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Helicobacter pylori isolated from stomach corpus and antrum: Comparison of DNA patterns
Journal of Infection (1996) 32, 219-221
Helicobacter pylori Isolated from Stomach Corpus and Antrum: Comparison of DNA Patterns L. Cellini, N. AIIocati, E. Di Campli, M. Masulli, S. Di Bartolomeo and B. Dainelli Istituto di Medicina Sperimentale, Facoltd~ di Medicina, Universitd~ 'G. D'Annunzio', Via dei Vestini 31, 6 6 1 0 0 Chieti, Italy Accepted for publication 13 January 1 9 9 6
The genomic DNA of Helicobacter pylori was studied in strains isolated from two different sites of the stomach: the corpus and the antrum. 70 strains of H. pylori were found in 36 patients; 34 out of the 36 patients harboured the strain in both districts analysed. Restriction endonuclease analysis with Hae III and Hind III was used to compare the DNA patterns of strains isolated from the anatomical sites studied. Two pairs of DNA samples u~ere not digested by these enzymes. 27 of the 32 pairs of the digested DNA appeared similar to each other. The analysis of chromosomal DNA in the remaining five pairs showed different electrophoretic patterns. These results indicate that the gastric mucosa can be colonized, at the same time, by strains of H. pylori with different genomic patterns, and this aspect can be important for epidemiologicaI studies.
Introduction
Materials and Methods
The discovery of Helicobacter pylori, 1 has led to the recognition that the aetiology of gastritis and peptic ulcer disease m a y involve infectious a g e n t s Y Therefore, it is possible to support a role for H. pylori in the development of gastric ulceration 4 and gastric cancer, s The source of the organism is humans, but the mode of transmission is unknown. Both faecal-oral and oral-oral routes have been supposed. 6 Bacterial DNA restriction endonuclease analysis, ribopatterns and the plasmid profile were more reproducible and sensitive methods to discriminate a m o n g H. pylori isolates from different gastric sites. 7-9 The genetic heterogeneity of H. pylori is wide spread from different strains isolated 8 and is probably due to the presence of N-nitrosamine compounds in vivo (in the stomach environment) when transient achlorhydria is found, n Since relapses of the illness have demonstrated the persistence of the same strain of It. pylorl 1° or reinfection of different newly acquired strains, 12 the results so far have been discordant. The aim of this work was to assess the presence of different DNA patterns of H. pylori in two anatomical sites of the stomach (the a n t r u m and the corpus) and to attempt the detection of the mode of transmission, not yet known, for this microorganism.
Specimens
Address correspondence to Luigina Cellini, Istituto di Medicina Sperimentale, Facolt~di Medicina, Universit&'G. D'Annunzio',Via dei Vestini, 31-66100 Chieti, Italy. 0163-4453/96/030219 + 03 $12.00/0
A total of 42 outpatients (24 M, 18 F, mean age 43.5 years, range 2 5 - 6 0 years) with dyspeptic symptoms were examined. Six biopsies, three from the a n t r u m and three from the corpus were taken from each patient. One biopsy, from each location, was drawn for CLO test (Delta West, Pry Ltd, Bentley, Western Australia), one biopsy from the a n t r u m and the corpus for bacterial culture was placed in 0.4 ml of Brucefia Broth (BB, Biolife Italiana, Italy). The other specimens were fixed in 10% buffered formalin and embedded in paraffin for routine histopathological examination. 5 m m thick sections stained by Hematoxylin and Eosin (H & E) and Giemsa procedure were examined. Tissue sections were evaluated by a pathologist without prior clinical information.
