DNA patterns of Helicobacter pylori isolated from gastric antrum, body, and suodenum
GASTROENTEROLOGY
1992:102:829-633
DNA Patterns of Helicobacter pylori Isolated From Gastric Antrum, Body, and Duodenum EMILY J. PREWETT, JANE BICKLEY, ROBERT J. OWEN, and ROY E. POUNDER Royal Free Hospital School of Medicine, and National Collection of Type Cultures, Central Public Health Laboratory, London, England
Biopsy specimens for culture of HeZicobacterpyZori were obtained from two different sites in the antrum, gastric body, and duodenal cap in 20 patients during endoscopic investigation of dyspepsia. H. pyZori was identified in 64 isolates obtained from 15 of the 20 patients. Analysis of chromosomal DNA from these isolates of H. pylori showed that 13 of 15 patients harbored a single strain of I-Z. pylori throughout their stomach and duodenum. Two differing If. pylori types were found in two patients. Unique DNA patterns were shown in each of the 15 patients. The genetic heterogeneity of H. pylori is unexplained but it could be of considerable value for epidemiological studies. elicobacter pylori is a microaerophilic bacteH rium frequently found in the gastric antrum of both asymptomatic subjects and patients with peptic ulcer disease.‘*’ It is usually detected in the antrum, but it also occurs in the body and fundus of the stomach and, in association with metaplastic gastric mucosa, in the duodenal cap. It does not colonize nongastric epithelium. The route of acquisition of H. pylori and other aspects of its epidemiology are not established.3 Humans are the probable reservoir, but strains similar to and possibly related to H. pylori have been recovered from the pig, baboon, and various species of monkey.4 Documentation of person-to-person spread has been hindered by the lack of bacterial typing methods. Recent studies have shown that DNA fingerprinting based on DNA digest patterns and ribopatterns provides a reproducible and sensitive method of discriminating between isolates of H. pyIori.5-7 A high level of genomic heterogeneity has been shown among H. pylori isolated from different patients in the United Kingdom, Netherlands, Australia, and Canada.5-” In this study we examined multiple isolates from different anatomical sites in a group of dyspeptic patients. The aim was to detect whether DNA heterogeneity is present in organisms found at different sites within an individual patient, or whether H. pylori with the same genomic DNA is present throughout the gastroduodenum in an individual.
Materials and Methods Twenty patients presenting for routine endoscopic assessment of their dyspepsia were investigated. Potential carriers of H. pylori were identified by history of duodenal ulceration, age, or childhood in a developing country. None had taken bismuth, antibiotics, or omeprazole within the previous 6 weeks. From these patients, biopsy specimens for culture of H. pylori were obtained from two different sites in the antrum, gastric body, and duodenal cap. An additional antral biopsy specimens was taken and examined histologically for organisms and inflammation. Each biopsy specimen was taken with a different pair of sterilized biopsy forceps. Between patients, the endoscope and biopsy forceps were washed automatically, with a cycle including a soak in 2% (vol/vol) glutaraldehyde for at least 4 minutes. The biopsy specimens for culture were placed directly in a selective enrichment medium. This consisted of Brucella broth (Difco Laboratories, East Molesey, Surrey, England) supplemented with 10% fetal calf serum, 1% (vol/ vol) Isovitalex (BBL Microbiology Systems, Becton Dickinson, Cowley, Oxford, England), 1000 U/mL polymyxin B 10 pg/mL vancomycin, and 2 yg/mL amphoteritin B.” Specimens were transported to the microbiology laboratory within 5 hours. Each biopsy specimens was then placed in 5 mL of selective enrichment medium and incubated at 37°C on a gyratory platform in a variable atmosphere incubator (Don Whitley Scientific Ltd., Shipley, Yorks, England) under microaerobic conditions (5% oxygen, 5% carbon dioxide, 2% hydrogen, and 88% nitrogen). A sample from each flask was subcultured on to Oxoid brain-heart infusion agar (Oxoid, Basingstoke, England), supplemented with 5% horse blood and 1% Isovitalex, after 48 hours. Positive growth was identified by Gram stain and production of urease, and cultures were preserved at -196°C on glass beads in Oxoid nutrient broth no. 2 containing 10% (vol/vol) glycerol.
