Download - Wiley Online Library

Journal of Applied Microbiology 2001, 90, 80S±84S
Helicobacter in water and waterborne routes of transmission
L. Engstrand
Bacteriology unit, Swedish Institute for Infectious Disease Control, Solna, Sweden
12
1 1.
2.
3.
4.
Summary, 80S
Introduction, 80S
Epidemiological studies, 80S
Viable but non-culturable H. pylori in the environment, 81S
5. Detection of environmental H. pylori, 82S
5.1 Culture, 82S
1. SUMMARY
Helicobacter pylori is the causative agent of gastritis and
duodenal ulcers and plays a role in the development of gastic
cancer. The transmission of H. pylori remains unclear but
two different pathways have been suggested: faecal±oral and
oral±oral. Studies from developing countries with low socioeconomic status and poor management of the drinking water
suggest that environmental factors are important. Thus, the
water may serve as a reservoir for H. pylori that infect
humans. Viable but non-culturable bacteria are widespread
in marine environments. The coccoid form of H. pylori in
non-culturable by ordinary techniques and it remains to be
studied whether this form is viable. Several studies have
been performed to assess the viability of H. pylori in water
since it has been suggested that the coccoid form of H. pylori
is responsible for transmission in the environment, probably
via contaminated water. Different DNA-based methods
have been used to detect Helicobacter spp. in water. Target
genes have been selected to avoid cross-reactivity between
H. pylori and other bacteria. However, closely related
bacteria and unknown subspecies of Helicobacter spp. may
confound the `species-speci®c' DNA-based assays and must
be considered if controversial results appear in analyses of
environmental samples.
2. INTRODUCTION
Helicobacter pylori colonizes the human stomach and establishes a chronic infection associated with an in¯ammatory
response. H. pylori is the causative agent of gastritis and
peptic ulcers and plays a role in the development of gastric
Correspondence to: Dr L. Engstrand, Bacteriology Unit, Swedish Institute for
Infectious Disease Control, SE-171 82 Solna, Sweden
(e-mail: [email protected]).
5.2 Microscopy, 82S
5.3 Autoradiography, 82S
5.4 DNA-based techniques, 82S
6. Presence of Helicobacter species in water,
7. Conclusions, 83S
8. References, 83S
82S
cancer. However, only a subpopulation of infected individuals develop gastroduodenal disease. The prevalence of
H. pylori infection in the world is assumed to be approximately 50%, with higher prevalence in developing than in
developed countries. Genetic and socio-economic factors
contribute to acquisition of infection. There appears to be no
substantial reservoir of H. pylori aside from the human
stomach (Dunn et al. 1997).
The epidemiology and transmission pathways of H. pylori
infection are important for the understanding of this
common worldwide infection (Taylor and Blaser 1991).
The transmission of H. pylori remains unclear but two
different pathways have been suggested: faecal±oral and
2 oral±oral (Feldman et al. 1998). Different transmission
routes may be predominant in different geographical areas.
In developed countries, where sanitary procedures such as
water treatment are well managed, reports show a clustering
of H. pylori infection within families, which supports an
oral±oral transmission pathway (Brenner et al. 1999). The
source of H. pylori could be saliva and dental plaques since
H. pylori organisms have been isolated from these locations
(Ferguson et al. 1993; Nguyen et al. 1993). Studies from
developing countries with low socio-economic status and
poor management of drinking water suggest that environmental factors are more important than the oral±oral
transmission route (Hopkins et al. 1993).
3. EPIDEMIOLOGICAL STUDIES
An association between the prevalence of H. pylori in
Peruvian children and the source of drinking water has been
shown (Klein et al. 1991). Transmission by the municipal
water supply was suggested since children whose homes had
external water sources were three times more likely to be
infected with H. pylori than those whose homes had internal
water sources. Hence, children living in homes with no
ã 2001 The Society for Applied Microbiology
HELICOBACTER IN WATER
internal plumbing had a higher level of infection than
children living in homes with standard plumbing. In
addition, children from high-income families with a municipal water supply had a 12-fold increase in the risk of being
infected with H. pylori than children from high-income
families with a water supply from community wells. It was
concluded that socio-economic status in Peru was a less
important risk factor than water source in acquiring the
infection. In developed countries no association between
H. pylori infection and water was evident (HulteÂn et al.
1998). Thus, the environment may serve as a reservoir
for H. pylori that infect humans, at least in developing
countries.
