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Helicobacter pylori PDF Print E-mail
Tuesday, 17 February 2009 16:09
Authors
Andreas Nocker
Steven Percival

 

  • highly prevalent in humans, especially in developing countries
  • can proliferate in the highly acidic environment of the stomach
  • role of water as transmission vector remain unclear
  • can adopt two distinct morphological forms: spiral and coccoid
  • when transferred into water or when exposed to adverse environmental conditions, the spiral form converts into the coccoid form
  • the culturable spiral form is considered viable and infectious, but there is controversy about the viability and infectivity of the non-culturable coccoid form
  • able to form biofilms and to adhere to existing biofilms
  • can survive in water for several days
  • susceptible to disinfectants
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:

  • H. pylori was found in 42% of samples (n=138) collected from five water systems in Mexico City using nested PCR ( Mazari-Hiriart et al. 2001). Prevalence ranged from 0% to 68% in different systems studied. A later study also reported high prevalence of PCR-detectable H. pylori in groundwater from wells serving as drinking water supplies for Mexico City ( Mazari-Hiriart et al. 2003). H. pylori genomes were detected in 69% of samples (n=62), prior to chlorination, and 57% of post-chlorination samples (n=21). The mean residual chlorine concentration in treated water was detected at 2.76 mg per liter. In this study the contact time of chlorine was considered short. An additional study reported the presence of H. pylori genomes in 10% of 102 samples from 30 extraction wells and 22 pumping points in Mexico City underlining the susceptibility of the local groundwater distribution system to contamination ( Mazari-Hiriart et al. 2005).

  • Hegarty and colleagues reported presence of H. pylori in the majority of 62 surface and shallow groundwater samples collected in Pennsylvania and Ohio. Using a combined fluorescent antibody - CTC staining method (see detection methods below), actively respiring H. pylori cells were present in 60% of surface water samples (n= 42) and 65% of shallow groundwater samples (n=20) ( Hegarty et al. 1999).

  • A study in Cataluña, Spain, revealed the presence of H. pylori genomes (detected by semi-nested PCR) in 66% of wastewater samples (n=15), 11% of river water samples (n=19), and 33% of human fecal samples (n=36) from children in a gastroenterology hospital unit. The two positive river water samples were taken from a river with moderately fecal pollution. None of 19 spring water samples tested positive ( Queralt et al. 2005).

  • Using an IMS-PCR-hybridization approach (see detection methods), a study in Sweden showed that 37% (n=24) of private wells, 12% (n=25) of municipal tap water, and 12% (n=25) of wastewater tested positive for H. pylori. Although two PCR assays targeting the hpaA and 16S rRNA genes were used, the authors pointed out that non-specificity due to the presence of other Helicobacter spp. could not be ruled out ( Hultén et al. 1998).

  • H. pylori were detected in water distribution system biofilms. Braganra and others found evidence on coupons placed in a bypass of a drinking water distribution system for up to 72 days. Cells with similar morphology to H. pylori were detected using labeled peptide nucleic acid (PNA) probes ( Braganra et al. 2007). An earlier study investigated distribution systems of treated water in England applying PCR detection on 151 samples from domestic properties, schools, reservoirs, and hydrants. The overall detection rate was 26% for Helicobacter spp. and 15% for H. pylori specifically. The highest frequency of detection (42%) of Helicobacter spp. was obtained with biofilms samples ( Watson et al. 2004).

  • A study of five North American rivers of different size and with different land use characteristics found genomes of Helicobacter spp. in 55% of 33 samples using PCR detection. H. pylori genomes were found in 33% of these samples, whereas fecal indicator bacteria were detected in 96% of samples. The results indicated that fecal bacteria are of limited use to indicate the presence of Helicobacter spp. or H. pylori ( Voytek et al. 2005).

