International Journal of Food Microbiology 138 (2010) 112

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o

Review

Transmission pathway of Helicobacter pylori: Does food play a role in rural and urban areas?
F.F. Vale a,, J.M.B. Vtor b
a b

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

a r t i c l e

i n f o

a b s t r a c t
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 however unbalanced 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 transmission pathway 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 evidence supporting a gastrooral, oraloral and faecaloral transmission, but no predominant mechanism of transmission has been yet identied. 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 of presence of H. pylori in water and food, namely the detection of its DNA and survival studies after articial contamination 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 developing rural and developed urban areas appears to be different. In developed areas, person-to-person transmission within 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 contact between infants and non-parental caretakers may have a greater inuence than within-family transmission. 2010 Elsevier B.V. All rights reserved.

Article history: Received 18 October 2009 Received in revised form 13 January 2010 Accepted 14 January 2010 Keywords: Helicobacter pylori Transmission Food Water Vehicle Source Rural and urban areas

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Epidemiology: diseases and prevalence . . . . . . . . . . . . 1.2. Risk factors . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Geographic strain distribution and globalization . . . . . . . . 1.5. Infection in rural (developing) and urban (developed) environments 1.6. Mixed colonization . . . . . . . . . . . . . . . . . . . . . . 1.7. Coccoid forms: cell death or VBNC? . . . . . . . . . . . . . . 1.8. Concepts of the microbiology of H. pylori . . . . . . . . . . . . Routes of transmission . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Person-to-person transmission . . . . . . . . . . . . . . . . . 2.1.1. Gastrooral transmission . . . . . . . . . . . . . . . 2.1.2. Oraloral transmission . . . . . . . . . . . . . . . . 2.1.3. Faecaloral transmission . . . . . . . . . . . . . . . 2.1.4. A role for water in person-to-person transmission? . . . 2.1.5. A role for food in person-to-person transmission? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 2 2 2 2 3 3 3 3 3 4 4 4 4 4

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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: lipavale@fe.ucp.pt (F.F. Vale). 0168-1605/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.01.016

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

Food chain transmission . . . . . . . . . . . . . . 2.2.1. Water ingestion . . . . . . . . . . . . . 2.2.2. Food ingestion . . . . . . . . . . . . . . 3. Likelihood of transmission in rural and urban environments 4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Helicobacter pylori was rst described by Warren and Marshall in 1983 in association with chronic gastritis and peptic ulcer (Warren and Marshall, 1983; Marshall and Warren, 1984). This discovery was rather controversial at the time but ultimately in 2005, these two scientists were awarded the Nobel Prize for this discovery. Nowadays, a H. pylori search on Pubmed will retrieve thousands of publications, but unanswered questions still remain. The transmission route of H. pylori has yet to be ascertained, as well as its source. Presently, the only recognized and accepted reservoir is the human stomach, but the existence of extra-gastric reservoirs for H. pylori has been suggested. This paper reviews and discusses the possibility of the transmission of H. 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 world population. The colonization is not always associated with the development of pathology as more than 70% of the infected population remains asymptomatic. However, colonized individuals may develop gastritis, peptic ulcer, gastric cancer or Mucosa Associated Lymphoid Tissue (MALT) lymphoma (McColl, 1999; Das and Paul, 2007). The infection is mainly acquired during childhood (Sykora et al., 2006). The prevalence of infection is typically higher in developing countries (greater than 80%) and lower in the developed ones (typically less than 40%) 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 and Yilmaz, 2007; Perez-Perez et al., 2004); low education level; poor hygiene practices during childhood; crowded families; lack of a household bath; absence of sanitary drinking water; absence of a sewage disposal facility during childhood (Nouraie et al., 2009) and food handled inappropriately (van Duynhoven and de Jonge, 2001). Most of these factors are a consequence of and associated with socioeconomic development, which in epidemiology studies usually acts as a confounding factor. The improvement of general hygienic conditions decreases the prevalence of the infection (Fujimoto et al., 2007). This statement suggests the existence of an environmental pool to which children are exposed, especially in developing areas (Gomes and De Martinis, 2004b). However, it cannot be excluded that in areas with high populations, the transmission can be even higher compared to low populated areas due to limited human contact. Host genetic factors have also been associated with the infection, especially in the progression to a disease state (Ando et al., 2007; Sgouros and Bergele, 2006). 1.3. Therapy Once acquired, the infection is usually lifelong, unless treated. The Maastricht III Consensus report indicates that H. pylori should be eradicated 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%, or after gastric cancer resection. It should also be eradicated in rst degree relatives of patients with gastric cancer, and also in the presence of unexplained iron-deciency anaemia and idiopathic thrombocytopenic purpura (Vakil and Megraud, 2007). For eradicating H. pylori, a triple therapy is commonly used (Megraud and Lehours, 2007), which involves two antibiotics (usually amoxicillin and clarithromycin) and a proton pomp inhibitor (PPI) or ranitidine bismuth during seven days (Kusters et al., 2006; Megraud, 2004a,b; Vakil and Megraud, 2007). The most commonly used antibiotics are tetracycline, amoxicillin, imidazole (metronidazole and tinidazol) and macrolids (clarithromycin and azithromycin). Antibiotic therapy fails in about 20% of the patients (Parente et al., 2003; Kusters et al., 2006), mainly due to antibiotic resistance (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 among different strains (Suerbaum, 2000). Moreover, the map of H. pylori strain diversity appears to be similar to the one of humans. This similar pattern of H. pylori and human geographic diversity and distribution strongly suggests a co-evolution between this bacterium and man, which can be used to understand human migrations (Covacci et al., 1999). The H. pylori distribution pattern follows human migration roots, which suggests that the colonization of the human stomach 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-person transmission mode. Globalization and the rapid mobility of the population may disturb in the near future the six large clusters of H. pylori strains currently spread throughout the world (Achtman et al., 1999; Linz et al., 2007). But again, this will be a response to human migrations. However, population genetics tools that have allowed the assignment of individual isolates to one of six discrete clusters of bacterial populations are inadequate to address 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 rural developing areas in contrast to urban developed ones (Brown et al., 2002; Cheng et al., 2009; Aguemon et al., 2005). Different cultural habitats and an increased number of risk factors may contribute to this difference. Populations from rural environments are probably exposed to an increased number of infectious sources, which is compatible with different routes of infections according to the population's culture and environment (Azevedo et al., 2007b; Schwarz et al., 2008). Familial transmission as a mode of dissemination of H. pylori is generally accepted (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; Kivi et al., 2003). However, most of these studies were performed within urban families and using a single isolate from each family member. Recently, a study of three families revealed that H. pylori infection may be acquired by more diverse routes than previously expected. In this particular report the mother was not implicated in the transmission

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in two of these three families. Sibling-to-sibling transmission and acquisition of H. pylori outside the household appears to be involved in the transmission pathway (Raymond et al., 2008). Likewise, there is evidence 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 via intensive 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 on strain genotyping after culture isolation from gastric biopsies. In most cases, only one isolated culture is achieved. The detection of the presence of mixed colonization (presence of two or more strains colonizing the stomach) appears to depend on the material used. Genotyping results with DNA extracted from gastric biopsy specimens were inconsistent with those of bacterial DNA isolated from H. pylori cultured from gastric biopsies. Mixed infection was detected in 27% of cases when tissue DNA was used compared to only 9% when bacterial DNA was used (Park et al., 2003). This led to the question of how representative are the isolates that are studied in transmission studies. Several other works report the presence of mixed colonization (Fantry et al., 1996; Wong et al., 2001; Sheu et al., 2009). Information on the genetic similarity of strains from different family would benet from more thorough attempts to identify multiple strains in individuals, which would be of particular relevance for better understanding modes of transmission (Azevedo et al., 2009). However, the strains colonizing the 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-negative bacterium, measuring 2 to 4 m in length and 0.5 to 1 m in width. Although usually spiral-shaped, the bacterium can appear as a rod, while coccoid shapes appear after prolonged in vitro culture, a long inoculation period in water or milk samples, or antibiotic treatment (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 a viable, 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 pathway of H. pylori, especially by animals and food, is still controversial (Nabwera and Logan, 1999; Velazquez and Feirtag, 1999). 1.8. Concepts of the microbiology of H. pylori The size of the seven sequenced genomes of H. pylori varies between 1.6 and 1.7 Mb. H. pylori is genetically heterogeneous possibly as an adaptation 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 incubated at 37 C for 48 h in an anaerobic jar with a gas generator system (Vale and Vitor, 2007). H. pylori is microaerophilic, with optimal growth at 2 to 5% of O2, 5 to 10% of CO2 and high humidity. Growth occurs at 34 to 40 C, with an optimum of 37 C. Although its natural habitat is the acidic gastric mucosa, H. pylori is considered to be a neutralophile. The bacterium survives brief exposure to pHs of <4, but growth occurs only at the relatively narrow pH range of 5.5 to 8.0, with optimal growth at neutral pH (Kusters et al., 2006).

