Helicobacter pylori virulence genotypes in Bogotá River and wastewater treatment plants in Colombia

Helicobacter pylori is a bacterium that infects 70%–80% of the population in Colombia, causing chronic gastritis in all those infected and gastric cancer in 1%–2% of those infected. In Colombia, some studies have identified the presence of vacA and cagA genes in environmental samples such as treated, surface, and wastewater, but they have not been evaluated in the Bogotá River. For this, the aim of this study was to identify the virulence genotypes of H. pylori present in samples from the Bogotá River and domestic wastewater treatment plants (WWTPs).

10] In Colombia, according to Resolution 2115 of 2007, 11 water disposed for human consumption should be free of fecal microorganisms including total coliforms, Escherichia coli (E.coli), heterotrophs, Cryptosporidium sp.oocysts, and Giardia sp. 11On the other hand, considering the regulations for the use of treated wastewater (resolution 1207/ 2014 and 1256/ 2021, Environmental Ministry), 12,13 wastewater discharges (resolution 0631/2015, Environmental Ministry) 14 and discharges of domestic-treated wastewater (resolution 699/ 2021, Environmental Ministry), 15 as a quality criteria for these categories the evaluation of thermotolerant coliforms, and other microorganisms such as fecal enterococci, human parasitic helminths, human parasitic protozoa and Salmonella sp., is only required.Nevertheless, recent studies [16][17][18] showed that the determination of E. coli and other fecal contamination indicators are not enough to assure free-pathogens water.
Thus, the U.S. Environmental Protection Agency (USEPA) since 2015 included a new list of pathogenic microorganisms which can avoid conventional treatments of wastewater and be transmitted through water to humans. 19,20One of those pathogenic microorganisms is Helicobacter pylori (H.pylori), which can colonize the human gastric mucous producing one of the most frequent infections worldwide with a prevalence rate of 50% around the world 19,21,22 and of 77%-80% in Colombia. 23H. pylori infection has been associated with several gastric diseases including the development of chronic gastritis, peptic ulcers, atrophic gastritis, MALT gastric lymphoma, and gastric adenocarcinoma, which is the main reason why the International Agency for Research on Cancer-WHO (IARC) included H. pylori as a type I carcinogen in 1994. 24en if the importance of H. pylori is well described, its exact transmission mechanism remains unknown.As H. pylori infects human beings and colonizes the gastric mucosa, after shedding of gastric mucosa cells H. pylori is transferred to the intestine and is delivered through the feces releasing it into the environment reaching different environmental repositories including agricultural irrigation water, raw water disposed for sanitation processes, and drinking water, which makes water sources a plausible vehicle of transmission to human beings.9][30][31][32] Previous studies have reported that H. pylori loses its ability to be cultured in contact with chlorine for short periods of time, although it remains potentially pathogenic in the VBNC state.Other studies have confirmed that H. pylori survives for about 4 days in water at 25°C and for 7 days at 4°C.This microorganism also resists and survives the usual wastewater disinfection procedures. 29,30[32] Several H. pylori genes have been associated with virulence and increased risk of severe gastric disease.The H. pylori virulence genes most frequently determined and of greatest genotypic importance are vacA and cagA, 33,34 coding for vacuolizing cytotoxin and a protein associated with cytotoxin A, respectively.The vacA gene functions as an excellent marker since it is present in all the isolates of this bacterium, [35][36][37] so amplifying this gene functions as an indicator of the presence of this microorganism. 38Additionally, this gene presents polymorphic regions called signal region (s), intermediate region (i), and middle region (m), involved in the development of the disease. 39,40The s region is involved in the efficiency of channel formation and the middle region (m) affects tropism toward host cells. 41The intermediate region (i) was recently described, and it has been observed that the vacuolizing activity of the vacA i1 variant is stronger than that of the vacA i subtype 2 41,42 which determine its activity, with the most active being the s1m1i1 combinations and the least active being the s2m2i2 combinations.The most active combinations are associated with the appearance of different symptomatology. 33,40,43,44e cagA gene is also polymorphic and has been detected in only 60% of the isolates, and its presence has been associated with the development of gastric cancer.The association of this gene and gastric cancer depends on the presence of sequences capable of phosphorylating intracellular proteins related to morphological changes and survival of gastric cells.These sequences are called EPIYA and the greater the number of these sequences found in the gene, the greater the number of intracellular effects that will be evident in the gastric mucosal cells. 45At the environmental level, it is interesting to detect the presence of the gene and allelic combinations of vacA to characterize in depth the presence of the bacterium in water sources.The United States Environmental Protection Agency has included H. pylori in the list of candidate contaminants of water (CCL), with the aim of incorporating its control into the legislation governing environmental pathogen control regulations; 46 however, limited data are available on the presence of H. pylori and the type of drinking water treatment, so a formal quantitative microbial risk assessment (QMRA) for H. pylori in water 47 has not yet been carried out, probably because of the low concentration at which it is detected.9][50][51][52][53] In addition, the detection of reservoirs and sources of contamination is important to understand and control the persistence and waterborne transmission of H. pylori, particularly in those geographic areas with high infection rates and water supplies coming directly from rivers or other untreated water sources. 21 Colombia, studies have identified the presence of vacA and cagA genes in environmental samples such as treated, surface, and wastewater 28,38,[54][55][56][57][58] However, these studies have only evaluated the allelic variability of vacA in s, m. 16,58 Therefore, determining and evaluating the presence of H. pylori in wastewater and surface water samples, as is the case in the Bogotá River, is of greater relevance, since this river receives, along its 313 km length, various untreated domestic discharges and effluents from WWTPs.
This water is subsequently used in different human activities as agricultural, livestock and industrial, and in some areas, it is used as drinking water and for potabilization. 4,59Currently, Colombian regulations allow treated wastewater to be reused in some activities such as crop irrigation; 12,13 nevertheless, there is a need to continue evaluating the risk of reusing this type of water in crops because they are currently associated with a potential danger of infection with H. pylori. 60,61though Colombian regulations 11,14 do not include the determination of H. pylori, its control is necessary to advance in its use as a new indicator to evaluate and monitor water quality, since the absence of bacterial DNA in water may be due to the levels of quality assurance and water treatment. 62The objective of this study was to identify the virulence genotypes of H. pylori present in samples from the Bogotá River and wastewater treatment plants (WWTPs), to identify the distribution and behavior of this pathogen at the environmental level, which could become a basis for research, environmental.

