|Tuesday, 17 February 2009 16:09|
H. pylori infestation is relatively common in humans with prevalence of infection estimated to be as high as 90% in developing countries and ranging from 25 to 50% in developed countries ( Gomes and De Martinis 2004; Hayes et al. 2006). Many carriers of H. pylori are asymptomatic but many individuals who present with chronic gastritis and stomach cancer have been found to be Helicobacter pylori positive ( Atherton JC 2006; Parsonnet et al. 1994). Age and acquisition of H. pylori has been linked to the hygiene hypothesis in children ( Percival and Thomas 2009).
H. pylori is the only known bacterium that can propagate in the highly acidic environment of the stomach. This is made possible by the production of urease that creates an alkaline microenvironment. Although the acquisition of H. pylori by humans remains to be elucidated, direct human-to-human contacts have been suggested as the primary source of transmission in developed countries ( Magalhães Queiroz and Luzza 2006). In developing countries, water has been suggested as a possible major source of transmission ( Goodman et al. 1996; Klein et al. 1991; Hulten et al. 1996). Acquisition of the pathogen by water might also occur to some minor extent in countries with good hygiene standards: In a recent German study 91 individuals who consumed well water contamined with H. pylori tested positive for this pathogen. Based on this study a positive correlation was found between contaminated well water and acquisition of H. pylori infection (odds ratio: 8.3; 95% confidence; Rolle-Kampczyk et al. 2004). In addition to this, a Japanese study involving 162 individuals from 41 families reported a positive correlation between H. pylori infection and the consumption of well water ( Karita et al. 2003). Within this study the prevalence of infection increased with age and with the duration of the history of drinking well water. A further example of a positive correlation between symptoms of gastritis and H. pylori-contaminated water was reported following an epidemiological study conducted on 147 farmworkers in Southeast Georgia. The conclusion drawn from this study was that farmworkers who drank contaminated water were 2.6 times more likely to develop gastritis ( Reavis C 2005). A case study with children in Lima, Peru, found that the consumption of water from the municipal water supply might present an important source of infection ( Klein et al. 1991). Children in Peru acquire H. pylori early in life with the number of infected individuals increasing rapidly with age. The overall prevalence in the population is 48%. The source of the water was more important than the socioeconomic status of the families. Infection was more prevalent among children whose homes had external water sources, compared to children from homes with internal water sources. As more homes from low-income families depended on external water sources, the prevalence was higher than in high-income families. However, there was no socioeconomic difference in infection prevalence among families with internal water sources: Within high-income families, children were 12 times more likely to acquire H. pylori with municipal water than with community well water. The fact that households with higher incomes generally have access to better water and on average can afford better sanitation and hygiene, might explain socioeconomic differences in H. pylori infection prevalence observed in other studies. A study with 1815 Chileans under 35 years of age suggested that consumption of uncooked vegetables grown with sewage-contaminated irrigation water might be an important factor of H. pylori transmission ( Hopkins et al. 1993). H. pylori seroposivity correlated with increased age, low socioeconomic status, and the consumption of uncooked vegetables. Also a recent study in India found a higher infection rate in lower socioeconomic groups ( Ahmed et al. 2007). The prevalence was higher among people drinking untreated well water than among people drinking tap water. The risk of infection was found to be higher in households with a lower clean water index and in overcrowded houses. To lower the risk, the authors recommended improved household hygiene practices, proper waste disposal measures, and regular use of boiling water for drinking purposes. A positive correlation between infection and age, crowding, type of water for drinking purposes, hygiene, and socioeconomic factors was confirmed in a study performed in Brazil ( Zaterka et al. 2007). For further information, epidemiological data are summarized in a review by Magalhães Queiroz and Luzza 2006. Another review by Bellack et al. presented a conceptual model of the role of water as a reservoir outlining features of a possible transmission cycle ( Bellack et al. 2006). It should be pointed out that within the studies mentioned above and other studies alike relatively small numbers of people were involved suggesting the need for further investigations in this area of public health.
