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MICROBIAL ECOLOGY IN HEALTH AND DISEASE VOL 1: 1-16 (1988) Campylobacter pylori in health and disease: An ecological perspective ADRIAN LEE and STUART L. HAZELL School of Microbiology, University of New South Wales, Sydney, Australia, 2033. Received 30 October 1987 The successful isolation by Marshall in 1984 of a spiral-shaped bacterium, at present called Campylobacter pylori, has sparked a resurgence of interest in the role of bacteria in stomach disease. C. pylori has been shown to be the causative agent of active chronic gastritis in humans. There is also accumulating evidence that this organism plays a significant role in the initiation of duodenal ulcers. The association of this organism with the human stomach is reviewed with an ecological bias. It is suggested that C. pylori is a member of a group of highly adapted mucus colonising bacteria that inhabit the gastrointestinal surfaces of all animals studied. The natural niche of C. pylori is the human stomach; over the period of evolution the organism has become well suited to the environment of gastric mucus and gastric epithelium. However C. pylori is only ‘almost’ normal flora as is always associated with a pathology albeit mild and most often asymptomatic. Understanding the factors that allow colonisation of the gastric mucosa, e.g. possibly morphology, special motility, microaerophilism, urease activity etc., provides a rational basis for the selection of appropriate methods of treatment and prophylaxis. There is a need to identify the ‘triggers’ to C. pyloriassociated symptomatology. An ecological approach to the microbiology, histopathology, symptomatology, and epidemiology of C.pyloriinfection in humans could resolve some of the controversy over the role of this organism in peptic and duodenal disease. CONTENTS Introduction Evidence for a n aetiological role of Campylobacter pylori in gastroduodenal disease The controversy Histological gastritis Duodenal ulcer Gastric ulcer N o n ulcer dyspepsia The epidemiology of Campylobacterpyloriinfection Incidence The source Transmission Carnpylobacter pylori as a member of a highly adapted group of mucus colonising bacteria The mucus associated bacteria of the gastrointestinal tract Common factors involved in colonisation of intestinal mucus Spiral morphology Motility Microaerophilism Special features of the colonisation of the stomach by Carnpylobacter pylori Localisation at intercellular junctions 089 1~60X/88/01000 1-1 6 $08.00 0 1988 by John Wiley & Sons, Ltd. Urease Specific receptor sites An ability to induce inflammation and escape the consequences Campylobacter pylori as ‘almost’ normal flora Other stomach associated spiral shaped bacteria? The triggers to Campylobacter pylori associated symptomatology Acute symptomatology Dyspepsia Ulceration Conclusion References INTRODUCTION ‘Dyspepsia: The remorse of a guilty stomach’ Alexander Kerr During the early 1980’s, Warren, a West Australian histopathologist, had noted the presence of large numbers of spiral-shaped bacteria o n the gastric epithelium in cases of active chronic gastritis. H e became convinced that these organisms were 2 A. LEE AND S. L. HAZELL associated with inflammation in the human stomach EVIDENCE FOR AN AETIOLOGICAL but failed to interest colleagues in the finding until ROLE O F CAMPYLOBACTER PYLORI IN he persuaded Dr Barry Marshall to try and culture GASTRODUODENAL DISEASE the bacteria from gastric biopsies. Attempts at culture were unsuccessful, until one Easter holiday The controversy when plates were given a longer than usual incuGiven that all previous papers suggesting a role bation. It was the publication of the successful for microorganisms in gastric pathology and duoisolation of this organisms2 in conjunction with denal u1cers14~20~38~47~80~83 had been ignored and Warren’s description of the association of the bac- that an extensive literature had accumulated on the terium with gastritis87 that sparked a resurgence of non microbial aetiology of these condition^,^' it was interest in the role of bacteria in stomach disease, a not surprising that the C. pylori story was received topic that had been the focus of some very early with a great deal of scepticism by both gastroenterinvestigations. In 1896 S a 1 0 m o n ~had ~ reported ologists and microbiologists. Indeed even today, sightings of ‘spirochaetes’ in the mucus layer of the nearly six years after the isolation of the bacterium, stomachs of humans and several animal species. C. pylori receives scant mention in reviews on Luger and N e ~ b e r g e noted r ~ ~ the presence of these stomach disease. Thus it is the opinion of many ‘spirochaetes’ in the stomachs of patients with specialist gastroenterologists and interested general ulcerating gastric carcinoma and reported that these physicians that the role of C. pylori in the aetiology organisms were rarely seen in normal individuals. of stomach disease is ‘not proven’. Steer” in 1975 published some excellent electron One aim of this review is to suggest that all too micrographs of bacteria, presumably Campylo- often it is assumed that the association of C. pylori bacter pylori, on gastric mucosae and claimed an with human disease, particularly ulcerative lesions, association between the presence of polymorpho- is at the exclusion of all those other factors that have nuclear leucocytes and the presence of these organ- been implicated in such pathology. The thesis of this isms, but a mistaken assumption that they were review is that C. pylori is an organism specially Pseudomonas aeruginosa resulted in little