| Wastewater and Bogotá River sampling
A total out of 75 samples were collected between 2016 and 2017.
Fifty-one samples from the Bogotá River and 24 samples of effluents from three wastewater treatment plants (WWTPs), mainly of domestic origin, whose discharges reach the Bogotá River directly or subsequently.[65] In the Bogotá River, three sampling stations were selected for each of the three basins (high, medium, and low, as it is geographically divided), for a total of nine points (Figure 1).Of the 51 samples evaluated, 15 were in the upper basin, which is characterized by few discharges or contamination, but around this basin, water from industrial and agricultural activities is discharged.Eighteen samples belonged to the middle basin, which receives pollution inputs from the Bogotá metropolitan area followed by small towns, agricultural, livestock, and industrial activities.Finally, 18 samples from places with high contamination correspond should be to the lower part of the river, receiving wastewater from 12 municipalities and the previous basins. 10TPs evaluated correspond to the treatment of wastewater from three municipalities bordering the Bogotá River, and which discharge their effluents into it.Briefly, the WWTP Guasca has pretreatment (coarse and fine grids) and addition of aluminum chloride to end up in a sedimentation tank and then discharged into the river.
WWTP Cajicá the treatment consists of pre-treatment with coarse and fine grids, passage through a grit remover and grease trap and finally passage to an anaerobic, facultative, and aerobic lagoon.In addition, the WWTP Salitre has primary treatment that removes suspended solids and organic matter through sedimentation, coagulation, flocculation, and filtration (Figure 1).

