Transmission Pathway of H.pylori

7/30/2019 Transmission Pathway of H.pylori

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Review

Transmission pathway of Helicobacter pylori: Does food play a role in rural andurban areas?

F.F. Vale a,, J.M.B. Vtor b

a Faculty of Engineering Catholic University of Portugal, Estrada Octvio Pato, 2635-631 Rio de Mouro, Portugalb iMed.UL (MedChem Division), Faculty of Pharmacy, University of Lisbon, Av. das Foras Armadas, 1649-003 Lisboa, Portugal

a b s t r a c ta r t i c l e i n f o

Article history:

Received 18 October 2009Received in revised form 13 January 2010

Accepted 14 January 2010

Keywords:

Helicobacter pylori

Transmission

Food

Water

Vehicle

Source

Rural and urban areas

Helicobacter pylori is a Gram-negative microaerophilic bacterium that has colonized the human gastric mucosa.This infection is very common and affects more than half of the human population. The prevalence is howeverunbalanced between rural developing areas (more than 80%) and urban developed areas (less than 40%). H.

pylori is responsible for several pathologies, such as gastritis, peptic ulcer and gastric cancer but its transmissionpathway is still not clear. The risk factors for H. pylori infection include poor social and economic development;

poor hygienic practices; absence of hygienic drinking water; and unsanitary prepared food. There is evidencesupporting a gastrooral, oraloral and faecaloral transmission, but no predominant mechanism oftransmission has been yet identified. Transmission may occur in a vertical mode (e.g. from parents to child)

or in a horizontal mode (across individuals or from environmental contamination). In either case, the

involvement of water and food cannot be excluded as vehicles or sources of infection. Indirect evidence ofpresence of H. pylori in water and food, namely the detection of its DNA and survival studies after artificialcontamination of food and water has been described. This paper reviews data both favourable and against the

role of water and food in the transmission of H. pylori, exploring their role as a potential transmission vehicle

for person-to-person and food-chain transmission. The likelihood of the transmission pathway in developingrural and developed urban areas appears to be different. In developed areas, person-to-person transmissionwithin families appears to be dominant, while in the rural developing areas the transmission pathway appears

to be more complex. In this later case, the transmission by contaminated food, water, or via intensive contactbetween infants and non-parental caretakers may have a greater influence than within-family transmission.

2010 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.1. Epidemiology: diseases and prevalence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2. Risk factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.3. Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.4. Geographic strain distribution and globalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.5. Infection in rural (developing) and urban (developed) environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.6. Mixed colonization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.7. Coccoid forms: cell death or VBNC? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.8. Concepts of the microbiology ofH. pylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2. Routes of transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.1. Person-to-person transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.1.1. Gastrooral transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.1.2. Oraloral transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.1.3. Faecaloral transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.1.4. A role for water in person-to-person transmission? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.1.5. A role for food in person-to-person transmission? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

International Journal of Food Microbiology 138 (2010) 112

Corresponding author. Present address: Faculty of Engineering. Catholic University of Portugal. Estrada Octvio Pato. 2635-631 Rio de Mouro (Lisboa), Portugal. Tel.:

+351214269770; fax: +351214269800.

E-mail address: [email protected] (F.F. Vale).

0168-1605/$ see front matter 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.ijfoodmicro.2010.01.016

Contents lists available at ScienceDirect

International Journal of Food Microbiology

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2.2. Food chain transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.2.1. Water ingestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.2.2. Food ingestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73. Likelihood of transmission in rural and urban environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1. Introduction

Helicobacter pylori was first described by Warren and Marshall in1983 in association with chronic gastritis and peptic ulcer (Warren andMarshall, 1983; Marshall and Warren, 1984). This discovery was rathercontroversial at the time but ultimately in 2005, these two scientists

were awarded the Nobel Prize for this discovery. Nowadays, a H. pylorisearch on Pubmed will retrieve thousands of publications, butunanswered questions still remain. The transmission route of H. pylorihas yet to be ascertained, as well as its source. Presently, the only

recognized and accepted reservoir is the human stomach, but theexistence of extra-gastric reservoirs for H. pylori has been suggested.This paper reviews anddiscusses the possibility of the transmissionofH.

pylori by food and water in developed and developing countries.

1.1. Epidemiology: diseases and prevalence

H. pylori colonizes the human stomach of about half of the worldpopulation. The colonization is not always associated with thedevelopment of pathology as more than 70% of the infected population

remains asymptomatic. However, colonized individuals may developgastritis, peptic ulcer, gastric cancer or Mucosa Associated LymphoidTissue (MALT) lymphoma (McColl, 1999; Das and Paul, 2007). Theinfection is mainly acquired during childhood (Sykora et al., 2006). The

prevalence of infection is typically higher in developing countries(greaterthan 80%) and lower in the developed ones (typically less than40%) with a declining pattern worldwide (Perez-Perez et al., 2004;Kusters et al., 2006). However, as socioeconomic level varies within

subpopulations of the same country, the prevalence in these subgroups

can be rather different (Bruce and Maaroos, 2008).