An important component of the response of H. pylori to acid is acid acclimation, which is dened as the ability of H. pylori to maintain periplasmic pH close to neutrality in the presence of extra bacterial acidity, to maintain cytoplasmic pH at physiological levels. The maintenance of a near neutral periplasmic pH makes cytoplasmic pH relatively neutral, allowing the organism to grow in gastric acidity as if it were in a neutral environment (Scott et al., 2007, 2010). Several acid acclimation genes are required for Helicobacter colonization. These genes include urease, those involved in its biosynthesis, and UreI, the proton gated urea channel essential for adequate access of medium urea to intrabacterial urease and periplasmic -carbonic anhydrase (Scott et al., 2007). Urease is a nickel-containing enzyme that consists of 12 UreA and 12 UreB subunits. The urease gene cluster contains a second operon downstream of the ureAB genes which encodes the UreIEFGH proteins. The UreEFGH accessory proteins probably function in subunit assembly and incorporation of nickel in the active sites of urease, whereas the UreI protein functions as an acidactivated urea channel (Kusters et al., 2006). Urease, present in the cytoplasm, produces 2NH3 + CO2 from urea. UreI is able to transport NH3 to the periplasm allowing rapid neutralization of entering protons, forming NH+. The pKa of the NH+/NH3 4 4 couple is 9.2 and does not buffer the periplasm effectively at a relatively neutral pH. Consequently, NH3 alone would not account for the nding that the periplasmic pH is relatively constant at 6.1 in the presence of urea. In contrast, pH 6.1 is the effective pKa of HCO, 3 allowing to buffer the periplasm. The periplasmic -carbonic anhydrase is able to produce HCO from CO2 (obtained from urea 3 breakdown) and water, working as a periplasmic buffer (Sachs et al., 2005). Considering the metabolism of H. pylori several biosynthetic pathways are missing, suggesting an adaptation to host. As a consequence, H. pylori can be grown only in chemically dened medium with the additional amino acids arginine, histidine, isoleucine, leucine, methionine, phenylalanine and valine, and some strains also require alanine and/or serine. H. pylori can catabolize glucose, but appears that other sugars cannot be catabolized by H. pylori. These aspects taken together with the fact that H. pylori is a microaerophilic bacterium that does not tolerate high oxygen conditions clearly limit the habitats that H. pylori can colonize (Kusters et al., 2006). 2. Routes of transmission The route of transmission of H. pylori is not completely understood. The only known reservoir of H. pylori is the human stomach (Megraud and Broutet, 2000) and since H. pylori appears to have a narrow host range, new infections are thought to occur as a consequence of direct human-to-human transmission or environmental contamination. There is evidence supporting a gastrooral, oraloral and faecaloral transmission, but no conclusive data addressing the predominance of transmission via any of these routes exist (Kusters et al., 2006; Azevedo et al., 2009). The minimum H. pylori infectious dose in non colonized rhesus monkey is 104 bacteria, but the bacterial load for secondary infection after antibiotic therapy decrease by a 10- to 100fold (Solnick et al., 2001). In humans the minimum dose have not been determined, but in human volunteers a dose ranging study (from 104 to 1010) suggested that 105 colony forming units might be near the minimal infectious dose (Graham et al., 2004). 2.1. Person-to-person transmission Person-to-person transmission can be subdivided in two main categories: vertical and horizontal transmission. The vertical transmission is infection spread from ascendants to descendents within the same family, while horizontal transmission involves contact with individuals outside the family but does not exclude environment contamination (Schwarz et al., 2008). Type of strains circulating

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within-family members are often similar (Kivi et al., 2003; Kivi and Tindberg, 2006; Bamford et al., 1993; Delport et al., 2006), namely between 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 sequences in 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 acquisition appeared to play a major role in the transmission pathway. Even the presence of common strains within the same family does not exclude acquisition of H. pylori from a common (external) source. H. pylori infection may be acquired by more diverse routes than previously expected (Raymond et al., 2008). The person-to-person transmission may occur by three possible pathways: the gastrooral, the oraloral and the faecaloral, which are described below. 2.1.1. Gastrooral transmission H. pylori is acquired in early life and the vomiting of achlorhydric mucus may serve as a vehicle for transmission. The transmission route could be by gastric juice, especially as a result of epidemic vomiting in childhood (Axon, 1995). For instance, H. pylori infection is easily transmitted from person-to-person by gastric intubation. H. pylori appears to survive outside the human body in unbuffered gastric juice. Culture of H. pylori was possible in 62% of the 21 patients following 2 h of gastric juice collection, 42% after 6 h and 10% after 24 h (Galal et al., 1997). Another study reported a similar isolation percentage from gastric juice of symptomatic patients (Okuda et al., 1996). Others reports suggest that a sibling history of vomitus (Luzza et al., 2000) or exposure 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 from vomitus (Leung et al., 1999a; Parsonnet et al., 1999). H. pylori was often present in high quantities in vomitus, with as many as 30,000 CFU/mL of sample, capable of being cultured from air samples collected after vomiting, but not before. However, the short duration of 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 gastro oral transmission, especially during childhood, coupled with poor hygiene practices, with the vomitus working as the putative vehicle of transmission. 2.1.2. Oraloral transmission Saliva is another possible source of H. pylori transmission, since the gastric ora can reach and colonize the mouth after regurgitation or vomiting. H. pylori DNA has been frequently amplied from saliva, subgingival biolm and dental plaque (Burgers et al., 2008; Souto and Colombo, 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 after vomiting and quantities of H. pylori in saliva tended to be low (Parsonnet et al., 1999). Likewise, other studies report that H. pylori cannot 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 include the frequent absence of related strains infecting spouses (Gisbert et al., 2002; Vale and Vitor, 2007; Luman et al., 2002; Suzuki et al., 1999), but this is controversial given reports that demonstrate the presence of common strains infecting couples (Kivi et al., 2003). Common strains with couples suggest person-to-person transmission or common source exposure within couples (Singh et al., 1999; Georgopoulos et al., 1996). Recently, a study of the primers used for the PCR detection of H. pylori suggested that the primers may provide unreliable results, due to limitations in sensitivity and specicity

(Sugimoto et al., 2009). These may explain controversial PCR results, since numerous species related to H. pylori may be present in the oral microbiota (Megraud and Broutet, 2000). These data suggest that although saliva might work as a vehicle of transmission, the oraloral transmission is not the main mode of transmission of H. pylori, at least in adults (Luman et al., 1996). 2.1.3. Faecaloral transmission H. pylori DNA has been frequently detected in human faeces (Queralt et al., 2005; Sen et al., 2005; Fujimura et al., 2004b; Monteiro et al., 2001a,b; Gramley et al., 1999; Oyofo et al., 1992). Attempts to culture H. pylori from faeces have had limited success (Falsa et al., 2007; Liang and Redlinger, 2003; Dore et al., 2000) as the bacterium exists there predominantly 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 with twelve noninoculated mice in a grated cage (to test oraloral transmission). A similar experiment was performed, but the housing was in a cage without grating on the oor (to test faecaloral transmission). 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 sacriced weekly after birth and serum and mother's milk from the stomach and gastric tissues were obtained from pups. H. pylori was not identied in cultures from the gastric mucosa of pups delivered by infected mothers, but H. pylori 16S rRNA was detected 4 weeks after birth, suggesting that Mongolian gerbil pups become infected via faecaloral maternal H. pylori transmission (Oshio et al., 2009). Transmission via faecal contaminants is also supported by the occurrence of H. pylori infections among institutionalized young people during outbreaks of gastroenteritis (Laporte et al., 2004). Taken together, these experiments support faecaloral transmission in the transmission 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 than vomitus, saliva or gastric juice. When hygienic conditions are poor, household contamination of treated water cannot be ruled out. H. pylori is easily controlled by chlorination (Johnson et al., 1997) (the principal disinfection agent of water), but recontamination of treated water is a widespread problem (Ashbolt, 2004). Poor hygienic practices during childhood, absence of a household bath, non-hygienic drinking water and absence of a sewage disposal facility (Nouraie et al., 2009) may lead to re(contamination) of drinking water, that in this way may serve as a vehicle of transmission. Even in treated water, the survival of H. pylori is possible, at least for short periods of time. When comparing the prevalence 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 this ecologic comparison limit its value (Azevedo et al., 2009). Recently, a study reports that it was possible to culture a reference strain after ve minutes of inoculation in chlorinated water (0.96 mg/l), and viable cells were detected by FISH up to 3 h post inoculation. The percentage of coccoid forms was higher than spiral forms after 40 s of chlorine exposure, but even after 24 h, FISH detection revealed the presence of spiral cells. After 24 h, amplication of the specic H. pylori 16S rDNA gene was possible. These results imply that H. pylori could survive drinking 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 food products from street vendors in Peru (Begue et al., 1998), suggesting that food may act as a vehicle rather than a reservoir (van Duynhoven and 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 food may 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 transmission from person-to-person. There is a single report of detection of H. pylori DNA in 6.1% (4/66) of breast milk samples, collected after delivery from H. pylori antibody-positive pregnant women (Kitagawa et al., 2001). However, this could be due to nipples or ngers contaminating the milk (Azevedo et al., 2007b). A community based study in Brazil revealed that there was no signicant difference in the prevalence of H. pylori and the history of infant breastfeeding (Braga et al., 2007). Other studies conducted in Vietnam (Nguyen et al., 2006), Germany (Rothenbacher et al., 2002) and Japan (Ueda et al., 2003) provided similar results. It has even been suggested that breastfeeding for more than 6 months (Nguyen et al., 2006; Pearce et al., 2005), and yogurt consumption (Ornelas et al., 2007) is associated with reduced seropositivity 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 human stomach is unknown. Indirect evidence of presence of H. pylori in water and food has been reported, namely the detection of H. pylori DNA and VBNC forms. H. pylori may enter as VBNC under conditions where growth is not possible (van Duynhoven and de Jonge, 2001). 2.2.1. Water ingestion The 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 potential pathogens (Alsam et al., 2006). H. pylori DNA (vacA and ureAB genes) has also been amplied 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. pylori playing an important role in the bacterial reinoculation of stomach or transmission to a new host (Salmanian et al., 2008). The association between H. pylori and Acanthamoeba and Candida should be further investigated. Indirect evidence that the transmission of H. pylori is waterborne is based upon four sets of data: i) presence of DNA in water samples; ii) observation of coccoid forms in water samples; iii) survival of H. pylori in articially contaminated water; iv) and growth of H. pylori from water samples (Table 1). This last mode of transmission is only briey discussed in the current literature. H. pylori DNA has been identied in several water sources (Table 1) using diverse gene targets. Drinking, river, sea, ground and wastewater 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 to conventional treatment (Johnson et al., 1997). However, H. pylori appears to be more resistant to chlorine and ozonation than E. coli (mandatory indicator microorganism to evaluate water quality in most countries), and equally resistant to monochlorine. Traditional indicator 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. pylori DNA were also negative (Queralt et al., 2005). Likewise, upstream river water samples were negative (Fujimura et al., 2004a). Taken together, these results suggest that faecal contamination is responsible for the contamination of river water. However, DNA isolation alone 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. In fact, the 16S rRNA gene sequence has been described as more suitable for the discrimination at the genus level. Even for Helicobacter species discrimination 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 and undergoing apoptosis is still an unanswered question. Coccoid H. pylori appears to conserve the capacity to produce proteins for at least 100 days when stored at 4 C, in either phosphate-buffered saline (PBS) or distilled water (Mizoguchi et al., 1999). It has been suggested that although the virulence of coccoid H. pylori induced by water decreases, the coccoids still retain a considerable urease activity and preserve adhering ability to epithelial cells. These coccoids induced by water have been capable of colonizing the gastric mucosa, causing gastrititis in mice (She et al., 2003). The VBNC state could be responsible for the difculty in isolating H. pylori from water samples (She et al., 2003). To our knowledge there are only two studies that report successful isolation of H. pylori from environmental water samples. In one case, the bacterium was isolated from wastewater (using immunomagnetic separation and culture), which is compatible with a faecaloral route of contamination (Lu et al., 2002). In the second case, H. pylori was isolated from a seawater sample (Cellini et al., 2005). Colonies were only obtained in residues on 200 m lters but not in residues on 0.64 and 0.22 m lters. H. pylori could only be isolated from fractionated seawater samples, containing large zooplanktonic organisms; without zooplankton, H. pylori cells could not be recovered from any other fractions by growth-dependent detection protocols (Cellini et al., 2004). This suggests that H. pylori requires an organic or proteic matrix to remain culturable. This should be further investigated and other studies attempting to isolate H. pylori from water sources using similar approaches are needed. Survival studies in water samples demonstrate that H. pylori can be cultured for a limited period of time in a temperature dependent manner (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 biolms 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). Biolms are slimy lms of bacteria, other microbes and organic materials that cover underwater surfaces, particularly inside plumbing. This makes them rather inaccessible and provides a matrix difcult to be reached by disinfectants. The detachment of bioms is the principal form of contamination of treated water (Gouider et al., 2009; Stoodley et al., 2001). H. pylori adherence to biolms appears to be

