Helicobacter pylori

1.0 Epidemiology of the Disease and Pathogen

1.1 Global Burden of Disease and Distribution

Helicobacter pylori is a human-associated bacterium colonizing the human stomach. It is estimated that H. pylori infection affects more than half of the worldwide adult population (Table 1). H. pylori has a major role in promoting the development of peptic ulcer disease and non-cardia adenocarcinoma of the stomach. H. pylori is estimated to be responsible for 75% of all gastric cancer cases (Graham, 2014). It is therefore of major public health interest.

The reasons for different trends are not fully understood. Low socioeconomic conditions in childhood are known to be the most important risk factors for H. pylori infection. This suggests that the transmission is mostly oral-oral and is associated with close contact within families and transmission of the infection from parents to children and between children. Attending daycare has also been shown increase the risk of transmission. Also, poor sanitary and hygienic conditions are reported risk factors, suggesting a potential role of water (or lack thereof) in transmission. Although the way the infection is transmitted is still unclear, interpersonal transmission appears to be the main route (Mentis et al., 2015), but there is also epidemiologic evidence for the possible role of waterborne transmission (Aziz et al., 2015; Bellack et al., 2006).

1.1.1 Symptomatology

H. pylori infection usually starts in early childhood with gastritis and remains as a chronic, persistent infection for decades. Interestingly, only a minority of infected individuals actually develop gastric diseases, which can include: chronic gastritis, peptic ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, non-Hodgkin’s lymphoma and gastric adenocarcinoma in adults (Graham, 2014). It is still not clear whether early childhood colonization causes any specific symptoms. Studies have shown that the onset of colonization in adults can trigger symptoms such as hypochlorhydria, nausea or gastric pain (Atherton and Blaser, 2009).

1.2 Taxonomic Classification of the Agent

H. pylori belongs to the family Helicobacteraceae and class epsilonproteobacteria.The genus Helicobacter contains 36 described species that have been validated at this point in time (http://www.bacterio.net/index.html) and is divided according to their major colonization sites as gastric or lower intestinal tract associated bacterial species.

H. pylori is microaerophilic, catalase-positive, and has specialized mechanisms for pH homeostasis, including a potent urease enzyme. The ability to maintain the internal pH enables its survival in the gastric mucus and to colonize the stomach. Glucose appears to be the only carbohydrate utilized by this obligate microaerophilic bacterium. Amino acids are potential sources of carbon, nitrogen, and energy (Mobley, 2001). It is a genetically-diverse gastric pathogen, carrying a range of antibiotic resistance patterns, and varies in geographic occurrence (Secka et al., 2013).

1.2.1 Physical description of the agent

H. pylori is a Gram-negative helical or spiral-shaped microaerophilic bacterium with multiple sheathed flagella at each pole of the bacterial cell. The cells are 2-4 µm long and 0.2-1.0 µm wide. In aged cultures and in water or other stressful conditions (inside and outside of gastric mucosa), viable but non-culturable (VBNC) coccoid forms of the cells dominate (Fernández-Delgado et al., 2016; Loke et al., 2016). The genome size is small, approximately 1.6 Mbp (G+C 39%). It was cultivated and described for the first time in 1982 (Marshall and Warren, 1984).

1.3 Transmission

1.3.1 Routes of transmission

Studies carried out over the last three decades indicate that there are two possible routes of transmission of H. pylori: oral-oral and fecal-oral. The oral-oral route is well-established and is the most likely route of infection in childhood. A large number of epidemiological studies have indicated that crowed family conditions, low socioeconomic status and poor hygienic practices, especially in childhood, are risk factors associated with acquisition of the infection. The presence of H. pylori in the mouth, saliva and vomit of infected persons supports the oral transmission. Moreover, several epidemiological studies have shown that the bacterium is transmitted by persons close to the children, especially mothers (Mentis et al., 2015).

1.3.2 Reservoirs: human, animal, and environmental

There are no confirmed data on zoonotic transmission of Helicobacter pylori from animals to humans.

H. pylori can cause a chronic infection. The infection is acquired during childhood, but symptoms generally do not appear until adulthood and vary from person to person (Graham, 2014; Mentis et al., 2015).