1.2. Risk factors

Several risk factors for H. pylori infection have been highlighted.These include, poor social and economic development (Lehours andYilmaz, 2007; Perez-Perez et al., 2004); low education level; poor

hygiene practices during childhood; crowded families; lack of ahousehold bath; absence of sanitary drinking water; absence of asewage disposal facility duringchildhood (Nouraie et al., 2009) and food

handled inappropriately (van Duynhoven and de Jonge, 2001). Most ofthese factors are a consequence of and associated with socioeconomicdevelopment, which in epidemiology studies usually acts as aconfounding factor. The improvement of general hygienic conditions

decreases the prevalence of the infection (Fujimoto et al., 2007). Thisstatement suggests the existence of an environmental pool to whichchildren are exposed, especially in developing areas (Gomes and DeMartinis, 2004b). However,it cannot be excludedthat inareas withhigh

populations, the transmission can be even higher compared to lowpopulated areas due to limited human contact. Hostgenetic factors havealsobeen associated with the infection,especiallyin the progression to adisease state (Ando et al., 2007; Sgouros and Bergele, 2006).

1.3. Therapy

Once acquired, the infection is usually lifelong, unless treated. TheMaastricht III Consensus report indicates that H. pylori should be eradi-cated in case of presence of associated pathologies, such as duodenal

ulcer, gastric ulcer, atrophic gastritis, MALT lymphoma, nonulcer dys-

pepsia, uninvestigated dyspepsia in areas with prevalence >10%, orafter gastric cancer resection. It should also be eradicated infirst degreerelatives of patients with gastric cancer, and also in the presence ofunexplained iron-deficiency anaemia and idiopathic thrombocytopenic

purpura (Vakil and Megraud, 2007). For eradicating H. pylori, a tripletherapy is commonly used (Megraud and Lehours, 2007), whichinvolves two antibiotics (usually amoxicillin and clarithromycin) anda proton pomp inhibitor (PPI) or ranitidine bismuth during seven days(Kusters et al., 2006; Megraud, 2004a,b; Vakil and Megraud, 2007). Themost commonly used antibiotics are tetracycline, amoxicillin, imidazole

(metronidazole and tinidazol) and macrolids (clarithromycin andazithromycin). Antibiotic therapy fails in about 20% of the patients(Parente et al., 2003; Kusters et al., 2006), mainly due to antibioticresistance (Megraud, 2004a), but also because the bacteria may be

present in a protective environment like the stomach mucus or even

intracellularly (Dubois and Boren, 2007).

1.4. Geographic strain distribution and globalization

H. pylori is characterised by extensive genetic variability amongdifferent strains (Suerbaum, 2000). Moreover, the map of H. pyloristrain diversity appears to be similar to the one of humans. Thissimilar pattern of H. pylori and human geographic diversity anddistribution strongly suggests a co-evolution between this bacteriumand man, which can be used to understand human migrations

(Covacci et al., 1999). The H. pylori distribution pattern follows humanmigration roots, which suggests that the colonization of the humanstomach occurred before modern man left East Africa (Linz et al.,

2007; Cavalli-Sforza, 2001; Covacci et al., 1999; Falush et al., 2003;

Vale et al., 2008, 2009). This observation points to a person-to-persontransmission mode.

Globalizationand the rapid mobility of the populationmay disturbin

the near future the six large clusters ofH. pylori strains currently spreadthroughout the world (Achtman et al.,1999; Linzet al.,2007). But again,this will be a response to human migrations. However, populationgenetics tools that have allowed the assignment of individualisolates to

one of six discrete clusters of bacterial populations are inadequate toaddress issues of individual transmission (Schwarz et al., 2008).

1.5. Infection in rural (developing) and urban (developed) environments

The prevalence of infection is especially higher in the ruraldeveloping areas in contrast to urban developed ones (Brown et al.,

2002; Cheng et al., 2009; Aguemon et al., 2005). Different culturalhabitats and an increased number of risk factors may contribute to this

difference. Populations from rural environments are probably exposedto an increased number of infectious sources, which is compatible withdifferent routes of infections according to the population's culture andenvironment (Azevedo et al., 2007b; Schwarz et al., 2008). Familial

transmission as a mode of dissemination of H. pylori is generallyaccepted (Kivi et al., 2003, 2007; Raymond et al., 2008; Fujimoto et al.,2007), especially transmission from mother to child (Kivi and Tindberg,2006; Weyermann et al., 2006; Kiviet al.,2003). However, most of these

studies were performed within urban families and using a single isolatefrom each family member.

Recently, a study of three families revealed that H. pylori infectionmay be acquired by more diverse routes than previously expected. In

this particular report the mother was not implicated in the transmission

2 F.F. Vale, J.M.B. Vtor / International Journal of Food Microbiology 138 (2010) 112


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in two of these three families. Sibling-to-sibling transmission and ac-quisition ofH. pylori outside the household appears to be involved in thetransmission pathway (Raymond et al., 2008). Likewise, there isevidence that familial transmission only plays a minor role in rural

environments (Karita et al., 2003; Schwarz et al., 2008). In these cases,horizontal transmission, through contaminated food, water, or viaintensive contact between infants and non-parental caretakers, may

jointly play a more important role than within-family transmission

(Schwarz et al., 2008).

1.6. Mixed colonization

The majority of the typing studies of H. pylori strains are based onstrain genotyping after culture isolation from gastric biopsies. In mostcases, only one isolated culture is achieved. The detection of the

presence of mixed colonization (presence of two or more strainscolonizing the stomach) appears to depend on the material used.Genotyping results with DNA extracted from gastric biopsy specimenswere inconsistent with those of bacterial DNA isolated from H. pyloricultured from gastric biopsies. Mixed infection was detected in 27% ofcases when tissue DNA was used compared to only 9% when bacterialDNA was used (Park et al., 2003). This led to the question of howrepresentative are the isolates that are studied in transmission studies.

