Isolation and identification of Helicobacter pylori by PCR from drinking water supplies in Erbil city

Out of the 200 samples of drinking water, which were collected from different source in Erbil city, from the period November 2014 until April 2015 . In this study, 36 isolates of Helicobacter pylori were isolated from samples as well as from biofilms, this study directed for the isolation of Helicobacter pylori by culturing in different media and identified by biochemical tests and PCR technique, in order to evaluate the potential role of drinking water in transmission of H. pylori . Water samples cultured on Brain heart infusion agar with supplement, Colombia agar base with supplement and Brucella agar; the results showed that The total number and percentage of the isolates of H. pylori from all tested water samples were 36 (18%), and these distributed as follow: 12 (24%), 11(22%), 8(16%) and 5(10%) from biofilms, reservoir, groundwater and tap water respectively. All H. pylori isolates were identified by using cultural , morphological , biochemical characteristics, from all(36) of isolates:30(83%) gave positive reaction for urease indicates that the isolates had the ability to produce the urease as well as 24(67%) of the total isolates; oxidase and catalase gave positive reaction while 17(61%) of the total isolates are motile. 35(97%) of the total samples confirmed by using primers for identification of H. pylori based on 16SrRNA this was the first report on using 16SrRNA amplification and confirmation of H. pylori isolates from water samples in Iraq. The ureA genotype was expected to be present in nearly all 34(94%) Helicobacter positive isolates. The PCR assay employed in this work specifically targets a region of the ureC ( glmM ) gene which has been shown to be unique and essential for the growth of H. pylori and the results appeared that 28(78%) of the total isolates have this gene This study was succeeding to detect the ure A and C gene in most of the isolates which was already confirmed by 16SrRNA .


Introduction
Helicobacter pylori colonizes the gastric mucosa of approximately half the world population, (Vyse et al. 2002). Most infected individuals are asymptomatic but in ca 10-20% of carriers chronic infection is associated with the development of gastric diseases ranging from ulcer to adenocarcinoma (Suerbaum and Michetti 2002). Infection is usually acquired in childhood and thought to be primarily spread from person to person via the faecal-oral route (Brown 2000;Go 2002). Nevertheless, the precise route and mode of transmission remain unproven and no reservoir of the organism, outside the human stomach, has been so far identified although H. pylori-like organisms can cause natural infections in some nonhuman primates (Suerbaum and Michetti 2002). The genus Helicobacter has expanded considerably over the past decade with the identification of at least 24 other species which are found in the stomach or lower bowel of various nonhuman primates, dogs, cats, rodents and birds as well as vegetables (yahaghi et al.,2014), prompted researchers to look at environmental sources as 151 vectors to humans. Previous studies suggested that H. pylori is present in groundwater, surface water, and other drinking water (Mazari et al., 2001).Implying that there is a waterborne route of transmission to humans. When hygienic conditions are poor, household contamination of treated water cannot be ruled out, H. pylori is easily controlled by chlorination which the principal disinfection agent of water (Johnson et al., 1997). Poor hygienic practices, absence of a household bath, non-hygienic drinking water and absence of a sewage disposal facility may lead to recontamination of drinking water that in this way may serve as a vehicle of transmission (Watson et al., 2004). Even in treated water, the survival of H. pylori is possible, at least for short periods of time. Recently, a study reports that it was possible to culture a reference strain after five minutes of inoculation in chlorinated water (0.96 mg/l), and viable cell coccoid forms was higher than spiral forms after 40 sec. of chlorine exposure amplification of the specific H. pylori 16S rRNA gene was possible (Moreno et al., 2007). H. pylori is easily controlled by chlorination which the principal disinfection agent of water (Lee,1994;Johnson et al.,1997), but recontamination of treated water is a widespread problem.
H. pylori DNA has been identified in several water sources using various gene targets. Drinking, river, sea, ground and wastewater have provided positive results by PCR analysis ( Cellini et al., 2004;Queralt et al., 2005) . However, H. pylori appears to be more resistant to chlorine and ozonation than E. coli, and same resistant to monochlorine. Traditional indicator organisms may fail to protect the consumer from exposure to H. pylori (Baker et al., 2002;Moreno et al., 2007). Regarding spring water, the results for presence of H. pylori DNA were also negative (Queralt et al., 2005). Likewise, upstream river water samples were negative (Fujimura et al., 2008), these results suggest that faecal is responsible for the contamination of water suppliers.
For the reasons above the study aimed to evaluated drinking water samples, biofilms, reservoirs and water storage tanks in Erbil city fed by different water distribution systems were tested for the presence of Helicobacter pylori by culture and molecular detection methods in order to provide an understanding of their potential role in transmission of H. pylori, the accuracy of results varies according to the sensitivities and specificities of the detection methods employed. Objectives of this research were (i) to isolate H. pylori from drinking water by utilizing culturing techniques, (ii) to identify H. pylori by classical microbiological tests and PCR (16S rRNA), (iii) to genotype detection of gene (ureA) and UreC gene in H. pylori isolates.