DNA Isolation, Digestion and Electrophoresis, Vacublotting, and RNA Gene Hybridization Chromosomal DNA samples from isolates of H. pyIori were prepared using the guanidium thiocyanate reagent method13 and were incubated with restriction enzymes, HaeIII and HindIII, to produce DNA restriction digestion patterns. Sixty-four isolates were obtained from 0 1992 by the American
Gastroenterological 0016-5085/92/$3.00
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the culture of biopsy specimens from the antrum, body, and duodenum of the patients in this study, and NCTC 11637 and NCTC 11638 (National Collection of Type Cultures strains) were also included as references on all gels. Agarose gel electrophoresis of all isolates was followed by transfer of the digested DNA onto nylon membranes by means of vacublotting (Vacu-Gene XL; Pharmacia LKB Biotechnology, Uppsala, Sweden). The membranes were hybridized using a biotinylated complementary DNA probe prepared from 16s and 23s ribosomal RNA (rRNA) of H. pylori NCTC 11638. ‘,14Plasmid DNA was prepared according to the method of Kado and Liu” and was then electrophoresed and stained with ethidium bromide.
Results H. pylori was identified by culture in at least one biopsy specimen from 15 of the 20 patients, and a total of 64 isolates were obtained from the 120 biopsy samples. H. pylori-like organisms were identified on routine histology of an antral biopsy from the same 15 patients. One additional patient was found to have H. pylori-like organisms on histology, but all the culture specimens were contaminated. H. pylori was cultured from at least one of the two antral biopsies in all 15 patients; positive cultures were obtained from the gastric body of 13 of these 15 patients, and from the duodenal cap of 9 patients. The 15 patients with culture positive for H. pylori had a median age of 39 years (range, 25-75 years); 11 were male. At endoscopy, 6 of the 15 patients had an active duodenal ulcer, 5 patients had endoscopic duodenitis, and 4 patients had normal endoscopic appearances of their esophagus, stomach, and first and second parts of the duodenum. DNA from 75% (48/64) of the isolates was cut by HaeIII, which was the enzyme that gave the best-resolved digest patterns and hybridization patterns for analysis. DNA types were defined on the basis of clearly distinct DNA fingerprints with multiple band differences, and subtypes were defined when only one or two bands differed. Figure 1 shows the DNA fingerprints from 3 patients, each having one type of H. pylori in their gastroduodenum. Figure 2 shows the rRNA blot patterns for the same 3 patients’ isolates; minor subtypic variation is seen in the bacterium isolated from patient I. Thirteen different HaeIII DNA types were identified among the sets of isolates from 15 individual patients. These included 2 patients with more than one DNA type among their isolates, and the 4 patients in whom DNA from one or more isolate was not cut by HaeIII and could therefore not be designated a type. Seven patients’ groups contained more than one DNA subtype, ranging from two to six subtypes within a patient-set. However, all isolates withina patient-set were far more similar to each other in visual comparisons than to any other
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Figure 1. Agarose gel electrophoresis of Hue111 digest fragments of chromosomal DNA from H. pylori. Lanes I,9 and 26, bacteriophage-Hind111 digests; lanes 2-7, isolates from patient G; lane 8, NCTC 11637; lane IO, NCTC 11636; lanes 11-12, isolates from patient H; and lanes 13-15, isolates from patient I.
isolate from any other patient. This demonstrates the high degree of homogeneity amongst isolates from a given patient, regardless of site of biopsy. Only two patient sets in the study contained isolates of a completely different DNA type. One such patient set (patient A in Figure 3) contained an isolate from a biopsy of the body of the stomach, which differed completely from all of the other five isolates in the set. In the second patient, patient P, an antral isolate differed completely from a duodenal isolate; no growth was obtained from this patient’s remaining four biopsy specimens. Four of the 15 patients in the study had H. pylori with DNA not digested by HaeIII. The DNA from these 16 strains was digested with HindIII, but the patterns were generally not as well-resolved as those from digestion with HaeIII. Hind111 patterns showed that strain sets from 3 patients comprised isolates of the same DNA type with some subtypic variation, whereas the fourth patient set contained two DNA
March 1992
Figure 2. Ribopatterns (HaeIII digests) of DNA from the following strains of H.pylori. Lanes 2-4, isolates from patient I; lanes 5-6, isolates from patient H; lane 7, NCTC 11638; lane 9, NCTC 11637; lanes 10-25, isolates from patient G; lanes 1, 8, and 26, bacteriophage-Hi&III digests.