Table 1 Examples of studies that present evidence of presence of H. pylori in water samples. Study Presence of H. pylori DNA Water type Drinking water (Bangladesh) Wastewater (Mexico) Method RT-PCR PCR + Southern blot N. samples 75 42 S 57 Gene ID hpaA and glmM 16S rRNA and cagA Observations Ref. Janzon et al. (2009) Mazari-Hiriart et al. (2008) Bockelmann et al. (2009) Queralt et al. (2005)

Articial recharge systems (Belgium, Italy, Spain) qPCR Wastewater Semi-nested PCR River water Spring water (Spain) Seawater (Italy) Nested PCR River water from 4 rivers (Japan) Nested PCR

S 24

Groundwater Surface water Wastewater (Mexico) Water system (drinking water, Mexico) 10 tap water 6 well water 10 river water 10 seawater (Japan) Drinking water (Peru) Spiked bottled mineral water Spiked groundwater Spiked ltered autoclaved stream water Spiked chlorinated ltered water (drinking water) Survival of H. pylori in articially Autoclaved distilled water contaminated water Spiked bottled mineral water (drinking water) Spiked chlorinated ltered water (drinking water) Presence of coccoid forms

Nested PCR

139

PCR RT-PCR

212

PCR SEM SEM Epiuorescence microscopy FISH Culture hybridization Culture Epiuorescence microscopy PCR Culture FISH PCR RT-PCR Culture Culture

48 S S S S S S

0% positive 14% positive 16S rRNA 6% both positive 16S rRNA 0% positive ureA 66% positive wastewater 11% positive river water 0% positive spring water glmM (ureC) H. pylori DNA only detected in fractionated water samples containing zooplaktonic organisms ureA and cagA 50% positive urea 45% positive both genes Positive results only from the middle and downstream samples 16S rRNA and cagA 68% surface water 100% untreated and 2% in treated groundwater 20% wastewater 16S rRNA and cagA 21% positive 16RRNA 9% both positive 16S rRNA and ureA 0% tap water 33% positive 16S RNA only 0% river water 0% seawater 16S rRNA and adhesin 50% adhesin positive 23% both positive Na Coccoid forms after 14 days Na Coccoid form after 24 and 72 h at 15 and 4 C Na 75% Coccoid forms after 10 days Na Coccoid forms maioritary after 40 s 16S rRNA ureA Cultivability >96 h in the dark at 25 C). More than 196 h reported for some of the H. pylori strains Culture until 5 days Cell viability until 14 days PCR detection at month 3. Culture until 5 min FISH viable cells until 3 h PCR positive after 24 h RT-PCR after 24 h Cultivability until 10 days H. pylori is more resistant to chlorine and ozonation than E. coli and equally resistant to monochlorine Culture up to 48 h

Cellini et al. (2004) F.F. Vale, J.M.B. Vtor / International Journal of Food Microbiology 138 (2010) 112 Fujimura et al. (2004a)

Mazari-Hiriart et al. (2001a)

Mazari-Hiriart et al. (2001b) Horiuchi et al. (2001)

Hulten et al. (1996) Queralt and Araujo (2007) Nayak and Rose (2007) Adams et al. (2003) Moreno et al. (2007) Azevedo et al. (2008) Queralt and Araujo (2007)

16S rRNA and vacA

Moreno et al. (2007)

Spiked ltered autoclaved stream water Spiked disinfected water (chlorine, monochlorine, ozone) Spiked autoclaved river water Growth of H. pylori Seawater (Adriatic sea) Wastewater (Mexico)

S S

Na Na Na vacA and cagA 16S rRNA and vacA

Culture S Autoradiography Filtration (200 mm, lter), culture S and PCR IMS, culture and PCR S

Adams et al. (2003) (Baker et al., 2002; Johnson et al., 1997) Shahamat et al. (1993)

H. pylori was only isolated from fractionated water Cellini et al. (2005) samples containing large zooplaktonic organisms H. pylori isolates from wastewater demonstrated Lu et al. (2002) vacA gene heterogeneity

S several samples, number not specied. Na not applicable. IMS Imunomagnetic separation. SEM scanning electron microscopy. FISH Fluorescence in situ hybridization.

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

independent of temperature (Azevedo et al., 2006). An ecological explanation for its presence in biolms is provided by the microaerophilic 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, which may 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 of true 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, and the formation of H. pylori biolm cannot be excluded (Andersen and Rasmussen, 2009; Watson et al., 2004). Karita et al. report that in 41 families, H. pylori prevalence signicantly increased with a history of drinking well water. For ve families whose wells were screened for H. pylori DNA presence, all wells gave positive results. (Karita et al., 2003). However these authors have only considered drinking the water from the well as an external source of H. pylori. The unequivocal proof would be the detection of H. pylori DNA in the water with the same genotype as of human isolates. 2.2.2. Food ingestion Several 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 mostly consumed during this period. 2.2.2.1. Milk. There is indirect evidence of H. 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 of 42 shepherds and 28 members of their families, whose H. pylori status was determined by the 13Curea breath test, revealed that H. pylori prevalence reached 97.6% in shepherds, 86% in their family members, but signicantly 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 urban areas (Plonka et al., 2006). These authors propose that H. pylori infection in shepherd children may originate from sheep. These studies led to the hypothesis of H. pylori infection being considered a zoonosis (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 of detection of H. pylori DNA is higher in raw milk when compared with pasteurized milk, and this percentage decreases when two genes are assayed (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 for short periods in milk (Quaglia et al., 2007, 2009; Bohmler et al., 1996), which is also a food product with a short shelife. 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, being eliminated as viable forms in faeces and could then contaminate the milk during the milking process (Megraud and Broutet, 2000). The genetic relationship among strains isolated from milk and human patients should be further characterised to clarify if these are different Helicobacter sp., which are similar to H. pylori, or if it is indeed H. pylori. In fact, Helicobacter spp. have been detected in the abomasums of sheep and cattle (De Groote et al., 1999b; Haesebrouck et al., 2009). Considering survival studies, contaminated milk (by humans for certain and not clear if by animals) may certainly be a vehicle for H. pylori. 2.2.2.2. Meat, vegetables and other food products. Studies on the detection of H. pylori in food products other than milk are quite sparse. However, it has been described that individuals who consume raw vegetables are more likely to acquire H. pylori (Goodman et al., 1996; Hopkins et al., 1993; Chen et al., 2005). The association of the infection with consumption of raw vegetables is an additional indirect evidence of the presence of H. 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 of H. pylori DNA in chopsticks after being used by sixty-nine asymptomatic volunteers was very low (2%) (Leung et al., 1999b), the sharing of chopsticks may contribute to crossinfection (Wong et al., 2005). There are few studies that address the survival of H. pylori in food products other than milk containing complex microbial ora (Table 3).

Table 2 Examples of studies that present evidence of presence of H. pylori in milk samples. Study Presence of H. pylori DNA Milk type Raw goat's milk Raw sheep's milk Raw cow's milk Raw cow's milk Pasteurized cow's milk Raw sheep's milk Raw sheep's milk Method Nested PCR N. samples Gene ID 160 130 110 18 20 63 51 13+13 S 3 S 18 20 63 51 glmM Observations Ref. Quaglia et al. (2008)

Semi-nested-PCR PCR PCR

Survival of H. pylori in articially Spiked pasteurized milk Culture contaminated milk and UHT milk Cow's milk Culture Raw sheep's, goat's and cow's milk Fresh milk without preservatives Raw cow's milk Pasteurized cow's milk Raw sheep's milk Raw sheep's milk Culture NestedPCR Culture Culture Culture Culture

26% positive goat's milk 43% positive sheep's milk 55% positive cow's milk ureA 72% positive raw milk 55% positive pasteurized milk 16 rRNA and vacA 60% positive 16S rRNA 8% both positive 16 rRNA and vacA 60% positive 9% both positive Na Median survival of 9 days in raw milk and 12 days in UHT milk Na Reisolated until 6 days cooled milk; 3 to 4 days 37 C milk glmM Limit of detection by PCR 3 cfu/ml Na Na Na Na Survival until 10 days at 4 C and 3 days at 25 C Positive for one raw milk Positive from raw sheep's milk Positive from raw sheep's milk

Fujimura et al. (2002) Dore et al. (2001) Dore et al. (1999) Quaglia et al. (2007) Bohmler et al. (1996) Quaglia et al. (2009) Fan et al. (1998) Fujimura et al. (2002) Dore et al. (2001) Dore et al. (1999)

Growth of H. pylori

UHT ultrahigh temperature milk.