Material and Methods
Samples were collected from different geographical locations in Erbil city, properties served by three different water distribution systems: reservoir, tap water, groundwater and biofilms. Fifty sample representative of the incoming supply water was collected from each area. Samples were collected in sterile bottles, also fifty biofilm samples from the header tank, water closet cistern (where possible, an area of 50 cm 2 was swabbed using a sterile cotton swaps), and the showerhead, were collected using a sterile swab. Deposit samples from the domestic water supply were collected by securing a 25-μm pore size sterile nylon tap net and flushing for 2 min following tap surface decontamination with 10% hypochlorite solution. Samples were transported to the laboratory within 24 h at ambient temperature.
The water samples were filtered through a 0·25-μm pore size nylon membrane filters (Nyflo; Pall Life Sciences, Poole, UK) and precipitates were by gently removed from the membrane with a disposable cell scraper and re suspended in 25 ml sterile phosphate-buffered saline (PBS). Swabs taken from biofilm were suspended in 10 ml PBS was used in this study.

Culturing of samples
Subsamples (25 μl) of each final concentrate of all 200 water and biofilm samples were cultured on three culture media: the first, Brain Heart Infusion Agar plus 7% HRBC plus antibiotic supplement contains (Vancomycin 5 mg, Trimethoprim lactate 25 mg, Polymyxin B 0.05 mg.), the second media was, Columbia Agar plus 7% HRBC plus growth supplement contains: a. Sodium pyruvate 0.125 g, b. Sodium metabisulphate 0.125 g and c. Ferrous sulphate 0.125 g. and Brucella agar. Small smooth colonies resembling H. pylori were subcultured, and were subsequently identified by Gram-stain and tests for rapid urease, cytochrome b oxidase and catalase activity (Owen 1998). The plates were incubated under microaerophilic conditions at 37 0 C, using an anaerobic jar and anaerobic Gas Pak kit without catalyst to generate a microaerophilic environment. Plates were inspected first on day 3 and then after one-day interval for a total of ten days. GasPak was changed whenever the jar was opened. H. pylori were identified by the following criteria: 1. Colonial morphology, Motility test, Catalse, oxidase and Urease Tests.