types. It was observed that this patient (patient P) yielded two isolates, one of which had DNA that was not cut by HaeIII; digestion of the DNA of the two isolates with HindIII showed two distinct types. Electrophoresis of plasmid extractions showed that 41% (26/64) of strains contained plasmids within the 2-7 megadalton range (Table 1). Different plasmids were present in the two patient sets that contained more than one DNA type, reinforcing the restriction digest and hybridization results. One patient set contained a single isolate with a plasmid that was not present in any of the other isolates in the set. However, this had no effect on the digest or hybridization patterns, because all isolates in this set were of the same DNA type. Discussion This study has confirmed the evidence for the considerable genomic diversity of H. pylori, i.e., unique DNA patterns were shown in each of 15 patients. Majewski and Goodwin,” Langenberg et al.,’ and Owen et a1.5have also shown unique patterns in each subject of the groups that they examined. The
DNA PATTERNS
OF GASTRODUODENAL
H. PYLORI
831
only report of identical DNA patterns found in different subjects was that of the 8 family members described by Rauws et a1.16 Other investigators who have looked at H. pylori from family contacts have not found identical isolates.‘0,17 We showed that 13 of 15 patients harbored a single strain of H. pylori throughout their stomach and duodenum. However, we identified two patients in whom two different H. pylori types were found. Beji et al. described one patient who had three different H. pylori DNA patterns from organisms cultured from three biopsy specimens at one endoscopy; this patient had an additional different strain on another occasion.l’ When H. pylori is isolated from an individual subject at different times, the organism usually has the same DNA type.5,‘0*1’Simor et al. identified different H. pylori strains by DNA restriction digests in each of 40 subjects; isolates obtained 1 year apart in three subjects gave identical chromosomal DNA restriction patterns [but one differed in the plasmid DNA content).” Forty-one percent of the isolates in the present study contained plasmids-a rate similar to that reported by Majewski and Goodwin who found plasmids in 40 of 84 isolates.” The presence of plasmids alone is therefore not particularly useful in typing H. pylori for epidemiological studies. In our study, minor subtypic DNA differences were seen in H. pylori isolated from 7 patients. These minor genomic variations may have arisen from point mutations or inversions occurring with time. In 13 of 15 patients, one DNA type was found throughout the gastroduodenum. This suggests the following three possibilities: firstly, that the subject is exposed to, and colonized by, one particular strain at one point in time; secondly, the presence of one strain in a patient may inhibit colonization by a different strain upon subsequent exposure; and thirdly, patients are exposed to infection by different strains, but usually one strain becomes dominant, thereby excluding less successful strains. Although H. pylori DNA fingerprints are highly stable in the laboratory, it is possible that H. pylori exhibits genetic instability in vivo; years of unremitting infection may result in the survival of (usually) one dominant or successful strain. We found no evidence that the selective enrichment medium used was completely inhibitory to some strains. Complete agreement was observed between successful culture and positive histology. However, the use of culture media in this and in previous studies’-” introduces a potential for bias in mixed populations of strains. DNA restriction digest data for single colonies showed that isolates of H. pylori were monocultures, and remained so after repeated subculture. *,’However, studies on strains isolated without selective enrichment are needed.
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Figure 3. Ribopatterns (HaeIII digests) of DNA from the following strains of H. pylori. Lanes 2-7, isolates from patient E; lanes 8-11, isolates from patient D; lane 12, NCTC 11636; lane 24, NCTC 11637; lanes 25-19, isolates from patient B; lanes 2-25, isolates from patient A; lanes I, 13, and 26, bacteriophageHindI digests.
Why should such genetic heterogeneity exist in H. pylori isolates? One possibility is that successful infection with the organism may itselfplace that organism under genetic stress. If acute infection with H. pylori is associated with a transient achlorhydria lg-‘* this will in turn result in luminal bacterial oveigrowth that may facilitate the formation of N-ni-
Table 1. Plasmid Patients Patient A B D E G H I K L M N P R S T
Results in H. pylori Isolates From 15 Proportion plasmid-bearing
of strains
6/6" O/5 O/4 ‘J/6 6/6 O/2 O/3 4/4 o/3 o/5 2/2 2/2b O/5 5/5 l/6
aOne isolate contained a different plasmid from the other five isolates in the set. This isolate was also a different DNA type. bThe two isolates in this set contained two different sized plasmids, and were also different DNA types.