  • A study investigated the prevalence of H. pylori DNA in four Japanese rivers using nested PCR. Positive results were obtained from the middle and downstream reaches of all four rivers, but not in the upper reaches which were not within the human biosphere. Culturing of H. pylori from the PCR-positive samples was not successful. When examining stool samples from children from nursery schools and kindergartens along one of the rivers, the prevalence of infection was 9.8% (n=61) for children living near the middle reaches and 23.8% (n=101) for children nearby downstream. In areas distant from the river, none of 62 tested children tested positive. The researchers concluded that H. pylori DNA is frequently to be found in river water from the middle and downstream reaches, which are under the influence of human settlement. The water could be a risk factor for H. pylori transmission although further studies are needed to confirm this correlation ( Fujimura et al. 2004). 

  • Azevedo and colleagues found that water-suspended (water-stressed) H. pylori could rapidly adhere and colonize different abiotic coupon surfaces: stainless steel 304 and 316, copper, polyvinylchloride (PVC), polypropylene (PP), and glass. The adhesion was not very dependent on the type of substratum. Colonization reached a steady state level after 96 hours with cell densities in the range between 2.3 x 106 and 3.6 x 106 total cells cm-2. Compared to planktonic bacteria, the sessile bacteria overall retained their spiral shapes longer. Substrate-dependent differences in cell morphology were however observed with higher proportions of spiral morphologies on nonpolymeric substrata: Whereas approx. 95% of cells showed coccoid or U-shaped morphology on PP and PVC after 192 hours, the proportion was only app. 50% on copper. For glass and the stainless steel surfaces the proportion of coccoid cells ranged between 70 and 85%. Different substrata were also found to cause different degrees of aggregate formation and aggregate sizes. Large aggregates (>50 cells) were mostly found on copper surfaces. However, staining of cells with PI and SYTO 9 suggested that most H. pylori cells on copper coupons had compromised cell membranes after only 48 hours. Aggregate formation occurred in general substantially faster after surface colonization compared to the planktonic phase ( Azevedo et al. 2006a). In another study looking at the effect of different physical parameters on H. pylori adhesion to stainless steel 304 and PP, it was shown that high shear stress negatively influenced the adhesion to the substrata. Temperature and inoculation concentration, on the other hand, had no effect on adhesion ( Azevedo et al. 2006b). The authors suggested that H. pylori has the ability to incorporate into preconditioned biofilms mainly under conditions of low shear stress.

  • Based on PCR results from diluted samples, municipal drinking water from a city in the southeastern U.S. was estimated to have 102 H. pylori genomes ml-1 at the time of sampling ( Benson et al. 2004).

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:

  • Moreno et al. studied the effect of chlorinated water on H. pylori. When exposing cells to 0.96 mg/l of free chlorine (1.16 mg/l total chlorine), culturability decreased progressively, until it was lost at 5 minutes (in non-chlorinated water, the counts decreased from an initial 4.5 x 106 to 2.3 x 105 cells at 24 hours). Total DAPI counts and 16S rRNA content remained constant over the study time of 24 hours. The latter observation suggested that chlorine did not result in ribosome degradation. Also vacA mRNA and genomic DNA (based on PCR amplification of 16S rRNA genes) were detected at all time points. Using a DVC-FISH method optimized for H. pylori (see detection methods below), viable elongated cells could still be seen after 3 hours of chorine exposure. The method suggested that the mean number of viable (non-culturable) cells decreased from 7.24 log10 units at time point zero to 4.88 log10 units after 3 hours and zero after 24 hours. The percentage of coccoid morphologies increased from approximately 20-30% at time point zero) to 30-40% (10 sec), 60-70% (40 sec), and 80-90% (1 min). At time points of 5 min, 3 h, or 24 h the proportion of coccoid cells was more than 90%, but spiral forms could still be seen until the end of the study period of 24 h. The authors concluded that H. pylori might survive disinfection practices normally used for drinking water treatment in a viable non-culturable form for some time ( Moreno et al. 2007).
  • Hayes et al. assessed the effect of UV light on H. pylori culturability. One clinical isolate and two ATCC (43504 and 49503) strains were suspended in phosphate buffered saline and exposed to light from two 15-watt low-pressure UV lamps. All three H. pylori strains were effectively inactivated with a decrease culturability of >4 log10 units CFU per ml at fluences of less than 8 mJ cm-2 ( Hayes et al. 2006).