Microbiological technique
Stomach samples were processed microbiologically within I h. Biopsies were trimmed with a razor, homogenized and cultured on Chocolate agar plus 1% IsoVitaleX (CA, Becton Dickinson & Co., Cockeisville, MD, U.S.A.) and Campylobacter selective medium (CSM, Unipath Ltd, Basingstoke U.K.). Plates were incubated in a microaerophylic atmosphere at 37°C for 5 days. Typical H. pylotq colonies were identified by standard methods. 13,14 © 1996 The British Society for the Study of Infection
DNA Patterns of Heficobacter pylori
220
1100[
Results H. pylori was isolated in at least one biopsy specimen from 36 patients out the 42 studied (85.7%). The strains were recovered from 16 females to 18 (88.9%) and from 20 males to 24 (83.3%). Figure 1 compares the incidence of H. pylori to the sex and to the age of the patients studied. When /-/. pylori was isolated by culture from one site, all investigating methods were positive for those anatomical sites; in one case /-/. pylori was not found in the culture and in CLO test but spiral shaped bacteria were recognized with histopathological examination. A total of 70 H. pylori were cultured from 84 biopsy samples. Thirty-four patients showed the microorganism from both the a n t r u m and the corpus, two patients were H. pylori-positive in the a n t r u m only. Table I shows the results of laboratory investigations in the patients studied. Hae III was the most discriminatory restriction endonucleases enzyme for digestion of the 34 pairs of chromosomal DNA. In fact, 25 out the 34 pairs of DNA
80 65 6O ¢9
40
-~ 2o
o
<35
35-50
>50
Age Figure 1. Percentage of patients H. pylori positive compared with the age and the sex. ( , ) male; (D) female.
Table I. Results of laboratory investigation in dyspeptic patients for H. pylori. Clinical condition (number of patients)
Active gastritis (13) Duodenal ulcer (24) Gastric ulcer (2) No gastritis (3)
H. pylori detection (%) with Histology
Culture of biopsy
CLO test
Antrum
Corpus
Antrum
Corpus
Antrum
Corpus
10 (77) 23 (96) 2 (100) 1 (33)
9 (69) 22 (92) 2 (100) 1 (33)
10 (77) 23 (96) 2 (100) 1 (33)
9 (69) 22 (92) 2 (100) 1 (33)
10 (77) 23 (96) 2 (100) 1 (33)
9 (69) 23 (96) 2 (100) 1 (33)
Subcultures of a single colony of the bacteria isolated from each pair of biopsy were used for DNA fingerprinting.
DNA isolation, digestion and electrophoresis Chromosomal DNA samples from H. pylori isolated from gastric corpus and a n t r u m of 34 patients and H. pylori NCTC 11639 were extracted as previously described.9 The purified DNA was incubated with restriction endonucleases Hae III (Boehringer Mannheim Italia SpA, Milano, Italy) for 4 h at 37°C and the digests were electrophoresed at 30V for 16 h in horizontal agarose. DNA of the strains which did not cut with Hae IfI were subjected to Hind III (Boehringer) digestion. After electrophoresis the gels were stained with ethidium bromide and photographed. Plasmid profiles demonstrated by electrophoresis of undigested DNA according to S ambrook et al. is
samples were cut by this enzyme, Hind III cut seven pairs of DNA patterns and only two pairs of samples were not cut by these enzymes. Twenty-seven patients harboured a unique strain of H. pylori from the a n t r u m and the corpus. The strains isolated from the a n t r u m and the corpus of the remaining five patients showed different electrophoretic patterns. Two DNA patterns were considered unlike when multiple bands were different, DNA subtypes were defined w h e n only one or two bands differed. Figure 2 shows the pair of DNA fingerprints from the five patients with unlike DNA patterns. These pairs of unlike DNA patterns from the same patient were different from each other but more similar t h a n chromosomal DNA fingerprints obtained a m o n g the other patients. In particular, the strains isolated from patients 2 and 7 showed a few differences between DNA fingerprints, but
L. Cellini et al.
Kb
¢q
eq
23.1--~ 9.4--~ 6.6~-~ 4.4-~
2.3 ---~ 2.0---~
221
isolated in the same patients compared with the differences among H. pylori isolated in different patients. This last aspect can support the hypothesis of a genetic variability in vivo due to the selective environmental pressure 8 but multicolonization of the gastric mucosa cannot be excluded. Many epidemtologic studies were found to determine the ecology, means of transmission, and natural history of H. pylori infection but, at the present time, the mode of transmission of this microorganism is doubtful. More studies on this aspect should clarify the clinical significance of these different strains in the same patient. Therefore, for epidemiological studies, an analysis of the genomic patterns of strains isolated from biopsies drawn in different anatomical sites of the stomach is needed.