An increased risk for gastric cancer among sewage workers
has been described in several studies (La¯eur and Vena
3 1991; Friis et al. 1993). These workers are often engaged at
both municipal drinking water plants and sewage plants.
Since H. pylori has been associated with an increased risk
for cancer of the stomach, Friis et al. (1996) tested the
hypothesis that sewage workers were at risk of infection by
H. pylori in their work. The study design was cross-sectional
and IgG antibodies against H. pylori were analyzed in 151
sewage workers and 138 referents (matched for age and
socio-economic status). The previously described increase in
the prevalence of IgG antibodies against H. pylori with
increasing age was observed in both groups but the exposure
to sewage work in Sweden did not cause an increased risk of
infection with H. pylori.
Another study, which casts some doubts on the theory of
faecal±oral transmission via a common source, was per4 formed among foreign travellers (Lindkvist et al. 1995).
This group studied seroconversion to H. pylori among 133
initially H. pylori seronegative young Swedes travelling to
developing countries for a total of 16á4 years abroad. Serum
samples were drawn before travelling. Gastroenteritis was
considered as a marker of exposure to faecally contaminated
food or water. Of the subjects, 66% reported one or more
episodes of gastroenteritis. Not one of the 133 travellers
coming for their second blood sample, drawn on average
4 months after returning to Sweden, seroconverted in the
H. pylori ELISA. Thus, these two epidemiological studies
could not provide any evidence for waterborne routes of
H. pylori transmission.
81S
the transmission of the coccoid form in the environment
have been discussed. However, the coccoid form of H. pylori
is non-culturable by ordinary techniques and it remains to
be studied whether the coccoid form is viable. Several
studies have been performed to assess the viability of
H. pylori in water (West et al. 1992; Shahamat et al. 1993;
Enroth et al. 1999). During the conversion from the rodshaped into the coccoid form, numerous different morphological forms of H. pylori are evident (Bode et al. 1993)
(Fig. 1). This transformation is a transient process that
occurs during prolonged culture in vitro. The morphology is
also affected by variations in the environment (oxygen stress,
temperature and presence of antibiotics). Analyses show that
the majority of coccoid bacteria have an intact cell wall, cell
membrane and cytoplasm (Bode et al. 1993; Moshkowitz
et al. 1994). Nutrient supplementation to coccoid forms has
been tested but no conversion from coccoid to rod-shaped
forms was observed (SoÈrberg et al. 1996). However, the
addition of fresh medium somewhat increased the intracellular ATP-content (SoÈrberg et al. 1996). Changes in morphology, intracellular composition and surface properties in
H. pylori during conversion from rod-shaped to coccoid
forms have been studied (Enroth et al. 1999); rods have a
higher density than cocci and bacteria stored in water have
the lowest density. If the coccoid forms are viable yet smaller
in size and volume, the density should theoretically be the
same. The quantitative DNA, RNA and ATP content are
reduced in coccoid bacteria and these forms also express
fewer surface-related proteins. The degenerative changes
found in this study point to dead bacteria and not to a viable
but non-culturable form. However, it was not ascertained
whether all coccoid forms have an overall low DNA, RNA
and ATP content, or if a few cells are viable with full energy
supplies and the remainder are empty, dead spheres. In
addition, the response to stress such as starvation and ageing
seems to be different from the response to acid stress as
4 . V I A B L E B U T N O N -C U L T U R A B L E
H. PYLORI IN THE ENVIRONMENT
Viable but non-culturable bacteria such as Salmonella spp.,
Campylobacter spp., and Vibrio spp. are widespread in
marine environments (Byrd et al. 1991). It has been
suggested that the coccoid form of H. pylori is responsible
for transmission in the environment, probably via contaminated water (HulteÂn et al. 1998). Possible mechanisms for
Fig. 1 The coccoid form of H. pylori. Note some rod-shaped bacteria.