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

Table 3 Survival of H. pylori in articially inoculated food products, other than milk. Food product Spiked leaf lettuce, tofu, yogurt and raw chicken Yogurt, ker, curd cheese, chicken Method Culture Culture Observations Survival until 5 days in tofu; 2 days in lettuce and raw chicken; not recovered from yogurt At 37 C survival until 3 h in yogurt; 24 h in ker; 10 h in curd cheese; At 7 C survival superior to 72 h in these 3 products. Survival until 48 h in chicken Survival until 3 days in autoclaved ground beef; 7 days in irradiated ground beef Survival until 72 h in sanitized lettuce and carrot; up to 96 h in sterilized carrots. Ref. Poms and Tatini (2001) Bohmler et al. (1996)

Autoclaved and irradiated (10 kGy) spiked ground beef Spiked lettuce and carrots

Culture Culture

Jiang and Doyle (2002) Gomes and De Martinis (2004a)

Foods with water activity higher than 0.97 and pH from 4.9 to 6.0 theoretically provide conditions for the survival of H. pylori. Also, general lack of efcient sanitation in removing or killing pathogens on raw fruits and vegetables may contribute to harbor pathogens (Gomes and De Martinis, 2004b; Beuchat, 2002). H. pylori is unlikely to grow in most food products, but it is able to survive in a low acid, high moisture environment for extended periods of time, especially if stored refrigerated (Bohmler et al., 1996; Gomes and De Martinis, 2004b; Jiang and Doyle, 1998). Likewise, the isolation of H. pylori from food products is extremely difcult due to the presence of accompanying microora 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 infected personnel, we cannot exclude the transmission of H. pylori by these foods. An infection of the consumer by this route is not very likely, but it cannot be completely ruled out (Bohmler et al., 1996). Proof of the ability of H. pylori to survive in common foods supports the hypothesis that primary contamination of a food product (animal reservoir) or secondary contamination 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, makes them 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 and sheep, 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 been isolated from the stomachs of pigs, is the most prevalent gastric nonpylori Helicobacter species in humans. The Candidatus Helicobacter bovis (De Groote et al., 1999b) is highly prevalent in the abomasums of cattle but has only occasionally been detected in the stomachs of humans. There are clear indications that gastric non-pylori Helicobacter infections in humans originate from animals and it is likely that transmission occurs through direct contact (Haesebrouck et al., 2009). As referred H. pylori can be found inside yeasts. Yeasts are present in the human oral cavity and different foods. Thus measurements regarding control of yeast content of foods might be the important to prevent the transmission of H. pylori (Salmanian et al., 2008). 3. Likelihood of transmission in rural and urban environments As described above, the prevalence of infection by H. pylori clearly differs between developing and developed countries, ranging from less than 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 these subgroups, with rural environments presenting a higher prevalence (Bruce and Maaroos, 2008). Contributing to this difference, we have the higher exposure to risk factors in rural environments, namely poor hygiene practices during childhood; crowded families; lack of household 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 direct consequence of a low socioeconomic background. This makes personto-person transmission more probable in urban environments, especially between family members (Kivi et al., 2003, 2007; Raymond et al., 2008; Fujimoto et al., 2007). When studying families from rural environments, horizontal transmission, such as through contaminated food, water or via intensive contact between infants and non-parental caretakers, may jointly play a more important role than within-family transmission (Karita et al., 2003; Schwarz et al., 2008). Unsanitary practices during food preparation might also be involved in vertical transmission, with water and food acting as vehicles of transmission. On the other hand, coupled with the extreme sensitivity of H. pylori to atmospheric oxygen pressure, lack of nutrients and outside temperatures in the 34 to 40 C range, direct person-to-person transmission remains the most likely transmission route (Kusters et al., 2006). H. pylori survive in articially contaminated samples of water and food for short periods of time (Tables 13), making the transmission feasible in case of exposure to these products. The contamination of water and food probably occurs by a faecaloral route. The major limitation in attributing a role for the transmission of H. pylori to water and food products in rural environments lies in the difculty to culture H. pylori from natural samples. The microbiologic culture of H. pylori from water and food would be the gold standard for attributing a role in transmission to these products. Moreover, the matching of the genotypes between human and food isolates would be the denitive proof of transmission 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 difculty in interpreting their presence, for instance in water and milk products (Tables 1 and 2). Finally, in the cases of horizontal transmission without identication of the source of infection, we cannot disregard the fact that other persons with close contact with children other than the parents were not considered for analysis. They would be important to characterise, since the traditional family model is changing. 4. Conclusions The principal transmission route of H. pylori is not clearly dened and two main models exist: i) the vertical transmission from parents to children within the framework of the same family, by direct person-toperson contact, probably by gastrooral or faecaloral route; and ii) the horizontal transmission, by ingestion of contaminated food or water, or via intensive contact between infants and non-parental caretakers. Most likely in urban (developed) areas the vertical transmission represents the main form of transmission, while in rural (non-developed) areas, the horizontal form appears to play a major role, but not to the exclusion of the rst. Horizontal transmission also includes person-to-person transmission, but does not exclude ingestion of contaminated water and food. However, the presence of H. pylori in water and food have only been veried 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 of H. pylori in raw food products, such as strawberries,

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

and fresh cut salads. Due to improving food preparation and farm hygiene during the last decade it would also be of interest to proceed with further comparison studies of the presence of H. pylori in food products.

References
Achtman, M., Azuma, T., Berg, D.E., Ito, Y., Morelli, G., Pan, Z.J., Suerbaum, S., Thompson, S.A., van der, E.A., Van Doorn, L.J., 1999. Recombination and clonal groupings within Helicobacter pylori from different geographical regions. Molecular Microbiology 32, 459470. Adams, B.L., Bates, T.C., Oliver, J.D., 2003. Survival of Helicobacter pylori in a natural freshwater environment. Applied and Environmental Microbiology 69, 74627466. Aguemon, B.D., Struelens, M.J., Massougbodji, A., Ouendo, E.M., 2005. Prevalence and risk-factors for Helicobacter pylori infection in urban and rural Beninese populations. Clinical Microbiology and Infection 11, 611617. Alsam, S., Jeong, S.R., Sissons, J., Dudley, R., Kim, K.S., Khan, N.A., 2006. Escherichia coli interactions with Acanthamoeba: a symbiosis with environmental and clinical implications. Journal of Medical Microbiology 55, 689694. Andersen, L.P., Rasmussen, L., 2009. Helicobacter pylori-coccoid forms and biolm formation. FEMS Immunology and Medical Microbiology 56, 112115. Ando, T., Goto, Y., Ishiguro, K., Maeda, O., Watanabe, O., Ohmiya, N., Niwa, Y., Hamajima, N., El-Omar, E., Goto, H., 2007. The interaction of host genetic factors and Helicobacter pylori infection. Inammopharmacology 15, 1014. Ashbolt, N.J., 2004. Microbial contamination of drinking water and disease outcomes in developing regions. Toxicology 198, 229238. Axon, A.T., 1995. Review article: is Helicobacter pylori transmitted by the gastrooral route? Alimentary Pharmacology & Therapeutics 9, 585588. Azevedo, N.F., Vieira, M.J., Keevil, C.W., 2003. Establishment of a continuous model system to study Helicobacter pylori survival in potable water biolms. Water Science and Technology 47, 155160. Azevedo, N.F., Pinto, A.R., Reis, N.M., Vieira, M.J., Keevil, C.W., 2006. Shear stress, temperature, and inoculation concentration inuence the adhesion of waterstressed Helicobacter pylori to stainless steel 304 and polypropylene. Applied and Environmental Microbiology 72, 29362941. Azevedo, N.F., Almeida, C., Cerqueira, L., Dias, S., Keevil, C.W., Vieira, M.J., 2007a. Coccoid form of Helicobacter pylori as a morphological manifestation of cell adaptation to the environment. Applied and Environmental Microbiology 73, 34233427. Azevedo, N.F., Guimaraes, N., Figueiredo, C., Keevil, C.W., Vieira, M.J., 2007b. A new model for the transmission of Helicobacter pylori: role of environmental reservoirs as gene pools to increase strain diversity. Critical Reviews in Microbiology 33, 157169. Azevedo, N.F., Almeida, C., Fernandes, I., Cerqueira, L., Dias, S., Keevil, C.W., Vieira, M.J., 2008. Survival of gastric and enterohepatic Helicobacter spp. in water: implications for transmission. Applied and Environmental Microbiology 74, 18051811. Azevedo, N.F., Huntington, J., Goodman, K.J., 2009. The epidemiology of Helicobacter pylori and public health implications. Helicobacter 14 (Suppl 1), 17. Baker, K.H., Hegarty, J.P., Redmond, B., Reed, N.A., Herson, D.S., 2002. Effect of oxidizing disinfectants (chlorine, monochloramine, and ozone) on Helicobacter pylori. Applied and Environmental Microbiology 68, 981984. Bamford, K.B., Bickley, J., Collins, J.S., Johnston, B.T., Potts, S., Boston, V., Owen, R.J., Sloan, J.M., 1993. Helicobacter pylori: comparison of DNA ngerprints provides evidence for intrafamilial infection. Gut 34, 13481350. Begue, R.E., Gonzales, J.L., Correa-Gracian, H., Tang, S.C., 1998. Dietary risk factors associated with the transmission of Helicobacter pylori in Lima, Peru. American Journal of Tropical Medicine and Hygiene 59, 637640. Beuchat, L.R., 2002. Ecological factors inuencing survival and growth of human pathogens on raw fruits and vegetables. Microbes and Infection 4, 413423. Bizri, A.R., Nuwayhid, I.A., Hamadeh, G.N., Steitieh, S.W., Choukair, A.M., Musharraeh, U.M., 2006. Association between hepatitis A virus and Helicobacter pylori in a developing country: the saga continues. Journal of Gastroenterology and Hepatology 21, 16151621. Bockelmann, U., Dorries, H.H., yuso-Gabella, M.N., Salgot de, M.M., Tandoi, V., Levantesi, C., Masciopinto, C., Van, H.E., Szewzyk, U., Wintgens, T., Grohmann, E., 2009. Quantitative PCR monitoring of antibiotic resistance genes and bacterial pathogens in three European articial groundwater recharge systems. Applied and Environmental Microbiology 75, 154163. Bohmler, G., Gerwert, J., Scupin, E., Sinell, H.J., 1996. The epidemiology of helicobacteriosis in humans; studies of the survival capacity of the microbe in food. DTW. Deutsche Tierrztliche Wochenschrift 103, 438443. Braga, A.B., Fialho, A.M., Rodrigues, M.N., Queiroz, D.M., Rocha, A.M., Braga, L.L., 2007. Helicobacter pylori colonization among children up to 6 years: results of a community-based study from Northeastern Brazil. Journal of Tropical Pediatrics 53, 393397. Bragana, S.M., Azevedo, N.F., Simoes, L.C., Keevil, C.W., Vieira, M.J., 2007. Use of uorescent in situ hybridisation for the visualisation of Helicobacter pylori in real drinking water biolms. Water Science and Technology 55, 387393. Brown, L.M., Thomas, T.L., Ma, J.L., Chang, Y.S., You, W.C., Liu, W.D., Zhang, L., Gail, M.H., 2001. Helicobacter pylori infection in rural China: exposure to domestic animals during childhood and adulthood. Scandinavian Journal of Infectious Diseases 33, 686691. Brown, L.M., Thomas, T.L., Ma, J.L., Chang, Y.S., You, W.C., Liu, W.D., Zhang, L., Pee, D., Gail, M.H., 2002. Helicobacter pylori infection in rural China: demographic, lifestyle and environmental factors. International Journal of Epidemiology 31, 638645. Bruce, M.G., Maaroos, H.I., 2008. Epidemiology of Helicobacter pylori infection. Helicobacter 13 (Suppl 1), 16.