Acknowledgments This work was supported by grants no CU.93.04315 from the Consiglio Nazionale delle Ricerche and of the Ministero dell'Universitgt e delia Ricerca Scientifica e Tecnologica (60%). Figure 2. Agarose gel electrophoresis of Hae III digests of chromosomal DNA from five pairs of H. pyIori isolated in the antrum (a) and in the corpus (b). Size indicated are for bacteriophage )v Hind III digest and H. pylori NCTC 11639 (type strain) was included as a reference.
for patient 7 there was a different plasmid profile supporting the differences between the strains. Analysing the undigested chromosomal DNA, 22 out the 68 strains contained plasmids of 2.3-23.1 Kb. From the five pairs of unlike strains isolated in the same patients, three pairs of DNA were without plasmids, in one pair only one strain contained a plasmid and in the last pair three plasmids were recovered in one strain and one unlike plasmid was recovered in the other strain.
Discussion This study confirms the presence in the gastric mucosa, of the same patient and at the same time, of different strains of H. pylori. Beji et al. described, in one patient, four different strains isolated over 1 year 12 and Owen et al. described single patients with unlike H. pylori, s and also patients examined at different times with the same stable strains of H. pyIorL 1° Our data shows the presence of the same strains of H. py!ori in the gastric mucosa and in the a n t r u m in 27 out of 34 patients isolated in the two anatomical sites. Five patients harboured H. pylori in the a n t r u m and in the corpus with different chromosomal DNAs and two of them had different plasmids. These differences were less marked between H. pylori
References 1 Marshall B. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1983; 1: 1273-1275. 2 Lee A. The microbiology and epidemiology of Helicobacter pylori infection. Scand] Gastroenterol 1994; 29 (Snppl, 201): 2-6. 3 Marshall BJ. Helicobacterpylori. Am J Gastroentero11994; 89: $116$122. 4 Dick ]D. Helicobacter (Caropylobacter) pylori: a twist on an old disease. Ann Rev Microbiol 1990; 108: 70-90. 5 Parsonnet J, Friedman GD, Vandersteen DP et al. Helicobacter pylori infection and the risk of gastric carcinoma. New Engl J Med 1991; 325: 1127-1131. 6 Mendall MA, Northfiels TC. Transmission of Helicobacter pylori. Gut 1995; 37: 1-3. 7 Ferguson DA Jr, Li C, Patel RN, Mayberry RW. Chi SD, Thomas E. Isolation of Helicobacter pylori from saliva. J Clin Microbiol 1.993; 31: 2802-2804. 8 0 w e n RJ, Bickley J, Costas M, Morgan DR. Genomic variation in Helicobacter pylori: application to identification of strains. Scand J Gastroenterol 1991; 26 (Suppl. 181): 43-50. 9 Simor AE, Shames B, Drumm P, Sherman P, Low DE, Penner JL. Typing of Caropglobacter pylori by bacterial DNA restriction endonuclease analysis and determination of plasmid profile. J Clin Microbiol 1990; 28: 83-86. 10 Fraser AG, Bicldey J, Owen RJ, Pounder RE. DNA fingerprints of Helicobaeter pylori before and after treatment with omeprazole. J Clin Pathol 1992; 42: 785-791. 11 Walter CL. Gastric juice N-nitrosocompounds. In: Reed PI, Hill MJ, eds. Gastric carcinogenesis. New York: Elsevier, 1988: 163-173. 12 Beji A, Vincent P, Darchis I, Husson MO, Cortot A, Leclerc H. Evidence of gastritis with several Helicobacter pylori strains (letter). Lancet 1989: ii: 1402-1403. 13 Cellini L, Allocati N, Piccolomini R, Di Campli E, Dainelli B. New plate medium for growth and detection of urease activity of Helicobacter pylori. J Clin Microbial 1992; 30: 1351-1353. 14 Hazell SL. Cultural techniques for the growth and isolation of Helicobacter pglori. In: Goodwin CS, Worsley BW, eds. Helicobacter pylori: biology and clinical practice. Boca Raton, Florida: CRC Press, 1993; 2 73-283. 15 8ambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.