Genomic DNA Extraction and PCR amplifications
The subjected (36) isolates to the Genomic DNA Extraction was according to the kit manufacturing company information procedures was done as follows; 0.1ml. of activated bacteria in the Luria Brittani broth transferred to sterilized test tube and centrifuged at 8000 rpm for (1) min. added 180 μl of Lysis Buffer (T1) and 25ml Proteinase K solution Vortex to mix, Incubated at 56c 0 until complete lysis was obtained (at least 1-3h). Added 200μl Buffer B3 ,vortex vigorously and incubate at 70c for 10 min .Vortex was done briefly , Dissolved Wash Buffer ( B5) 7 μl in 210 μl ethanol (96-100%)Added 500 μl Buffer BW, centrifuge for 1 min at 11,000xg . Discard flow-through and place the column back into the Collection Tube.*2 nd wash added 600 μl Buffer B5 to the column and centrifuge for 1 min at 11,000xg, Discard flow-through and place the column back into the Collection Tube. Centrifuged the column for 1 min 11, 000 x g. residual ethanol is removed during this step. Placed the NucleoSpin Tissue Column into a 1.5 μl microcentrifuge tube and added 100ml prewarmed Buffer BE (70c 0 ). Incubate at room temperature for 1 min, Centrifuged 1 min at 11,000xg (MACHERTY NaGeL). Stored the DNA in the freezing at -20c (Boom et al. 1990). The primers sequencing which used for the identification were appeared in table 1 Detection of 16srRNA gene and Urea gene and Hp/ Urec gene A sterile amplification tube was used; the following orders were mixed at final volume 20 μl for each primer (Bioneer kit ),As shown the PCR reaction mixture(master mix 20 μl, forward primer 1 μl, reverse primer1 μl, nuclease free water16 μl and template DAN 2 μl. The master mix composed from the following dNTP, 250μM each: (dATP,dCTP,dGTP,dTTP; Taq DNA Polymerase, 1U/ml; Tris-HCl(pH 9.0), 10μM; KCl,30mM; Mgcl 2 , 1.5mM. DNA amplification was carried out for 35 cycles in 20ml total reaction mixture as follows: Temperature cycling by using 16S rRna, denaturation at 94c 0 for1 min., annealing at 55c 0 for 30 s extension at 72c 0 for 2 min., final extension at 72c 0 for 7min , hold at 4c 0 . Temperature cycling of UreaA gene :Denaturation at 94c 0 for 1 min. annealing at 45c 0 for 1min, extension at 72c 0 for 2 min , final extension at 72c 0 for 7min , hold at 4c 0 Temperature cycling of HP ureC gene :Denaturation at 94c 0 for 1 min. Annealing at 55c 0 for 1min .extension at 72c 0 for 2 min final extension at 72c 0 for 7min , hold at 4c 0 . Aliquots of each PCR product were separated by electrophoresis in a 1% (w/v) agarose gel in TBE buffer (90 mm Tris-HCl, 90 mm boric acid, 2·0 mm EDTA) and stained in ethidium bromide at 0·5 μg ml −1 . Assays on all samples were repeated in duplicate. Samples were interpreted as being positive for the presence of Helicobacter DNA if one or more of the assays produced a fragment comparable in size to that of the positive control DNA (Al-Sulami et al., 2012).

Results and Discussion
In this study 200 samples from different source of drinking water and biofilms (smooth layer) in Erbil governorate were collected ,the samples examined for the presences of H. pylori by using special selective media for this bacteria as appeared in table 1 , the collected samples included 50 samples from each one. The total number and percentage of a doubted isolates of H. pylori from all tested water samples were 36 (18%), and these distributed as follow: 12 (24%), 11(22%), 8(16%) and 5(10%) from biofilms (smooth Layer), reservoir, 154 groundwater and tap water respectively. As shown different source of water contaminated with a suspected isolates of H. pylori from total samples as appeared positive and contaminated with this bacteria , this result agree with (Al-Sulami et al.,2010;Al-Sulami et al., 2012) in basrah Iraq studies indicated that H. pylori found in tap water and samples from tankers supplying, Reverse osmosis (RO),the water pipes suffer from breaks and corrosion at many sites, which cause drinking water contamination by sewage infiltration and rain leakage into the system. Sartory and Holmes, (1997) needed to establish which factors affect the ability of H. pylori to survive in distribution systems and be isolated from drinking water, such as the bacterial strains, density of bacteria in the distribution systems, type of water pipe materials, efficiency of disinfection process and the techniques and materials used for culture, also Degnan et al., (2003) in America studies suggested that H. pylori was present in ground water, surface water, and other drinking water as well as Mazari et al., (2001) ;Yingzhi et al.,( 2002), they found positive result for isolation H .pylori from drinking water.