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 90, 80S±84S
82S L . E N G S T R A N D
determined by assessing protein synthesis under various
conditions (Mizoguchi et al. 1998). If coccoid H. pylori
exhibits diversity in viability following exposure to different
stresses, this may be of importance in the colonization
process of the gastric mucosa. Only one report has shown a
successful reversion of coccoid H. pylori to the rod shaped
culturable form. This study was performed in mice but
contradictory results have been reported in pigs (Eaton et al.
5 1995; Wang et al. 1995).
5 . D E T E C T I O N O F E N V I R O N M E N TA L
H. PYLORI
5.1. Culture
So far, no report has been published on successful culture of
H. pylori from environmental samples. All bacillary H. pylori
organisms change morphology gradually over 10 d of culture
and enter a non-culturable stage after 7±10 d on an agar
plate. It has been shown that such coccoid forms did not
convert into the bacillary form during passage through eggs
(Enroth and Engstrand 1996). Egg passage has been used for
the isolation and growth of slow-growing organisms such as
Rickettsiae spp. and Chlamydia spp., among others, and is a
simple and rapid method of culturing and evaluating
culturability of bacteria (Cox 1938; Fei Fan et al. 1957). It
is, however, theoretically possible to grow the organism
within a few days in water if the rod-shaped form is
transferred to the water source. This is illustrated by patients
with gastroenteritis and vomiting who may transfer the
bacteria through a gastric±oral route (Parsonnet et al. 1999).
5.2. Microscopy
H. pylori was found in a majority of the surface and shallow
groundwater samples examined in Pennsylvania and Ohio
6 (Hegarty et al. 1999; see section 6). Fluorescent antibody
staining was used with a H. pylori-speci®c monoclonal
antibody with no cross-reactivity to closely related bacterial
species. However, at least 24 species within the genus
Helicobacter are described in the literature and there is a
possibility of cross-reactivity among these Helicobacter
species or other bacteria. The result in Hegarty's study
supports a waterborne route of transmission of the organism
although no evidence of viability was shown.
5.3. Autoradiography
Findings based on an autoradiographic approach gave
evidence supporting the hypothesis that there is a
waterborne route of infection for H. pylori (Shahamat et al.
1993). Tritium-labelled cells of H. pylori revealed aggregations of silver grains associated with uptake by H. pylori of
radiolabelled substrate. Temperature was a signi®cant
environmental factor associated with the viability of the
organism in water. H. pylori remained viable as determined
by autoradiography at temperatures of 4°C for 26 months.
However, sterile water does not re¯ect the natural environment in which competition with natural populations of
microorganisms can occur. The persistent coccoid form of
H. pylori detected by autoradiography may play an important role in the transmission of disease.
5.4. DNA-based techniques
Different PCR methods have been used to detect Helicobacter spp. in environmental samples, gastric juice, faeces
and gastric biopsy samples (Mapstone et al. 1993; Enroth
and Engstrand 1995; Li et al. 1995). Target genes for PCR
have been selected to avoid cross-reactivity between
H. pylori and other bacteria. Such genes are the urease
encoding gene ureA, the adhesin encoding gene hpaA and
the cytotoxin associated geneA, cagA. Nonetheless, closely
related bacteria and unknown subspecies of Helicobacter
spp. could confound the `species-speci®c' PCR assays and
must be considered if controversial results appear in
analyses of environmental samples. Several different animal
species are infected by unique Helicobacter species and at
least 24 named Helicobacter spp. as well as closely related
unnamed bacteria have been reported so far. In addition,
some features are shared between almost all Helicobacter
spp. such as strong urease activity. Thus, the speci®city of
a DNA-based assay is not consistent and relies on the
availability of up to date accurate sequence data. For
detection of H. pylori organisms in stool and water
samples, a pre-PCR step involving immunomagnetic
separation (IMS) has been developed (Enroth and Engstrand 1995 Fig. 2). Rabbit hyperimmune antiserum was
produced and magnetic beads were coated with puri®ed
IgG which bound to both coccoid and bacillary forms of
H. pylori. Spiked faecal and water samples and a patient's
stool specimen all scored positive with the IMS-PCR
technique.