Helicobacter pylori Isolated from Stomach Corpus and Antrum: Comparison of DNA Patterns L. Cellini, N. AIIocati, E. Di Campli, M. Masulli, S. Di Bartolomeo and B. Dainelli Istituto di Medicina Sperimentale, Facoltd~ di Medicina, Universitd~ 'G. D'Annunzio', Via dei Vestini 31, 6 6 1 0 0 Chieti, Italy Accepted for publication 13 January 1 9 9 6
The genomic DNA of Helicobacter pylori was studied in strains isolated from two different sites of the stomach: the corpus and the antrum. 70 strains of H. pylori were found in 36 patients; 34 out of the 36 patients harboured the strain in both districts analysed. Restriction endonuclease analysis with Hae III and Hind III was used to compare the DNA patterns of strains isolated from the anatomical sites studied. Two pairs of DNA samples u~ere not digested by these enzymes. 27 of the 32 pairs of the digested DNA appeared similar to each other. The analysis of chromosomal DNA in the remaining five pairs showed different electrophoretic patterns. These results indicate that the gastric mucosa can be colonized, at the same time, by strains of H. pylori with different genomic patterns, and this aspect can be important for epidemiologicaI studies.
Introduction
Materials and Methods
The discovery of Helicobacter pylori, 1 has led to the recognition that the aetiology of gastritis and peptic ulcer disease m a y involve infectious a g e n t s Y Therefore, it is possible to support a role for H. pylori in the development of gastric ulceration 4 and gastric cancer, s The source of the organism is humans, but the mode of transmission is unknown. Both faecal-oral and oral-oral routes have been supposed. 6 Bacterial DNA restriction endonuclease analysis, ribopatterns and the plasmid profile were more reproducible and sensitive methods to discriminate a m o n g H. pylori isolates from different gastric sites. 7-9 The genetic heterogeneity of H. pylori is wide spread from different strains isolated 8 and is probably due to the presence of N-nitrosamine compounds in vivo (in the stomach environment) when transient achlorhydria is found, n Since relapses of the illness have demonstrated the persistence of the same strain of It. pylorl 1° or reinfection of different newly acquired strains, 12 the results so far have been discordant. The aim of this work was to assess the presence of different DNA patterns of H. pylori in two anatomical sites of the stomach (the a n t r u m and the corpus) and to attempt the detection of the mode of transmission, not yet known, for this microorganism.
Specimens
Address correspondence to Luigina Cellini, Istituto di Medicina Sperimentale, Facolt~di Medicina, Universit&'G. D'Annunzio',Via dei Vestini, 31-66100 Chieti, Italy. 0163-4453/96/030219 + 03 $12.00/0
A total of 42 outpatients (24 M, 18 F, mean age 43.5 years, range 2 5 - 6 0 years) with dyspeptic symptoms were examined. Six biopsies, three from the a n t r u m and three from the corpus were taken from each patient. One biopsy, from each location, was drawn for CLO test (Delta West, Pry Ltd, Bentley, Western Australia), one biopsy from the a n t r u m and the corpus for bacterial culture was placed in 0.4 ml of Brucefia Broth (BB, Biolife Italiana, Italy). The other specimens were fixed in 10% buffered formalin and embedded in paraffin for routine histopathological examination. 5 m m thick sections stained by Hematoxylin and Eosin (H & E) and Giemsa procedure were examined. Tissue sections were evaluated by a pathologist without prior clinical information.
