Presence of Helicobacter pylori in drinking water of Karachi , Pakistan

Background: Prevalence of Helicobacter pylori (H. pylori) infection is an increasing problem in developing countries. Several environmental factors such as overcrowding, poverty, contaminated drinking water and food are considered to contribute toward transmission of infection; however, little is known about their definitive roles. The aim of this study is to investigate the presence of H. pylori in drinking water samples of Karachi, Pakistan. Methodology: Samples of drinking water were collected from 18 different towns located in the metropolitan area of Karachi. Samples were concentrated by membrane filtration method and subjected to PCR for the detection of H. pylori. Results: Two out of 50 (4%) samples collected from two different densely populated town areas were found to be positive for H. pylori. Conclusion: The study provides evidence for the presence of H. pylori in municipal drinking water of Karachi.


Introduction
It is widely accepted that Helicobacter pylori (H.pylori) is a stomach colonizer of half of the world's population.The organism is often associated with a number of gastric diseases including gastric ulcer, peptic ulcer, gastritis and gastric adenocarcinoma.Despite widespread presence, little is known about the transmission of H. pylori.Among well-known modes, iatrogenic transmission of the organism due to invasive endoscopy is proven [1], while a previous study also presents positive association of H. pylori colonization with direct person-to-person contact [2].Moreover, a high incidence rate among hospitalassociated individuals and clustering of infection within families provide a valid basis for person-toperson transmission of infection.A number of human body secretions including vomitus, saliva, gastric juice and feces have been predicted as possible modes of transmission [3].Several reports showed the prevalence of infection among infants younger than two years of age that correlate the transmission of infection through infected mothers to infants [4,5].Studies conducted on possible zoonotic involvement show the ability of H. pylori to colonize in stray cats and sheep for long periods; however, strong evidence about their roles in human transmission are lacking [6,7].
Another important factor of infection transmission is contaminated water.Due to fecal contamination, water might be an important and neglected source of Helicobacter transmission.Evidence of high rates of infection in those nations that rely on untreated water support this hypothesis.In 1991, a retrospective study in Peru showed the prevalence of infection in those individuals who rely on municipal water.The observation was further confirmed by a thorough investigation in which municipal water was tested positive for H. pylori n those areas with high prevalence of Helicobacter associated gastritis [8,9].H. pylori have also been observed in ground water, drinking water storage reservoirs, and sewage samples, providing more suspicion toward the involvement of water in transmission of infection; however, no direct evidence was provided [10][11][12].
H. pylori is present in a coccoid and uncultivable form in water, which has different antigenicity than the cultivable form; therefore, PCR assay is considered the most appropriate detection method.It is believed that H. pylori are not often detected in water because of the high amount of polymerase inhibitors present in water.However, pre-PCR treatment of water samples might increase the sensitivity of the test thus making it useful in identifying the mechanism of transmission.Although inconsistencies are present in research data, the presence of H. pylori in water samples from different geographical areas of the world indicates its likely involvement in the transmission of the organism.
Karachi is the largest city of Pakistan and among the largest metropolitan areas in the world with an estimated population of 16 million.We have already observed the high rate of Helicobacter infections among the residents of the city (unpublished data).Therefore, it is worth investigating the factors that are likely associated with the spread of infection in local communities.
In this study, we investigate the presence of H. pylori in drinking water supplied to different regions of Karachi, Pakistan.

Sample collection and processing
As shown in figure 1, the metropolitan city of Karachi is divided into 18 administrative sectors that are known as towns.A total of 50 drinking water samples were collected from the taps of public drinking water supplies located in 18 towns.Sample collection spots were identified on the basis of frequency of use, the properly installed tap and water supply system, and average sanitary conditions around the collection area.Sampling was avoided from the places that were located in the close proximity of public toilets.The number of samples from each town varied according to the socioeconomic status of the people and population size, which helped us to identify high-risk populations.Table 1 shows the geographical distribution of areas from where samples were collected.Samples were collected as follows: first the surface of tap was cleaned with water; then water was flushed away from the taps for five minutes and one liter of the midstream sample was collected directly from the tap into clean plastic bottles.The bottles were capped and transported to the lab at room temperature, where the water was filtered through a filter assembly using a 0.45µm membrane.Two to four membranes were used for each sample depending on the quality of water.All membranes from each sample were immersed in 5 ml of sterile Milli-Q water (Millipore, Billerica, USA) for 30 minutes and centrifuged for 10 minutes at 9000 rpm.Supernatant and membranes were discarded while sediment was resuspended in 200 µl sterile Milli-Q water and stored at -80 o C.