When released into water or when exposed to detrimental and stressful environmental conditions, the culturable and infectious rod or spiral-shaped H. pylori cells acquire coccoid forms ( Azevedo et al. 2007; Nayak and Rose 2007). The spiral form typically measures app. 2-5 μm in length and 0.5-0.8 μm in width and the coccoid form has a diameter of app. 0.8 μm ( Konishi et al. 2007; Percival et al. 2004a; Percival and Thomas 2009). The transition occurs via an intermediate U- and V-shaped form, but the process of coccoid conversion, the underlying reason, and the consequences are still obscure. The morphological conversion goes along with modification of the outer membrane protein profiles ( Citterio et al. 2004). In laboratory systems, the changes can be caused by factors like nutrient deprivation ( West et al. 1990), aging ( Cellini et al. 2004b), or pH adjustment ( Catrenich and Makin 1991). Azevedo reported that the spiral-coccoid transformation only occurs under mild circumstances, whereas under extreme conditions the bacteria are unable to undergo the morphological transition ( Azevedo et al. 2007). This finding supports the view that the change in shape is a result of an active, biological process that is initiated as part of a bacterial protection mechanism. There is controversy, however, about the viability and infectivity status of the coccoid form. Both morphological forms have been observed in the human stomach. Some scientists believe that the latter seems to represent a viable but not culturable state as part of a survival strategy ( Adams et al. 2003; Avezedo NF 2007; Kurokawa et al. 1999), others think it is a morphologic manifestation of degeneration and loss of viability ( Bumann et al. 2004; Kusters et al. 1997). Both views are backed by scientific evidence, although currently the adaptation theory might have more support. If the coccoid form were viable and infectious, there would be no doubt about the importance of water for transmission of H. pylori ( Hultén et al. 1998). There are certain indications that ageing H. pylori populations are physiologically diverse ( Adams et al. 2003; Cellini et al. 2004b) and some coccoid forms can still be viable and potentially infectious (see section infectious dose). Only one study has reported successful reversion (transformation from coccoid to spiral) according to our knowledge ( Andersen et al. 1997). Transformation was observed with a 4-day-old culture upon transfer to fresh medium.
H. pylori can be detected in wastewater, surface waters, seawater, and drinking water ( Percival et al. 2000; Percival et al. 2001; Bitton G 2005) however most of the data generated is not quantitative. Several studies have concluded that fecal indicators cannot be used to reliably predict H. pylori presence ( Hegarty et al. 1999; Mendoza et al. 2004; Voytek et al. 2005). Despite the frequent detection of H. pylori in environmental waters, the viability status of the cells in different water types is very unclear (see survival section). So far, only one study has reported the isolation of H. pylori from raw municipal wastewater after immunomagnetic capture ( Lu et al. 2002). The population in the isolation area along the U.S.-Mexico border in Ciudad Juárez has a H. pylori prevalence rate of 74%. Genotyping revealed that some of the isolated strains belonged to the clinically relevant vacA classes, which are associated with advanced disease. The Rio Grande at which Ciudad Juárez is located has been shown to have a high prevalence of H. pylori genomes in a later study ( Mendoza et al. 2004). As H. pylori loses its culturability rapidly in water, the success of cultivation in this study might be explained by the possibility that the cells were introduced into the wastewater only shortly before sampling and isolation ( Adams et al. 2003). The low temperatures during the season when isolation was successful might also have been beneficial for obtaining culturable cells.