| Detection and genotyping of H. pylori
Each water Bogotá River sample was centrifuged at 3000 × g for 20 min and the pellet was resuspended in 2 mL of phosphatebuffered saline (PBS 1×: 130 mmol/L sodium chloride, 10 mmol/L sodium phosphate, pH 7.2), as described Vesga et al. 58 and wastewater sample DNA was directly extracted from 2 mL as described by Sánchez-Alfonso et al. 10  For determination of the presence of the cagA gene and of the vacA s1/s2 genotypes; m1/m2 and i1/i2 the primer sequences described in Table 1 were used.
For PCR analysis, the final reaction volume of 10 μL contained

| Prevalence
The prevalence of allelic variants of the vacA gene and the predominant genotype in the Bogotá River and the WWTPs were determined by dividing the total number of positive samples by the total number of samples analyzed (p = No.events/total events).

| RE SULTS
A total out of 75 samples were processed, of which 51 samples correspond to the Bogotá River and 24 samples from domestic wastewater treatment plants (WWTPs) for the detection of H. pylori, through the amplification of the vacA gene.A total of 44% positive samples (33/75) were obtained, of which 41.2% (21/51) corresponded to the Bogotá River and 50% (12/24) to samples from the

| DISCUSS ION
Currently, the USEPA has included H. pylori on its CCL, which includes chemical contaminants and microorganisms known to be present in drinking water systems and are suspected of posing a risk to public health 19,20 ; however, the available data on the presence of H. pylori and the type of treatment carried out on the water are limited.Additionally, a quantitative microbial risk assessment (QMRA) for H. pylori in water has not yet been performed, 47 and, therefore, this pathogen remains a candidate while a method for its detection and identification in environmental samples is standardized and implemented.
Multiple studies have confirmed the presence of H. pylori in surface and drinking waters worldwide. 16,17,58,68,69The present investigation was carried out to determine the presence of H. pylori and the variety of genotypes of the H. pylori vacA genes and presence cagA TA B L E 1 Primers sequence for genes cagA y vacA (s1/s2; m1/m2; i1/i2).gene, considering all the allelic variants reported for the latter (s, m, i) 70  The middle basin is supplied by wastewater with and without treatment in different proportions.While the discharges from these treatment plants begin at the first sampling point and a low positivity for H. pylori is obtained, for the last point it is found in a higher proportion.This directly associated with the increase in population and therefore of wastewater domestic that in some cases are taken directly to the Bogotá River, but in others they exceed the treatment capacity. 71,72nally, the lower basin is considered the point of highest contamination in the Bogotá River 71 ; this could also be evident in the results of this study.At the seven point (toll Nuevo Salto), the river has received domestic wastewater with and without treatment, industrial wastewater, and discharges of other wastewater, coming from approximately 10 million inhabitants and industries, 10,71 where the greatest contamination results from the wastewater from the country's capital.

Gen
The sampling point eight (La Guaca) a lower presence of H. pylori was obtained (1/6) (Figure 2), one of the reasons to explain this is the presence of a wastewater treatment system of the generating plants of the energy, this shows the great impact that wastewater treatment has on reducing pollutants discharged into the Bogotá River and therefore risks to both environmental and public health.Even so, according to the above, it was possible to find H. pylori in one of the samples, which shows that this process is not very efficient for its elimination.This also agrees with the study carried out by Vesga et al., 58 where it is highlighted that the chlorination process is scarce for the elimination of this pathogen.
During the 1-year sampling period, H. pylori DNA was detected in 41.2% of the samples from the Bogotá River and in 50% of the samples from WWTPs (

| CON CLUS IONS
To the best of our knowledge, this is the first long-term sampling study to determine H. pylori contamination in water from wastewater treatment plants and in the Bogotá River (Colombia).The presence of H. pylori was confirmed both in the Bogotá River and in the effluents of the wastewater treatment plants, which shows that the treatment carried out on wastewater of domestic origin in these plants is not sufficient for the elimination of this pathogen.Our findings agree with other studies that have shown that H. pylori can survive disinfection practices commonly used in water plants. 17,73e genotype distribution was highly heterogeneous; however, the vacAs1/m1 y vacAs1/s2/m1/i1 genotypes were the most prevalent in the samples evaluated.The presence of this pathogen in the waters can be represent a risk to the health of the surrounding population since these waters are reused by the communities for different purposes.