1.3.4 Population susceptibility

With the exception that most of the infections are acquired during childhood, there are no clear trends in population susceptibility. The incidence is mostly affected by living conditions, such as low economic status, poor sanitation, and crowded household conditions. A few studies have demonstrated that transmission can occur between adults, especially within couples or persons living together (Kivi et al., 2003; Perez-Perez et al., 1991).

Variable prevalence within social groups living in the same area may exist. In Brazil, different infection rates have been observed among children of low versus high socioeconomic status. In Colombia, people living in the Andes Mountains have a much higher gastric cancer incidence compared to the low costal region. In both areas, the incidence of H. pylori, the origin of the gastric cancer, is the same. Notably, differences in cancer occurrence rate have been related to the ancestral origin of H. pylori strains in both populations. The prevalence of the highly-virulent European strains is more dominant among people inhabiting the mountain area, while the African strains are more frequently detected in coastal areas, likely explaining the higher incidence of the gastric cancer (de Sablet et al., 2011).

1.4 Population and Individual Control Measures

1.4.1 Vaccines

No vaccines are currently available, but there has been some effort towards development (Blanchard et al., 2004).

1.4.2 Hygiene measures

All epidemiological data show that the living standards associated with improved hygiene (toilets, running water, safe drinking water, possibility for hand washing within families) are associated with distinct reduction in the infection prevalence (Gomes and De Martinis, 2004). In most of the high standard of living countries, the prevalence is nowadays low and a decreasing trend has been noted (Graham, 2014; Mentis et al., 2015).

2.0 Environmental Occurrence and Persistence

2.1 Detection Methods

The gold standard for H. pylori detection from clinical samples is culture. However, the applicably of this method to H. pylori detection from environmental samples is problematic due to the predominance of VBNC states (including coccoidal cells) in the environment. Most attempts to isolate H. pylori from the aquatic environment have failed. Nonetheless, other methods based on molecular techniques have demonstrated that it does exist as potentially active cells in the aquatic environment (Table 2, 3, and 4).

Isolates have been obtained from clinical samples, which present the advantage of enabling bacterial strain characterization. H. pylori is a fastidious microorganism, requiring a complex growth medium supplemented by blood or derivatives (Velázquez and Feirtag, 1999). While there is not a standard method, Brucella or Columbia agar supplemented with 5% to 15% of horse or sheep blood or fetal calf serum and antibiotics, such as Skirrow or Dent supplements, are commonly used. Specific growth media can be obtained from commercial companies.

Culture conditions are critical for successful isolation of H. pylori. As it is a microaerophilic bacterium, an adequate atmosphere between 3% to 7% of O₂ and 5% to 10% of CO₂ is needed. After an incubation time between 3 days to 7 days at 37ºC with humidity, small, translucent colonies should appear, about 0.5 mm to 2 mm in diameter. Bacterial identification has been based on a battery of phenotypic tests, the most important being morphology of the cells, staining gram-negative, and positive reaction for urease, catalase and oxidase tests.

H. pylori have been isolated from cathartic human feces (Parsonnet and Shmuely, 1999) and from probably malnourished children from Gambia (Thomas et al., 1992) but not from routine culture fecal samples, where the bacteria is frequently in a VBNC state. As a consequence, its presence is mostly detected with other methods, such as PCR, or especially with stool antigens enzyme immunoassay.

H. pylori has been repeatedly detected in the aquatic environment, applying other techniques such as microscopy (Hegarty et al., 1999), autoradiography, ATP, or molecular techniques. Molecular techniques that have successfully been applied to detect H. pylori in the environment include fluorescence in situ hybridization (FISH), PCR or qPCR (Moreno-Mesonero et al., 2016). Microarrays to detect its presence jointly with other pathogens from food and in sewage have been also designed (Kostić et al.2010; Li et al., 2015).

Often a pre-treatment before bacterial detection, such as sample concentration by filtering or immunomagnetic beads, is applied to reduce inhibitors and /or competitors. Monoclonal-based immunomagnetic separation has been very useful to enable detection both by molecular methods (Flanigan and Rodgers, 2003; Hulten et al., 1996; Monteiro et al., 2001; Sasaki et al., 1999) and culture (Lu et al., 2002; Moreno and Ferrús, 2012; Watson et al., 2004).