Bunn, J.E., MacKay, W.G., Thomas, J.E., Reid, D.C., Weaver, L.T., 2002. Detection of Helicobacter pylori DNA in drinking water biolms: implications for transmission in early life. Letters in Applied Microbiology 34, 450454. Burgers, R., Schneider-Brachert, W., Reischl, U., Behr, A., Hiller, K.A., Lehn, N., Schmalz, G., Ruhl, S., 2008. Helicobacter pylori in human oral cavity and stomach. European Journal of Oral Sciences 116, 297304. Carroll, I.M., Ahmed, N., Beesley, S.M., Khan, A.A., Ghousunnissa, S., Morain, C.A., Habibullah, C.M., Smyth, C.J., 2004. Microevolution between paired antral and paired antrum and corpus Helicobacter pylori isolates recovered from individual patients. Journal of Medical Microbiology 53, 669677. Cavalli-Sforza, L.L., 2001. Genes, Peoples and Languages. Penguin Books, Peoples and Languages. Cellini, L., Allocati, N., Campli, E.D., Masulli, M., DiBartolomeo, S., Dainelli, B., 1996. Helicobacter pylori isolated from stomach corpus and antrum: comparison of DNA patterns. Journal of Infection 32, 219221. Cellini, L., Dainelli, B., Angelucci, D., Grossi, L., Di, B.S., Di, C.E., Marzio, L., 1999. Evidence for an oralfaecal transmission of Helicobacter pylori infection in an experimental murine model. Acta Pathologica, Microbiologica et Immunologica Scandinavica 107, 477484. Cellini, L., Del, V.A., Di, C.M., Di, C.E., Favaro, M., Donelli, G., 2004. Detection of free and plankton-associated Helicobacter pylori in seawater. Journal of Applied Microbiology 97, 285292. Cellini, L., Di, C.E., Grande, R., Di, B.S., Prenna, M., Pasquantonio, M., Pane, L., 2005. Detection of Helicobacter pylori associated with zooplankton. Aquatic Microbial Ecology: International Journal 40, 115120. Chen, T.S., 2004. Is the coccoid form of Helicobacter pylori viable and transmissible? Journal of the Chinese Medical Association 67, 547548. Chen, S.Y., Liu, T.S., Fan, X.M., Dong, L., Fang, G.T., Tu, C.T., Gu, X.Y., Wang, J.Y., 2005. Epidemiological study of Helicobacter pylori infection and its risk factors in Shanghai. Zhonghua Yi Xue Za Zhi 85, 802806. Cheng, H., Hu, F., Zhang, L., Yang, G., Ma, J., Hu, J., Wang, W., Gao, W., Dong, X., 2009. Prevalence of Helicobacter pylori infection and identication of risk factors in rural and urban Beijing, China. Helicobacter 14, 128133. Chow, T.K., Lambert, J.R., Wahlqvist, M.L., Hsu-Hage, B.H., 1995. Helicobacter pylori in Melbourne Chinese immigrants: evidence for oraloral transmission via chopsticks. Journal of Gastroenterology and Hepatology 10, 562569. Covacci, A., Telford, J.L., Del, G.G., Parsonnet, J., Rappuoli, R., 1999. Helicobacter pylori virulence and genetic geography. Science 284, 13281333. Das, J.C., Paul, N., 2007. Epidemiology and pathophysiology of Helicobacter pylori infection in children. Indian Journal of Pediatrics 74, 287290. De Groote, D., Van Doorn, L.J., Ducatelle, R., Verschuuren, A., Haesebrouck, F., Quint, W.G., Jalava, K., Vandamme, P., 1999a. Candidatus Helicobacter suis, a gastric helicobacter from pigs, and its phylogenetic relatedness to other gastrospirilla. International Journal of Systematic Bacteriology 49 (Pt 4), 17691777. De Groote, D., Van Doorn, L.J., Ducatelle, R., Verschuuren, A., Tilmant, K., Quint, W.G., Haesebrouck, F., Vandamme, P., 1999b. Phylogenetic characterization of Candidatus Helicobacter bovis, a new gastric helicobacter in cattle. International Journal of Systematic Bacteriology 49 (Pt 4), 17071715. De, S.L., Vasquez, L., Velasco, J., Parlapiano, D., 2006. Isolation of Helicobacter pylori in gastric mucosa, dental plaque and saliva in a population from the Venezuelan Andes. Investigacin Clnica 47, 109116. Delport, W., Cunningham, M., Olivier, B., Preisig, O., van der Merwe, S.W., 2006. A population genetics pedigree perspective on the transmission of Helicobacter pylori. Genetics 174, 21072118. Dewhirst, F.E., Shen, Z., Scimeca, M.S., Stokes, L.N., Boumenna, T., Chen, T., Paster, B.J., Fox, J.G., 2005. Discordant 16S and 23S rRNA gene phylogenies for the genus Helicobacter: implications for phylogenetic inference and systematics. Journal of Bacteriology 187, 61066118. Dimola, S., Caruso, M.L., 1999. Helicobacter pylori in animals affecting the human habitat through the food chain. Anticancer Research 19, 38893894. Dore, M.P., Vaira, D., 2003. Sheep rearing and Helicobacter pylori infectionan epidemiological model of anthropozoonosis. Digestive and Liver Disease 35, 79. Dore, M.P., Sepulveda, A.R., Osato, M.S., Realdi, G., Graham, D.Y., 1999. Helicobacter pylori in sheep milk. Lancet 354, 132. Dore, M.P., Osato, M.S., Malaty, H.M., Graham, D.Y., 2000. Characterization of a culture method to recover Helicobacter pylori from the feces of infected patients. Helicobacter 5, 165168. Dore, M.P., Sepulveda, A.R., El-Zimaity, H., Yamaoka, Y., Osato, M.S., Mototsugu, K., Nieddu, A. M., Realdi, G., Graham, D.Y., 2001. Isolation of Helicobacter pylori from sheepimplications for transmission to humans. American Journal of Gastroenterology 96, 13961401. Dubois, A., Boren, T., 2007. Helicobacter pylori is invasive and it may be a facultative intracellular organism. Cellular Microbiology 9, 11081116. Falsa, T., Valizadeh, N., Naja, M., Ehsani, A., Khani, A., Landarani, Z., Falahi, Z., 2007. Culture of Helicobacter pylori from stool samples in children. Canadian Journal of Microbiology 53, 411416. Falush, D., Wirth, T., Linz, B., Pritchard, J.K., Stephens, M., Kidd, M., Blaser, M.J., Graham, D.Y., Vacher, S., Perez-Perez, G.I., Yamaoka, Y., Megraud, F., Otto, K., Reichard, U., Katzowitsch, E., Wang, X., Achtman, M., Suerbaum, S., 2003. Traces of human migrations in Helicobacter pylori populations. Science 299, 15821585. Fan, X.G., Chua, A., Li, T.G., Zeng, Q.S., 1998. Survival of Helicobacter pylori in milk and tap water. Journal of Gastroenterology and Hepatology 13, 10961098. Fantry, G.T., Zheng, Q.X., Darwin, P.E., Rosenstein, A.H., James, S.P., 1996. Mixed infection with cagA-positive and cagA-negative strains of Helicobacter pylori. Helicobacter 1, 98106. Ferguson Jr., D.A., Li, C., Patel, N.R., Mayberry, W.R., Chi, D.S., Thomas, E., 1993. Isolation of Helicobacter pylori from saliva. Journal of Clinical Microbiology 31, 28022804.

