1194
Isolation of Helicobacter pylori from human faeces
Helicobacter pylori is arguably the commonest chronic infection in man. However, its route of transmission is unknown. We have isolated viable H pylori from the faeces of an infected individual from The Gambia. The organism was cultured on selective media after concentration of faecal bacteria by centrifugation in a buffer equilibrated with a
microaerophilic gas mixture. Growth characteristics, microscopic appearances, and enzyme activities were the same as those of a typical gastric isolate of H pylori. Protein preparations derived from the new isolate and the typical strain were antigenically similar, and had very similar electrophoretic profiles (including two major protein bands of 62 and 26 kDa, corresponding to the urease enzyme subunits). With the same technique, organisms with the colony morphology, growth requirements, enzyme activities, and microscopic appearances of H pylori were isolated from the faeces of 9 of 23 randomly selected childen aged 3-27 months from a Gambian village with a high prevalence of H pylori infection in early life. Faecal-oral transmission is probably important in the spread of infection in such communities. Lancet 1992; 340: 1194-95.
Helicobacter pylori is arguably the commonest chronic bacterial infection in man; in parts of Africa it is almost universal among adults,l and in The Gambia close to 50% of infants are infected, the percentage being 90% by age 5 years.2 H pylori colonises and has been isolated from the gastric mucosa.3 Infective bacteria have also been isolated from dental plaquewhich indicates that the infection could be transmitted by oral secretions. The suggestion that the organism may be excreted in the stool5 could account for the relation between a high prevalence of infection and poor domestic amenities.6 The aim of this study was to isolate viable H pylori from the faeces of an infected person. Stool samples from a 24-year-old Gambian man with an established H pylori infection diagnosed by 13C-urea breath test were processed immediately after collection.7 The man had not received antibiotics for at least 3 months before the study, had no dyspeptic symptoms, and was healthy. A faecal slurry (25% weight/volume) was prepared by homogenising faeces in sodium phosphate buffer (0.mol/1, pH 7-0) that had been autoclaved and equilibrated with a microaerophilic gas mixture (5% O2, 10% CO2, 85% N2) for 24 h. The slurry was then sieved (250 µm pore size) and the resulting suspension was centrifuged and washed once at 20 000 g for 30 min. Samples of the resultant bacterial layer, a loopful of which produced an instant violet colour with 200 ul of urease indicator (30 mmol/1 urea with phenol red, acidified to the point of yellow colour change), were plated onto selective media (5% horse blood, Colombia agar base
Coomassie brilliant blue G250 stained SDS-polyacrylamide gel of antigen preparations derived from isolates of H pylori obtained from a gastric biopsy (A) and faeces (B).
MR are shown in kDa. with either Dent or Skirrow’s antibiotic supplements) and incubated at 37°C in a microaerophilic atmosphere for 1 week. Primary incubation produced a mixed growth of bacteria. On almost all plates examined, tiny colonies that produced an immediate violet colour when a small inoculum was introduced into 200 µ1 of urease indicator were detected after 5 days. Bacteria from these colonies had the typical appearance of H pylori on gram staining. On two separate occasions colonies were subcultured and a. pure growth of bacteria was obtained with 5% blood Colombia agar base with Dent or Skirrow’s antibiotic supplements. These colonies were catalase positive. Pure subcultures were harvested from plates into distilled water and washed twice. Antigens were prepared from these subcultures and from an isolate of H pylori obtained from a gastric biopsy specimen and examined with an IgG enzyme-linked immunosorbent assay (ELISA).8 Serum samples from 40 patients who had undergone endoscopy, representing a range of IgG reactivities against H pylori, were used to compare antigen preparations. ELISA revealed a significant association between IgG concentrations obtained with either antigen preparation for all 40 serum samples (correlation, R= 0-695). The electrophoretic protein profiles of the crude antigens prepared from the stool isolate were compared with those from a strain of Hpylori from a gastric biopsy specimen. The supernatant obtained after centrifugation was separated by electrophoresis in a sodium dodecyl sulphate/10% polyacrylamide gel using a discontinuous buffer system.9 Both preparations contained major bands at 62 kDa and 26 kDa (figure), which correspond to the subunits of H pylori urease.8 Other major bands common to both preparations occurred at 14-2 kDa and 45 kDa, whereas differences in minor protein bands were most apparent at 18 kDa, 25 kDa, and 50 kDa. The same cultural techniques were used to examine faeces from 23 randomly selected children (aged 3-27 months, mean age 13-8 months) from a village in The Gambia, West