6. PRESENCE OF HELICOBACTER
SPECIES IN WATER
7 The IMS-PCR technique described in section 5.4 was used
to provide an epidemiological link between Helicobacter
spp. isolated from drinking water and the community
(HulteÂn et al. 1996). Although non-speci®c ampli®cation
cannot be ruled out, the results were consistent with
conclusions from the previous epidemiological study; for
example, an indication for waterborne transmission of
Helicobacter spp. in some environments was found. The
study was performed in 1995 and several reports of closely
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 90, 80S±84S
HELICOBACTER IN WATER
83S
7. CONCLUSIONS
Fig. 2 Immunomagnetic separation (IMS) with coccoid and rodshaped H. pylori adhered to the beads.
related bacteria and other Helicobacter spp. have been
published since then.
Tap and well water and ®eld soil samples were collected in
a region of Japan with a high H. pylori infection rate and
examined using a similar IMS-PCR technique to that
8 described in section 5.4, but with a different target gene for
the PCR reaction (Sasaki et al. 1999). Nucleotide sequencing of the nested PCR products was also performed.
Although the authors observed that the conserved region in
ureA is more speci®c than the target genes used in the
Peruivian study (16S rRNA and hpaA adhesin gene), no
further evidence of these conclusions has been published.
The presence of Helicobacter spp. DNA in Swedish water
was detected with the same primer set as that applied in the
Peruvian study (HulteÂn et al. 1998). However, non-speci®city of the PCR methods due to the presence of unknown
or closely related bacteria could not be totally ruled out.
This was illustrated when sequence alignment of the
H. pylori hpaA gene showed high similarity to H. nemestrinae
and H. acinonyx which indicates that subspecies of Helicobacter could confound the results. One possible explanation
could be that surface water leaking into countryside
household wells was contaminated with such subspecies.
The same problem must be considered in the study
performed in the US (Hegarty et al. 1999). Although 10
Helicobacter subspecies were tested for cross-reactivity with
the H. pylori-speci®c monoclonal antibody, it is impossible
to state that the antibody is speci®c for H. pylori when there
are at least seven known additional subspecies that have not
been tested. In conclusion, future studies of routes of
H. pylori infection must consider the possibility of controversial results due to the presence of unknown Helicobacter
species in the environmental samples. The only H. pylorispeci®c assay is culture.
Evidence for waterborne transmission routes of H. pylori has
been provided by a number of investigators. Despite the
epidemiological evidence supporting a waterborne route of
infection, there is little information available regarding the
occurrence of the organism in groundwater, which is the
main source of drinking water to private households.
Detection of H. pylori in environmental samples is confounded by the lack of a standard procedure for the
detection of this organism. Attempts to culture H. pylori
directly from water samples have been unsuccessful, probably due to its conversion into a coccoid, non-culturable
form. However, overgrowth of other microorganisms may
provide false-negative results even if selective media is used.
Other tools for detection of H. pylori in water, such as
DNA-based techniques, microscopy using monoclonal
antibodies and autoradiography, all have a potential for
false-positive results.
H. pylori does not appear to exist in a culturable form
outside the stomach and even though signs of H. pylori are
detected in water or other environmental samples, there is
still no evidence for transmission by this route. Future
research in this ®eld should focus on development of
H. pylori-speci®c diagnostic tools such as identi®cation of a
H. pylori-speci®c gene as target a for PCR. Furthermore,
attempts to optimize culture conditions for H. pylori in
environmental samples could be one additional challenge.
Studies of transmission pathways of H. pylori infection need
to continue and are important for the understanding of this
common infection. To prevent spread of H. pylori-associated
gastroduodenal diseases, especially in the developing countries, we need an improved understanding of how H. pylori
is transmitted between hosts and if there is any reservoir for
the bacteria aside from the human stomach. Extended
eradication strategies will lead to increased use of antibiotics
in the community that will increase the risk of development
and spread of resistance among H. pylori and other bacteria.