DNA extraction
DNA extraction was performed using QIAamp Mini DNA Mini Kit (Qaigen, Valencia, USA) as previously described for H. pylori detection from water samples [13].A total of 200 μl of each sample was added to the tubes containing 20 μl of protease followed by the addition of 4μl of RNase A stock solution and 200 μl of buffer to get RNA-free genomic DNA.After 10 minutes of incubation at 56°C, the mixture was carefully applied to the QIAamp Mini Spin Column (Qiagen, Valencia, USA) for subsequent cleaning and rinsing steps.DNA elution was achieved in 100μl of sterile Milli-Q water and purified DNA was stored at -20 o C.

Detection of H. pylori by PCR
Samples were subjected to PCR for the presence of H. pylori using primer pair HP1: 5'-CTGGAGAGACTAAGCCCTCC-3'' and HP2: 5'-ATTACTGACGCTGATTGTGC-3' targeting the16srRNA gene of H. pylori.Primers were used at a final concentration of 0.4 µM in a reaction mixture containing 1.5 mM MgCl 2 , 1 U of Taq polymerase, 2.5µl PCR buffer, 200µM each dNTPs and 2 µl of genomic DNA as the template.Amplification conditions were as follows: initial denaturation at 95.0°C for 5 minutes; 35 cycles for 30 seconds each of 95.0°C; annealing temperature of 60°C; and an elongation temperature of 72°C with a final extension of 72°C for 5 minutes.PCR products were resolved by electrophoresis on 1.5% gels with positive and negative controls and anamplicon of 109 bp was considered positive [14].

Results and discussion
Out of 50 samples collected from 18 different towns of Karachi, two (4%) were successfully amplified (Figure 1).Positive samples belonged to two different densely populated town areas, specifically Landhi and Malir.Landhi Town is comprised of 13 union councils with a population of 564,505 people living in low socioeconomic conditions, whereas Malir Town is inhabited by 398,289 people who belong to an average socioeconomic level (Figure 2).

Figure 1. PCR amplification of 16S rRNA gene of H. pylori in water samples
The product were electrophoresed on 1.5% agarose gel and stained with ethidium bromide.Lane N: negative control; Lane P: positive control;Lanes 1-17: water samples;Lane L: molecular size marker (100 bp ladder) Approximately 25% of Karachi's total population of 16 million people live below the poverty line.Overcrowding, poverty and low nutrition status are usually considered important factors in the transmission of gastric diseases; therefore, the presence of H. pylori DNA in drinking water samples of densely populated area is interesting.
Although, water-borne infections and gastrointestinal outbreaks have been reported earlier [15], no data is available regarding the presence of H. pylori in drinking water supplies of Karachi.In this study, the samples were collected from public water supplies.These public spots provide treated water through a central distribution system of the city.The presence of H. pylori was observed in 4% of the treated water samples from Karachi.A recent study reports the presence of H. pylori in 40% of water samples collected from Lahore, a city located in a different province of Pakistan [16].The sharp contrast in the positivity rates of these two cities is surprising and raises serious concerns about the quality of water and the water distribution system.
Increased rates of H. pylori infection in developing nations such as Pakistan, Vietnam, Bangladesh and India, which combat poverty, poor sanitary conditions and overcrowding, indicate the role of environmental factors in the transmission of infection [17][18][19][20].In 2007, Ahmed et al. indicated that the risk of H. pylori infection can be reduced by improving personal hygiene and regular use of boiled drinking water [20].On the other hand, Janzon et al. reported the less likely involvement of drinking water in a South Asian region [21].
The ability of H. pylori to form biofilms is an area of growing concern.It has been reported earlier that chlorine disinfectant can inactivate the organism, which thus hampers the detection of H. pylori in treated water [22,23].However, the organism can form a biofilm on the surface of water supplies and can invade the hetertrophic biofilms of treated water [10].Therefore, the detection of H. pylori by PCR also indicates the possible presence of viable bacteria in drinking water supplies.However, it is necessary to adapt molecular methods to detect the viability of H. pylori in drinking water supplies.Recently, Buck et al. reported the presence of a viable but uncultivable (VBNC) form of H. pylori in raw vegetables that indicates the possible contribution of water or soil [24].In conclusion, this study provides evidence of the presence of H. pylori in drinking water samples in Karachi, Pakistan.Further investigations of treated and untreated water supplies needs to be performed.

Figure 2 .
Figure 2. Map of Karachi presenting locations of sample collection

Table 1 .
List of water samples collected from different geographical locations of Karachi S.