Review article: have we found the source of Helicobacter pylori?

Transcription

1 Aliment Pharmacol Ther 2000; 14 (Suppl. 3): 7±12 Review article: have we found the source of Helicobacter pylori? F. ME GRAUD & N. BROUTET Laboratoire de BacteÂriologie, Universite Victor Segalen Bordeaux 2, Bordeaux, France SUMMARY Besides the well established Helicobacter pylori reservoir, i.e. the human stomach, numerous other sources have been hypothesized. However, none has been de nitely proven. In some instances (pig, sheep), Helicobacter species closely related but different from H. pylori were detected but the results were misleading because culture of suf ciently discriminating molecular techniques were not used. In other cases, the strain was really H. pylori (cat) but the case was anecdotal or the animal species (monkey) has so little contact with humans that the possible source has no epidemiological consequence. This is also the case for house ies which theoretically can be a vehicle, but practically speaking are not because of too fewviable bacteria present in faeces. Molecular epidemiology studies demonstrating the route of transmission (faecal±oral, oral±oral or gastro±oral) are still lacking but recent studies have con rmed the presence of viable H. pylori in vomitus and in faeces in the event of diarrhoea. INTRODUCTION To make public health recommendations to limit the transmission of a pathogenic micro-organism, its source and route of transmission must be well known. Besides the well-known reservoir of Helicobacter pylori, i.e. the stomach of humans, several other potential sources have been highlighted in the last fewyears. This paper reviews the arguments for and against these sources as well as the possible route of transmission from one human stomach to another. SUSPECTED ANIMAL RESERVOIRS OF H. PYLORI A number of animals, mostly living in a human environment, have been suspected of harbouring H. pylori in their stomach and therefore to be involved in the transmission of this bacterium. Correspondence to: Dr F. MeÂgraud, Laboratoire de BacteÂriologie, Universite Victor Segalen, Bordeaux 2, 146, rue LeÂo Saignat, Bordeaux, France. francis.megraud@chu-bordeaux.fr The pig Historically, the rst animal incriminated was the pig. At the beginning of the 1990s, Jones and Elridge were able to isolate a strain of H. pylori from the stomach of a laboratory pig. 1 This strain was studied in depth and considered to be a true H. pylori, suggesting that pigs could be a reservoir for this bacterium. Other data supported this hypothesis: (a) the pig was one of the fewanimal models for H. pylori infection. Several groups succeeded in infecting gnotobiotic piglets which subsequently developed gastritis; 2,3 (b) when studying histological preparations of gastric mucosa from pigs, with antibodies directed against H. pylori by immuno uorescence, Vaira et al. observed spiral bacteria; 4 and (c) abattoir workers were found to have a signi cantly higher prevalence of H. pylori infection than clerks working in the same enterprise. 5,6 Where do we stand now? Recently, an improved understanding of stomach ora has allowed the identi cation in pigs of a spiral bacterium morphologically different from H. pylori. 7 Although this bacterium has not been cultured, molecular biology techniques, especially 16S rdna sequencing, allowed its inclusion in the genus Ó 2000 Blackwell Science Ltd 7