Several other works report the presence of mixed colonization (Fantryet al., 1996; Wong et al., 2001; Sheu et al., 2009). Information on thegenetic similarity of strains from different family would benefit frommore thorough attempts to identify multiple strains in individuals,

which would be of particular relevance for better understanding modesof transmission (Azevedo et al., 2009). However, the strains colonizingthe same individual appear to be quite similar (Lee et al., 2005; Cellini

et al., 1996; Carroll et al., 2004).

1.7. Coccoid forms: cell death or VBNC?

H.pylori is a Gram-negativebacterium,measuring2 to 4 m inlengthand 0.5 to1 m in width. Although usually spiral-shaped, the bacteriumcan appear as a rod,while coccoid shapes appear after prolonged in vitro

culture, a longinoculation period in water or milk samples, or antibiotictreatment (Kusters et al., 2006). These coccoid forms cannot be cultured

in vitro and are thought to represent dead cells (Kusters et al., 1997),although it has been suggested that coccoid forms may represent aviable, but nonculturable state (VNBC) (Azevedo et al., 2007a; Chen,

2004). Coccoid forms are repeatedly observed in several environments,but since it is not known if they represent cell death or a resistant state,their role in the transmission pathwayofH. pylori, especially by animalsandfood, is still controversial (Nabwera andLogan,1999; Velazquez and

Feirtag, 1999).

1.8. Concepts of the microbiology of H. pylori

The size of the seven sequenced genomes ofH. pylori varies between

1.6 and 1.7 Mb. H. pylori is genetically heterogeneous possibly as anadaptation of the bacteria to the gastric conditions of its host (Kusters

et al., 2006).Typically H. pylori is grown on H. pylori selective agar (Wilkins

Chalgren agar supplemented with 10% horse blood, with antibiotics,such as vancomycin [10 mg/l], cefsulodin [5 mg/l], trimethoprim

[5 mg/l], and cycloheximide [100 mg/l]) and incubatedat 37 C for 48 hin an anaerobic jar with a gas generator system (Vale and Vitor, 2007).

H.pyloriis microaerophilic, with optimalgrowthat 2 to 5% of O2,5to10%of CO2 and high humidity. Growth occurs at 34 to 40 C, with anoptimum of 37 C. Although its natural habitat is the acidic gastricmucosa, H. pylori is considered to be a neutralophile. The bacterium

survives brief exposure to pHs of


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within-family members are often similar (Kivi et al., 2003; Kivi andTindberg, 2006; Bamford et al., 1993; Delport et al., 2006), namelybetween mother and child (Weyermann et al., 2006; Raymond et al.,2004). In populations with low H. pylori prevalence, the infected

mother is likely to be the primary source for infant H. pylori infection(Weyermann et al., 2009). Recently, a study of 1590 coding sequencesin several strains among three families using micro-array revealed

that the mother was not involved in the transmission in two of those

families. Transmission among siblings as well as outside acquisitionappeared to play a major role in the transmission pathway. Even thepresence of common strains within the same family does not exclude

acquisition of H. pylori from a common (external) source. H. pyloriinfection may be acquired by more diverse routes than previouslyexpected (Raymond et al., 2008). The person-to-person transmissionmay occur by three possible pathways: the gastrooral, the oraloral

and the faecaloral, which are described below.

2.1.1. Gastrooral transmissionH. pylori is acquired in early life and the vomiting of achlorhydric

mucus may serve as a vehicle for transmission. The transmissionroutecould be by gastric juice, especially as a result of epidemic vomiting inchildhood (Axon, 1995). For instance, H. pylori infection is easily

transmitted from person-to-person by gastric intubation. H. pyloriappears to survive outside the human body in unbuffered gastric juice.Culture ofH. pylori was possible in 62% of the 21patients following2 hof gastric juice collection, 42% after 6 h and 10% after 24 h ( Galal et al.,

1997). Another study reported a similar isolation percentage fromgastric juice of symptomatic patients (Okuda et al., 1996). Othersreports suggest that a sibling history of vomitus (Luzza et al., 2000) orexposure to an infected household member with gastroenteritis

(particularly with vomiting) is a risk factor for H. pylori infection(Perry et al., 2006). Others have reported the culture of H. pylori fromvomitus (Leung et al., 1999a; Parsonnet et al., 1999). H. pylori wasoften present in high quantities in vomitus, with as many as

30,000 CFU/mL of sample, capable of being cultured from air samplescollected after vomiting, but not before. However, the short durationof the contamination and the limited dispersion of organisms (less

than 1.2 m) makes aerosol exposure an unlikely source of infection(Parsonnet et al., 1999). These results are favourable to the gastrooral transmission, especially during childhood, coupled with poorhygiene practices, with the vomitus working as the putative vehicle oftransmission.

2.1.2. Oraloral transmissionSaliva is another possible source ofH. pylori transmission, since the

gastric flora can reach and colonize the mouth after regurgitation orvomiting. H. pylori DNA has been frequently amplified from saliva,

subgingival biofilm and dental plaque (Burgers et al., 2008; Souto andColombo, 2008). H. pylori has also been cultured directly from saliva(Ferguson et al., 1993; Parsonnet et al., 1999). Based on these reports,the mouth might be a reservoir of H. pylori (Gebara et al., 2006).