trosamine compounds.22 The N-nitrosamine compounds may damage the DNA of the original infecting H. pylori. The surviving H. pylori with altered DNA will be subjected to selection pressures, with one organism finally dominating the ecological niche, and that organism will probably have a different genetic make-up to the original infecting organism. This hypothesis is amenable to testing in humans. The genetic heterogeneity of H. pylori is difficult to explain, but it could be of considerable value in epidemiological studies, for example, in the investigation of the spread of H. pylori within institutions or between family members. However, the finding of two strains of H. pylori colonizing the gastroduodenal mucosa of 2 of 15 patients (13%) means that multiple mucosal biopsies will be required for epidemiological studies. References Malfertheiner P, Ditschuneit H. Helicobacter pylori, gastritis and peptic ulcer. Berlin, Germany: Springer-Verlag, 1990. Menge H, Gregor M, Tytgat GNJ, Marshall NJ, McNulty CAM. Helicobacter pylori 1990.Berlin, Germany: Springer-Verlag, 1990. Graham DY. Helicobacter pylori in human populations the present and predictions of the future based on the epidemiology of polio. In: Menge H, Gregor M, Tytgat GNJ, Marshall BJ, McNulty CAM, eds. Helicobacter pylori 1990. Berlin, Germany: Springer-Verlag, 1990:97-103. Lee A, Fox J. Helicobacter pylori and other gastric spirilla: similarities and differences. In: Menge H, Gregor M, Tytgat
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GNJ, Marshall BJ, McNulty CAM, eds. Helicobacter pylori 1990.Berlin, Germany: Springer-Verlag, 1990:52-62. 5. Owen RJ, Fraser J, Costas M, Morgan D, Morgan DR. Signature patterns of DNA restriction fragments of Helicobacter pylori before and after treatment. J Clin Path01 1990;43:646-649. 6. Morgan DD, Owen RJ. Use of DNA restriction endonuclease digest and ribosomal RNA gene probe patterns to fingerprint Helicobacter pylori and Helicobacter mustelae isolated from human and animal hosts. Mol Cell Probes 1999;4:321-334. 7. Owen RJ, Bickley J, Costas M, Morgan DR. Genomic variation in Helicobacter pylori: application to identification of strains. Stand J Gastroenterol 1991;26(Suppl 181):43-50. 8. Langenberg W, Rauws EAJ, Widjojokusumo A, Tytgat GNJ, Zanen HC. Identification of Campylobacter pyloridis isolates by restriction endonuclease DNA analysis, J Clin Microbial 1986;24:414-417. 9. Oudbier JH, Langenberg W, Rauws EAJ, Bruin-Mosch C. Genotypical variation of Campylobacter pylori from gastric mucosa. J Clin Microbial 1990;28:559-565. 10. Majewski SIH, Goodwin CS. Restriction endonuclease analysis of the genome of Campylobacter pylori with a rapid extraction method: evidence for considerable genomic variation. J Infect Dis 1988;157:465-471, 11. Simor AE, Shames B, Drumm B, Sherman P, Low DE, Penner JL. Typing of Campylobacter pylori by bacterial DNA restriction endonuclease analysis and determination of plasmid profile. J Clin Microbial 1990;28:83-86. 12. Morgan DR, Mathewson JJ, Freedman R, Kraft WG. Evaluation of a selective enrichment technique for the isolation of Campylobacter pylori. FEMS Microbial Lett 1990;66:303-306. 13. Pitcher DC, Saunders NA, Owen RJ. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 1989;8:151-156. 14. Owen RJ, Costas M, Dawson C. Application of different chromosomal DNA restriction digest fingerprints to specific and subspecific identification of Campylobacter isolates. J Clin Microbial 1989;27:2338-2343.
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15. Kado CI, Liu ST. Rapid procedure for detection and isolation of large and small plasmids. J Bacterial 1981;145:1365-1373. 16. Rauws EAJ, Langenberg W, Oudbier J, Mulder CJJ, Tytgat GNJ. Familial clustering of peptic ulcer disease colonised with C. pylori of the same DNA composition (abstr). Gastroenterology 1989;96:A409. 17. Shames B, Krajden S, Babida C, Gurgis RY, Kurjanczyk L, Penner JL. First Meeting European Campylobacter Pylori Study Group, Bordeaux, France 1988:124. 18. Beji A, Vincent P, Darchis I, Husson MO, Cortot A, Leclerc H. Evidence of gastritis with several Helicobacter pylori strains (letter). Lancet 1989;2:1402-1403, 19. Petersen W, Lee E, Skoglund M. The role of Campylobacter pyloridis in epidemic gastritis with hypochlorhydria (abstr). Gastroenterology 1987;92:1575. 20. Graham DY, Smith JL, Albert LC, Yoshimure HH. Iatrogenic Campylobacter pylori infection is a cause of epidemic achlorhydria. Am J Gastroenterol 1988;83:974-980. 21. Hunt RH. Campylobacter pylori and spontaneous hypochlorhydria. In: Rathbone BJ, Heatley RV, ed. Campylobacter pyIori and gastroduodenal disease. Oxford, England: Blackwell Scientific, 1989:176-184. 22. Walter CL. Gastric juice N-nitrosocompounds. In: Reed PI, Hi11 MJ, eds. Gastric carcinogenesis. New York: Elsevier, 1988:163-173.
Received February 15,1991. Accepted August 9,1991. Address requests for reprints to: Roy E. Pounder, M.A., M.D., F.R.C.P., University Department of Medicine, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, England. The authors thank the Procter & Gamble Company, Cincinnati, Ohio, for a research grant to the National Collection for Type Cultures Doris Elliott prepared the manuscript.