2 8 F. MEÂ GRAUD & N. BROUTET Helicobacter. This bacterium has been recently named `candidatus Helicobacter suis'. 8 It corresponds to the previous description of Gastrospirillum suis and Helicobacter heilmannii type 1. In our experience, however, although H. heilmannii type 1 is the main Helicobacter species found in the pig stomach, it is also possible to nd H. heilmannii type 2. 9 We nowknowthat pigs are not the reservoir for H. pylori as all of the studies focusing on this bacterium in pigs have failed to nd it. The bacterium detected by Vaira was probably H. suis, and the pig strain described by Jones was probably acquired from humans. The higher incidence of H. pylori infection in abattoir workers vs. clerks has been explained by these two groups being of a different socio-economic status. It is, however, possible that H. heilmannii can be transmitted from pigs to humans. The frequence of this infection in humans is lowbut not nil. The pig has been claimed to be the major source of this infection. 10 The cat In 1994 Handt et al. isolated H. pylori from the stomach of six cats from the same closed colony of laboratory cats sold by commercial vendors. 11 Identi cation was con rmed by 16S rdna sequencing and fatty acid analysis. The bacteria were present in large numbers and accompanied by a lymphocytic in ltrate. The fact that all of the cats from the same commercial facility harboured morphologically similar gastric organisms implies that these bacteria are ef cient colonizers. In addition, H. pylori could be grown from gastric juice samples from 11 of 12 cats, salivary secretions from six of 12 cats (50%) and faeces from four of ve cats. H. pylori speci c IgG and IgA were also present in saliva. 12 These data are strong arguments in favour of a feline reservoir of H. pylori. This is potentially important for the transmission to humans because cats are popular pets, with a signi cant amount of human contact. Furthermore, they occasionally vomit and are continually grooming themselves. Where do we stand now? Five years later, no con rmation of these ndings has been made. We knowthat a number of Helicobacter species, different from H. pylori, can colonize the cat stomach: H. felis, H. heilmannii, and possibly H. salomonis. 13 These species are constantly found in cat stomach but in no study was H. pylori able to be grown, indicating again that the ndings from this cat colony were probably anecdotal, and that possibly a human contamination was the cause. Furthermore, in epidemiological studies, having cats at home was not found to be a risk factor. 14 In some studies it was even found to be a protective factor which disappeared after adjustment of socio-economic status. The house y It is well known that pathogens which are excreted in faeces such as Salmonella sp. can be transmitted from faeces to food by house ies. 15 Based on this fact, GruÈ bel et al. designed an experiment where caged house ies (Musca domestica) were exposed to freshly grown H. pylori on agar plates. 16 After a 6-h feeding period, the plates were removed and were replaced at regular intervals by sterile Petri dishes. H. pylori was isolated from the external surfaces of the ies for up to 12 h and from gut and excreta for up to 30 h after the feeding period. Helicobacter-like organisms were also found attached to intestinal epithelial cells of the ies. Flies that can only ingest liquid must regurgitate their gastric content full of enzymes on to food to facilitate feeding. Therefore, if H. pylori can survive in the mid gut, food could be contaminated either by defecation or regurgitation. Such experimental results constitute a good argument for a faecal±oral transmission, with ies being the vehicle between faeces and food. This theory ts with the particular worldwide prevalence of the infection, i.e. there is a very high prevalence in developing countries where outdoor toilets are common, while in developed countries this is no longer the case and the prevalence has decreased. The same group also carried out a search for H. pylori in house ies obtained from countries on different continents (USA, Egypt and Poland). Using PCR, they were able to detect H. pylori in all of these countries. 17 Where do we stand now? The problem is that H. pylori may not be in a viable form in faeces. The experiment previously reported is very arti cial in the sense that house ies will never be exposed to billions of H. pylori organisms under normal conditions. When house ies were exposed for 24 h to fresh human faeces of an H. pylori-positive subject, or to arti cially seeded faeces from an H. pylori-negative subject, H. pylori was not recovered from the insects. 18

3 REVIEW:HAVEWEFOUNDTHESOURCEOFH. PYLORI? 9 The PCR results obtained from house ies caught in different countries must also been interpreted cautiously. With Helicobacter species, we are dealing with a group of bacteria that has only partially been explored. The consequence is that, when PCR is applied to detect H. pylori outside of the niche of this bacterium, stringent conditions are necessary before concluding its absence or presence, i.e. either sequencing of the complete 16S rdna or several positive PCR, based on the sequence of several supposedly speci c genes. The sheep A very high prevalence of H. pylori infection was reported in Sardinian shepherds. 19 The prevalence was close to 100% among 123 shepherds while it was lower in 22 family members with no direct animal contact (73%) and in 221 unrelated blood donors (43%). The results of H. pylori serology were also con rmed using CagA serology, an antigen that is not found in other bacteria and therefore excludes the possibility of false positive results due to cross-reacting antigens. In addition, the H. pylori infection was con rmed in 49 of 51 shepherds who consented to endoscopy. This result, along with that showing that H. pylori can survive in milk, 20 is an indirect argument for sheep being a possible source of infection for humans. In further studies, the same authors reported the presence of H. pylori DNA in 6% of rawsheep milk samples collected from different ocks in northern Sardinia. They used PCR to amplify a 500-bp fragment of the 16S rdna which was sequenced in four cases giving a 99% identity with H. pylori. They also detected vaca sequences in four cases and were able to grow one strain. 21 This result implies that H. pylori would be living in the stomach of sheep, would be eliminated as viable forms in faeces and then would contaminate the milk during the milking process. Where do we stand now? These results are strong arguments for the possible role of sheep as a reservoir of H. pylori and a source of contamination for humans. However, more recently, new data have been presented on the detection of a new Helicobacter sp. in the abomasum of sheep. 22 This organism has the same morphology as H. pylori, is urease positive, and crossreacts with antibodies directed against H. pylori by immunochemistry. However, the 16S rdna sequencing excludes its identi cation as H. pylori (De Groote, personal communication). It is closely related to the Helicobacter species found in cattle abomasum. 23 Therefore, it seems that we are dealing with a Helicobacter sp. very similar to, but different from H. pylori and its role in human disease remains to be identi ed. The epidemiological data from the study on shepherds were subject to methodological problems, especially in terms of adjustment of the controls for socio-economic factors. The monkey Monkeys also carry Helicobacter sp. in their stomach. Some of them seem to be H. heilmannii-like organisms. However, there are some reports showing that H. pylori can also be present. 24,25 This is relatively unusual and it has been hypothesized that these animals were contaminated by humans. In any case, contact between humans and monkeys is so limited that this possible transmission can only be anecdotal. TRANSMISSION FROM THE HUMAN RESERVOIR Having reviewed the possible animal reservoirs for H. pylori, it appears that none have been con rmed, and so the human stomach of possibly three billion people remains the only proven source. However, there are still doubts concerning the vehicle involved. The faeces Since everything from the stomach is eliminated in faeces, it is logical to think that H. pylori is present in human faeces. However, the con rmation of its constant presence in faeces came only when the antigen stool test was developed. An excellent correlation was found between the presence of H. pylori both in the stomach and faeces. 26,27 Detection by PCR was not always successful. The rst publication using this technique by Mapstone et al. in , was only recently con rmed. 26 The presence of PCR inhibitors in stools are probably the reason 29 and they are dif cult to eliminate. To have a role in transmission, H. pylori must be in a viable form in stools and this viability is highly controversial, as previously stated. Successful cultures