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F.F. Vale, J.M.B. Vtor / International Journal of Food Microbiology 138 (2010) 112 Kivi, M., Rodin, S., Kupershmidt, I., Lundin, A., Tindberg, Y., Granstrom, M., Engstrand, L., 2007. Helicobacter pylori genome variability in a framework of familial transmission. BMC Microbiology 7, 54. Kurosawa, M., Kikuchi, S., Inaba, Y., Ishibashi, T., Kobayashi, F., 2000. Helicobacter pylori infection among Japanese children. Journal of Gastroenterology and Hepatology 15, 13821385. Kusters, J.G., Gerrits, M.M., Van Strijp, J.A., Vandenbroucke-Grauls, C.M., 1997. Coccoid forms of Helicobacter pylori are the morphologic manifestation of cell death. Infection and Immunity 65, 36723679. Kusters, J.G., van Vliet, A.H., Kuipers, E.J., 2006. Pathogenesis of Helicobacter pylori infection. Clinical Microbiology Reviews 19, 449490. Laporte, R., Pernes, P., Pronnier, P., Gottrand, F., Vincent, P., 2004. Acquisition of Helicobacter pylori infection after outbreaks of gastroenteritis: prospective cohort survey in institutionalised young people. BMJ 329, 204205. Lee, Y.C., Lee, S.Y., Pyo, J.H., Kwon, D.H., Rhee, J.C., Kim, J.J., 2005. Isogenic variation of Helicobacter pylori strain resulting in heteroresistant antibacterial phenotypes in a single host in vivo. Helicobacter 10, 240248. Lehours, P., Yilmaz, O., 2007. Epidemiology of Helicobacter pylori infection. Helicobacter 12 (Suppl 1), 13. Leung, W.K., Siu, K.L., Kwok, C.K., Chan, S.Y., Sung, R., Sung, J.J., 1999a. Isolation of Helicobacter pylori from vomitus in children and its implication in gastrooral transmission. American Journal of Gastroenterology 94, 28812884. Leung, W.K., Sung, J.J., Ling, T.K., Siu, K.L., Cheng, A.F., 1999b. Use of chopsticks for eating and Helicobacter pylori infection. Digestive Diseases and Sciences 44, 11731176. Liang, S., Redlinger, T., 2003. A protocol for isolating putative Helicobacter pylori from fecal specimens and genotyping using vacA alleles. Helicobacter 8, 561567. Linz, B., Balloux, F., Moodley, Y., Manica, A., Liu, H., Roumagnac, P., Falush, D., Stamer, C., Prugnolle, F., van der Merwe, S.W., Yamaoka, Y., Graham, D.Y., Perez-Trallero, E., Wadstrom, T., Suerbaum, S., Achtman, M., 2007. An African origin for the intimate association between humans and Helicobacter pylori. Nature 445, 915918. Lu, Y., Redlinger, T.E., Avitia, R., Galindo, A., Goodman, K., 2002. Isolation and genotyping of Helicobacter pylori from untreated municipal wastewater. Applied and Environmental Microbiology 68, 14361439. Luman, W., Alkout, A.M., Blackwell, C.C., Weir, D.M., Plamer, K.R., 1996. Helicobacter pylori in the mouth-negative isolation from dental plaque and saliva. European Journal of Gastroenterology & Hepatology 8, 1114. Luman, W., Zhao, Y., Ng, H.S., Ling, K.L., 2002. Helicobacter pylori infection is unlikely to be transmitted between partners: evidence from genotypic study in partners of infected patients. European Journal of Gastroenterology & Hepatology 14, 521528. Luzza, F., Mancuso, M., Imeneo, M., Contaldo, A., Giancotti, L., Pensabene, L., Doldo, P., Liberto, M.C., Strisciuglio, P., Foca, A., Guandalini, S., Pallone, F., 2000. Evidence favouring the gastrooral route in the transmission of Helicobacter pylori infection in children. European Journal of Gastroenterology & Hepatology 12, 623627. Malaty, H.M., Tanaka, E., Kumagai, T., Ota, H., Kiyosawa, K., Graham, D.Y., Katsuyama, T., 2003. Seroepidemiology of Helicobacter pylori and hepatitis A virus and the mode of transmission of infection: a 9-year cohort study in rural Japan. Clinical Infectious Diseases 37, 10671072. Marshall, B.J., Warren, J.R., 1984. Unidentied curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1, 13111315. Mazari-Hiriart, M., Lopez-Vidal, Y., Calva, J.J., 2001a. Helicobacter pylori in water systems for human use in Mexico City. Water Science and Technology 43, 9398. Mazari-Hiriart, M., Lopez-Vidal, Y., Castillo-Rojas, G., Ponce de, L.S., Cravioto, A., 2001b. Helicobacter pylori and other enteric bacteria in freshwater environments in Mexico City. Archives of Medical Research 32, 458467. Mazari-Hiriart, M., Ponce-de-Leon, S., Lopez-Vidal, Y., Islas-Macias, P., Mieva-Fernandez, R.I., Quinones-Falconi, F., 2008. Microbiological implications of periurban agriculture and water reuse in Mexico City. PLoS ONE 3, e2305. McColl, K.E., 1999. Helicobacter pylori 19881998. European Journal of Gastroenterology & Hepatology 11, 1316. Megraud, F., 2004a. H pylori antibiotic resistance: prevalence, importance, and advances in testing. Gut 53, 13741384. Megraud, F., 2004b. Basis for the management of drug-resistant Helicobacter pylori infection. Drugs 64, 18931904. Megraud, F., Broutet, N., 2000. Review article: have we found the source of Helicobacter pylori? Alimentary Pharmacology & Therapeutics 14 (Suppl 3), 712. Megraud, F., Lehours, P., 2007. Helicobacter pylori detection and antimicrobial susceptibility testing. Clinical Microbiology Reviews 20, 280322. Mizoguchi, H., Fujioka, T., Nasu, M., 1999. Evidence for viability of coccoid forms of Helicobacter pylori. Journal of Gastroenterology 34 (Suppl 11), 3236. Monteiro, L., Gras, N., Megraud, F., 2001a. Magnetic immuno-PCR assay with inhibitor removal for direct detection of Helicobacter pylori in human feces. Jornal of Clinical Microbiology 39, 37783780. Monteiro, L., Gras, N., Vidal, R., Cabrita, J., Megraud, F., 2001b. Detection of Helicobacter pylori DNA in human feces by PCR: DNA stability and removal of inhibitors. Journal of Microbiological Methods 45, 8994. Moreira Jr., E.D., Nassri, V.B., Santos, R.S., Matos, J.F., de Carvalho, W.A., Silvani, C.S., Santana e Sant'ana, 2005. Association of Helicobacter pylori infection and giardiasis: results from a study of surrogate markers for fecal exposure among children. World Journal of Gastroenterology 11, 27592763. Moreno, Y., Piqueres, P., Alonso, J.L., Jimenez, A., Gonzalez, A., Ferrus, M.A., 2007. Survival and viability of Helicobacter pylori after inoculation into chlorinated drinking water. Water Research 41, 34903496. Nabwera, H.M., Logan, R.P., 1999. Epidemiology of Helicobacter pylori: transmission, translocation and extragastric reservoirs. Journal of Physiology and Pharmacology 50, 711722.