PCR is the most widely-applied technique, with several gene fragments targeted: urease (ureA) or (ureC), adhesion (hpsA), citotoxin CagA (cagA), vaculizing (vacA), glmM (Labigne et al., 1991; Shahamat et al., 2004) and also 16S rRNA of genus and species specific (Chisholm et al., 2001; Ho et al., 1991; Logan et al., 2000). The sensitivity and specificity of these techniques has not been established, in addition they might be subject to variability: according to the characteristics of the strains, samples, type of matrix, presence of inhibitors, concentration of the bacteria, extraction method, etc. Often more complementary techniques have been applied. For example, its 16S rRNA gene has been confirmed along with the presence of ureA or vacA (Mazari-Hiriart et al., 2001; Nayak and Rose, 2007) or by sequencing (Twing et al., 2011).

More research is needed to develop techniques that allow determination of both the presence and infectivity of water-associated H. pylori. Such techniques should be effective for both chlorinated and unchlorinated water samples and should also take into consideration possible protective niches of H. pylori, such as biofilms and/or protozoa. In particular, new media better adapted to isolate the bacteria from different matrices, and new molecular techniques such as PMA qPCR that distinguish viable cells, would be very useful.

2.2 Data on Occurrence

Isolation of H. pylori by cultivation from water has rarely been achieved. Lu et al. (2002) isolated for the first time the bacteria from untreated sewage water. Since then few groups have isolated the bacteria, probably because in water or in biofilms the bacteria are mostly in a VBNC state or, if not, they are fastidious slow-growing microorganisms (Lu et al., 2002). In the last two years H. pylori has been cultured from drinking water, but not in pure culture, with its presence among accompanying microbiota confirmed by PCR (El-Sharouny et al., 2015; Santiago et al., 2015). Recently Ranjbar et al. (2016) published the isolation of H. pylori from 8 out of 450 bottled mineral water samples in Iran, with a low-level of co-culture of non-target microbiota (Ranjbar et al., 2016).

2.2.1 Human feces

H. pylori can be detected in human feces using different techniques. Most of the analyses include the stool antigen test with a mean sensitivity and specificity of 92% and 92.2% respectively (Gisbert and Pajares, 2004). PCR has also been used to identify the presence of Helicobacter in feces (Mapstone et al., 1993; Queralt et al., 2005). However, the presence of the antigen or DNA does not prove that infective organisms are present. Some researchers have successfully cultured H. pylori from feces (Kelly et al. 1994; Parsonnet and Shmuely, 1999; Thomas et al., 1992), but the recovery percentages are very low, because media were not appropriate to isolate this fastidious organism (Dore et al., 2005) or because many cells in stool are transformed into coccoid or VBNC cells (Kabir, 2001). The presence of H. pylori in feces reinforces the possibility of a fecal-oral transmission route.

2.2.2 Sewage

The presence of H. pylori in sewage water has been confirmed by some studies. In 2007, Nayak and Rose detected by qPCR this species in 87% of the analyzed samples with a concentration between 2.0×10³ to 2.8×10⁴ cells/L from a wastewater plant in Michigan (Nayak and Rose, 2007) (Table 2). More recently, Moreno and Ferrús (2012) cultivated the bacterium in six out of 23 positive wastewater plant samples in Spain (Moreno and Ferrús, 2012) and in a range of wastewater and receiving waters in Michigan, USA (Bai et al., 2016).

2.2.3 Surface, ground and drinking waters

Helicobacter has been detected in water aqueducts 3.6 x10⁴ copies/L and 3.3x10³ copies/L (Montero-campos et al., 2015) (Table 3).

Isolations were first reported from polluted canal water in 2002 (Lu et al., 2002). Since then, there have been a few reports of cultivation from water. For example, isolation from municipal drinking water in Iraq (Al-Sulami et al., 2010), in Spain from drinking water using an elaborate isolation technique (Moreno and Ferrús, 2012; Santiago et al., 2015) and in Iran from tap water and dental unit water (Bahrami et al., 2013).