However, the recovery of H. pylori does not seem to increase aftervomiting and quantities of H. pylori in saliva tended to be low(Parsonnet et al., 1999). Likewise, other studies report that H. pyloricannot be isolated from saliva or dental plaque in patients with

positive gastric biopsy cultures (De et al., 2006; Luman et al., 1996 ).Other negative arguments against the oraloral transmission includethe frequent absence of related strains infecting spouses (Gisbertet al., 2002; Vale and Vitor, 2007; Luman et al., 2002; Suzuki et al.,

1999), but this is controversial given reports that demonstrate thepresence of common strains infecting couples (Kivi et al., 2003).Common strains with couples suggest person-to-person transmissionor common source exposure within couples (Singh et al., 1999;

Georgopoulos et al., 1996). Recently, a study of the primers used forthe PCR detection ofH. pylori suggested that the primers may provide

unreliable results, due to limitations in sensitivity and specificity

(Sugimoto et al., 2009). These may explain controversial PCR results,since numerous species related to H. pylori may be present in the oralmicrobiota (Megraud and Broutet, 2000). These data suggest thatalthough saliva might work as a vehicle of transmission, the oraloral

transmission is not the main mode of transmission ofH. pylori, at leastin adults (Luman et al., 1996).

2.1.3. Faecaloral transmission

H. pylori DNAhasbeen frequently detectedin human faeces(Queraltet al., 2005; Sen et al., 2005; Fujimura et al., 2004b; Monteiro et al.,2001a,b; Gramleyet al., 1999; Oyofoet al., 1992). Attempts to culture H.

pylori from faeces have had limited success (Falsafi et al., 2007; Liangand Redlinger, 2003; Dore et al., 2000) as the bacterium exists therepredominantly in a nonculturable (coccoid) form (Kabir, 2001).

Mice were used as an experimental animal model to evaluate the

transmission of H. pylori. Twelve inoculated mice were housed withtwelve noninoculated mice in a grated cage (to test oraloraltransmission). A similar experiment was performed, but the housingwas in a cage without grating on the floor (to test faecaloral trans-mission). H. pylori was only recovered in mice housed in the cage

without grating, supporting faecaloral route (Cellini et al., 1999).Another group infected four-week-old female Mongolian gerbils with H.

pylori, which were then mated with uninfected males two months after

the infection. The offspring were sacrificedweekly after birth and serumand mother's milk from the stomach and gastric tissues were obtainedfrom pups. H. pylori was not identified in cultures from the gastricmucosa of pups delivered by infected mothers, but H. pylori 16S rRNA

was detected 4 weeks after birth, suggesting that Mongolian gerbil pupsbecome infected via faecaloral maternal H. pylori transmission (Oshioet al., 2009). Transmission via faecal contaminants is also supported by

the occurrence of H. pylori infections among institutionalized youngpeople during outbreaks of gastroenteritis (Laporte et al., 2004). Takentogether, these experiments support faecaloral transmission in thetransmission of H. pylori, especially when the hygienic conditions are

poor.

2.1.4. A role for water in person-to-person transmission?

Person-to-person transmission may involve vehicles other thanvomitus, saliva or gastric juice. When hygienic conditions are poor,household contamination of treated water cannot be ruled out.H. pyloriis easily controlled by chlorination (Johnson et al., 1997) (the principaldisinfection agent of water), but recontamination of treated water is a

widespread problem (Ashbolt, 2004). Poor hygienic practices duringchildhood, absence of a household bath, non-hygienic drinking waterand absence of a sewage disposal facility (Nouraie et al., 2009) may leadto re(contamination) of drinking water, that in this way may serve as a

vehicle of transmission. Evenin treated water, the survivalofH. pylori ispossible, at least for short periods of time. When comparing theprevalence of H. pylori in three Japanese populations with different

drinking water sources (two with river water, one with groundwater)the population with the groundwater source had a much lower

prevalence (Fujimura et al., 2008), but the small numbers in thisecologic comparison limit its value (Azevedo et al., 2009). Recently, a

study reports that it was possible to culture a reference strain afterfiveminutes of inoculation in chlorinated water (0.96 mg/l), and viable cellswere detected by FISH up to 3 h post inoculation. The percentage ofcoccoid forms was higher than spiral forms after 40 s of chlorine

exposure, but even after 24 h, FISH detection revealed the presence ofspiral cells. After 24 h, amplification of the specific H. pylori 16S rDNAgene was possible. These results imply that H. pylori could survivedrinking water disinfection practices as VNBC (Moreno et al., 2007).