1195
Africa, where 90% of children are infected with H pylori by the age of 5 years.2 In 9 of the 23 children tiny colonies of bacteria with strong urease activity were detected within a week after culture under the conditions described above. Bacteria from these colonies had the typical microscopic appearances of H pylori. The subjects from whom we have successfully grown H pylori normally live in the same Gambian village. The faecal isolate examined in detail had the same microbiological and antigenic characteristics as isolates obtained from the gastric mucosa, and electrophoretic analysis showed that it contained the same
major proteins as a typical gastric isolate. Differences in minor protein bands between the faecal isolate and the control isolate were within the limits of normal variation.1o There are two important implications of this report. First, the isolation of viable H pylori from faeces shows that infective organisms are present in the colon of some individuals; and second, the high prevalence of H pylori infection in West African children2is most probably due to faecal-oral spread of infection. The intraluminal environment of the gastrointestinal tract of West Africans, by supporting the passage of H pylori from stomach to colon, and into the faeces, may account for differences in clinical picture of H pylori infection between the developed and developing worlds.1 Exposure of small intestinal lymphoid tissue to bacterial antigen in early life may modify the immune response, which could explain why the clinical features of the infection in adults in West Africa 5 are milder than those in the developed world.5 We thank the Thrasher Research Fund for a grant, and Dr T. D. Bohane for helpful suggestions during the preparation of this manuscript.
REFERENCES 1. Holcombe C, Omotara BA, Eldridge J, Jones DM. Helicobacter pylori, the most common bacterial infection in Africa: a random serological study. Am J Gastroenterol 1992; 87: 28-30. 2. Thomas JE, Downes RM, Lunn PG, et al. Seroepidemiology of Helicobacter pylori infection in early childhood. Gut 1991; 32: A1231
(abstr).
BJ, Royce H, Annear DI, et al. Original isolation of Helicobacter pyloridis from human gastric mucosa. Microbiol Lett 1984; 25: 83-88. 4. Shames B, Krajden S, Fuksa M, et al. Evidence for the occurrence of the same strain of Campylobacter pylori in the stomach and dental plaque. J Clin Microbiol 1989; 27: 2849-50. 5. Mapstone NP, Lynch DA, Axon ATR, Dixon MF, Quirke P. The detection of Helicobacter pylori in faeces by the polymerase chain reaction. Ir J Med Sci 1992; 161 (suppl 10): 29. 6. Klein PD, Graham DY, Gaillour A, et al. Water source as a risk factor for Helicobacter pylori infection in Peruvian children. Lancet 1991; 337: 3. Marshall
1503-06. 7. Klein PD, Graham DY. Detection of Campylobacter pylori by the 13C-urea breath test. In: Rathbone BJ, Heatley RV, eds. Campylobacter pylori and gastroduodenal disease. Oxford: Blackwell, 1989: 83-94. 8. Thomas JE, Whatmore AM, Barer MR, et al. Serodiagnosis of Helicobacter pylori infection in childhood. J Clin Microbiol 1990; 28: 2641-46. 9. Miller L, Gray L, Beachey E, Kehoe M. Antigenic variation among group A streptococcal M proteins: nucleotide sequence of the serotype 5 m protein gene and its relationship with genes encoding types 6 and 24 m proteins. J Biol Chem 1988; 263: 5668-73. 10. Costas M, Owen RJ, Bickley J, Morgan DR. Molecular techniques for studying the epidemiology of infection by Helicobacter pylori. Scand J Gastroenterol 1991; 26 (suppl 181): 20-32.
ADDRESS: MRC Dunn Nutrition Centre, Cambridge, UK (J. E. Thomas, MRCP, G. R. Gibson, PhD, M. K. Darboe, L. T. Weaver, MD) and The Gambia, West Africa (A. Dale, MRCP). Correspondence to Dr J. E. Thomas, MRC Dunn Nutrition Centre, Milton Road, Cambridge CB4 1 XJ.