1992:102:829-633
DNA Patterns of Helicobacter pylori Isolated From Gastric Antrum, Body, and Duodenum EMILY J. PREWETT, JANE BICKLEY, ROBERT J. OWEN, and ROY E. POUNDER Royal Free Hospital School of Medicine, and National Collection of Type Cultures, Central Public Health Laboratory, London, England
Biopsy specimens for culture of HeZicobacterpyZori were obtained from two different sites in the antrum, gastric body, and duodenal cap in 20 patients during endoscopic investigation of dyspepsia. H. pyZori was identified in 64 isolates obtained from 15 of the 20 patients. Analysis of chromosomal DNA from these isolates of H. pylori showed that 13 of 15 patients harbored a single strain of I-Z. pylori throughout their stomach and duodenum. Two differing If. pylori types were found in two patients. Unique DNA patterns were shown in each of the 15 patients. The genetic heterogeneity of H. pylori is unexplained but it could be of considerable value for epidemiological studies. elicobacter pylori is a microaerophilic bacteH rium frequently found in the gastric antrum of both asymptomatic subjects and patients with peptic ulcer disease.‘*’ It is usually detected in the antrum, but it also occurs in the body and fundus of the stomach and, in association with metaplastic gastric mucosa, in the duodenal cap. It does not colonize nongastric epithelium. The route of acquisition of H. pylori and other aspects of its epidemiology are not established.3 Humans are the probable reservoir, but strains similar to and possibly related to H. pylori have been recovered from the pig, baboon, and various species of monkey.4 Documentation of person-to-person spread has been hindered by the lack of bacterial typing methods. Recent studies have shown that DNA fingerprinting based on DNA digest patterns and ribopatterns provides a reproducible and sensitive method of discriminating between isolates of H. pyIori.5-7 A high level of genomic heterogeneity has been shown among H. pylori isolated from different patients in the United Kingdom, Netherlands, Australia, and Canada.5-” In this study we examined multiple isolates from different anatomical sites in a group of dyspeptic patients. The aim was to detect whether DNA heterogeneity is present in organisms found at different sites within an individual patient, or whether H. pylori with the same genomic DNA is present throughout the gastroduodenum in an individual.
Materials and Methods Twenty patients presenting for routine endoscopic assessment of their dyspepsia were investigated. Potential carriers of H. pylori were identified by history of duodenal ulceration, age, or childhood in a developing country. None had taken bismuth, antibiotics, or omeprazole within the previous 6 weeks. From these patients, biopsy specimens for culture of H. pylori were obtained from two different sites in the antrum, gastric body, and duodenal cap. An additional antral biopsy specimens was taken and examined histologically for organisms and inflammation. Each biopsy specimen was taken with a different pair of sterilized biopsy forceps. Between patients, the endoscope and biopsy forceps were washed automatically, with a cycle including a soak in 2% (vol/vol) glutaraldehyde for at least 4 minutes. The biopsy specimens for culture were placed directly in a selective enrichment medium. This consisted of Brucella broth (Difco Laboratories, East Molesey, Surrey, England) supplemented with 10% fetal calf serum, 1% (vol/ vol) Isovitalex (BBL Microbiology Systems, Becton Dickinson, Cowley, Oxford, England), 1000 U/mL polymyxin B 10 pg/mL vancomycin, and 2 yg/mL amphoteritin B.” Specimens were transported to the microbiology laboratory within 5 hours. Each biopsy specimens was then placed in 5 mL of selective enrichment medium and incubated at 37°C on a gyratory platform in a variable atmosphere incubator (Don Whitley Scientific Ltd., Shipley, Yorks, England) under microaerobic conditions (5% oxygen, 5% carbon dioxide, 2% hydrogen, and 88% nitrogen). A sample from each flask was subcultured on to Oxoid brain-heart infusion agar (Oxoid, Basingstoke, England), supplemented with 5% horse blood and 1% Isovitalex, after 48 hours. Positive growth was identified by Gram stain and production of urease, and cultures were preserved at -196°C on glass beads in Oxoid nutrient broth no. 2 containing 10% (vol/vol) glycerol.