AUTH O R CO NTR I B UTI O N S
ARBB and AMMD participated in the molecular determination.
DNA was purified from a 1 mL aliquot of each concentrated sample using the DNeasy Blood & Tissue kit (Qiagen), according to the manufacturer's instructions.The DNA was preserved at −80 (±10)°C.Initially, the detection of H. pylori was carried out by conventional PCR amplification of the vacA gene using the sequences of the primers proposed by Nilsson et al. 66 (F: 5′-GGC ACA CTG GAT TTG TGG CA -3′ y R: 5′-CGC TCG CTT GAT TGG ACA GA -3′) that amplify a 372 bp fragment.The amplification conditions used were those established by Piqueres et al., 67 with annealing temperature of 57°C.The specificity of all the primers used in this study was verified in silico, using the database of the NCBI (www.ncbi.nlm.nih.gov) and the BLAST program (www.ncbi.nlm.nih.gov/BLAST) and by means of DNA amplification of the reference strains H. pylori NCTC 11637 and 11638, H. pylori 301A, and E. coli ATCC 25922.

5
μL of GoTaq® Green Master Mix (Promega, M7123), 0.5 μL of each primer (10 mM), and 1 μL of DNA template.A positive control with H. pylori DNA strain NCTC 11637 and a control of external contamination consisting of PCR mix without DNA were included in each PCR analysis, and as negative control used E. coli DNA strain ATCC 25992.All water samples and controls were run in triplicate.The PCR products were analyzed in 2% (w/v) agarose gel electrophoresis prepared with 1× TAE Buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0 ± 0.2), and stained with 0.02% SYBR® Safe-DNA Gel F I G U R E 1 Sampling points of the Bogotá River and Wastewater Treatment Plants (WWTPs).Stain (Invitrogen), at run 80 V for 1 h.The gel was visualized through the Gel DocTM XR+ Imaging System Molecular Imager (BIO-RAD).100 bp Plus DNA ladder (Invitrogen™ by Life Technologies™) was used as a molecular weight marker.
-GAT AAC AGG CAA GCT TTT GAG G-3′ 349 pb Yamaoka et al. (1999) 34 R: 5′-CTG CAA AAG ATT GTT TGC GAG A-3′ vacA s1/s2 F: 5′-ATG GAA ATA CAA CAA ACA CC-3′ s1: 259 pb s2: 286 pb Atherton et al. (1995) 40 and Erzin et al. (2006) 44 Yamaoka et al. (1999) 34 R: 5′-CTG CTT GAA TGC GCC AAA C-3′ vacA m1/m2 F: 5′-CAA TCT GTC CAA TCA AGC GAG-3′ m1: 570 pb m2: 645 pb R: 5′-GCG TCT AAA TAA TTC CAA GG-3′ vacA i1 F:5′-GTT GGG ATT GGG GGA ATG CCG-3′ I1: 426 pb Melo-Narváez et al. (2018) 70 R:5′-TTA ATT TAA CGC TGT TTG AAG-3′ vacA i2 F:5′-GTT GGG ATT GGG GGA ATG CCG-3′ I2: 432 pb R:5′-GAT CAA CGC TCT GAT TTG A-3′ in samples of environmental origin, specifically from the Bogotá River and domestic wastewater treatment plants (WWTPs) that were positive for the presence of this pathogen.The general results showed the presence of H. pylori (vacA gen) in 33 of the 75 samples analyzed (44%); of these, 41.2% (21/51) belong to the Bogotá River; this high percentage is due to the number of samples studied (51/75) (Figure2).The basin with the highest prevalence of H. pylori was the lower basin with 18% (9/51) followed by the middle and upper basin with 12% (6/51) for each.The results obtained in the upper basin of the Bogotá river are of special interest, since it is considered one of the areas with the least anthropogenic intervention; however, at the source of the river, only one sample was positive for H. pylori and as reported by Sánchez in F I G U R E 2 Determination of the prevalence of cagA and vacA genes and genotyping of the vacA gene.(), prevalence rates; C-WWTP, Cajicá WWTP; G-WWTP, Guasca WTTP; LRB, Lower River Basin; MRB, Middle River Basin; S-WWTP, Salitre WWTP; TRB, Total Basin River; TS, total samples; TWWTP, total WTTP; URB, Upper River Basin.2017 10 this area has no contact with the urban area of any municipality, but there are settlements dedicated to various agricultural activities, which can be related to direct wastewater discharges into the Bogotá river.10,71At the sampling points 2 and 3 (Saucio and Puente Florencia), where a total of four positive samples were obtained altogether, the river receives domestic wastewater from the municipality of Villapinzón and the provinces of Almeidas and Sabana Centro in the north of the department of Cundinamarca no prior treatment.It should be noted that the municipality of Villapinzón and the provinces do not have a plant to decontaminate the wastewater generated, which reinforces the possibility of finding H. pylori in these geographical areas of the river.