Smoking and decreased fertilisation rates in vitro
To examine possible mechanisms for the association between cigarette smoking and reduced fertility, we have measured the concentration of the nicotine metabolite cotinine in ovarian follicular fluid collected at the time of oocyte recovery during treatment for in-vitro fertilisation. In a group of women in whom follicular fluid cotinine could not be detected (limit of accurate measurement 20 ng/ml) 116 oocytes were collected, of which 84 became fertilised (72%), whereas among women with cotinine concentration greater than 20 ng/ml 20/45 (44%) oocytes did so (p
Isolation of Helicobacter pylori from human faeces
Helicobacter pylori is arguably the commonest chronic infection in man. However, its route of transmission is unknown. We have isolated viable H pylori from the faeces of an infected individual from The Gambia. The organism was cultured on selective media after concentration of faecal bacteria by centrifugation in a buffer equilibrated with a
microaerophilic gas mixture. Growth characteristics, microscopic appearances, and enzyme activities were the same as those of a typical gastric isolate of H pylori. Protein preparations derived from the new isolate and the typical strain were antigenically similar, and had very similar electrophoretic profiles (including two major protein bands of 62 and 26 kDa, corresponding to the urease enzyme subunits). With the same technique, organisms with the colony morphology, growth requirements, enzyme activities, and microscopic appearances of H pylori were isolated from the faeces of 9 of 23 randomly selected childen aged 3-27 months from a Gambian village with a high prevalence of H pylori infection in early life. Faecal-oral transmission is probably important in the spread of infection in such communities. Lancet 1992; 340: 1194-95.
Helicobacter pylori is arguably the commonest chronic bacterial infection in man; in parts of Africa it is almost universal among adults,l and in The Gambia close to 50% of infants are infected, the percentage being 90% by age 5 years.2 H pylori colonises and has been isolated from the gastric mucosa.3 Infective bacteria have also been isolated from dental plaquewhich indicates that the infection could be transmitted by oral secretions. The suggestion that the organism may be excreted in the stool5 could account for the relation between a high prevalence of infection and poor domestic amenities.6 The aim of this study was to isolate viable H pylori from the faeces of an infected person. Stool samples from a 24-year-old Gambian man with an established H pylori infection diagnosed by 13C-urea breath test were processed immediately after collection.7 The man had not received antibiotics for at least 3 months before the study, had no dyspeptic symptoms, and was healthy. A faecal slurry (25% weight/volume) was prepared by homogenising faeces in sodium phosphate buffer (0.mol/1, pH 7-0) that had been autoclaved and equilibrated with a microaerophilic gas mixture (5% O2, 10% CO2, 85% N2) for 24 h. The slurry was then sieved (250 µm pore size) and the resulting suspension was centrifuged and washed once at 20 000 g for 30 min. Samples of the resultant bacterial layer, a loopful of which produced an instant violet colour with 200 ul of urease indicator (30 mmol/1 urea with phenol red, acidified to the point of yellow colour change), were plated onto selective media (5% horse blood, Colombia agar base
Coomassie brilliant blue G250 stained SDS-polyacrylamide gel of antigen preparations derived from isolates of H pylori obtained from a gastric biopsy (A) and faeces (B).
MR are shown in kDa. with either Dent or Skirrow’s antibiotic supplements) and incubated at 37°C in a microaerophilic atmosphere for 1 week. Primary incubation produced a mixed growth of bacteria. On almost all plates examined, tiny colonies that produced an immediate violet colour when a small inoculum was introduced into 200 µ1 of urease indicator were detected after 5 days. Bacteria from these colonies had the typical appearance of H pylori on gram staining. On two separate occasions colonies were subcultured and a. pure growth of bacteria was obtained with 5% blood Colombia agar base with Dent or Skirrow’s antibiotic supplements. These colonies were catalase positive. Pure subcultures were harvested from plates into distilled water and washed twice. Antigens were prepared from these subcultures and from an isolate of H pylori obtained from a gastric biopsy specimen and examined with an IgG enzyme-linked immunosorbent assay (ELISA).8 Serum samples from 40 patients who had undergone endoscopy, representing a range of IgG reactivities against H pylori, were used to compare antigen preparations. ELISA revealed a significant association between IgG concentrations obtained with either antigen preparation for all 40 serum samples (correlation, R= 0-695). The electrophoretic protein profiles of the crude antigens prepared from the stool isolate were compared with those from a strain of Hpylori from a gastric biopsy specimen. The supernatant obtained after centrifugation was separated by electrophoresis in a sodium dodecyl sulphate/10% polyacrylamide gel using a discontinuous buffer system.9 Both preparations contained major bands at 62 kDa and 26 kDa (figure), which correspond to the subunits of H pylori urease.8 Other major bands common to both preparations occurred at 14-2 kDa and 45 kDa, whereas differences in minor protein bands were most apparent at 18 kDa, 25 kDa, and 50 kDa. The same cultural techniques were used to examine faeces from 23 randomly selected children (aged 3-27 months, mean age 13-8 months) from a village in The Gambia, West