interest in adapted to a particular niche in the human stomach this paper. and that its presence is an essential prerequisite for The bacterium that Marshall cultured was micro- certain stomach diseases, although these diseases aerophilic, Gram-negative, had a spiral mor- are not an inevitable consequence of C. pylori infecphology and grew on media commonly used to tion. The challenge to us as microbial ecologists is to isolate Campylobacter jejuni. Because of its spiral determine how variations in the local environment morphology the newly isolated organism was allow this bacterium to influence gastroduodenal thought to belong to the genus Campylobacter and, disease. What then is the evidence for C . pylori as a due to its localization in the pyloric region of the contributor to gastric or duodenal pathology? stomach, was initially named Campylobacter pyl~ridis.~’Realization that this name was linguistiHistological gastritis cally incorrect resulted in its being renamed All investigators have found a very high correCampylobacter py10t-i.~~ As the characteristics of this bacterium became known, many major differ- lation of 8&95 per cent between the presence of C . ences were observed which made it clear that it pylori and the presence of active chronic antral could not belong to the genus Campylobacter gastritis, inflammation associated with both polydespite having a close G + C content ( - 36 per cent). morphonuclear and mononuclear cells and varying For example, the organism had multiple sheathed degrees of damage to the superficial epithelium of flagella compared to campylobacters with a single the a n t r U m , 9 , 2 3 , 3 0 , 3 2 , 3 6 , 5 0 , 5 8 , 6 3 , 7 4 , 7 7 ~ 8 2 At first unsheathed polar flagellum, was strongly urease sceptics considered the bacterium was colonising positive, and had a unique fatty acid profile. 19,43,66 the pre-existing inflammation i.e. the gastritis proRomaniuk, Trust and colleague^'^ at the University vided a suitable environment for c o l o n i ~ a t i o n . ~ ~ of Victoria, confirmed by comparison of partial 16s However studies with antimicrobial agents have rRNA sequences that C. pyfori is not a true consistently shown that administration of an agent Campylobacter species. Due to the intricacies of that removes the organism results in a resolution of ~ ’r, example, ~~ McNulty cleared international nomenclature it is likely to be several the g a ~ t r i t i s . ~ ,Fo years before the organism is assigned to a different patients of both bacteria and acute inflammatory cells with bismuth ~ a l i c y l a t eThat . ~ ~ this was due to genus or given a name of its own. CAMPYLOBACTER PYLORI IN HEALTH AND DISEASE the proven antimicrobial activity of the bismuth relapse rate, in the order of 17 per cent per month.48 c o m p ~ u n and d ~ not ~ ~any ~ ~ cytoprotective effect is In duodenal ulcer patients treated with bismuth suggested by studies with antibiotics. Langenberg4' salts (bismuth salicylate or subcitrate), agents with had some success with amoxycillin; once again, known antibacterial activity, the relapse rate is sig~ these , ~ ' patients, both the ulcer when the organisms were removed, the polymorphs nificantly l e ~ s . ~In disappeared. All attempts to clear C .pylorifrom the and C. pylori associated duodenitis resolve. Thus in human stomach have shown that the organism is a double blind trial over one year involving I00 ulcer very difficult to remove on a long term basis, i.e. the patients with successful ulcer treatment, Marshall majority of both McNulty's and Langenberg's and colleagues56 found that 80 per cent of their patients relapsed. In all cases when the organism proven ' C . pylori positive patients relapsed comrecolonised the stomach, active gastritis returned, pared to 20 per cent of persons in whom ulcer further evidence for a causal role. resolution was accompanied by elimination of C . Two volunteers have ingested C . p y l ~ r i ;the ~ ~ pylori , ~ ~ and resolution of the associated duodenitis. A more controlled 'swallow' was that of Morris64 in number of other trials all show the same trend, supNew Zealand. He demonstrated stomach acidity porting the hypothesis that the presence of C .pyfori and normal histology in both the antrum and body is an essential prerequisite for ulcer formation, of the stomach, 87 and 30 days prior to ingestion of although other factors need to be present for ulcer 105 cells of a fresh clinical isolate of C . pylori. For initiation. These observations suggesting a role for 2 4 days after ingestion acute symptomatology was C. pyfori in ulcerogenesis have important impliexperienced including nausea, epigastric pain and cations, provided a satisfactory regimen of antivomiting. On day 5, an antral biopsy showed active microbials can be found, the incidence of relapse gastritis and the presence of large numbers of C. may be significantly reduced. pylori while the body biopsy was normal. By day 1 1, the body biopsy was also showing active inflammation and the presence of bacteria. Over eighteen Non ulcer dyspepsia months, the organism has remained and the histological picture now resembles the active chronic gasIn any population, the incidence of persons with tritis commonly associated with C.pylori. As would dyspeptic symptoms e.g. heartburn, wind, epigastric be predicted from clinical trials, the organism is pain, is high. 1 , 3 9 The appearance of these symptoms proving very difficult to remove from this individ- in volunteers deliberately or accidently inoculated ual; until an effective regimen for clearing C .pylori is with C. pylori'6,'7,28,64,73 suggests that