DNA Isolation, Digestion and Electrophoresis, Vacublotting, and RNA Gene Hybridization Chromosomal DNA samples from isolates of H. pyIori were prepared using the guanidium thiocyanate reagent method13 and were incubated with restriction enzymes, HaeIII and HindIII, to produce DNA restriction digestion patterns. Sixty-four isolates were obtained from 0 1992 by the American
Gastroenterological 0016-5085/92/$3.00
Association
830 PREWETT ET AL.
GASTROENTEROLOGY Vol. 102, No. 3
the culture of biopsy specimens from the antrum, body, and duodenum of the patients in this study, and NCTC 11637 and NCTC 11638 (National Collection of Type Cultures strains) were also included as references on all gels. Agarose gel electrophoresis of all isolates was followed by transfer of the digested DNA onto nylon membranes by means of vacublotting (Vacu-Gene XL; Pharmacia LKB Biotechnology, Uppsala, Sweden). The membranes were hybridized using a biotinylated complementary DNA probe prepared from 16s and 23s ribosomal RNA (rRNA) of H. pylori NCTC 11638. ‘,14Plasmid DNA was prepared according to the method of Kado and Liu” and was then electrophoresed and stained with ethidium bromide.
Results H. pylori was identified by culture in at least one biopsy specimen from 15 of the 20 patients, and a total of 64 isolates were obtained from the 120 biopsy samples. H. pylori-like organisms were identified on routine histology of an antral biopsy from the same 15 patients. One additional patient was found to have H. pylori-like organisms on histology, but all the culture specimens were contaminated. H. pylori was cultured from at least one of the two antral biopsies in all 15 patients; positive cultures were obtained from the gastric body of 13 of these 15 patients, and from the duodenal cap of 9 patients. The 15 patients with culture positive for H. pylori had a median age of 39 years (range, 25-75 years); 11 were male. At endoscopy, 6 of the 15 patients had an active duodenal ulcer, 5 patients had endoscopic duodenitis, and 4 patients had normal endoscopic appearances of their esophagus, stomach, and first and second parts of the duodenum. DNA from 75% (48/64) of the isolates was cut by HaeIII, which was the enzyme that gave the best-resolved digest patterns and hybridization patterns for analysis. DNA types were defined on the basis of clearly distinct DNA fingerprints with multiple band differences, and subtypes were defined when only one or two bands differed. Figure 1 shows the DNA fingerprints from 3 patients, each having one type of H. pylori in their gastroduodenum. Figure 2 shows the rRNA blot patterns for the same 3 patients’ isolates; minor subtypic variation is seen in the bacterium isolated from patient I. Thirteen different HaeIII DNA types were identified among the sets of isolates from 15 individual patients. These included 2 patients with more than one DNA type among their isolates, and the 4 patients in whom DNA from one or more isolate was not cut by HaeIII and could therefore not be designated a type. Seven patients’ groups contained more than one DNA subtype, ranging from two to six subtypes within a patient-set. However, all isolates withina patient-set were far more similar to each other in visual comparisons than to any other
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Figure 1. Agarose gel electrophoresis of Hue111 digest fragments of chromosomal DNA from H. pylori. Lanes I,9 and 26, bacteriophage-Hind111 digests; lanes 2-7, isolates from patient G; lane 8, NCTC 11637; lane IO, NCTC 11636; lanes 11-12, isolates from patient H; and lanes 13-15, isolates from patient I.
isolate from any other patient. This demonstrates the high degree of homogeneity amongst isolates from a given patient, regardless of site of biopsy. Only two patient sets in the study contained isolates of a completely different DNA type. One such patient set (patient A in Figure 3) contained an isolate from a biopsy of the body of the stomach, which differed completely from all of the other five isolates in the set. In the second patient, patient P, an antral isolate differed completely from a duodenal isolate; no growth was obtained from this patient’s remaining four biopsy specimens. Four of the 15 patients in the study had H. pylori with DNA not digested by HaeIII. The DNA from these 16 strains was digested with HindIII, but the patterns were generally not as well-resolved as those from digestion with HaeIII. Hind111 patterns showed that strain sets from 3 patients comprised isolates of the same DNA type with some subtypic variation, whereas the fourth patient set contained two DNA
March 1992
Figure 2. Ribopatterns (HaeIII digests) of DNA from the following strains of H.pylori. Lanes 2-4, isolates from patient I; lanes 5-6, isolates from patient H; lane 7, NCTC 11638; lane 9, NCTC 11637; lanes 10-25, isolates from patient G; lanes 1, 8, and 26, bacteriophage-Hi&III digests.
