The Prevalence of Helicobacter pylori in Drinking Drinking Water Samples in Egypt

Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Microbiology journal is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers related to the research across the whole spectrum of the subject. These including bacteriology, virology, mycology and parasitology. In addition, the journal promotes research on the impact of living organisms on their environment with emphasis on subjects such a resource, depletion, pollution, biodiversity, ecosystem.....etc www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


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Detection of H. pylori in drinking water strengthens the argument for a possible route of transmission.It has been suggested that H. pylori can be acquired through different transmission routes, including water (Yingzhi et al., 2002).In fact, several epidemiological studies have concluded that the drinking water source, or drinking waterrelated conditions, was a risk factor for H. pylori acquisition (Guimarães et al., 2014).
An important stumbling block for H. pylori detection is the difficulty of isolating it from environmental samples using traditional culture methods.This is probably a consequence of the transformation of H. pylori in water samples from a culturable spiral-shaped form to a non-culturable coccoid one.To overcome this problem, a variety of culture-independent methods have been developed for the analysis of clinical and environmental samples, such as enzyme immunoassays, the urease activity test, and different DNA-based methods, including fluorescence in situ hybridization (FISH) and PCR (Yanez et al., 2009).
Although PCR permits detection of single cells in a sample, environmental samples must be concentrated to detect concentrations of microorganisms of <1cell/ml.Target genes must not be lost or destroyed during the concentration of cells from the environment, and substances used in the concentration procedure must not interfere with DNA amplification or detection.
In the present study, we used the filtration-PCR assay and Fluoropore filters (FHLP) described by Bej et al. (1991).The primary objective of this study was to determine, for a population in which H. pylori is common, the potential environmental sources of infection by both filtration PCR and filtration culture PCR for water samples.Also we aimed to study the cagA and vacA genotypes of these strains.

MATERIALS AND METHODS
Collection of water samples.Samples of 100 ml of tap water were collected from two different regions of urban and rural areas in Alexandria, Egypt.Samples were collected in glass bottles containing 0.1% (w/v) of sodium thiosulphate to dechlorinate the water.Filtration of water samples.Filtration was done through Millipore Fluoropore FHLP membranes (Millipore Corp) and a Swinnex filter holder.Each filter was soaked first in ethanol until it became transparent (≤1 minute) and then transferred with the laminated surface to the Swinnex filter holder to collect the bacterial cells under vacuum.In order to facilitate the filtration process, prefiltration of dechlorinated water samples was done by using filter with 0.8 µm pore size and 25 mm diameter.For PCR amplification, the water samples were filtered through membrane with 0.5 µm pore size and 13 mm diameter, whereas with 0.5 µm and 25 mm diameter for bacterial culture.After filtration, the filters were folded and transferred with a sterile forceps to 0.5 ml GeneAmp reaction tubes, containing 73 µl sterile distilled water, with the cell-coated surface facing the inside of the tubes, and were stored at -20 o C until PCR processing.
Cultivation of H pylori.After filtration, the filters were transferred with a sterile forceps to Dents media (Campygen CN0025, Oxoid Ltd) with the cell-coated surface facing the media and incubated at 37 o C for 72 hours under microaerophilic conditions.DNA extraction from samples for PCR of ure C gene.Boiling method was applied to extract the DNA from culture as described by Englen and Kelley (2000).It was used for bacterial colonies which isolated through Dent's medium.A loopful of bacterial growth was suspended in 300µl of sterile distilled water in a microcentrifuge tube.The tube was boiled for 10 minutes and immediately cooled in ice for 30 minutes.After that, to precipitate PCR inhibiting particles, the tube was centrifuged at 5,000 rpm for 10 min and the supernatant was used as DNA templates for PCR amplification.Boiling method was also used to extract The Prevalence of Helicobacter pylori in Water Samples in Egypt 95 crude DNA from water samples as described by Bej et al. (1991).After filtration (without removing the filters), the bacterial cells in the tubes were lysed and the DNAs were released by heating at 95º C for 5 minutes.The tubes were then cooled in ice for 30 minutes before the addition of PCR mixture.PCR amplification for detection of H. pylori DNA (Labigne et al., 1991).The PCR was performed using primers for urease gene Ure C (294 bp) 5' -AAG CTT TTA GGG GTG TTA GGG GTT T-3' and 5' -AAG CTT ACT TTC TAA CAC TAA CGC -3' indicative of H. pylori infection.Amplification was performed in a final volume of 150µl (for filter PCR) and 25µl (for bacterial culture DNA) of PCR mixture containing 0.8 µm of each primer, 10 mM of each deoxy nucleotide triphosphate (dATP, dGTP, dTTP and dCTP), 10 mM tris HCl, 50 mM KCl, 0.1% triton X-100, 1.5 mM MgCl2, and 1 unit of DNA polymerase (Bioline).DNA amplification was applied as follow: denaturation at 94°C for 5 minutes, annealing at 55°C for 2 minutes and extension at 72°C for 2 minutes (for 35 PCR cycles), followed by a final 5 minutes extension at 72 º C. Amplifications were performed with a thermal cycler (Genius Techne, England).PCR products were loaded on agarose (2%) in TBE (Tris/Borate/EDTA) buffer containing 0.5µg/ml ethidium bromide (Figure 1).After electrophoresis, the gels were photographed under UV light using black and white Polaroid film.Booster PCR assay was used for PCR negative samples with final volume 25 µl PCR mixture and 3 µl PCR products.Multiplex PCR for detection of H. pylori genotypes (Atherton et al., 1999;Mukhopadhyay et al., 2002;Chattopadhyay et al., 2004).PCR was performed to detect CagA and VacA (s1/s2, m1/m2) genes using primers shown in Table (1).Amplification was performed in a final volume of 50µl of PCR mixture containing 0.5µM of each primer, 10mM of each deoxynucleotide triphosphate (dATP, dGTP, dTTP and dCTP).Ten mM tris HCl, 50 mM KCl. 0.1% triton X-100.1.5 mM MgCl 2 , 1 unit of Taq DNA polymerase (Maxima Hot Start Green PCR Master Mix, Thermo Scientific) and 10 µl of template DNA.DNA amplification was carried out under the following general conditions: 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min.Statistical analysis was conducted by two ways ANOVA analysis.