infection found, further volunteer studies should be deferred. with this organism could be one of the causes of non Despite these difficulties, the case for C. pylori as ulcer dyspepsia. Early studies had claimed gastritis the causative agent for the majority of episodes of was a factor in non ulcer dyspepsia.31 A number of active chronic gastritis in the human stomach is very workers have claimed symptom relief in their convincing. patients following uncontrolled treatment studies for C. p y l ~ r i . ~There , ' ~ have also been some controlled studies which all show a trend towards relief Duodenal ulcer of symptoms following eradication of the organIn contrast, the case for C. pylori as a causative ism5 ,4 1,49 but they remain unconvincing, relying on agent of duodenal ulcer remains controversial but statistical analysis of relatively small differences in supportive evidence is accumulating as more clini- symptom scores. The difficulty in linking C . pylori cal trials are reported. Up to 100 per cent of duo- with non ulcer dyspepsia becomes clear following denal ulcers have been reported to be associated examination of the data in Figure 1. Firstly a large with the presence of C . pylori, both at the mar- control population, i.e. blood donors, are found to gins of the ulcer itself and in the gastric have evidence of C . pylori associated g a s t r i t i ~ . ~ , ~ ~ antrum.17,30,32,56,58,81,82 If the ulcer is successfully The second group in this figure, a large number of treated with, for example the histamine H, receptor patients presenting to a clinic for endoscopy, i.e. a antagonists (cimetidine or ranitidine) which are symptomatic group, have a higher incidence of C. known to have no antibacterial activity, the ulcer pylori associated gastritis compared to blood crater disappears due to reduction in acid secretion; donors, especially in the younger age group, howhowever, the underlying C . pylori associated duo- ever, at least 3 W O per cent are negative for the denitis remains. Such ulcer patients have a high ~ r g a n i s m . ~Thus ' a large number of people have C. 4 A. LEE AND S. L. HAZELL 214 e 2 c3 d 3 + 50 E 02 10 0 N = 767 kd 0 30 20 2 pr: 40 z w V 4 pr: w 100 : 3 z wzz 0 23.27 28-32 33-37 311.42 43.47 18-52 53-51 AGE GROUP Figure I . Campylobacterpylori infection in two populations in New South Wales. E3 A group of767 blood donors. Infection was defined by a very high cut off level of antibody using an ELISA which has been shown to be 98 per cent predictive of Campylohacter pylori associated gastritis. EZ A group of 1200 persons with gastric symptomatology who had attended for endoscopy. Campylobacrer pylori associated gastritis was determined by microscopy of biopsy specimens and rapid urease testing. 31 455 879 1302 1726 ABSORBANCE (405 nm)(X 1000) Figure 2. Distribution of antibody against Campylobacter pylori in a population of 767 normal blood donors in New South Wales as measured by an ELISA against sonicates of whole cell suspensions of Campylobacter pylori. same population is shown in Figure 1, where it can be seen that over the age of 40 over 40 per cent of py/ori without dyspepsia while a proportion of per- blood donors were actively infected. Morris62 sons with dyspeptic symptoms have no C . pylori. looked at a group of persons who had died of Dyspepsia is such a vague group of symptoms pre- trauma unrelated to stomach pathology and found sumably with a variety ofcauses that assuming there 37 per cent were infected with C . pyfori and the is only a proportion of subjects having symptoms majority had histological gastritis. Populations with different ethnic origins also due to the presence of the bacterium, clinical trials appear to have significantly different colonisation are unlikely to show up highly significant differences. A priority is to identify the group whose rates; thus Graham et af.'l using their CI3-urea symptoms are caused by C. pylori. Even though breath test showed differences between the colonismany specialists gastroenterologists are convinced ation of Chinese Americans compared to Americans from their own experience that there is a place for of Anglo Saxon origin. Similarly, in Australia, treatment of non ulcer dyspepsia with anti- C . Carrick found in a series of 424-dyspeptic patients, pylori agents, general recommendations to this 55 per cent overall had C .pylori associated gastritis. effect are not at present warranted, particularly as In this wholly adult population, frequency of C. pylori did not vary significantly within any given age satisfactory regimens are yet to be found. grouping. Significant differences were however detected among the various ethnic groups. Whereas THE EPIDEMIOLOGY OF only 81/213 (38 per cent) of Anglo Saxons had C. CAMPYLOBACTER PYLORI INFECTION pyfori infection, 50/57 (87 per cent) of Greeks, 54/83 (67 per cent) of Italians and 91/21 (90 per cent) of Incidence Arabs were found to be infected (Dr Jane Carrick, Reports from many countries show that active personal communication). infection with C .pylori is common. Serological tests have been developed that are highly predictive of, not only the presence of C .pylori, but also the pres- The source ence of active chronic g a ~ t r i t i s . ~ ~ The " . ~dis~,~~~~~ Animals have been suggested as a possible resertribution of antibody measured by an ELISA assay in a group of 767 normal blood donors in New voir for C . pylori, although with the two exceptions South Wales3 is shown in Figure 2 . The second peak described below, the organism has only been isoof the curve represents the group of actively infected lated from the human stomach. The proposal put persons. Thus, overall about 20 per cent of this forward in this article is that this is a highly population has C . pyfori associated gastritis. This adapted organism well suited to the niche of gastric CAMPYLOBACTER PYLORI IN HEALTH A N D DISEASE 5 epithelium. Given that more than half of the popu- The mucus associated bacteria of the gastrointestinal lation forty years of age or over are c ~ l o n i s e d , ~ tract .