98. REFERENCES
Bode, G., Mauch, F. and Malfertheiner, P. (1993) The coccoid forms
of Helicobacter pylori. Criteria for their viability. Epidemiology and
Infection 111, 483±490.
Brenner, H., Rothenbacher, D., Bode, G., DieudonneÂ, P. and Adler, G.
(1999) Active infection with Helicobacter pylori in healthy couples.
Epidemiology and Infection 122, 91±95.
Byrd, J.J., Xu, H.-S. and Colwell, R.R. (1991) Viable but nonculturable bacteria in drinking water. Applied and Environmental Microbiology 57, 875±878.
Cox, H.R. (1938) Use of yolk sac of developing chick embryo as
medium for growing Rickettsiae of Rocky Mountain spotted
10 fever and typhus group. Public Health Report Washington 53,
2241±2247.
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 90, 80S±84S
84S L . E N G S T R A N D
Dunn, B.E., Cohen, H. and Blaser, M.J. (1997) Helicobacter pylori.
Clinical Microbiology Reviews 10, 720±741.
Eaton, K.A., Catrenich, C.E., Makin, K.M. and Krakowka, S. (1995)
Virulence of coccoid and bacillary forms of Helicobacter pylori in
gnotobiotic piglets. Journal of Infectious Diseases 171, 459±462.
Enroth, H. and Engstrand, L. (1995) Immunomagnetic separation and
PCR for detection of Helicobacter pylori in water and stool
specimens. Journal of Clinical Microbiology 33, 2162±2165.
Enroth, H. and Engstrand, L. (1996) Egg passage of rodshaped and
coccoid forms of Helicobacter pylori: preliminary studies. Helicobacter
1, 183±186.
Enroth, H., Wreiber, K., Rigo, R., Risberg, D., Uribe, A. and
Engstrand, L. (1999) In vitro ageing of Helicobacter pylori: changes in
morphology, intracellular composition and surface properties.
Helicobacter 4, 7±16.
Fei Fan, T., Hsiao-Lou, C., Yuan-Tung, H. and KoÂ-Chien, W. (1957)
Studies on the etiology of trachoma with special reference to isolation
of the virus in chick embryo. Chinese Medical Journal 75, 429±446.
Feldman, R.A., Eccersley, A.J.P. and Hardie, J.M. (1998) Epidemiology of Helicobacter pylori: acquisition, transmission, population
prevalence and disease-to-infection ratio. British Medical Bulletin 54,
39±53.
Ferguson, D.A. Jr, Li, C., Patel, N.R., Mayberry, W.R., Chi, D.S. and
Thomas, E. (1993) Isolation of Helicobacter pylori from saliva.
Journal of Clinical Microbiology 31, 2802±2804.
Friis, L., Edling, C. and Hagmar, L. (1993) Mortality and incidence of
cancer among sewage workers: a retrospective cohort study. British
Journal of Industrial Medicine 50, 563±567.
Friis, L., Engstrand, L. and Edling, C. (1996) Prevalence of
Helicobacter pylori infection among sewage workers. Scandinavian
Journal of Work, Environment & Health 22, 364±368.
Hegarty, J.P., Dowd, M. and Baker, K.H. (1999) Occurrence of
Helicobacter pylori in surface water in the United States. Journal of
Applied Microbiology 87, 697±701.
Hopkins, R.J., Vial, P.A., Ferreccio, C., Ovalle, J., Prado, P.,
Sotomayor, V., Russell, R.G., Wassermann, S.S. and Morris, J.G.
Jr (1993) Seroprevalence of Helicobacter pylori in Chile: vegetables
may serve as one route of transmission. Journal of Infectious Diseases
168, 222±226.
HulteÂn, K., Enroth, H., NystroÈm, T. and Engstrand, L. (1998)
Presence of Helicobacter species DNA in Swedish water. Journal of
Applied Microbiology 85, 282±286.