Microbiological technique
Stomach samples were processed microbiologically within I h. Biopsies were trimmed with a razor, homogenized and cultured on Chocolate agar plus 1% IsoVitaleX (CA, Becton Dickinson & Co., Cockeisville, MD, U.S.A.) and Campylobacter selective medium (CSM, Unipath Ltd, Basingstoke U.K.). Plates were incubated in a microaerophylic atmosphere at 37°C for 5 days. Typical H. pylotq colonies were identified by standard methods. 13,14 © 1996 The British Society for the Study of Infection
DNA Patterns of Heficobacter pylori
220
1100[
Results H. pylori was isolated in at least one biopsy specimen from 36 patients out the 42 studied (85.7%). The strains were recovered from 16 females to 18 (88.9%) and from 20 males to 24 (83.3%). Figure 1 compares the incidence of H. pylori to the sex and to the age of the patients studied. When /-/. pylori was isolated by culture from one site, all investigating methods were positive for those anatomical sites; in one case /-/. pylori was not found in the culture and in CLO test but spiral shaped bacteria were recognized with histopathological examination. A total of 70 H. pylori were cultured from 84 biopsy samples. Thirty-four patients showed the microorganism from both the a n t r u m and the corpus, two patients were H. pylori-positive in the a n t r u m only. Table I shows the results of laboratory investigations in the patients studied. Hae III was the most discriminatory restriction endonucleases enzyme for digestion of the 34 pairs of chromosomal DNA. In fact, 25 out the 34 pairs of DNA
80 65 6O ¢9
40
-~ 2o
o
<35
35-50
>50
Age Figure 1. Percentage of patients H. pylori positive compared with the age and the sex. ( , ) male; (D) female.
Table I. Results of laboratory investigation in dyspeptic patients for H. pylori. Clinical condition (number of patients)
Active gastritis (13) Duodenal ulcer (24) Gastric ulcer (2) No gastritis (3)
H. pylori detection (%) with Histology
Culture of biopsy
CLO test
Antrum
Corpus
Antrum
Corpus
Antrum
Corpus
10 (77) 23 (96) 2 (100) 1 (33)
9 (69) 22 (92) 2 (100) 1 (33)
10 (77) 23 (96) 2 (100) 1 (33)
9 (69) 22 (92) 2 (100) 1 (33)
10 (77) 23 (96) 2 (100) 1 (33)
9 (69) 23 (96) 2 (100) 1 (33)
Subcultures of a single colony of the bacteria isolated from each pair of biopsy were used for DNA fingerprinting.
DNA isolation, digestion and electrophoresis Chromosomal DNA samples from H. pylori isolated from gastric corpus and a n t r u m of 34 patients and H. pylori NCTC 11639 were extracted as previously described.9 The purified DNA was incubated with restriction endonucleases Hae III (Boehringer Mannheim Italia SpA, Milano, Italy) for 4 h at 37°C and the digests were electrophoresed at 30V for 16 h in horizontal agarose. DNA of the strains which did not cut with Hae IfI were subjected to Hind III (Boehringer) digestion. After electrophoresis the gels were stained with ethidium bromide and photographed. Plasmid profiles demonstrated by electrophoresis of undigested DNA according to S ambrook et al. is
samples were cut by this enzyme, Hind III cut seven pairs of DNA patterns and only two pairs of samples were not cut by these enzymes. Twenty-seven patients harboured a unique strain of H. pylori from the a n t r u m and the corpus. The strains isolated from the a n t r u m and the corpus of the remaining five patients showed different electrophoretic patterns. Two DNA patterns were considered unlike when multiple bands were different, DNA subtypes were defined w h e n only one or two bands differed. Figure 2 shows the pair of DNA fingerprints from the five patients with unlike DNA patterns. These pairs of unlike DNA patterns from the same patient were different from each other but more similar t h a n chromosomal DNA fingerprints obtained a m o n g the other patients. In particular, the strains isolated from patients 2 and 7 showed a few differences between DNA fingerprints, but
L. Cellini et al.
Kb
¢q
eq
23.1--~ 9.4--~ 6.6~-~ 4.4-~
2.3 ---~ 2.0---~
221
isolated in the same patients compared with the differences among H. pylori isolated in different patients. This last aspect can support the hypothesis of a genetic variability in vivo due to the selective environmental pressure 8 but multicolonization of the gastric mucosa cannot be excluded. Many epidemtologic studies were found to determine the ecology, means of transmission, and natural history of H. pylori infection but, at the present time, the mode of transmission of this microorganism is doubtful. More studies on this aspect should clarify the clinical significance of these different strains in the same patient. Therefore, for epidemiological studies, an analysis of the genomic patterns of strains isolated from biopsies drawn in different anatomical sites of the stomach is needed.