~~ then it is reasonable to propose that the natural The gut surface is coated with mucus arising from reservoir for C .pylori is the human. mucus secreting cells within the intestinal epithelium. This layer of mucus and the mucus filled intestinal crypts in the majority of animal species Transmission studied are full of distinct populations of bacteria.44 There have been examples of direct spread from Importantly, in the depths of these crypts these stomach to stomach via endoscopes and pH elec- populations in any one site appear to be of only one t r o d e ~ . ’ ~ , ~ ~However, , ~ * * ’ ~ if the human is the morphologic type. Even though the outer mucus natural reservoir then one needs to look for facing the intestinal lumen may harbour many instances of person-to-person spread. Institutions, different species, close examination reveals the particularly where the inmates are intellectually organisms closest to the surface are the same as handicapped, are known to have a higher incidence those in the depths of the crypts. Examples of some of those diseases spread by direct contact.6 Thus it of these mucus associated populations are shown in was important that a significantly increased infec- Figures 3 and 4. These observations are consistent tion rate of C . pylori was found in an institution for with the hypothesis that these specialist colonisers the handicapped in New South Wales.3 Seventy-five have a selective advantage in this environment of per cent of inmates aged 33-47 years showed evi- intestinal mucus. The mucus colonisers are different dence of active infection whereas only 18 per cent of at different locations along the length of the intesthe control blood donor population of this age were tine but they appear to share certain characteristics. positive. Also the institutionalised persons were These properties, listed below, are shared by C . infected at a much younger age than the normal pylori. An understanding ofthese factors responsible population consistent with an overall heavier for colonisation may provide a rational basis for loading within the community. developing appropriate therapeutic or prophylactic Further evidence for person-to-person spread strategies. comes from the study of the families of four children with significant dyspeptic symptoms and proven C . Commonfactors involved in colonisation of intestinal pylori associated gastritis.61 The family members mucus were found to have a significantly greater incidence of C . pylori infection (64 per cent) as measured by Spiral morphology serology compared to 166 aged matched controls All the populations naturally colonising mucus in (12 per cent). Contrary to these findings, Jones et the depths of the crypts have a spiral m ~ r p h o l o g y . ~ ~ found no evidence of interpersonal spread. Here the description spiral is used in the loose sense These workers looked at the family contacts of adult as a number of them appear helical rather than a patients which may explain the differences simple spiral. This common morphology must be observed. important in habitation in the mucus environment as the organisms are very different in many other ways and clearly belong to different genera. CAMPYLOBACTER PYLORI AS A MEMBER OF A HIGHLY ADAPTED GROUP OF MUCUS COLONISING Motility BACTERIA The spiral/helical morphology has been proThe evidence to date would suggest C . pylori is an posed to give these organisms a selective advantage extremely common human pathogen which pro- in intestinal mucus by better preparing them for duces a spectrum of disease from the insignificant to motility in the viscous mucus environment.” the severe. We would suggest that the organism’s Certainly experiments in solutions of methyl cellunatural ecological niche is in the gastric surface, lose of different concentrations and viscosities and is thus likely to share many characteristics with demonstrated that these bacteria can move much the specialised populations of bacteria that are more rapidly in higher viscosities than rod shaped known to occupy similar niches in other parts of the b a ~ i l l i . ’ The ~ . ~ same ~ result has been found with C . gastrointestinal tract. p y l ~ r iHowever, .~~ close examination of mucus in 6 A. LEE A N D S. L. HAZELL Figure 3. Surface associated bacteria on the caecal mucosa of a normal mouse. A. A light micrograph of a section of caecal epithelium. Two populations of bacteria are seen in the area of intestinal mucus. The outer layer of fusiform shaped bacilli and the inner layer of spiral bacteria which are also seen in the crypt (bar = 20 pn),B. A scanning electron micrograph of the caecal surface showing the fusiform bacilli with a few spiral organisms (arrow) visible underneath (bar = 2 pm). C . A scanning electron micrograph of the opening of a caecal crypt with the outer mucus displaced showing the large population of spiral bacteria that colonise the mucus in the depth of the crypt (bar= 3 pm). Micrographs courtesy of J. ORourke, University of New South Wales. fixed clinical specimens and observation of these bacteria in wet preparations of mucus scrapings shows motility may involve more than just a corkscrew like action. The