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:

  • Adams and co-workers studied changes of H. pylori in membrane diffusion chambers exposed to the natural fluctuations of a small natural freshwater stream. Within this study chambers were anchored in a stream at a depth of about 0.3 m. Culturability of cells in the 9°C creek lost culturability by 42 hours. Despite the loss of culturability, a large number of cells were reported to have intact cell membranes as indicated by a green staining in the BacLight assay. The authors reported that in contrast to other laboratory studies there was no statistically significant change in the proportions of rod, O-U-, or coccoid form over the study period of approximately 4 days. The coccoid morphology was not found to be dominant as was expected indicated by the loss of cultivation. The authors suggested that all the cells, independent of their morphology, had entered a viable-non culturable form under these conditions. In laboratory experiments, cells in a viable non-culturable state continued to transcribe several genes (unpublished data). Another part of the study was to place H. pylori into the creek at different seasons. An effect of temperature and  time on the culturability of the long cells was reported.It was found that culturability lasted longest at colder water temperatures (20?C (e.g. in the range of a few hours at 23°C) ( Adams et al. 2003).
  • Konishi et al. studied morphological changes and viability of H. pylori (ATCC 43504) kept in deep ground water and natural seawater at 4°C for seven days. Both natural waters were not treated with abiocides allowing for the  natural flora of the water source to proliferate. Scanning electron microscopy showed that the majority of bacteria (≥70%) in seawater, and even more so in ground water, maintained their spiral forms until day 7, whereas bacteria in culture medium (Brucella broth) rapidly transformed into coccoid forms after day 2. Almost no spiral cells could be seen on day 7. H.pylori preserved in the broth could be cultivated until day 4 followed then by a sharp drop in culturability and complete loss in culturability after day 5. Bacteria preserved in ground- or seawater, could be cultured until day 7. Survival was especially good in seawater with a loss of culturability of only approximately 1 log10 unit over the study period, compared to a loss of approximately 3 log10 units in groundwater ( Konishi et al. 2007).

  • Nayak and Rose applied a SYBR Green qPCR assay (see detection methods) to studying H. pylori survival in seeded groundwater samples which were UV-exposed prior to seeding to reduce naturally occurring microbes. The seeding dose was 107 CFU per ml, the survival was monitored over 12 days using cultivation, qPCR, and MPN-qPCR. Selected results are summarized in the following table. H. pylori signals could be detected longest using the qPCR assay. Scanning electron microscopy showed that at 15°C all cells were coccoid after 24 hours and at 4°C coccoid forms started appearing after 72 hours. After applying the qPCR assay on raw sewage samples, 86% of the samples (n=23) were found to be H.pylori positive with concentrations ranging between 2 and 28 cells per ml ( Nayak and Rose 2007).
  • Beneduce and colleagues studied the survival of the clinical H. pylori strain SR55 in well water in direct comparison with the E. coli strain FU 08 (isolated from surface water). Persistence of seeded cells (initial concentration of about 106 CFU per ml) was tested in untreated well water, filtered well water, and autoclaved well water. Whilst H. pylori had lost culturability after 48 hours at 25°C, it could be cultured until day 12 when kept at 5°C. It was however undetectable after 18 days at a temperature of 5°C. For E. coli, the temperature effect was not very apparent within the study period of 18 days. The culturable population decreased only slowly, especially in filtered water. In general, both strains survived better in filtered or autoclaved water than in untreated water, with persistence being highest in filtered water ( Beneduce et al. 2003).

  • West et al. studied the effects of physical conditions on the survival of different H. pylori strains in aquatic environments. The optimal pH range for survival (based on cultivation) was found to be between pH 5.8 and 6.9. The optimal ionic strength (NaCl) was met at physiological salinity (0.15 M). With the addition of urea (final concentrations of 100 μM and 5 mM) to neutral unbuffered 0.15 M NaCl a reduction of H.pylori culturability was reported ( West et al. 1992).