FJV
and CV drafted the manuscript.FJV and AAT conceived of, conducted the study, and reviewed the paper.CV edited and prepared the graphs of the article.All authors read and gave their approval to the final version of the manuscript.ACK N OWLED G M ENTS We acknowledge the Ministerio de Ciencias, Tecnología e Innovación (MinCiencias, Colombia) and the Vicerrectoría de Investigación of the Pontificia Universidad Javeriana (VRI) for the financial assistance that was provided for Project 120380763025, CTO 807-2018: "Búsqueda de nuevas alternativas terapéuticas para erradicar la infección por H. pylori: Una estrategia para reducir el riesgo de cáncer gástrico."We greatly acknowledge the collaboration of the Andrea C. Sánchez-Alfonso by the sampling from the Bogotá River and treatment plants (WWTPs) used in this study.
Detection of H. pylori DNA from water simples of Bogotá River and effluents wastewater treatment plants.
TA B L E 2

Table 2
The results of this research may indicate that the treatment processes in the WWTPs, as well as the use of traditional indicators, are not sufficient to evaluate the efficiency of the process, nor the elimination of pathogens, which is why the EPA proposes the need to use other indicators, and within them, it has proposed H. pylori, which has demonstrated its high resistance to the disinfection processes used in water treatment.Thus, our results strengthen the possibility of using H. pylori as an indicator of the effectiveness of wastewater treatment; however, more data on the presence of H. pylori and traditional indicators should be compared to determine whether this is the case.
), using PCR, suggesting constant contamination from the Bogotá River with H. pylori.Regarding the distribution of the genotypes, in this study it was possible to describe for the first time in the world in water samples the i allele of the vacA gene.A high F I G U R E 3 Prevalence of H. pylori genotypes in the Bogotá River and WWTPs.prevalence of the vacAs1 variant was found with 87.8% (29/33), followed by the vacAm1 variants with 81.8% (27/33) and vacAi1 with 66.6%(22/33).The variants, vacAs2, vacAm2, and vacAi2, include a prevalence of 27.3% (9/33), 0% and 6.1% (2/33), respectively.(Figure3).Likewise, it is the first study in Colombia that performs a complete genotyping of the H. pylori vacA gene, identifying the i vari-Finally, the results of this study are very important because the presence of H. pylori DNA has been demonstrated in residual and surface waters, such as the Bogotá River, for which it is necessary to continue with this type of study in order to verify the hypothesis that H. pylori can be carried by water, and additionally, in vivo studies are lacking to determine whether it is possible to present H. pylori infection with the consumption of these contaminated waters.