Association with biofilms. Biofilm constitute a potential haven for the growth and persistence of waterborne pathogens in water ( Percival et al. 1998; Percival and Walker 1999; Percival et al. 1999b; Percival et al. 2000; Flemming et al. 2002) and as a potential reservoir of H. pylori ( Percival and Thomas 2009). Initial evidence that H. pylori was capable of adhering to a mature heterotrophic mixed-species biofilm in a continuous culture chemostat was reported by Mackay et al. 1998. Within this study H. pylori was detected in biofilms for up to 192 hours (8 days) post-challenge. A later study showed the ability of H. pylori to attach to potable water biofilms generated on stainless steel coupons ( Azevedo et al. 2003). After successful incorporation into the biofilms, H. pylori could be detected for up to five days after inoculation using peptide nucleic acid (PNA) probes. Later studies showed the ability of H. pylori to form biofilms itself at the air-liquid interface of batch cultures ( Cole et al. 2004; Stark et al. 1999). Monospecies biofilms contained polysaccharides and channels for nutrient flow suggesting that biofilms might represent an alternate lifestyle of the bacterium ( Cole et al. 2004). Indeed, H. pylori could be detected by PCR in biofilm collected from a section of old cast iron pipe that was removed from an urban drinking water distribution system in Scotland during routine maintenance work ( Park et al. 2001). This correlates with the finding that H. pylori is able to adhere to different plumbing materials used in drinking water distribution systems ( Azevedo et al. 2006a, see below). Adhered cells retained their spiral morphology longer than the planktonic counterparts. Most attached bacteria, however, were found to have compromised cell membranes and seemed not to be viable. Although these findings indicate that biofilms might play a role in environmental H. pylori reservoirs, however the question about their viability remains like for planktonic cells suspended in water. In seawater, association of H. pylori with plankton has been suggested ( Cellini et al. 2004a). From an infectivity perspective the finding that mucin influenced the biofilm – planktonic ratio of H. pylori is of great interest. Using glass surfaces, it was shown that 10% mucin greatly increased the number of planktonic cells suggesting that in a mucus-rich stomach, the planktonic form might be favored over the adhesion to epithelial cells ( Cole et al. 2004).
Examples of studies on occurrence in water are summarized in the following:
It was generally believed that H. pylori was susceptible to disinfectants commonly used in the treatment of drinking water ( Health Canada 2006). The presence of disinfectants is known to greatly accelerate the unculturability of H.pylori. For example, Johnson and colleagues studied the resistance of three H. pylori strains to 0.5 ppm chlorine in chlorine demand-free buffer adjusted to pH 6, 7, or 8 ( Johnson et al. 1997). A temperature of 5°C was chosen to minimize the biocidal activity of chlorine. With an initial inoculum of approximately 104 cells per ml, a >3.5 log10 reduction occurred in all instances after 80 sec of exposure. The fact that E. coli in comparison was slightly more sensitive under the same conditions, was explained by the culture preparation procedure. Nevertheless, disinfection has to be performed carefully as suggested by a hospital report- when examining 128 endoscopes used for diagnosis of H. pylori-positive patients, 54 were contaminated before cleaning and disinfection. One endoscope was still found contaminated after manual cleaning and disinfection with 2% glutaraldehyde ( Nürnberg et al. 2003). It also has to be considered that although culturability can be destroyed quickly, disinfection might not be as efficient to destroy the viability of H.pylori. Once having entered a distribution system, H. pylori may be able to tolerate the disinfectant residuals in a viable-non culturable form ( Baker et al. 2002).
Selected studies on disinfection are summarized in the following:
Data on H. pylori survival in water are of great interest based on the principal that persistence for prolonged times would support the suspicion that acquisition could potentially be by water consumption. Under laboratory conditions, H. pylori can be cultured for days, up to weeks ( Shahamat et al. 1993), when kept in sterile river water, saline solution, and distilled water ( Health Canada 2006; West et al. 1992). Water temperature has been reported to be a significant environmental stress factor to affect cell viability. It has been reported that colder temperatures (less that 350C) generally favor survival ( Shahamat et al. 1993; Beneduce et al. 2003; Nayak and Rose 2007). In tap water at 4°C, H. pylori can be cultured for up to 4 days but levels do show a steady decrease in CFU over time ( Fan et al. 1998). However in this study electron microscopy revealed that the coccoid form of H. pylori was still present after 7 days. More recent publications have reported that although culturability and the bacillar spiral morphology of H. pylori get lost relatively rapidly over time in water, cells with intact membranes, active transcription of mRNA, and with metabolic activity can be detected for much longer. This observation correlates with an early study by Shahamet and colleagues who showed that aged, nonculturable H. pylori were capable of uptake of metabolites using autoradiography ( Shahamat et al. 1993). Data from Ren et al. indicated that whilst urease activity and mRNA activity decreased in aging H. pylori cultures between day 0 and 10, the mRNA of a 26kDa protein and 16S rRNA were expressed unchanged for up to 14 and 21 days. Continued transcription of several genes (including those of virulence factors) has also been mentioned in other studies ( Adams et al. 2003; Cellini et al. 2004b). Support of the view that the coccoid morphology might represent a survival strategy under adverse conditions is also the finding that coccoid H. pylori takes up propidium iodide slower than spiral forms with different morphologies developed under the same experimental conditions ( Azevedo et al. 2006a). One study indicated that nonculturable H. pylori which transformed from the spiral to the coccoid form by exposure to sterile tap water, retained their ability to infect mice ( She et al. 2003). Morphology and viability might have to be seen as two separate, but interlinked, entities. The relationships between morphology, viability, and infectivity remains at present controversial. Genomic DNA tends to be relatively persistent in water and can be detected in the range of months. Queralt and Araujo concluded that culturability underestimates the presence of infective H. pylori cells, whereas PCR-detection results in overestimation ( Queralt and Araujo 2007).