types. It was observed that this patient (patient P) yielded two isolates, one of which had DNA that was not cut by HaeIII; digestion of the DNA of the two isolates with HindIII showed two distinct types. Electrophoresis of plasmid extractions showed that 41% (26/64) of strains contained plasmids within the 2-7 megadalton range (Table 1). Different plasmids were present in the two patient sets that contained more than one DNA type, reinforcing the restriction digest and hybridization results. One patient set contained a single isolate with a plasmid that was not present in any of the other isolates in the set. However, this had no effect on the digest or hybridization patterns, because all isolates in this set were of the same DNA type. Discussion This study has confirmed the evidence for the considerable genomic diversity of H. pylori, i.e., unique DNA patterns were shown in each of 15 patients. Majewski and Goodwin,” Langenberg et al.,’ and Owen et a1.5have also shown unique patterns in each subject of the groups that they examined. The
DNA PATTERNS
OF GASTRODUODENAL
H. PYLORI
831
only report of identical DNA patterns found in different subjects was that of the 8 family members described by Rauws et a1.16 Other investigators who have looked at H. pylori from family contacts have not found identical isolates.‘0,17 We showed that 13 of 15 patients harbored a single strain of H. pylori throughout their stomach and duodenum. However, we identified two patients in whom two different H. pylori types were found. Beji et al. described one patient who had three different H. pylori DNA patterns from organisms cultured from three biopsy specimens at one endoscopy; this patient had an additional different strain on another occasion.l’ When H. pylori is isolated from an individual subject at different times, the organism usually has the same DNA type.5,‘0*1’Simor et al. identified different H. pylori strains by DNA restriction digests in each of 40 subjects; isolates obtained 1 year apart in three subjects gave identical chromosomal DNA restriction patterns [but one differed in the plasmid DNA content).” Forty-one percent of the isolates in the present study contained plasmids-a rate similar to that reported by Majewski and Goodwin who found plasmids in 40 of 84 isolates.” The presence of plasmids alone is therefore not particularly useful in typing H. pylori for epidemiological studies. In our study, minor subtypic DNA differences were seen in H. pylori isolated from 7 patients. These minor genomic variations may have arisen from point mutations or inversions occurring with time. In 13 of 15 patients, one DNA type was found throughout the gastroduodenum. This suggests the following three possibilities: firstly, that the subject is exposed to, and colonized by, one particular strain at one point in time; secondly, the presence of one strain in a patient may inhibit colonization by a different strain upon subsequent exposure; and thirdly, patients are exposed to infection by different strains, but usually one strain becomes dominant, thereby excluding less successful strains. Although H. pylori DNA fingerprints are highly stable in the laboratory, it is possible that H. pylori exhibits genetic instability in vivo; years of unremitting infection may result in the survival of (usually) one dominant or successful strain. We found no evidence that the selective enrichment medium used was completely inhibitory to some strains. Complete agreement was observed between successful culture and positive histology. However, the use of culture media in this and in previous studies’-” introduces a potential for bias in mixed populations of strains. DNA restriction digest data for single colonies showed that isolates of H. pylori were monocultures, and remained so after repeated subculture. *,’However, studies on strains isolated without selective enrichment are needed.
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Figure 3. Ribopatterns (HaeIII digests) of DNA from the following strains of H. pylori. Lanes 2-7, isolates from patient E; lanes 8-11, isolates from patient D; lane 12, NCTC 11636; lane 24, NCTC 11637; lanes 25-19, isolates from patient B; lanes 2-25, isolates from patient A; lanes I, 13, and 26, bacteriophageHindI digests.
Why should such genetic heterogeneity exist in H. pylori isolates? One possibility is that successful infection with the organism may itselfplace that organism under genetic stress. If acute infection with H. pylori is associated with a transient achlorhydria lg-‘* this will in turn result in luminal bacterial oveigrowth that may facilitate the formation of N-ni-
Table 1. Plasmid Patients Patient A B D E G H I K L M N P R S T
Results in H. pylori Isolates From 15 Proportion plasmid-bearing
of strains
6/6" O/5 O/4 ‘J/6 6/6 O/2 O/3 4/4 o/3 o/5 2/2 2/2b O/5 5/5 l/6
aOne isolate contained a different plasmid from the other five isolates in the set. This isolate was also a different DNA type. bThe two isolates in this set contained two different sized plasmids, and were also different DNA types.