‫٭‬positive samples with suspected H. pylori
In addition to the previous mentioned , samples as a contaminated with suspected H. pylori the characters of isolated bacteria were determined to all positive samples after being stained with gram stain the cells shows Gram negative spiral to coccobacilli, rod shaped , and the colonies appear small to middle in size, rounded, and creamy in color, raised and smooth , the morphology characters of isolated H .pylori from different sources appeared in the results were comparative to that found by Giao et al.,( 2010) found that morphology of the majority of cells was spiral shaped and did change during the duration of the experiment under any of the conditions tested ,as well as Eshraghian (2014), had been demonstrated in vitro that H. pylori cells can transform from a cultivatable spiral-shaped form to a non-cultivatable coccoid form, in which the recovery of the bacterium is very difficult by routine culture methods. on the other side, Ali khan et al ., (2007) and Yahaghi et al.,(2014) Iranians studies showed that H. pylori growth in spiral and coccoid forms in the human gut, also Azevedo et al.,( 2007) and Ranjbar et al., (2016) isolated H. pylori with different shapes changes from the normal spiral-shaped bacillary form into the coccoid form when it is exposed to water or to other adverse conditions.

Identification of isolated bacteria by Biochemical characterization
The isolates examined to Urease production, which was the key for more confirmation a positive rate as shown in table3, from all(36) of the isolates30(83%) gave positive reaction for urease the color changing to dark pink indicates that the isolates had the ability to produce the urease; were 24(67%) of the total isolates; oxidase and catalase 155 gave positive reaction while 17(61%) of the total isolates are motile. the results were comparative to that found by Virginia et al,. (2014) as shown Urease is one of the key enzymes in H. pylori pathogenesis. also urease is strongly immunogenic and chemotaxic for phagocytes (Al-Sulami et al.,2010), while Amieva and omar,(2008) indicated that catalase, oxidase and urease positive, three aspect of the bacterium were essential to colonization, including urease, motility and adherence, infection depends up on a combination of these bacterial. This study succeeded in isolating Helicobacter pylori from water samples. Few studies were done on the presences H. pylori in water and drinking water.  (Liu et al.,2008),the identification of H. pylori which isolated from water samples by PCR has been reported35(97%) of the total samples(36) as appeared in table 4. Studies in several countries (Mazari-Hiriart et al.,2001;Hegarty et al.,1999) demonstrated the presence of H. pylori in surface water.
Identification of the isolates confirmed by using primers specifically designed for the identification of H. pylori based on 16SrRNA gene, and ureA gene, based primers gave bands on agarose gel corresponding to a 500 base pair and 411bp respectively, when compared to the molecular ladder, this was the first report on using 16SrRNA amplification and confirmation of H. pylori isolates from water samples in Iraq. The ureaA genotype was expected to be present in nearly all 34(94%) Helicobacter pylori positive isolates. However, our study was succeeding to detect the ureA gene in most of the isolates which was already confirmed by16SrRNA. This result agrees with Percival and Thomas, (2009) who used ureA gene. On the other hand, the detection of ureC gene also was done for H. pylori isolates and the results appeared that 28(78%) have this gene as well as appeared as band 294bp on agarsoe gel electrophoresis as shown in figure 3 .
Studies results of Ahmad et. al., (2013) they extracted the genomic DNA and amplification for ureC (g/mM) HP. Extracted genomic DNA was amplified for the ureC (glmM) gene and detected with the specific primers, the gene product was 294 bp. Vale and Vito(2010);  they found in their studies, ureC gene of H. pylori was detected in tap water, dental units' water, refrigerated water with filtration ,and public cooler water samples. H. pylori DNA has been identified in several water sources using diverse gene targets. The PCR assay employed in this work specifically targets a region of the ureC (glmM) gene which 156 has been shown to be unique and essential for the growth of H. pylori (Safaei et al., 2011;.  Electrophoresis was performed on 1% agarose gel .Lane M is (100 bp) ladder. S1 S2 S3 S4 S5 S6 S7 are negative control and S8 shows positive control. Based on this figure the 8 th ladder was positive and completed other samples M S 1 S 2 S 3 S 4 S 5 S 6 S 7 S 8 S 9 S 10 S 11 S 12 Figure 2: Gel electrophoresis for amplification of ureA Gene for H. pylori (product size (411 bp). Electrophoresis was performed on 1% agarose gel .Lane M is (100 bp) ladder , S1,S4,S7 is negative and S2, S3, S5,S6,S8,S9,S10,S11,S12 is positive.