The difficulty in obtaining colonies on agar plates from environmental samples principally lies on the fact that H. pylori is a fastidious bacterium with a relatively slow growth rate. Therefore, its isolation from polluted water can be easily masked by outcompeting microbiota with simpler nutritional requirements and faster growth rates. Also, as discussed above, VBNC coccoid cells rapidly form with exposure to aquatic environments.

Biofilms and protozoa are of special interest as protective microhabitats for viable H. pylori in the aquatic environment. The presence of H. pylori in biofilms has been demonstrated. In this habitat the bacteria are protected from harsh conditions and may persist longer than in water (Azevedo et al., 2006; Gião et al., 2008). As well, survival within the free-living amoeba, Acanthamoeba, has been demonstrated under lab conditions (Moreno-Mesonero et al., 2016; Winiecka-Krusnell et al., 2009). In any case more evidence is needed to understand the importance of these habitats.

It is difficult to relate the presence of H. pylori with specific water characteristics. However, its presence has generally been related with fecal pollution, accordingly the most probable sources of the bacteria in the environment (Lu et al., 2002; Moreno and Ferrús, 2012; Queralt et al., 2005). Thus, adequate sanitary conditions are likely to pose benefits for reducing transmission of H. pylori.

2.2.4 Seawaters

H. pylori has been detected in a few studies from seawater (Carbone et al., 2005; Cellini et al., 1994; Holman et al., 2014). Nonetheless, viable H. pylori cells appear to persist in seawater to an even lesser degree than they do for freshwater (West et al., 1990).

2.3 Persistence

The persistence and survival of H. pylori in the aquatic environment has been studied applying various techniques, but there are numerous knowledge gaps. The bacteria lose their cultivability within 1 to 14 days, with reductions between 5 up to 7 log₁₀ in all the studies, but survive as VBNC cells for much longer. The survival of different strains has been essentially studied in controlled lab studies such as inoculating into autoclaved distilled water (Azevedo et al., 2008), bottled mineral water (Queralt and Araujo, 2007), chlorinated water (Moreno et al., 2007) or river water (Adams et al., 2003).

Environmental conditions can trigger morphological changes, but, only temperature has been directly associated with survival. Specifically, the bacterium survives longer at cold temperatures. Water above 20ºC induces a quick decrease of cultivability (Adams et al., 2003; Nayak and Rose, 2007). Other factors such as white light rapidly induced the VBNC state (Buck and Oliver, 2010). When the persistence has been analyzed with molecular techniques, such as PCR or qPCR (Nayak and Rose, 2007; Queralt and Araujo, 2007), the DNA can remain detectable for much longer. However, its presence in the media is not always indicative of infective cells, which is the ultimate concern.

3.0 Reductions by Sanitation Management

3.1 Excreta and Wastewater Treatment

No available information could be found regarding waterless sanitation, water-based sanitation, coupled engineered and environmental-based systems, wastewater treatment facilities, sewage sludge treatment or by disinfection as a tertiary treatment.

There is no known surrogate for H. pylori reported in the literature, probably because it is difficult to find bacteria with similar characteristics. When H. pylori is in its active and free spiral form, its behavior may not be very different from that observed for other Gram-negative bacteria, particularly Campylobacter. However, their sensitivity in the face of sanitation treatments may change when under stress conditions, transforming to VBNC coccoid forms or when it is within biofilms or protozoa (Moreno-Mesonero et al., 2016; Percival and Suleman, 2014).

3.2 Disinfection

Research available on disinfectants has shown rapid inactivation of culturable cells, but, the comparison of the 2 log₁₀ reduction of one strain of H. pylori and one of E. coli showed that H. pylori is more resistant to chlorine and ozone but not monochloramine than E. coli, with a CT₉₉ of 0.299 mg/liter.min versus 0.119 mg/liter.min for chlorine and a CT₉₉ 0.24 mg/liter.min versus 0.09 mg/liter.min for ozone (Baker et al., 2002). A more recent work would indicate survival following normal UV wastewater treatment (Bai et al., 2016). But further research is needed in this area.