  • Shahamat et al. studied the survival of seeded H. pylori cells in sterile river water and distilled water microcosms using plate counts and acridine orange total counts (AODC) for enumeration. At 4°C, plate counts decreased from an initial concentration of 108 cells per ml to zero between 20 to 25 days. Total counts did not show any significant reduction for more than 2 years. A microcosm kept at 15°C showed culturability for up to 10 to 15 days, whereas at 22 and 37°C culturability was lost within 24 and 48 hours. AODC remained constant at the initial inoculum concentration. Viable counts decreased at the following rates (in log10/day): -0.31 at (4°C, -0.49 at 15°C, -2.57 at 22°C and -4.14 at 37°C. Persisting nonculturable cells appeared to have small metabolic activity indicated by the measurement of [3H] thymidine uptake ( Shahamat et al. 1993).

  • Queralt and Araujo studied the survival of seeded H. pylori in bottled mineral water stored at 7±1°C in the dark. Culturable cells decreased from an initial 105 CFU per ml to zero within 7 days. The culturability of E. coli serving as a control decreased only slowly at day 3 and could still be cultured after day 21.  Total cell counts of both species remained constant over the study period of 3 weeks. Gradual morphological changes of H. pylori from spiral to coccoid were observed from day 3 onward, but already at day 3 cells appeared more rounded than at day 0 although the bacillar spiral form was predominant. At days 14 and 21, most cells were reported to be cocci shaped with ultramicroscopic pictures revealing irregular surfaces. Using the LIVE/DEAD BacLight kit, all H. pylori cells until day 3 appeared to have an intact cell membrane as indicated by green staining. Until day 14, intact cells (green) and cells with damaged cell membranes (red) coexisted.The proportion of damaged red cells increased after day 14. After 21 days no green cells were visible with the majority of cells staining red and yellow. Testing for H. pylori genomic DNA by PCR was positive throughout the study period and also after 3 months ( Queralt and Araujo 2007).

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:

  • CTC-immunofluorescent microscopy: Samples were exposed to CTC, fixed with formalin, and detected using an indirect fluorescent antibody staining procedure. The primary antibody was specific for H. pylori. Actively respiring cells contained a red fluorescent formazan crystal. Cells which were capable of CTC reduction and gave a positive immunostaining signal, were counted as actively respiring H. pylori cells ( Hegarty et al. 1999).

  • PCR: Different PCR assay have been developed targeting different genes including ureA (urease subunit A; Benson et al. 2004), glmM (phosphoglucosamine mutase; Shahamat et al. 2004), and a conserved hypervariable region upstream of the 16S rRNA gene ( Shahamat et al. 2004). Primers targeting the latter were reported to be more specific for H. pylori than primers targeting the glmM gene. Detection limits were around 50-1000 genomes in the original sample. Sen et al. mentioned several regular PCR assays for detecting waterborne H. pylori with detection sensitivities from 2 to 100 cells per reaction. ( Sen et al. 2007)

  • IMS – PCR: Magnetic immunoseparation was used followed by PCR amplification of the adhesion-encoding gene hpaA. The method was tested on H. pylori from cultured samples detection ( Enroth and Engstrand 1995). The assay was reported to be influenced by the fact that different morphological states have different antigenicity and DNA content. Detection limits depended on the age of the cultures and varied between 102 cells per ml (for 3-day cultures, rod-shaped cells), 104 cells per ml (for 6-day cultures, mixtures of rods and cocci), and 106 cells per ml (for 10-day cultures dept in water for 4 weeks with coccoid forms only). The assay was successfully validated with seeded stool and water specimens. The IMS procedure was reported to readily remove inhibitory substances by washing steps. The assay was applied to H. pylori detection in municipal water, wastewater, and well water in a later study. Sensitivity was further improved by analyzing PCR products with hybridization using digoxigenin-labelled probes ( Hultén et al. 1998).