2.1.5. A role for food in person-to-person transmission?As it happens with water, food products may also be contaminated

while handling, under poor hygienic conditions. The application of a

questionnaire to a group of patients requiring an upper gastrointestinal



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endoscopy associated the H. pylori infection with consumption of foodproducts from street vendors in Peru (Begue et al., 1998), suggestingthat food may act as a vehicle rather than a reservoir ( van Duynhovenand de Jonge, 2001). Although it is unlikely that H. pylori grows on food,

it may survive as VNBC (van Duynhoven and de Jonge, 2001). The foodmay also serve as a vehicle in premastication of child's food by parents(Kurosawa et al., 2000). This last aspect is deeply associated with the

cultural background, and could never explain alone the route of trans-

mission from person-to-person.There is a single report of detection ofH. pylori DNA in 6.1% (4/66)of breast milk samples, collected after delivery from H. pyloriantibody-positive pregnant women (Kitagawa et al., 2001). However,this could be due to nipples or fingers contaminating the milk(Azevedo et al., 2007b). A community based study in Brazil revealedthat there was no significant difference in the prevalence of H. pyloriand the history of infant breastfeeding (Braga et al., 2007). Otherstudies conducted in Vietnam (Nguyen et al., 2006), Germany(Rothenbacher et al., 2002) and Japan (Ueda et al., 2003) providedsimilar results. It has even been suggested that breastfeeding for more

than 6 months (Nguyen et al., 2006; Pearce et al., 2005), and yogurtconsumption (Ornelas et al., 2007) is associated with reducedseropositivity for H. pylori. Moreover, most of the lactating women(60.2%) who were seropositive for H. pylori had some IgA in their

colostrum (Tanriverdi et al., 2006).

2.2. Food chain transmission

The exact mode by which H. pylori gains access to the humanstomach is unknown. Indirect evidence of presence of H. pylori inwater and food has been reported, namely the detection of H. pyloriDNA and VBNC forms. H. pylori may enter as VBNC under conditionswhere growth is not possible (van Duynhoven and de Jonge, 2001).

2.2.1. Water ingestionThe association of serum antibodies against H. pylori with serum

antibodies against two known waterborne pathogens (hepatitis A virus(Bizri et al., 2006) and Giardia (Moreira et al., 2005), suggests that the

infection may be waterborne or related to poor sanitary practices.However, these associations are not always observed (Malaty et al.,2003). Recently, the absence of association of Helicobacter with

Acanthamoeba, a free living amoeba that inhabits a wide range of

ecological niches, including river water was reported (Kawaguchi et al.,2009). A previous report had described the successful co-cultivation of

H. pylori with Acanthamoeba castellanii (Winiecka-Krusnell et al.,2002), which feeds on Gram-negative bacteria, and harbours potentialpathogens (Alsam et al., 2006). H. pylori DNA (vacA and ureAB genes)has also been amplified from the oral yeast Candida albicans total DNA,

showing the intracellular occurrence of H. pylori (Salmanian et al.,2008). The yeast Candida is found in food, water, oral cavity,gastrointestinal, genital and urinary tract of human (King et al., 1980;Restaino et al., 1995), and may be a reservoir/protective vehicle of H.

pyloriplayingan important role in thebacterialreinoculationof stomachor transmission to a new host (Salmanian et al., 2008). The associationbetween H. pylori and Acanthamoeba and Candida should be furtherinvestigated.

Indirect evidence that the transmissionofH. pylori is waterborne isbased upon four sets of data: i) presence of DNA in water samples; ii)observation of coccoid forms in water samples; iii) survival ofH. pyloriin artificially contaminated water; iv) and growth of H. pylori from

water samples (Table 1). This last mode of transmission is only brieflydiscussed in the current literature.

H. pylori DNAhas been identified inseveral water sources (Table 1)

using diverse gene targets. Drinking, river, sea, ground and wastewa-ter have provided positive results by PCR analysis (Hulten et al., 1996;Horiuchi et al., 2001; Mazari-Hiriart et al., 2001a,b; Fujimura et al.,

2004a;Cellini et al., 2004; Queralt et al., 2005). Treated drinking water

is not usually contaminated with H. pylori DNA (Janzon et al., 2009;Horiuchi et al., 2001), suggesting sensitivity of this bacterium toconventional treatment (Johnson et al., 1997). However, H. pyloriappears to be more resistant to chlorine and ozonation than E. coli(mandatory indicator microorganism to evaluate water quality inmost countries), and equally resistant to monochlorine. Traditionalindicator organisms may fail to protect the consumer from exposure

to H. pylori (Baker et al., 2002; Johnson et al., 1997; Moreno et al.,

2007). Regarding spring water, the results for presence of H. pyloriDNA were also negative (Queralt et al., 2005). Likewise, upstreamriver water samples were negative (Fujimura et al., 2004a). Taken

together, these results suggest that faecal contamination is respon-sible for the contamination of river water. However, DNA isolationalone does not provide any indication of the viability of the bacterium(Giao et al., 2008). Moreover, studies addressing the presence of H.

pylori DNA based on the detection of 16r RNA may not be suitable. Infact, the 16S rRNA gene sequence has been described as more suitablefor the discrimination at the genus level. Even for Helicobacterspeciesdiscrimination it is not a suitable marker (Dewhirst et al., 2005;

Vandamme et al., 2000).The H. pylori DNA present in water samples could be from dead H.

pylori cells or from VBNC forms, since culture is usually not possible.Water spiked with viable H. pylori cells rapidly led to the observation

of coccoid forms (Queralt and Araujo, 2007; Nayak and Rose, 2007;Adams et al., 2003; Moreno et al., 2007). Whether the coccoid form of

H. pylori is viable in the dormant state or is degenerative andundergoing apoptosis is still an unanswered question. Coccoid H.

pylori appears to conserve the capacity to produce proteins for at least100 days when stored at 4C, in either phosphate-buffered saline(PBS) or distilled water (Mizoguchi et al., 1999). It has been suggestedthat although the virulence of coccoid H. pylori induced by water

decreases, the coccoids still retain a considerable urease activity andpreserve adhering ability to epithelial cells. These coccoids induced bywater have been capable of colonizing the gastric mucosa, causinggastrititis in mice (She et al., 2003).