RESULTS
This study was carried out on fifty water samples (tap water).Fifty two percent (52%) of these samples were from rural areas and 48% were from urban regions in Alexandria.H. pylori ureC gene was screened by PCR after Filtration and after culture in all water samples, whereas only positive H. pylori samples were subjected to cagA and vacA genotypes (Table 2).In this study, ure C gene of H. pylori was detected in 4 (8%) of 50 samples of water.Furthermore, the source of water has no effect on the efficiency of PCR test since the p-value is larger than 0.05.
The efficiency of PCR test (after filtration against after culture) was evaluated using two ways ANOVA analysis.There was no statistical significance different between both ways of applying PCR test in detecting H. pylori, since the p-value of PCR test was larger than 0.05.

DISCUSSION
The detection of H. pylori in water samples could provide support for a waterborne route of H. pylori transmission and infection.In the present study, out of the 50 tested water samples in Alexandria city, H. pylori was detected in 4 (8%) of drinking water samples.Other nearly similar detection rates were reported by Khan et al. (2012), in Pakistan (Karachi) and by Bahrami et al. (2013) in Iran, which were 4%, 10%, respectively.
Higher results were reported by Hulten et al. (1998) in Sweden, by Watson et al. (2004) in England, by Samra et al. (2011) in Pakistan (Lahore) and by Hulten et al. (1996) in Peru, which were 12%, 15%, 40% and 50%, respectively.Increased rates of H. pylori infection in developing nations, which combat poverty, poor sanitary conditions and overcrowding indicate the role of environmental factors in the transmission of infection (Khan et al., 2012).
This variation in detection rates of H pylori in drinking water samples presented by different investigators may be explained by several factors including the incidence rate of the bacteria in different environments, hygienic status of the city, low socioeconomic factors such as a lack of public health education, poverty, overcrowding, poor sanitation and unsafe water supplies (Khan et al., 2012;Plonka et al., 2014).Other possible factor that may account for the variation in prevalence rates is the detecting methods used for the etiological agent.
Egypt had the highest prevalence of H. pylori in the healthy asymptomatic population both in adults and the pediatric age group.Low socioeconomic status, low body weight and height, living in rural areas and lower educational status were risk factors for the acquisition of H. pylori in Egyptian studies (Eshraghian, 2014).
On the other hand failure to identify H. pylori in drinking water has been reported by Horiuchi et al. (2001) in Japan and by Yanez et al. (2009) in Spain.The lack of H. pylori detection in drinking water may reflect the fact that H. pylori is sensitive to chlorination and should therefore be controlled by disinfection practices normally employed in the treatment of drinking water (Eshraghian, 2014).

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In this work, to compare the efficiency of PCR test directly after filtration against after culture, two ways ANOVA analysis was conducted on a sample of 50 observations of tap water.The result showed no significance different between both ways of applying PCR test on discovering H pylori gene, since the p-value of PCR test was larger than 0.05.In addition, there was no effect of the source of water sample, wither it is from rural or urban region, on the efficiency of PCR test since the p-value of the test of significance is larger than 0.05.
It is worth notice that in the present study, the positive H. pylori UreC genes of water samples yielded by PCR assay after direct filtration were 2 of 50 (4%), whereas these two samples gave negative results by PCR assay after culture.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.Guimarães et al. (2014) showed that after being exposed to water for 24 h at 25ºC, H. pylori was no longer culturable.Studies have reported that when exposed to water, H. pylori enter a viable, but non-culturable state as a response to unfavourable environmental conditions, which means that even though H. pylori cannot be recovered by plating techniques, bacterial cells might remain viable (Guimarães et al., 2014).
In conclusion, this study provides evidence of the presence of H. pylori in drinking water samples in Alexandria, Egypt.The development of PCR assay after filtration and after culture to detect H pylori from water samples may be a significant tool for evaluating the ecological and public health aspects of the transmission of H. pylori through the environment.

Table 2 :
Distribution of H. pylori Ure C gene detection among water samples by PCR method in relation to regions.