bacteria are consistently seen in characteristic parallel streams.27When examined live, C. pylori and other spiral bacteria are seen to move rapidly up and down as if on t r a ~ k s ~This ~, could be an alignment with the mucus strands. Whatever the mechanism of movement in mucus of these organism they are clearly better at it than bacteria of different morphologies. C. pylori is sensitive to acid,24 but once this organism contacts the gastric mucus it is very well equipped to quickly move away from the- hostile acid environment of the stomach lumen down into the protected environ~ment ~ . of gastric mucus. A characteristic feature of C. pylori in mucus can be seen in Figure 5a. When the organism is out on its own in the mucus and not 7 CAMPYLOBACTER PYLORIIN HEALTH A N D DISEASE predictable as they live away from the often reduced lumen at the edge of the highly oxygenated tissue from which presumably there is diffusion of small amounts of oxygen into the mucus. Thus the autochthonous spirals in the rodent ileum seen in Figure 4a are microaerophilic. The natural spiral organisms of the caecum (Figure 3c) are strictly anaerobic69 which is in apparent conflict to the idea of small amounts of oxygen being available in the intestinal mucus. However, the lumen of the caecum is highly reduced and possibly any oxygen is rapidly taken up. Interestingly, when the natural flora of the mouse caecum are removed by antibiotics or purgatives and the normal flora of these animals allowed to recolonise, the caecal crypts become packed with the microaerophilic spiral bacterium that normally inhabits the ileal crypts before the normal caecal bacteria r e t ~ r n ~i.e. * ,at~ this ~ stage the caecum is not highly reduced and so the oxygen availability at the surface resembles that of the ileum. Similarly, the stomach contents of humans are not highly reduced and thus the environment close to the gastric surface is likely to be microaerobic, as is the mouse ileum, and so the microaerophilic C. pylori thrives. Microaerophilism is an oft forgotten ecological determinant and may have particular significance for C. pylori as a contributing factor to the ineffectiveness of antimicrobial therapy, but more importantly it would ensure that the organism is living in an environment hostile to phagocytic cells; this property is an important determinant of pathogenicity. Figure 4. Surface associated bacteria in the mucus lining of the rodent ileum and colon showing distinctly different populations of bacteria. A. A transmission electron micrograph of a cross section of a crypt of the ileum of a mouse showing large numbers of bacteria with characteristic periplasmic fibrils (bar = 2 pm). B. A transmission electron micrograph of a section of colonic mucosa showing bacteria attaching to mucus filled goblet cells (bar= 1 pm). Both these populations in other sections are seen to have a spiral morphology. Micrographs courtesy of M. Phillips, University of New South Wales. associated with the tissue surface the spiral morphology is particularly evident. This can also be seen in vitro; the organism in solutions of liquid growth medium plus 1-1.5 per cent methyl cellulose is seen to be very curved and its auger like movement easily observed. Microaerophilisrn A common feature of these bacteria is a requirement for slightly reduced oxygen tension for optimal growth. Given their natural habitat, this is SPECIAL FEATURES O F COLONISATION O F THE STOMACH BY CAMPYLOBACTER PYLORI Gastric mucosa is a different environment from other parts of the intestinal tract and so even though organisms adapted to this site share common properties related to environmental similarities, stomach colonisers are likely to have a number of distinctive traits which explain their specificity of localisation. It is particularly important that these determinants be well defined. Localisation at intercellularjunctions C. pylori can be seen in two general locations in gastric biopsies examined under light microscopy, in the mucus as described above and secondly apparently adhering to the tissue surface. Careful observation of specimens cut at right angles to the 8 A. LEE AND S . L. HAZELL Figure 5. Cumpylobucter pylori in stomach biopsies of patients with active chronic gastritis. A. A backscattered scanning electron micrograph showing the typical spiral morphology of bacteria in the outer mucus and organisms penetrating down into an intercellular junction (bar = 5 pm). B. A transmission electron micrograph showing an organism (P) at each of three intercellular junctions (bar= 1 pm). C. A transmission electron micrograph of a cross section through a number of mucus secreting gastric cells showing the concentration of organisms at the cell junctions (bar = 5 pm). epithelium reveals the bacteria are not evenly distributed over the tissue surface, rather they appear to be localising close to intercellular junction^.'^ This is more clearly seen in transmission micrographs. Figure 5b and c shows this localisation. The scanning electron micrograph of Steer" shown in Figure 6 , best shows this distribution on the stomach surface. There must be a factor at this particular site on the epithelium which is particularly attractive or nutritive to C. pylori. Given that intercellular junctions are potential leaky sites for tissue substrates to enter the mucus layer, it is likely that localisation at this site is due to the presence of a preferred metabolite for the o r g a n i ~ m . ' ~ Urease C. pylori has an exceptionally high activity of the enzyme urease,' 1 3 2 7 , 4 3 , 6 6 which catalyses the break down of urea to ammonia and carbon dioxide. This activity has been shown to be almost 100 times greater than Proteus vulgaris, the archetypal urease positive bacterium and the enzyme is manufactured constitutively." So much enzyme is produced in vivo that a urease test direct on a biopsy specimen has proved to be a highly specific and rapid method of diagnosing C. pylori i n f e c t i ~ n . ' ~1,59 , ~ No organism would continually manufacture vast amounts of an enzyme without its being of special importance. 