1195
Africa, where 90% of children are infected with H pylori by the age of 5 years.2 In 9 of the 23 children tiny colonies of bacteria with strong urease activity were detected within a week after culture under the conditions described above. Bacteria from these colonies had the typical microscopic appearances of H pylori. The subjects from whom we have successfully grown H pylori normally live in the same Gambian village. The faecal isolate examined in detail had the same microbiological and antigenic characteristics as isolates obtained from the gastric mucosa, and electrophoretic analysis showed that it contained the same
major proteins as a typical gastric isolate. Differences in minor protein bands between the faecal isolate and the control isolate were within the limits of normal variation.1o There are two important implications of this report. First, the isolation of viable H pylori from faeces shows that infective organisms are present in the colon of some individuals; and second, the high prevalence of H pylori infection in West African children2is most probably due to faecal-oral spread of infection. The intraluminal environment of the gastrointestinal tract of West Africans, by supporting the passage of H pylori from stomach to colon, and into the faeces, may account for differences in clinical picture of H pylori infection between the developed and developing worlds.1 Exposure of small intestinal lymphoid tissue to bacterial antigen in early life may modify the immune response, which could explain why the clinical features of the infection in adults in West Africa 5 are milder than those in the developed world.5 We thank the Thrasher Research Fund for a grant, and Dr T. D. Bohane for helpful suggestions during the preparation of this manuscript.
REFERENCES 1. Holcombe C, Omotara BA, Eldridge J, Jones DM. Helicobacter pylori, the most common bacterial infection in Africa: a random serological study. Am J Gastroenterol 1992; 87: 28-30. 2. Thomas JE, Downes RM, Lunn PG, et al. Seroepidemiology of Helicobacter pylori infection in early childhood. Gut 1991; 32: A1231
(abstr).
BJ, Royce H, Annear DI, et al. Original isolation of Helicobacter pyloridis from human gastric mucosa. Microbiol Lett 1984; 25: 83-88. 4. Shames B, Krajden S, Fuksa M, et al. Evidence for the occurrence of the same strain of Campylobacter pylori in the stomach and dental plaque. J Clin Microbiol 1989; 27: 2849-50. 5. Mapstone NP, Lynch DA, Axon ATR, Dixon MF, Quirke P. The detection of Helicobacter pylori in faeces by the polymerase chain reaction. Ir J Med Sci 1992; 161 (suppl 10): 29. 6. Klein PD, Graham DY, Gaillour A, et al. Water source as a risk factor for Helicobacter pylori infection in Peruvian children. Lancet 1991; 337: 3. Marshall
1503-06. 7. Klein PD, Graham DY. Detection of Campylobacter pylori by the 13C-urea breath test. In: Rathbone BJ, Heatley RV, eds. Campylobacter pylori and gastroduodenal disease. Oxford: Blackwell, 1989: 83-94. 8. Thomas JE, Whatmore AM, Barer MR, et al. Serodiagnosis of Helicobacter pylori infection in childhood. J Clin Microbiol 1990; 28: 2641-46. 9. Miller L, Gray L, Beachey E, Kehoe M. Antigenic variation among group A streptococcal M proteins: nucleotide sequence of the serotype 5 m protein gene and its relationship with genes encoding types 6 and 24 m proteins. J Biol Chem 1988; 263: 5668-73. 10. Costas M, Owen RJ, Bickley J, Morgan DR. Molecular techniques for studying the epidemiology of infection by Helicobacter pylori. Scand J Gastroenterol 1991; 26 (suppl 181): 20-32.
ADDRESS: MRC Dunn Nutrition Centre, Cambridge, UK (J. E. Thomas, MRCP, G. R. Gibson, PhD, M. K. Darboe, L. T. Weaver, MD) and The Gambia, West Africa (A. Dale, MRCP). Correspondence to Dr J. E. Thomas, MRC Dunn Nutrition Centre, Milton Road, Cambridge CB4 1 XJ.
Smoking and decreased fertilisation rates in vitro
To examine possible mechanisms for the association between cigarette smoking and reduced fertility, we have measured the concentration of the nicotine metabolite cotinine in ovarian follicular fluid collected at the time of oocyte recovery during treatment for in-vitro fertilisation. In a group of women in whom follicular fluid cotinine could not be detected (limit of accurate measurement 20 ng/ml) 116 oocytes were collected, of which 84 became fertilised (72%), whereas among women with cotinine concentration greater than 20 ng/ml 20/45 (44%) oocytes did so (p