HulteÂn, K., Han, S.W., Enroth, H., Klein, P.D., Opekun, A.R.,
Gilman, R.H., Evans, D.G., Engstrand, L., Graham, D.Y. and
El-Zaatari, F.A.K. (1996) Helicobacter pylori in the drinking water in
Peru. Gastroenterology 110, 1031±1035.
Klein, P.D., Graham, D.Y., Gaillour, A., Opekun, A.R. and Smith,
E.O. (1991) Water source as risk factor for Helicobacter pylori
infection in Peruvian children. Lancet 337, 1503±1506.
La¯eur, J. and Vena, J.E. (1991) Retrospective cohort mortality study
of cancer among sewage plant workers. American Journal of Industrial
11 Medicine 19, 75±86.
Li, C., Musich, P.R., Ha, T., Ferguson, D.A. Jr, Patel, N.R., Chi, D.S.
and Thomas, E. (1995) High prevalence of Helicobacter pylori in
saliva demonstrated by a novel PCR assay. Journal of Clinical
Pathology 48, 662±666.
Lindkvist, P., WadstroÈm, T. and Giesecke, J. (1995) Helicobacter pylori
and foreign travel. Journal of Infection and Disease 172, 1135±1136.
Mapstone, N.P., Lynch, D.A.F., Lewis, F.A., Axon, A.T.R., Tomkins, D.S., Dixon, M.F. and Quirke, P. (1993) PCR identi®cation of
Helicobacter pylori in faeces from gastritis patients (Letter). Lancet
341, 447.
Mizoguchi, H., Fujioka, T., Kishi, K., Nishizono, A., Kodama, R. and
Nasu, M. (1998) Diversity in protein synthesis and viability of
Helicobacter pylori coccoid forms in response to various stimuli.
Infection and Immunity 66, 5555±5560.
Moshkowitz, M., Gorea, A., Arber, N., Konikoff, F., Berger, S. and
Gilat, T. (1994) Morphological transformation of Helicobacter pylori
during prolonged incubation: association with decreased acid
resistance. Journal of Clinical Pathology 47, 172±174.
Nguyen, A.-M.H., Engstrand, L., Genta, R.M., Graham, D.Y. and
El-Zaatari, F.A.K. (1993) Detection of Helicobacter pylori in dental
plaque by reverse transcription-polymerase chain reaction. Journal of
Clinical Microbiology 31, 783±787.
Parsonnet, J., Shmuely, H. and Haggerty, T. (1999) Fecal and oral
shedding of Helicobacter pylori from healthy infected adults. Journal
of the American Medical Association 282, 2240±2245.
Sasaki, K., Tajiri, Y., Sata, M., Fujii, Y., Matsubara, F., Zhao, M.,
Shimizu, S., Toyonaga, A. and Tanikawa, K. (1999) Helicobacter
pylori in the natural environment. Scandinavian Journal of Infectious
Diseases 31, 275±279.
Shahamat, M., Mai, U., Paszko-Kolva, C., Kessel, M. and Colwell,
R.R. (1993) Use of autoradiography to assess viability of Helicobacter
pylori in water. Applied and Environmental Microbiology 59,
1231±1235.
SoÈrberg, M., Nilsson, M., Hanberger, H. and Nilsson, L.E. (1996)
Morphologic conversion of Helicobacter pylori from bacillary to
coccoid form. European Journal of Clinical Microbiology and Infectious
Diseases 15, 216±219.
Taylor, D.N. and Blaser, M.J. (1991) The epidemiology of Helicobacter
pylori infection. Epidemiology Reviews 13, 42±59.
Wang, X., SturegaÊrd, E., Rupar, R., Nilsson, H.-O., Aleljung, P.A.,
CarleÂn, B., WilleÂn, R. and WadstroÈm, T. (1995) Infection of BALB/
cA mice by spiral and coccoid forms of Helicobacter pylori. Journal of
Medical Microbiology 46, 657±663.
West, A.P., Millar, M.R. and Tomkins, D.S. (1992) Effect of physical
environment on survival of Helicobacter pylori. Journal of Clinical
Pathology 45, 228±231.
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 90, 80S±84S