4 10 F. MEÂ GRAUD & N. BROUTET were reported by Thomas et al. 30 Conditions were very special: the study population was children from developing countries with a short transit time but, considering the number of diarrhoeal episodes in children in the world, such routes of transmission may be signi cant. Many other attempts to grow H. pylori in faeces failed. However, recently Shmuely et al. were able to grow H. pylori from stools obtained after induction of diarrhoea with a sodium phosphate solution in ve of 12 H. pylori-infected subjects. 31 Dore et al. also reported the isolation of H. pylori from three H. pylori-positive patients among 12 explored, by adding cholestyramin to the culture medium. 32 These results suggest that, at least under certain conditions, H. pylori can be viable in faeces and therefore faeces could be a source of H. pylori infection. The saliva Saliva is another potential source of H. pylori transmission since, following regurgitation or vomiting, gastric ora can reach the oral cavity. Controversial results have been obtained using PCR, again linked to the fact that numerous species related to H. pylori can be present in the mouth. This emphasizes the need to perform several PCR on different speci c genes or to sequence the whole 16S rdna. 33 The discrepancy between the results obtained in the stomach and in saliva (or dental plaque) should be viewed as an argument against the validity of the results rather than an argument for a different bacterial reservoir. Only anecdotal reports of positive culture have been published. 34 The vomitus Axon has suggested that vomitus could be the main source of contamination for H. pylori. 35 The point is that H. pylori is cultured from gastric juice very frequently. Recently, Galal et al. studied the survival of H. pylori outside the human body in the unbuffered gastric juice of 21 patients. 36 Culture was successful in 62% of the cases within 2 h of collection, 42% after 6 h and 10% after 24 h. In a similar study to that reported for faeces, Shmueli et al. also induced vomiting by administration of ipecac in their 12 H. pylori-positive subjects. H. pylori was cultured from all of the vomitus samples and, in ve instances, air sampled at 30 cm from the subjects vomiting was also contaminated with H. pylori. 37 Another epidemiological study found a sibling history of vomitus to be a risk factor for H. pylori infection. 38 These results are strong arguments in favour of a gastro-oral transmission given that organisms are found in high numbers in vomitus and the high rate of transmission among young children. The question of regurgitation in infants and young children has unfortunately never been explored. SUSPECTED ENVIRONMENTAL SOURCES Although it is clear, based on the physiology of H. pylori, that multiplication in the environment cannot occur 39 there are reports that H. pylori was found in water specimens. 40 However, the identi cation was not made by culture, but by PCR, and the remarks made previously on this approach are still valid for water specimens. The argument in favour of water as a source of infection comes from epidemiological studies such as one carried out in Peru where municipal water was incriminated 41 and another in Columbia where young boys bathing in ponds and rivers had a higher prevalence of infection than those who did not. 42 Consumption of rawvegetables, in an area where human faeces is used as fertilizer, was also found to be a risk factor for this infection. 43 The controversy remains, therefore, concerning the possible survival of H. pylori in the environment especially in water. This raises the highly debated question of the existence of viable but non-culturable forms of this bacterium. Numerous arguments have been brought forth against the viability of coccoidal forms 44 but such forms induced in vitro may be different from those induced in vivo. CONCLUSIONS Despite numerous attempts to nd an animal source for H. pylori transmission, the only proven reservoir remains the human stomach. Animals do not seem to play a signi cant role, if indeed any role at all. The limits of many studies to date are in relation to bacteriological or epidemiological methodology. The exquisite adaptation of H. pylori to its host implies that this species has evolved with its host from early times. We can hypothesize that there was a common ancestor to the Helicobacter species that are currently known, and a parallel evolution in the different animal species concerned. Given that the stomach environment of

5 REVIEW:HAVEWEFOUNDTHESOURCEOFH. PYLORI? 11 these different animal species is relatively similar, the different Helicobacter species still have many features in common. For this reason, the PCR data implying the presence of H. pylori DNA must be interpreted very cautiously, as previously stated, since a great amount of Helicobacter DNA from different sources may be released in nature. With regard to epidemiology, we lack proper longitudinal studies that could con rm the direction of the transmission, either from humans to animals or from animals to humans. The isolation of an organism in an animal or in the environment is not suf cient evidence to consider it as a reservoir. 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