Selected studies on survival are summarized in the following:
The infectious dose in humans is unknown. Barry Marshall, one of the first scientists experimenting with H. pylori, performed a self-experiment and developed gastritis after swallowing 109 bacteria ( Marshall et al. 1985). In a test with a human volunteer with normal gastric mucosa, the administration of 3 x 105 CFU in combination with an acid suppressant resulted in disease manifestation ( Morris and Nicholson 1987). In specific-pathogen (H. pylori)-free rhesus monkeys the minimum infectious dose was reported to be 104 bacteria ( Solnick et al. 2001). Nevertheless, the actual infectious dose is assumed to be much lower given the high prevalence of the bacterium worldwide. This is supported by accidental infections, such as ingestion during laboratory activities or improperly maintained endoscopes ( Health Canada 2006). Nevertheless, only a subpopulation (6-20%) of infected individuals develops gastroduodenal disease, with approximately 1% of those cases progressing to gastric cancer ( Health Canada 2006). The infectivity of the coccoid form remains to be elucidated. In an experiment with immunocompetent and immunodeficient BALB/cA mice which were fed orally with a dose of 108 CFU of H. pylori, both spiral cells (from a 2-day old culture) and coccoid cells (from an aged 12-day old culture) resulted in disease manifestation ( Aleljung et al. 1996). The capability of non-culturable coccoid cells from a 20-day old H. pylori culture to infect BALB/c mice was also suggested in a previous study by Cellini et al. ( Cellini et al. 1994). She et al. used H. pylori cells whose coccoid morphology was induced by exposure to sterile tap water ( She et al. 2003). Although the urease activity and the ability to adhere to Hep-2 cells were found lower in these coccoid cells than in spiral cells, they were capable of colonizing the gastric mucosa of BALB/c mice and of causing gastritis. It has to be considered though that there might still be a residual number of spiral cells in aged or stressed cultures that can be responsible for the effect in this type of studies. The difference in infectivity between the two morphologies needs further clarification.
Although optimized selective cultivation conditions have been tested for improved recovery of water-stressed H. pylori ( Azevedo et al. 2004; Degnan et al. 2003), these bacteria are still difficult to isolate from water due to their fastidious nature and the fact that they persist in a nonculturable coccoid form. The viable but non-culturable state of bacteria is a common occurrence with public health significant bacteria ( Hegarty et al. 2001). Only the spiral form can so far be grown under laboratory conditions.
Selected non-cultivation based methods tested on water samples are listed in the following:
The following pictures are part of the Public Health Image Library (PHIL) from the Centers for Disease Control and Prevention (CDC). The photos are in the public domain and thus free of copyright restrictions. We would like to express our appreciation for providing these images.
This scanning electron micrograph depicts a grouping of Gram-negative ”Flexispira rappini” bacteria, magnified 13,951x.
This scanning electron micrograph depicts two ”Flexispira rappini” bacteria, magnified 13,472x.
Links to useful external websites can be found in the following.
The Helicobacter pylori project: http://www.ceb.uminho.pt/biofilm/pylori.htmWikepedia: http://en.wikipedia.org/wiki/Helicobacter_pylori
|Last Updated on Tuesday, 03 April 2012 02:50|