  • qPCR: A SYBR Green qPCR assay targeting the vacuolating cyctotoxin (vacA) gene was applied to quantify H. pylori in 50 ml raw sewage samples and seeded groundwater samples. The qPCR assay was reported to be a 10- and 100-fold more sensitive than MPN-PCR and a conventional PCR assay, respectively, and to have a detection limit of as few as 2 bacteria per assay. The detection assay was combined with IMS or high-speed centrifugation for sample concentration. When applied to raw sewage samples, positive results were obtained in 86% of 23 samples, whereas they were negative by PCR targeting 16S rRNA genes ( Nayak and Rose 2007). McDaniels et al. reported a TaqMan® qPCR assay targeting a conserved region of the ureA gene (encoding subunit A of the urease gene). Samples were collected using membrane filters. A mean detection sensitivity of 10 cells per liter water was reported and a 95% confidence sensitivity when detecting 40 cells ( McDaniels et al. 2005). An internal amplification control for this assay was developed by Sen et al. ( Sen et al. 2007). The control consisted of a recombinant E. coli strain carrying the 135 bp H. pylori-specific ureA sequence targeted in the assay by McDaniels et al. The fragment was modified in the probe binding region to allow differentiation between a potential H. pylori signal and the control signal using 2 different probes. The recombinant E. coli control was added to water samples to be tested for H. pylori and was optimized to be used at a concentration of 10 cells per liter. The presence of the E. coli was shown not to compete with 5 to 3,000 cells of H. pylori in the sample. The assay with the incorporated internal control was suggested as the basis for a robust screening tool for the detection of H. pylori in water samples at low concentrations (5-10 cells per liter).

  • FISH: The FISH technique was proposed to have the potential as a sensitive method for H. pylori detection in environmental samples. The technique was successfully used to detect H. pylori in two river water samples and one wastewater sample, while PCR yielded only one positive result. ( Moreno et al. 2003)

  • DVC-FISH: The direct viable count method (DVC), which allows microscopic differentiation between cells with an active metabolism and inactive cells, was modified and adapted to H. pylori analysis. Incubation with 0.5 μg ml-1 of the DNA-gyrase inhibitor novobiocin for 24 hours was found optimal to produce distinguishable cell sizes. Treatment resulted in strongly elongated cells (15-25 μm in length after novobiocin treatment, compared to 3-4 μm before incubation). The treatment was combined with FISH for specific detection of H. pylori, cells of up to 30 μm in length were observed. The method was applied to detect H. pylori in environmental water samples. Viable elongated H. pylori cells were detected in 16 out of 45 total samples. Positive results were obtained with samples from wastewater, river water, seawater, and water from an irrigation channel. Although enumeration was not performed in this study, the developed technique would allow enumeration of viable H. pylori cells. The technique was applied to assess the effect of chlorinated water on H. pylori (see section ‘susceptibility to disinfection’: Moreno et al., 2007) ( Piqueres et al. 2006).
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.

Figure 1:

                                         Helicobacter_Photo 1   

This scanning electron micrograph depicts a grouping of Gram-negative ”Flexispira rappini” bacteria, magnified 13,951x.
Its name ”F. rappini” is considered provisional, for it was never formally proposed or accepted. Subsequently determined to be closely related to Helicobacter spp., it is referred to as Helicobacter sp. flexispira in the literature.

Source: http://phil.cdc.gov/phil/home.asp
Photo ID: 5715
Content provider(s): Centers for Disease Control/ Dr. Patricia Fields, Dr. Collette Fitzgerald
Photo Credit: Janice Carr

Figure 2:

                                           Helicobacter_Photo 2  

This scanning electron micrograph depicts two ”Flexispira rappini” bacteria, magnified 13,472x.

Source: http://phil.cdc.gov/phil/home.asp
Photo ID:  5709
Content provider(s):  Centers for Disease Control/ Dr. Patricia Fields, Dr. Collette Fitzgerald
Credit:  Janice Carr

Last Updated on Tuesday, 03 April 2012 02:50
 

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