Fujimoto, Y., Furusyo, N., Toyoda, K., Takeoka, H., Sawayama, Y., Hayashi, J., 2007. Intrafamilial transmission of Helicobacter pylori among the population of endemic areas in Japan. Helicobacter 12, 170176. Fujimura, S., Kawamura, T., Kato, S., Tateno, H., Watanabe, A., 2002. Detection of Helicobacter pylori in cow's milk. Letters in Applied Microbiology 35, 504507. Fujimura, S., Kato, S., Kawamura, T., 2004a. Helicobacter pylori in Japanese river water and its prevalence in Japanese children. Letters in Applied Microbiology 38, 517521. Fujimura, S., Kato, S., Nagai, K., Kawamura, T., Iinuma, K., 2004b. Detection of Helicobacter pylori in the stools of newborn infants. Pediatric Infectious Disease Journal 23, 10551056. Fujimura, S., Kato, S., Watanabe, A., 2008. Water source as a Helicobacter pylori transmission route: a 3-year follow-up study of Japanese children living in a unique district. Journal of Medical Microbiology 57, 909910. Galal, G., Wharburton, V., West, A., Mapstone, N., Axon, A.T.R., Hawkey, P., Moayyedi, P., 1997. Isolation of H-pylori from gastric juice. Gut 41, A40A41. Gebara, E.C., Faria, C.M., Pannuti, C., Chehter, L., Mayer, M.P., Lima, L.A., 2006. Persistence of Helicobacter pylori in the oral cavity after systemic eradication therapy. Journal of Clinical Periodontology 33, 329333. Georgopoulos, S.D., Mentis, A.F., Spiliadis, C.A., Tzouvelekis, L.S., Tzelepi, E., Moshopoulos, A., Skandalis, N., 1996. Helicobacter pylori infection in spouses of patients with duodenal ulcers and comparison of ribosomal RNA gene patterns. Gut 39, 634638. Giao, M.S., Azevedo, N.F., Wilks, S.A., Vieira, M.J., Keevil, C.W., 2008. Persistence of Helicobacter pylori in heterotrophic drinking-water biolms. Applied and Environmental Microbiology 74, 58985904. Gisbert, J.P., Arata, I.G., Boixeda, D., Barba, M., Canton, R., Plaza, A.G., Pajares, J.M., 2002. Role of partner's infection in reinfection after Helicobacter pylori eradication. European Journal of Gastroenterology & Hepatology 14, 865871. Gomes, B.C., De Martinis, E.C.P., 2004a. Fate of Helicobacter pylori articially inoculated in lettuce and carrot samples. Brazilian Journal of Microbiology 35, 145150. Gomes, B.C., De Martinis, E.C.P., 2004b. The signicance of Helicobacter pylori in water, food and environmental samples. Food Control 15, 397403. Goodman, K.J., Correa, P., Tengana Aux, H.J., Ramirez, H., DeLany, J.P., Guerrero, P.O., Lopez, Q.M., Collazos, P.T., 1996. Helicobacter pylori infection in the Colombian Andes: a population-based study of transmission pathways. American Journal of Epidemiology 144, 290299. Gouider, M., Bouzid, J., Sayadi, S., Montiel, A., 2009. Impact of orthophosphate addition on biolm development in drinking water distribution systems. Journal of Hazardous Materials 167, 11981202. Graham, D.Y., Opekun, A.R., Osato, M.S., El-Zimaity, H.M., Lee, C.K., Yamaoka, Y., Qureshi, W.A., Cadoz, M., Monath, T.P., 2004. Challenge model for Helicobacter pylori infection in human volunteers. Gut 53, 12351243. Gramley, W.A., Asghar, A., Frierson Jr., H.F., Powell, S.M., 1999. Detection of Helicobacter pylori DNA in fecal samples from infected individuals. Journal of Clinical Microbiology 37, 22362240. Haesebrouck, F., Pasmans, F., Flahou, B., Chiers, K., Baele, M., Meyns, T., Decostere, A., Ducatelle, R., 2009. Gastric helicobacters in domestic animals and nonhuman primates and their signicance for human health. Clinical Microbiology Reviews 22, 202223. Hopkins, R.J., Vial, P.A., Ferreccio, C., Ovalle, J., Prado, P., Sotomayor, V., Russell, R.G., Wasserman, S.S., Morris Jr., J.G., 1993. Seroprevalence of Helicobacter pylori in Chile: vegetables may serve as one route of transmission. Journal of Infectious Diseases 168, 222226. Horiuchi, T., Ohkusa, T., Watanabe, M., Kobayashi, D., Miwa, H., Eishi, Y., 2001. Helicobacter pylori DNA in drinking water in Japan. Microbiology and Immunology 45, 515519. Hulten, K., Han, S.W., Enroth, H., Klein, P.D., Opekun, A.R., Gilman, R.H., Evans, D.G., Engstrand, L., Graham, D.Y., El-Zaatari, F.A., 1996. Helicobacter pylori in the drinking water in Peru. Gastroenterology 110, 10311035. Janzon, A., Sjoling, A., Lothigius, A., Ahmed, D., Qadri, F., Svennerholm, A.M., 2009. Failure to detect Helicobacter pylori DNA in drinking and environmental water in Dhaka, Bangladesh, using highly sensitive real-time PCR assays. Applied and Environmental Microbiology 75, 30393044. Jiang, X., Doyle, M.P., 1998. Effect of environmental and substrate factors on survival and growth of Helicobacter pylori. Journal of Food Protection 61, 929933. Jiang, X., Doyle, M.P., 2002. Optimizing enrichment culture conditions for detecting Helicobacter pylori in foods. Journal of Food Protection 65, 19491954. Johnson, C.H., Rice, E.W., Reasoner, D.J., 1997. Inactivation of Helicobacter pylori by chlorination. Applied and Environmental Microbiology 63, 49694970. Kabir, S., 2001. Detection of Helicobacter pylori in faeces by culture, PCR and enzyme immunoassay. Journal of Medical Microbiology 50, 10211029. Karita, M., Teramukai, S., Matsumoto, S., 2003. Risk of Helicobacter pylori transmission from drinking well water is higher than that from infected intrafamilial members in Japan. Digestive Diseases and Sciences 48, 10621067. Kawaguchi, K., Matsuo, J., Osaki, T., Kamiya, S., Yamaguchi, H., 2009. Prevalence of Helicobacter and Acanthamoeba in natural environment. Letters in Applied Microbiology 48, 465471. King, R.D., Lee, J.C., Morris, A.L., 1980. Adherence of Candida albicans and other Candida species to mucosal epithelial cells. Infectous and Immunity 27, 667674. Kitagawa, M., Natori, M., Katoh, M., Sugimoto, K., Omi, H., Akiyama, Y., Sago, H., 2001. Maternal transmission of Helicobacter pylori in the perinatal period. The Journal of Obstetrics and Gynaecology Research 27, 225230. Kivi, M., Tindberg, Y., 2006. Helicobacter pylori occurrence and transmission: a family affair? Scandinavian Journal of Infectious Diseases 38, 407417. Kivi, M., Tindberg, Y., Sorberg, M., Casswall, T.H., Befrits, R., Hellstrom, P.M., Bengtsson, C., Engstrand, L., Granstrom, M., 2003. Concordance of Helicobacter pylori strains within families. Journal of Clinical Microbiology 41, 56045608.

F.F. Vale, J.M.B. Vtor / International Journal of Food Microbiology 138 (2010) 112 Nayak, A.K., Rose, J.B., 2007. Detection of Helicobacter pylori in sewage and water using a new quantitative PCR method with SYBR green. Journal of Applied Microbiology 103, 19311941. Nguyen, B.V., Nguyen, K.G., Phung, C.D., Kremp, O., Kalach, N., Dupont, C., Raymond, J., Vidal-Trecan, G., 2006. Prevalence of and factors associated with Helicobacter pylori infection in children in the north of Vietnam. American Journal of Tropical Medicine and Hygiene 74, 536539. Nouraie, M., Lati-Navid, S., Rezvan, H., Radmard, A.R., Maghsudlu, M., Zaer-Rezaii, H., Amini, S., Siavoshi, F., Malekzadeh, R., 2009. Childhood hygienic practice and family education status determine the prevalence of Helicobacter pylori infection in Iran. Helicobacter 14, 4046. Okuda, M., Tanaka, T., Nakamoto, H., Katai, A., Nakata, H., Koike, M., Miyamoto, H., 1996. Helicobacter pylori infection in childhood: H. pylori isolation rate in gastric juice in relation to positive serum antibody rates. Journal of Gastroenterology 31 (Suppl 9), 68. Ornelas, I.J., Galvan-Potrillo, M., Lopez-Carrillo, L., 2007. Protective effect of yoghurt consumption on Helicobacter pylori seropositivity in a Mexican population. Public Health Nutrition 10, 12831287. Oshio, I., Osaki, T., Hanawa, T., Yonezawa, H., Zaman, C., Kurata, S., Kamiya, S., 2009. Vertical Helicobacter pylori transmission from Mongolian gerbil mothers to pups. Journal of Medical Microbiology 58, 656662. Oyofo, B.A., Thornton, S.A., Burr, D.H., Trust, T.J., Pavlovskis, O.R., Guerry, P., 1992. Specic detection of Campylobacter jejuni and Campylobacter coli by using polymerase chain reaction. Journal of Clinical Microbiology 30, 26132619. Papiez, D., Konturek, P.C., Bielanski, W., Plonka, M., Dobrzanska, M., Kaminska, A., Szczyrk, U., Bochenek, A., Wierzchos, E., 2003. Prevalence of Helicobacter pylori infection in Polish shepherds and their families. Digestive and Liver Disease 35, 1015. Parente, F., Cucino, C., Bianchi, P.G., 2003. Treatment options for patients with Helicobacter pylori infection resistant to one or more eradication attempts. Digestive and Liver Disease 35, 523528. Park, S.R., MacKay, W.G., Reid, D.C., 2001. Helicobacter sp. recovered from drinking water biolm sampled from a water distribution system. Water Research 35, 16241626. Park, C.Y., Kwak, M., Gutierrez, O., Graham, D.Y., Yamaoka, Y., 2003. Comparison of genotyping Helicobacter pylori directly from biopsy specimens and genotyping from bacterial cultures. Jornal of Clinical Microbiology 41, 33363338. Parsonnet, J., Shmuely, H., Haggerty, T., 1999. Fecal and oral shedding of Helicobacter pylori from healthy infected adults. JAMA: The Journal of the American Medical Association 282, 22402245. Pearce, M.S., Thomas, J.E., Campbell, D.I., Parker, L., 2005. Does increased duration of exclusive breastfeeding protect against Helicobacter pylori Infection? The Newcastle Thousand Families Cohort Study at age 4951 years. Journal of Pediatric Gastroenterology and Nutrition 41, 617620. Percival, S.L., Thomas, J.G., 2009. Transmission of Helicobacter pylori and the role of water and biolms. Journal of Water and Health 7, 469477. Perez-Perez, G.I., Rothenbacher, D., Brenner, H., 2004. Epidemiology of Helicobacter pylori infection. Helicobacter 9 (Suppl 1), 16. Perry, S., de la Luz, S.M., Yang, S., Haggerty, T.D., Hurst, P., Perez-Perez, G., Parsonnet, J., 2006. Gastroenteritis and transmission of Helicobacter pylori infection in households. Emerging Infectious Diseases 12, 17011708. Plonka, M., Bielanski, W., Konturek, S.J., Targosz, A., Sliwowski, Z., Dobrzanska, M., Kaminska, A., Sito, E., Konturek, P.C., Brzozowski, T., 2006. Helicobacter pylori infection and serum gastrin, ghrelin and leptin in children of Polish shepherds. Digestive and Liver Disease 38, 9197. Poms, R.E., Tatini, S.R., 2001. Survival of Helicobacter pylori in ready-to-eat foods at 4 degrees C. International Journal of Food Microbiology 63, 281286. Quaglia, N.C., Dambrosio, A., Normanno, G., Parisi, A., Firinu, A., Lorusso, V., Celano, G.V., 2007. Survival of Helicobacter pylori in articially contaminated ultrahigh temperature and pasteurized milk. Food Microbiology 24, 296300. Quaglia, N.C., Dambrosio, A., Normanno, G., Parisi, A., Patrono, R., Ranieri, G., Rella, A., Celano, G.V., 2008. High occurrence of Helicobacter pylori in raw goat, sheep and cow milk inferred by glmM gene: a risk of food-borne infection? International Journal of Food Microbiology 124, 4347. Quaglia, N.C., Dambrosio, A., Normanno, G., Celano, G.V., 2009. Evaluation of a NestedPCR assay based on the phosphoglucosamine mutase gene (glmM) for the detection of Helicobacter pylori from raw milk. Food Microbiology 20, 119123. Queralt, N., Araujo, R., 2007. Analysis of the survival of H. pylori within a laboratorybased aquatic model system using molecular and classical techniques. Microbial Ecology 54, 771777. Queralt, N., Bartolome, R., Araujo, R., 2005. Detection of Helicobacter pylori DNA in human faeces and water with different levels of faecal pollution in the north-east of Spain. Journal of Applied Microbiology 98, 889895. Raymond, J., Thiberg, J.M., Chevalier, C., Kalach, N., Bergeret, M., Labigne, A., Dauga, C., 2004. Genetic and transmission analysis of Helicobacter pylori strains within a family. Emerging Infectious Diseases 10, 18161821. Raymond, J., Thiberge, J.M., Kalach, N., Bergeret, M., Dupont, C., Labigne, A., Dauga, C., 2008. Using macro-arrays to study routes of infection of Helicobacter pylori in three families. PLoS ONE 3, e2259. Restaino, L., Frampton, E.W., Hemphill, J.B., Palnikar, P., 1995. Efcacy of ozonated water against various food-related microorganisms. Applied and Environmental Microbiology 61, 34713475. Rothenbacher, D., Bode, G., Brenner, H., 2002. History of breastfeeding and Helicobacter pylori infection in pre-school children: results of a population-based study from Germany. International Journal of Epidemiology 31, 632637. Sachs, G., Weeks, D.L., Wen, Y., Marcus, E.A., Scott, D.R., Melchers, K., 2005. Acid acclimation by Helicobacter pylori. Physiology (Bethesda, Md.) 20, 429438.