The VBNC state could be responsible for the difficulty in isolating H.pylori from water samples (Sheet al., 2003). To our knowledge thereare

only two studies that report successful isolation of H. pylori fromenvironmental water samples. In one case, the bacterium was isolated

from wastewater (using immunomagnetic separation and culture),which is compatible with a faecaloralroute of contamination (Luet al.,2002). In the second case, H. pylori was isolated from a seawatersample

(Cellini et al., 2005). Colonies were only obtained in residueson 200 mfiltersbut not in residues on 0.64 and 0.22 m filters. H. pylori could onlybe isolated from fractionated seawater samples, containing largezooplanktonic organisms; without zooplankton, H. pylori cells could

not be recovered from any other fractions by growth-dependentdetection protocols (Cellini et al., 2004). This suggests that H. pylorirequires an organic or proteic matrix to remain culturable. This shouldbe further investigated and other studies attempting to isolate H. pylorifrom water sources using similar approaches are needed.

Survival studies in water samples demonstrate that H. pylori can becultured for a limited period of time in a temperature dependentmanner (Azevedo et al., 2008; Queralt and Araujo, 2007; Moreno et al.,2007; Adams et al., 2003; Baker et al., 2002; Johnson et al., 1997).Elevated temperature results in loss of culturability (Shahamat et al.,

1993). The presence of H. pylori associated with biofilms from wells,rivers and water distribution systems has been reported recently(Azevedo et al., 2003, 2007b; Percival and Thomas, 2009; Park et al.,2001; Giao et al., 2008; Bunn et al., 2002; Bragana et al., 2007; Watson

et al., 2004). Biofilms are slimy films of bacteria, other microbes andorganic materials that cover underwater surfaces, particularly insideplumbing. This makes them rather inaccessible and provides a matrixdifficult to be reached by disinfectants.The detachment of biofimsisthe

principal form of contamination of treated water (Gouider et al., 2009;

Stoodley et al., 2001). H. pylori adherence to biofilms appears to be

5F.F. Vale, J.M.B. Vtor / International Journal of Food Microbiology 138 (2010) 112


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independent of temperature (Azevedo et al., 2006). An ecologicalexplanation for its presence in biofilms is provided by the microaer-ophilic nature of this pathogen (Giao et al., 2008).

In geographic areas where there is access to safe drinking water,

the prevalence rates for H. pylori infection are clearly lower, whichmay suggest a transmission route to the host (Perez-Perez et al., 2004;Kusters et al., 2006). However, there are no established culture

methods for the detection of viable H. pylori in the environment, in

particular drinking water supplies, which prevent the development oftrue epidemiological and risk assessments (Percival and Thomas,2009). Epidemiological studies suggest that environmental water is a

risk factor for H. pylori infection when compared with tap water, andthe formation of H. pylori biofilm cannot be excluded (Andersen andRasmussen, 2009; Watson et al., 2004).

Karita et al. report that in 41 families, H. pylori prevalence

significantly increased with a history of drinking well water. For fivefamilies whose wells were screened for H. pylori DNA presence, all wellsgave positive results. (Karita et al., 2003). However these authors haveonly considered drinking the water from the well as an external source

ofH. pylori. The unequivocal proof would be the detection of H. pyloriDNA in the water with the same genotype as of human isolates.

2.2.2. Food ingestionSeveral studies address the role of food in the transmission of H.

pylori. Food products analysed are mainly milk, meat and vegetables.Among these, milk products are the most studied, probably because

the infection is mainly acquired during childhood and milk is mostlyconsumed during this period.

2.2.2.1. Milk. There is indirect evidence ofH. pylori transmission trough

milk, similar to that obtained for water, but less extensive (Table 2).

H. pylori prevalence among shepherds is almost 100%. A study of42 shepherds and 28 members of their families, whose H. pylori statuswas determined by the 13Curea breath test, revealed that H. pyloriprevalence reached 97.6% in shepherds, 86% in their family members,

but significantly less, 65.1%, in controls without contact with sheep(Papiez et al., 2003). Also, children having contact with sheep show

about twice higher H. pylori prevalence than children living in urbanareas (Plonka et al., 2006). These authors propose that H. pyloriinfection in shepherd children may originate from sheep. Thesestudies led to the hypothesis of H. pylori infection being considered azoonosis (Papiez et al., 2003; Plonka et al., 2006).

The isolation of H. pylori is not always associated with raw milk.For instance a study of 440 raw sheep milk samples did not yield any

H. pylori isolate (Turutoglu and Mudul, 2002). The percentage ofdetection ofH. pylori DNA is higher in raw milk when compared with

pasteurized milk, and this percentage decreases when two genes areassayed (Quaglia et al., 2008; Fujimura et al., 2002; Dore et al., 1999,2001). These data sustain previous comments on the use of 16S rRNA

to identify H. pylori.

Survival studies clearly demonstrate that H. pylori can survive forshort periods in milk (Quaglia et al., 2007, 2009; Bohmler et al., 1996),which is also a food product with a short shelflife. As was observed for

milk, the temperature increase is associated with loss of culturability(Fan et al., 1998).