9 CAMPYLOBACTER PYLORI IN HEALTH A N D DISEASE changes could make it easier for gastric acid to penetrate mucus and finally there is a possibility that the ammonia generated by the urease has a direct toxic effect on cells. Specijic receptor sites Figure 6. A scanning micrograph of the surface of the stomach of a human patient with gastritis. Taken from the paper of Steers1 with permission from the author and Gut. The Campylobacrer pylori are seen to be localised at the intercellular junctions. Possession of another enzyme, glutamate dehydrogenase,” would indicate that the organism has an ability to utilize extracellular ammonia, thus it is likely urea is a major nitrogen source for this bacterium. Leakage of urea through the intercellular junctions has been dem~nstrated;~’ 60 per cent of the non-protein nitrogen in blood is in the form of urea. There is plenty of precident for surface associated bacteria utilising blood urea; organisms lining the surface of the rumen have high urease activity and it has been suggested that this activity creates a urea gradient which encourages the passage of urea into the lumen.8,88Thus one of the preferred metabolites resulting in localisation of C . pylori at the stomach cell junctions could be urea. Goodwin et aI.,l7 have suggested that the urease plays a role by creating a cloud of ammonia around the organism in the lumen thus protecting it from gastric acidity. Given that many organisms can successfully pass through the stomach following meals, it would appear unlikely that production of such a large amount of this enzyme is an acid resistance mechanism, although this could be a secondary advantage. Though not of any ecological importance there is a possibility that the possession of this enzyme may contribute to the pathology of C. pylori associated di~ease.’~.’~ A major contributor to peptic ulceration is gastric acidity. Changes in the local environment due to the break down of large amounts of urea at the tissue surface could contribute to ulceration in a number of ways; disruption of the cellular Na+/K dependent ATP-ase sodium ion gradient could result in back diffusion of H + ions which in certain patients could cause tissue damage, ionic + There have been some suggestions that there are specific attachment sites on the gastric mucosa for C.pylori. Goodwin has commented on the presence of attachment ‘pedestals’ analagous to enterotoxigenic Escherichia coli although these have not been seen by many ~ 0 r k e r s . The l ~ type of localisation seen in Figure 5a, where the organism is penetrating part way down an intercellular junction indicates that classical attachment may not be the whole story. However, there is some important data on localisation which suggests that some form of very specific interaction with the tissue must occur. C . pylori will only colonise gastric type In some persons patches of intestinal tissue, i.e. small bowel mucosa, are found in the stomach. C . pylori will not colonise these areas of intestinal m e t a p l a ~ i a .Indeed ~~ examination of biopsies from an infected stomach will reveal heavy colonisation of the gastric cells which stops completely at the first adjacent intestinal metaplastic cell. Conversely, commonly in the duodenum particularly in the duodenal bulb, areas of gastric metaplasia are foundz9i.e. patches of gastric type epithelium in the intestinal tissue. In the duodenum, C . pylori will only colonise the areas of gastric metaplasia and is normally associated with active chronic inflammation at this site.67,61.82,83,89 Thus duodenitis induced by C .pylori is strictly ‘gastritis’ in the duodenum. Another example of the specificity of C.pylori association was found in a 14 year old girl with spina bifida and hydrocephalus who also had Barrett’s oesophagus i.e. the presence of acid secreting gastric epithelium in the oesophageal tissue. C .pylori was found to be colonising only the gastric type tissue in the child’s oesophagm61 The presence of the organism coincided with areas of oesophagitis. An ability to induce injiammation and escape the consequences One of the most fascinating and unique aspects of C. pylori associated gastritis, is that the organism continually induces an active component of the 10 inflammatory response, i.e. stimulation of infiltration by polymorphonuclear leucocytes, for year upon year and escapes the consequences of what is presumably a host defence mounted against the organism. How can a pathogen successfully survive in its ecological niche for such long periods of time? One possibility which has been referred to above is the microaerophilic growth optimum of the organism ensuring that it is living in an environment that would be hostile to polymorphs. However, this cannot be all. A strong immune response is found in infected and bacteria are found to be coated with a n t i b ~ d y ~although ~ . ~ ’ there is a suggestion that organisms deep down in the crypt are not; perhaps a consequence of protease activity.2 In addition the chronic component of active chronic gastritis is presumably due to an accumulation of committed mononuclear cells with specificity against the o r g a n i ~ m . ~ ~ . ~A’n*hypothesis ~~*~’ particularly relevant to an understanding of the ecology of C. pylori infection is that the inflammation is an important part of col~nisation.