11

Salmanian, A.H., Siavoshi, F., Akbari, F., Afshari, A., Malekzadeh, R., 2008. Yeast of the oral cavity is the reservoir of Heliobacter pylori. Journal of Oral Pathology & Medicine 37, 324328. Schwarz, S., Morelli, G., Kusecek, B., Manica, A., Balloux, F., Owen, R.J., Graham, D.Y., van der, M.S., Achtman, M., Suerbaum, S., 2008. Horizontal versus familial transmission of Helicobacter pylori. PLoS Pathogens 4, e1000180. Scott, D.R., Marcus, E.A., Wen, Y., Oh, J., Sachs, G., 2007. Gene expression in vivo shows that Helicobacter pylori colonizes an acidic niche on the gastric surface. Proceedings of the National Academy of Sciences of the United States of America 104, 72357240. Scott, D.R., Marcus, E.A., Wen, Y., Singh, S., Feng, J., Sachs, G., 2010. Cytoplasmic histidine kinase (HP0244)-regulated assembly of urease with UreI, a channel for urea and its metabolites, CO2, NH3, and NH(+), is necessary for acid survival of Helicobacter 4 pylori. Journal of Bacteriology 192, 94103. Sen, N., Yilmaz, O., Simsek, I., Kupelioglu, A.A., Ellidokuz, H., 2005. Detection of Helicobacter pylori DNA by a simple stool PCR method in adult dyspeptic patients. Helicobacter 10, 353359. Sgouros, S.N., Bergele, C., 2006. Clinical outcome of patients with Helicobacter pylori infection: the bug, the host, or the environment? Postgraduate Medical Journal 82, 338342. Shahamat, M., Mai, U., Paszko-Kolva, C., Kessel, M., Colwell, R.R., 1993. Use of autoradiography to assess viability of Helicobacter pylori in water. Applied and Environmental Microbiology 59, 12311235. She, F.F., Lin, J.Y., Liu, J.Y., Huang, C., Su, D.H., 2003. Virulence of water-induced coccoid Helicobacter pylori and its experimental infection in mice. World Journal of Gastroenterology 9, 516520. Sheu, S.M., Sheu, B.S., Lu, C.C., Yang, H.B., Wu, J.J., 2009. Mixed infections of Helicobacter pylori: tissue tropism and histological signicance. Clinical Microbiology and Infection 15, 253259. Singh, V., Trikha, B., Vaiphei, K., Nain, C.K., Thennarasu, K., Singh, K., 1999. Helicobacter pylori: evidence for spouse-to-spouse transmission. Journal of Gastroenterology and Hepatology 14, 519522. Solnick, J.V., Hansen, L.M., Caneld, D.R., Parsonnet, J., 2001. Determination of the infectious dose of Helicobacter pylori during primary and secondary infection in rhesus monkeys (Macaca mulatta). Infection and Immunity 69, 68876892. Souto, R., Colombo, A.P., 2008. Detection of Helicobacter pylori by polymerase chain reaction in the subgingival biolm and saliva of non-dyspeptic periodontal patients. Journal of Periodontology 79, 97103. Stoodley, P., Wilson, S., Hall-Stoodley, L., Boyle, J.D., Lappin-Scott, H.M., Costerton, J.W., 2001. Growth and detachment of cell clusters from mature mixed-species biolms. Applied and Environmental Microbiology 67, 56085613. Suerbaum, S., 2000. Genetic variability within Helicobacter pylori. International Journal of Medical Microbiology 290, 175181. Sugimoto, M., Wu, J.Y., Abudayyeh, S., Hoffman, J., Brahem, H., Al-Khatib, K., Yamaoka, Y., Graham, D.Y., 2009. Unreliability of results of PCR detection of Helicobacter pylori in clinical or environmental samples. Journal of Clinical Microbiology 47, 738742. Suzuki, J., Muraoka, H., Kobayasi, I., Fujita, T., Mine, T., 1999. Rare incidence of interspousal transmission of Helicobacter pylori in asymptomatic individuals in Japan. Journal of Clinical Microbiology 37, 41744176. Sykora, J., Pazdiora, P., Varvarovska, J., Pomahacova, R., Stozicky, F., Siala, K., 2006. Current epidemiological and clinical issues regarding Helicobacter pylori infection in childhood. Epidemiologie, Mikrobiologie, Imunologie 55, 316. Tanriverdi, H.A., Acun, C., Ustundag, G., Barut, A., Tekin, I.O., Ustundag, Y., 2006. Investigation of human colostrum Helicobacter pylori IgA content in lactating women. European Journal of Obstetrics, Gynecology, and Reproductive Biology 124, 5860. Turutoglu, H., Mudul, S., 2002. Investigation of Helicobacter pylori in raw sheep milk samples. Journal of Veterinary Medicine. B. Infectious Diseases and Veterinary Public Health 49, 308309. Ueda, M., Kikuchi, S., Kasugai, T., Shunichi, T., Miyake, C., 2003. Helicobacter pylori risk associated with childhood home environment. Cancer science 94, 914918. Vakil, N., Megraud, F., 2007. Eradication therapy for Helicobacter pylori. Gastroenterology 133, 9851001. Vale, F.F., Vitor, J.M., 2007. Genomic methylation: a tool for typing Helicobacter pylori isolates. Applied and Environmental Microbiology 73, 42434249. Vale, F.F., Encarnacao, P., Vitor, J.M., 2008. A new algorithm for cluster analysis of genomic methylation: the Helicobacter pylori case. Bioinformatics 24, 383388. Vale, F.F., Megraud, F., Vitor, J.M., 2009. Geographic distribution of methyltransferases of Helicobacter pylori: evidence of human host population isolation and migration. BMC Microbiology 9, 193. van Duynhoven, Y.T., de Jonge, R., 2001. Transmission of Helicobacter pylori: a role for food? Bulletin of the World Health Organization 79, 455460. Vandamme, P., Harrington, C.S., Jalava, K., On, S.L., 2000. Misidentifying helicobacters: the Helicobacter cinaedi example. Journal of Clinical Microbiology 38, 22612266. Velazquez, M., Feirtag, J.M., 1999. Helicobacter pylori: characteristics, pathogenicity, detection methods and mode of transmission implicating foods and water. International Journal of Food Microbiology 53, 95104. Warren, J.R., Marshall, B.J., 1983. Unidentied curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1, 12731275. Watson, C.L., Owen, R.J., Said, B., Lai, S., Lee, J.V., Surman-Lee, S., Nichols, G., 2004. Detection of Helicobacter pylori by PCR but not culture in water and biolm samples from drinking water distribution systems in England. Journal of Applied Microbiology 97, 690698. Webb, P.M., Knight, T., Elder, J.B., Newell, D.G., Forman, D., 1996. Is Helicobacter pylori transmitted from cats to humans? Helicobacter 1, 7981. Weyermann, M., Adler, G., Brenner, H., Rothenbacher, D., 2006. The mother as source of Helicobacter pylori infection. Epidemiology 17, 332334.

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F.F. Vale, J.M.B. Vtor / International Journal of Food Microbiology 138 (2010) 112 Hong Kong: metronidazole sensitivity and overall genotype. Alimentary Pharmacology & Therapeutics 15, 493503. Wong, K.K., Chung, P.H., Lan, L.C., Lin, S.C., Tam, P.K., 2005. Trends in the prevalence of Helicobacter pylori in symptomatic children in the era of eradication. Journal of Pediatric Surgery 40, 18441847.

Weyermann, M., Rothenbacher, D., Brenner, H., 2009. Acquisition of Helicobacter pylori infection in early childhood: independent contributions of infected mothers, fathers, and siblings. American Journal of Gastroenterology 104, 182189. Winiecka-Krusnell, J., Wreiber, K., von, E.A., Engstrand, L., Linder, E., 2002. Free-living amoebae promote growth and survival of Helicobacter pylori. Scandinavian Journal of Infectious Diseases 34, 253256. Wong, B.C., Wang, W.H., Berg, D.E., Fung, F.M., Wong, K.W., Wong, W.M., Lai, K.C., Cho, C.H., Hui, W.M., Lam, S.K., 2001. High prevalence of mixed infections by Helicobacter pylori in