The isolation of H. pylori from raw milk samples is rather rare(Fujimura et al., 2002) (Dore et al., 1999, 2001). This implies that H.

pylori would be living in the stomach of cows and sheeps, beingeliminated as viable forms in faeces and could then contaminate themilk during the milking process (Megraud and Broutet, 2000). Thegenetic relationship among strains isolated from milk and human

patients should be further characterised to clarify if these are different

Helicobactersp., which are similar to H. pylori, or if it is indeed H. pylori.In fact, Helicobacterspp. have been detected in the abomasums of sheepand cattle (De Groote et al., 1999b; Haesebrouck et al., 2009).

Considering survival studies, contaminated milk (by humans for certainand not clear if by animals) may certainly be a vehicle for H. pylori.

2.2.2.2. Meat,vegetables and otherfood products. Studies on the detectionofH. pylori in food products other than milk are quite sparse. However,it has been described that individuals who consume raw vegetables aremore likely to acquire H. pylori (Goodman et al., 1996; Hopkins et al.,

1993; Chen et al., 2005). The association of the infection withconsumption of raw vegetables is an additional indirect evidence ofthe presence ofH. pylori in water used for irrigation of these vegetables(Mazari-Hiriart et al., 2001b, 2008).

In Malaysia/Singapore, an association of the seroprevalence of H.

pylori with the use of chopsticks was found (Chow et al., 1995).Although the detection ofH. pylori DNA in chopsticks after being used

by sixty-nine asymptomatic volunteers was very low (2%) (Leunget al., 1999b), the sharing of chopsticks may contribute to cross-

infection (Wong et al., 2005). There are few studies that address thesurvival of H. pylori in food products other than milk containingcomplex microbial flora (Table 3).

Table 2

Examples of studies that present evidence of presence of H. pylori in milk samples.

Study Milk type Method N. samples Gene ID Observations Ref.

Presence ofH. pylori DNA Raw goat's milk Nested PCR 160 glmM 26% positive goat's milk Quaglia et al. (2008)

Raw sheep's milk 130 43% positive sheep's milk

Raw cow's milk 110 55% positive cow's milk

Raw cow's milk Se mi-nested-PCR 18 ureA 72% positive raw milk Fujimura et al. (2002)

Pasteurized cow's milk 20 55% positive pasteurized milkRaw sheep's milk PCR 63 16 rRNA and vacA 60% positive 16S rRNA Dore et al. (2001)

8% both positiveRaw sheep's milk PCR 51 16 rRNA and vacA 60% positive Dore et al. (1999)

9% both positive

Survival ofH. pylori in artificially

contaminated milk

Spiked pasteurized milk

and UHT milk

Culture 13+13 Na Median survival of 9 days in raw

milk and 12 days in UHT milk

Quaglia et al. (2007)

Cow's milk Culture S Na Reisolated until 6 days cooled milk;

3 to 4 days 37 C milk

Bohmler et al. (1996)

Raw sheep's, goat's and

cow's milk

Culture Nested-

PCR

3 glmM Limit of detection by PCR 3 cfu/ml Quaglia et al. (2009)

Fresh milk without

preservatives

Culture S Na Survival until 10 days at 4 C and

3 days at 25 C

Fan et al. (1998)

Growth ofH. pylori Raw cow's milk Culture 18 Na Positive for one raw milk Fujimura et al. (2002)

Pasteurized cow's milk 20

Raw sheep's milk Culture 63 Na Positive from raw sheep's milk Dore et al. (2001)Raw sheep's milk Culture 51 Na Positive from raw sheep's milk Dore et al. (1999)

UHT

ultrahigh temperature milk.



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Foods with water activity higher than 0.97 and pH from 4.9 to 6.0theoretically provide conditions for the survival ofH. pylori. Also, generallackof efficient sanitation in removing or killing pathogens on raw fruitsand vegetables may contribute to harbor pathogens (Gomes and De

Martinis,2004b; Beuchat, 2002). H. pylori is unlikely to grow inmost foodproducts, but it is able to survive in a low acid, high moistureenvironment for extended periods of time, especially if stored refriger-ated (Bohmler et al., 1996; Gomes and De Martinis, 2004b; Jiang and

Doyle, 1998). Likewise, the isolation of H. pylori from food products isextremely difficult due to the presence of accompanying microflora and

to the presumably very low H. pylori load (Quaglia et al., 2009).Considering contamination of food products within the industrial

facilities or in households by poor hygiene-management of infectedpersonnel, we cannot exclude the transmission of H. pylori by thesefoods. An infection of the consumer by this route is not very likely, but itcannot be completelyruled out(Bohmler et al., 1996). Proof of the ability

of H. pylori to survive in common foods supports the hypothesis thatprimary contamination of a foodproduct (animal reservoir)or secondarycontamination due to inappropriate handling(human reservoir)can be a

vehicle for H. pylori transmission (Quaglia et al., 2007).The isolation of other Helicobacter spp. from several animals

(Kusters et al., 2006), mostly living in human environments, makesthem suspects of harbouring H. pylori in their stomachs and hence

participating in the transmission of this pathogen.