~’ In summary, the organism creates a milieu more conducive to continued survival by inducing inflammation with its accompanying leakage of serum constituents thus making its preferred metabolites more accessible. This would explain why the presence of the organism is always accompanied by inflammation. A. LEE AND S. L. HAZELL OTHER STOMACH ASSOCIATED SPIRAL-SHAPED BACTERIA The concept of C .pylori as a highly adapted human organism has important consequences with respect to the production of animal models of C. pytori infection. I f the hypothesis above is correct, then it should prove difficult to establish the organism in the stomachs of other animal species and mimic the human infection; this has been proven to be the case. The only animals that C. pylori has colonised have been primates, presumably due to their similarity to man, and laboratory and gnotobiotic The organism will not establish in normal pigs, possibly as the natural pig stomach organisms are better adapted to the pig tissue and do not allow the human organism to colonise. However, in germ free piglets without the competing pig flora, C . pylori will inhabit the stomach. Although, the gnotobiotic pig has been proposed as an animal model for human infection, it is disappointing as the associated component of the human infection, active inflammation, is not sustained. In one respect the pig model could be important as there does appear to be tissue specificity as in the human i.e. in 616 piglets the stomach was colonised while 216 had the organism in the duodenum, and 016 had the organism elsewhere in the boweL4’ This could be a useful model for investigating the basis of tissue specificity particularly with respect to gastric type mucosa. CAMPYLOBACTER PYLORI AS ‘ALMOST’ Due to the host specificity of these stomach associNORMAL FLORA ated bacteria it is possible that investigation of their Proponents of the case against C . pylori as a patho- association with their natural host might provide gen argue that here is a bacteriumpresent in as high information relevant to our understanding of the as 80 per cent of some communities and yet causing association of C. pylori with the human stomach. Many animal species have naturally occurring minimal symptomatology in these persons. We propose the role of this organism in human disease is spiral-shaped organisms in their stomachs.Three more easily accepted if one looks at it from an eco- examples are shown in Figure 7. There has been an logical perspective. C . pylori is a natural inhabitant extensive literature on these bacteria over the years of the human stomach. Over the period of evolution but they have never been cultured. It is interesting it has acquired a number of adaptations that make it that the morphology of the organisms naturally particularly well suited to the environment of gastric found in the monkey, cat and dog are very similar to mucus and gastric epithelium. This concept of C . each other and are markedly different to C. pylori. pylori as an highly adapted organism with a unique Whether these bacteria cause pathology is not ecological niche in the human stomach is as consis- resolved. Yet another different bacterium has been tent with the idea of the bacterium being part of the found in the ferret and there have been claims of normal flora in the same way as we would consider both ulceration and no histopathology associated several species of Bacteroides being part of the nor- with its presen~e.~,’~,’’ The cat is showing promise as an interesting mal bowel flora even though they may not be isolated from every individual. However, C. pylori is animal model. A spiral shaped organism colonises a only ‘almost’ normal flora as it is always associated larger proportion of cats with increasing age as does with a pathology in the human albeit mild and often C . pylori in humans. We have been successful in culturing this organism and this should allow the asymptomatic. 11 CAMPYLOBACTER PYLORI IN HEALTH AND DISEASE f Figure 7 Transmission electron micrographs of sections of the gastric mucusa of a number of animal species showing the presence of populations of spiral shaped bacteria A Gastric pit of a dog (bar = 5 pm) B. Bacteria of the gastric pit of a dog (bar = 1 pm) C Gastric gland of a cat (bar= 3 pm) D Bactenum in the glandular lumen of a cat (bar = 1 pm) E Gastric gland of an ape (bar = 3 pm) F Bacterium in the glandular lumen of an ape (bar = 1 pm) Micrographs courtesy of J ORourke, University of New South Wales testing of a number of the hypotheses cited above. For example the organism is also very strongly urease positive and this will allow us to determine the role of this enzyme in colonisation. The distribution of the cat spiral in the tissue is different to C. pylori, the organism gets deeper into the crypts and is even found within parietal cells, an uncommon finding with C. pylori. However, organisms are found in the mucus and this probably explains why this organism is as hard to clear with antimicrobial agents as is the human organism. We consider the cat will be a useful model for screening anti- microbials for activity at the stomach epithelial surface and in mucus. Julie Dent and Cliodna McNulty recently published a letter" in which they report the presence of a spiral-shaped bacterium in the biopsies of three human patients. This organism was clearly different to C. pyfori. Close examination of their photographs and the original slides revealed that the morphology was similar to our cat isolate. Serology on the serum from these patients has shown that there is a high level of antibody to the cat organism compared to lower levels of antibody to C. pylori. 