trosamine compounds.22 The N-nitrosamine compounds may damage the DNA of the original infecting H. pylori. The surviving H. pylori with altered DNA will be subjected to selection pressures, with one organism finally dominating the ecological niche, and that organism will probably have a different genetic make-up to the original infecting organism. This hypothesis is amenable to testing in humans. The genetic heterogeneity of H. pylori is difficult to explain, but it could be of considerable value in epidemiological studies, for example, in the investigation of the spread of H. pylori within institutions or between family members. However, the finding of two strains of H. pylori colonizing the gastroduodenal mucosa of 2 of 15 patients (13%) means that multiple mucosal biopsies will be required for epidemiological studies. References Malfertheiner P, Ditschuneit H. Helicobacter pylori, gastritis and peptic ulcer. Berlin, Germany: Springer-Verlag, 1990. Menge H, Gregor M, Tytgat GNJ, Marshall NJ, McNulty CAM. Helicobacter pylori 1990.Berlin, Germany: Springer-Verlag, 1990. Graham DY. Helicobacter pylori in human populations the present and predictions of the future based on the epidemiology of polio. In: Menge H, Gregor M, Tytgat GNJ, Marshall BJ, McNulty CAM, eds. Helicobacter pylori 1990. Berlin, Germany: Springer-Verlag, 1990:97-103. Lee A, Fox J. Helicobacter pylori and other gastric spirilla: similarities and differences. In: Menge H, Gregor M, Tytgat
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GNJ, Marshall BJ, McNulty CAM, eds. Helicobacter pylori 1990.Berlin, Germany: Springer-Verlag, 1990:52-62. 5. Owen RJ, Fraser J, Costas M, Morgan D, Morgan DR. Signature patterns of DNA restriction fragments of Helicobacter pylori before and after treatment. J Clin Path01 1990;43:646-649. 6. Morgan DD, Owen RJ. Use of DNA restriction endonuclease digest and ribosomal RNA gene probe patterns to fingerprint Helicobacter pylori and Helicobacter mustelae isolated from human and animal hosts. Mol Cell Probes 1999;4:321-334. 7. Owen RJ, Bickley J, Costas M, Morgan DR. Genomic variation in Helicobacter pylori: application to identification of strains. Stand J Gastroenterol 1991;26(Suppl 181):43-50. 8. Langenberg W, Rauws EAJ, Widjojokusumo A, Tytgat GNJ, Zanen HC. Identification of Campylobacter pyloridis isolates by restriction endonuclease DNA analysis, J Clin Microbial 1986;24:414-417. 9. Oudbier JH, Langenberg W, Rauws EAJ, Bruin-Mosch C. Genotypical variation of Campylobacter pylori from gastric mucosa. J Clin Microbial 1990;28:559-565. 10. Majewski SIH, Goodwin CS. Restriction endonuclease analysis of the genome of Campylobacter pylori with a rapid extraction method: evidence for considerable genomic variation. J Infect Dis 1988;157:465-471, 11. Simor AE, Shames B, Drumm B, Sherman P, Low DE, Penner JL. Typing of Campylobacter pylori by bacterial DNA restriction endonuclease analysis and determination of plasmid profile. J Clin Microbial 1990;28:83-86. 12. Morgan DR, Mathewson JJ, Freedman R, Kraft WG. Evaluation of a selective enrichment technique for the isolation of Campylobacter pylori. FEMS Microbial Lett 1990;66:303-306. 13. Pitcher DC, Saunders NA, Owen RJ. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 1989;8:151-156. 14. Owen RJ, Costas M, Dawson C. Application of different chromosomal DNA restriction digest fingerprints to specific and subspecific identification of Campylobacter isolates. J Clin Microbial 1989;27:2338-2343.
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15. Kado CI, Liu ST. Rapid procedure for detection and isolation of large and small plasmids. J Bacterial 1981;145:1365-1373. 16. Rauws EAJ, Langenberg W, Oudbier J, Mulder CJJ, Tytgat GNJ. Familial clustering of peptic ulcer disease colonised with C. pylori of the same DNA composition (abstr). Gastroenterology 1989;96:A409. 17. Shames B, Krajden S, Babida C, Gurgis RY, Kurjanczyk L, Penner JL. First Meeting European Campylobacter Pylori Study Group, Bordeaux, France 1988:124. 18. Beji A, Vincent P, Darchis I, Husson MO, Cortot A, Leclerc H. Evidence of gastritis with several Helicobacter pylori strains (letter). Lancet 1989;2:1402-1403, 19. Petersen W, Lee E, Skoglund M. The role of Campylobacter pyloridis in epidemic gastritis with hypochlorhydria (abstr). Gastroenterology 1987;92:1575. 20. Graham DY, Smith JL, Albert LC, Yoshimure HH. Iatrogenic Campylobacter pylori infection is a cause of epidemic achlorhydria. Am J Gastroenterol 1988;83:974-980. 21. Hunt RH. Campylobacter pylori and spontaneous hypochlorhydria. In: Rathbone BJ, Heatley RV, ed. Campylobacter pyIori and gastroduodenal disease. Oxford, England: Blackwell Scientific, 1989:176-184. 22. Walter CL. Gastric juice N-nitrosocompounds. In: Reed PI, Hi11 MJ, eds. Gastric carcinogenesis. New York: Elsevier, 1988:163-173.
Received February 15,1991. Accepted August 9,1991. Address requests for reprints to: Roy E. Pounder, M.A., M.D., F.R.C.P., University Department of Medicine, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, England. The authors thank the Procter & Gamble Company, Cincinnati, Ohio, for a research grant to the National Collection for Type Cultures Doris Elliott prepared the manuscript.