Acknowledgments This work was supported by grants no CU.93.04315 from the Consiglio Nazionale delle Ricerche and of the Ministero dell'Universitgt e delia Ricerca Scientifica e Tecnologica (60%). Figure 2. Agarose gel electrophoresis of Hae III digests of chromosomal DNA from five pairs of H. pyIori isolated in the antrum (a) and in the corpus (b). Size indicated are for bacteriophage )v Hind III digest and H. pylori NCTC 11639 (type strain) was included as a reference.
for patient 7 there was a different plasmid profile supporting the differences between the strains. Analysing the undigested chromosomal DNA, 22 out the 68 strains contained plasmids of 2.3-23.1 Kb. From the five pairs of unlike strains isolated in the same patients, three pairs of DNA were without plasmids, in one pair only one strain contained a plasmid and in the last pair three plasmids were recovered in one strain and one unlike plasmid was recovered in the other strain.
Discussion This study confirms the presence in the gastric mucosa, of the same patient and at the same time, of different strains of H. pylori. Beji et al. described, in one patient, four different strains isolated over 1 year 12 and Owen et al. described single patients with unlike H. pylori, s and also patients examined at different times with the same stable strains of H. pyIorL 1° Our data shows the presence of the same strains of H. py!ori in the gastric mucosa and in the a n t r u m in 27 out of 34 patients isolated in the two anatomical sites. Five patients harboured H. pylori in the a n t r u m and in the corpus with different chromosomal DNAs and two of them had different plasmids. These differences were less marked between H. pylori
References 1 Marshall B. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1983; 1: 1273-1275. 2 Lee A. The microbiology and epidemiology of Helicobacter pylori infection. Scand] Gastroenterol 1994; 29 (Snppl, 201): 2-6. 3 Marshall BJ. Helicobacterpylori. Am J Gastroentero11994; 89: $116$122. 4 Dick ]D. Helicobacter (Caropylobacter) pylori: a twist on an old disease. Ann Rev Microbiol 1990; 108: 70-90. 5 Parsonnet J, Friedman GD, Vandersteen DP et al. Helicobacter pylori infection and the risk of gastric carcinoma. New Engl J Med 1991; 325: 1127-1131. 6 Mendall MA, Northfiels TC. Transmission of Helicobacter pylori. Gut 1995; 37: 1-3. 7 Ferguson DA Jr, Li C, Patel RN, Mayberry RW. Chi SD, Thomas E. Isolation of Helicobacter pylori from saliva. J Clin Microbiol 1.993; 31: 2802-2804. 8 0 w e n RJ, Bickley J, Costas M, Morgan DR. Genomic variation in Helicobacter pylori: application to identification of strains. Scand J Gastroenterol 1991; 26 (Suppl. 181): 43-50. 9 Simor AE, Shames B, Drumm P, Sherman P, Low DE, Penner JL. Typing of Caropglobacter pylori by bacterial DNA restriction endonuclease analysis and determination of plasmid profile. J Clin Microbiol 1990; 28: 83-86. 10 Fraser AG, Bicldey J, Owen RJ, Pounder RE. DNA fingerprints of Helicobaeter pylori before and after treatment with omeprazole. J Clin Pathol 1992; 42: 785-791. 11 Walter CL. Gastric juice N-nitrosocompounds. In: Reed PI, Hill MJ, eds. Gastric carcinogenesis. New York: Elsevier, 1988: 163-173. 12 Beji A, Vincent P, Darchis I, Husson MO, Cortot A, Leclerc H. Evidence of gastritis with several Helicobacter pylori strains (letter). Lancet 1989: ii: 1402-1403. 13 Cellini L, Allocati N, Piccolomini R, Di Campli E, Dainelli B. New plate medium for growth and detection of urease activity of Helicobacter pylori. J Clin Microbial 1992; 30: 1351-1353. 14 Hazell SL. Cultural techniques for the growth and isolation of Helicobacter pglori. In: Goodwin CS, Worsley BW, eds. Helicobacter pylori: biology and clinical practice. Boca Raton, Florida: CRC Press, 1993; 2 73-283. 15 8ambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.