12 A. LEE AND S. L. HAZELL Control sera from proven C. pylori positive patients showed a high level of antibody to C. pylori and low levels to the cat organism. It could be that these three rare human cases (3/1200) could represent infection from an animal source. Jones et and Newel1 and Ba~kerville~’ have both isolated organisms identical to C . pylori from primates in experimental colonies cared for by humans. Could it be that the monkeys have acquired C. pylori from their human handlers? THE TRIGGERS TO CAMPYLOBACTER PYLORI ASSOCIATED SYMPTOMATOLOGY Given that the case against C. pylori as a significant cause of stomach disease is proven and the concept of specific adaption is as outlined above, the future directions for research become clearer. While there is a need to better define the factors that allow the organism to colonise its specific niche, there is a much greater need to appreciate how changes in this local environment result in the appearance of overt symptoms. The triggers of symptomatology are yet to be identified. Acute symptomatology When Morris64inoculated himself with a live culture of C. pylori he suffered a few days of acute symptoms. We have investigated a patient of Frommer’ in Sydney, who showed serological evidence of early infection following an acute attack similar to Morris. In both these cases, the initial symptomatic phase resolved although infection persisted. Early histology showed large numbers of C. pylori and polymorphs with no mononuclear cells. One year later, they both remain symptom free with classical C .pylori associated active chronic gastritis. In the majority of persons the initial acute phase of C. pylori infection will usually be missed; dismissed as ‘gastroenteritis’. Patients with self-limiting episodes of epigastric pain do not usually come from endoscopic examination and hence acute acquisition of C. pylori infection passes uninvestigated. Marshall noted the descriptions of acute gastritis in the 1920 edition of Sir William Osler’s Textbook of Medicine, where vomiting was described as a common symptom. Marshall speculated that many first infections of this organism present in this way. One brief study of ours would suggest that acute symptomatology can be triggered in certain circumstances in persons with long standing active chronic ’ ga~tritis.~’ A group of obese females were endoscoped as a prelude to the insertion of a ‘Balobes’ intragastric balloon for weight reduction treatment. As would be expected a proportion, about 30 per cent, had C .pylori associated active chronic gastritis and were free of major dyspeptic symptoms. Following insertion of the balloon, 19 of 3 1 of the C. pylori positive patients experienced vomiting while none of the 60 C. pylori negative patients suffered this acute symptom. Clearly, in the presence of C. pylori, insertion of the balloon made a major change to the stomach environment, triggering off symptoms. Dyspepsia As mentioned above, anecdotal evidence of gastroenterologists with a specialist interest in C. pylori, suggests that ultimately the case for a causative role for this organism in non ulcer dyspepsia will be proven. However, many patients have the infection and no symptoms, while others only have symptoms occasionally. Analysis of the experiences of large numbers of C. pylori negative and positive dyspeptic patients, looking for various factors that could alter the stomach milieu and activate organism activity or host receptivity may allow us to better define that sub-group of C. pylori-related dyspepsias. Ulceration Wyatt” recently published an important paper in which she proposed a sequence of events that could result in ulceration particularly in the duodenum. Firstly there is a prerequisite for C. pylori gastritis in the stomach antrum. Organisms from this source infect areas of gastric metaplasia in the duodenum which also becomes inflamed. Then in a subset of these people other factors come into play which result in ulceration. C. pylori on its own is not the cause of duodenal ulcer. It is this perception of C. pylori as the cause of duodenal ulcers that has aroused scepticism of gastroenterologists, as it would seem to be contradictory or exclusive of the established ideas on ulcer aetiology. C. pylori is indeed another factor in the complex equation of ulcer formation but it is only part of the story. If we take as given, an essential prerequisite for duodenal ulcer as being the underlying colonisation of tissue with this well adapted bacterium, the question now becomes how can the established ideas of ulcer aetiology explain the different response in C . pylori CAMPYLOBACTER PYLORI IN HEALTH AND DISEASE positive ulcer positive patients, to those C. pylori positive ulcer negative persons. For example, what is different in the duodenal environment of a smoker, that may allow C. pylori to react in a certain way. All the literature on ulcer aetiology and pathogenesis needs to be completely re-examined in light of this assumption of C. pylori as an essential prerequisite for a majority of ulcers. CONCLUSION We believe that an ecological approach to the microbiology, histopathology, symptomatology, and epidemiology of C. pylori infection in humans could resolve some of the controversy over the role of this organism in peptic and duodenal disease. 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