Animals participating in the human food-chain, like the pig andsheep, have been considered as possible reservoirs for this bacterium(Dore and Vaira, 2003; Brown et al., 2001; Dimola and Caruso, 1999;

Webb et al., 1996). H. suis (De Groote et al., 1999a), which has beenisolated from the stomachs of pigs, is the most prevalent gastric non-pylori Helicobacter species in humans. The Candidatus Helicobacterbovis (De Groote et al., 1999b) is highly prevalent in the abomasums

of cattle but has only occasionally been detected in the stomachs ofhumans. There are clear indications that gastric non-pylori Helico-bacterinfections in humans originate from animals and it is likely thattransmission occurs through direct contact (Haesebrouck et al., 2009).

As referred H. pylori can be found inside yeasts. Yeasts are presentin the human oral cavity and different foods. Thus measurementsregarding control of yeast content of foods might be the important to

prevent the transmission ofH. pylori (Salmanian et al., 2008).

3. Likelihood of transmission in rural and urban environments

As described above, the prevalence of infection by H. pylori clearlydiffers between developing and developed countries, ranging from lessthan 40% to more than 80% (Perez-Perez et al., 2004; Kusters et al.,2006). The socioeconomic level varies in subpopulations within the

same country making the prevalence rather different in thesesubgroups, with rural environments presenting a higher prevalence(Bruce and Maaroos, 2008). Contributing to this difference, we have thehigher exposure to risk factors in rural environments, namely poor

hygiene practices during childhood; crowded families; lack of house-hold bath; absence of sanitized drinking water and sewage disposal

facilities (Nouraie et al., 2009) as well as unsafe preparation of food (van

Duynhoven and de Jonge, 2001). Most of these, if not all, are a directconsequence of a low socioeconomic background. This makes person-to-person transmission more probable in urban environments, espe-cially between family members (Kivi et al., 2003, 2007; Raymond et al.,

2008; Fujimoto et al., 2007). When studying families from ruralenvironments, horizontal transmission, such as through contaminatedfood, water or via intensive contact between infants and non-parentalcaretakers, may jointly play a more important role than within-family

transmission (Karita et al., 2003; Schwarz et al., 2008). Unsanitarypractices during food preparation might also be involved in vertical

transmission, with water and foodactingas vehicles of transmission. Onthe other hand, coupled with the extreme sensitivity of H. pylori to

atmospheric oxygen pressure, lack of nutrients and outside tempera-tures in the 34 to 40 C range, direct person-to-person transmissionremains the most likely transmission route (Kusters et al., 2006). H.pylori survive in artificially contaminated samples of waterand food for

short periods of time (Tables 13), making the transmission feasible incaseof exposureto these products. Thecontamination of water andfoodprobably occurs by a faecaloral route. The major limitation in

attributing a role for the transmission of H. pylori to water and foodproducts in rural environments lies in the difficulty to culture H. pylorifrom natural samples. The microbiologic culture ofH. pylori from waterand food would be the gold standard for attributing a role in trans-

mission to these products. Moreover, the matching of the genotypes

between human and food isolates would be the definitive proof oftransmission through food. The nature of the coccoid forms, dead forms(Kusters et al., 1997) or viable, but nonculturable states (Azevedo et al.,

2007a; Chen, 2004) is still not clear, which increases the difficulty ininterpreting their presence, for instance in water and milk products(Tables 1 and 2). Finally, in the cases of horizontal transmission withoutidentification of thesourceof infection,we cannot disregard thefact that

other persons with close contact with children other than the parentswere not considered for analysis. They would be important tocharacterise, since the traditional family model is changing.

4. Conclusions

The principal transmission route ofH. pylori is not clearly definedand

two main models exist: i) the vertical transmission from parents tochildren within the framework of the same family, by direct person-to-person contact, probably by gastrooral or faecaloral route; and ii) thehorizontal transmission, by ingestion of contaminated food or water, or

via intensive contact between infants and non-parental caretakers. Mostlikelyin urban (developed) areasthe vertical transmission representsthemain form of transmission, while in rural (non-developed) areas, thehorizontal form appears to play a major role, but not to the exclusion of

the first. Horizontal transmission also includes person-to-persontransmission, but does not exclude ingestion of contaminated waterand food. However, the presence ofH. pylori in water and food have onlybeen verified by indirect evidence, such as presence of DNA and survival

studies, which are a limitation in attributing a role in the transmission of

H. pylori to water and food. Future studies should include the presence

and the survival ofH. pylori in raw food products, such as strawberries,

Table 3

Survival ofH. pylori in artificially inoculated food products, other than milk.

Food product Method Observations Ref.

Spiked leaf lettuce, tofu, yogurt

and raw chicken

Culture Survival until 5 days in tofu; 2 days in lettuce and raw chicken;

not recovered from yogurt

Poms and Tatini (2001)

Yogurt, kefir, curd cheese, chicken Culture At 37 C survival until 3 h in yogurt; 24 h in kefir; 10 h in curd cheese;

At 7 C survival superior to 72 h in these 3 products.

Bohmler et al. (1996)

Survival until 48 h in chicken

Autoclaved and irradiated (10 kGy)

spiked ground beef

Culture Survival until 3 days in autoclaved ground beef; 7 days in irradiated

ground beef

Jiang and Doyle (2002)

Spiked lettuce and carrots Culture Survival until 72 h in sanitized lettuce and carrot; up to 96 h in

sterilized carrots.

Gomes and De Martinis (2004a)



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and fresh cut salads. Due to improving food preparation and farmhygiene during the last decade it would also be of interest to proceedwith further comparison studies of the presence of H. pylori in foodproducts.

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Transmission Pathway of H.pylori

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