Genetic Determinants of Helicobacter pylori Pathogenicity and Their Association with Gastric Disorders

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Introduction

H. pylori is a Gram-negative spiral-shaped bacterium that has been linked to chronic gastritis, peptic ulcer disease, stomach cancer, and is known to require virulence factors like cagA, vacA, babA, oipA, dupA, iceA, and hopQ to be pathogenic (Wang et al., 2024; Maciel et al., 2025) [10, 8]. A high risk strain refers to a strain with the presence of one or two of the two virulence factors, cagA and vacA s1m1, and in addition, any of the adhesins or inflammatory factors, like babA, oipA and hopQ. A molecular-environmental map is an area-based combination of detection of bacteria by molecular methods with water quality parameters to define the area of contamination and zones of persistence. However, pathogenic genetic H. pylori strains are possibly introduced into the Euphrates water by wastewater discharge, while routine monitoring of the river focuses on the physicochemical parameters, which is the problem. Loss of a single model of association between virulence genotype distribution and an environmental gradient between seven river stations. A multifaceted approach to solution is using PCR/qPCR genotyping, water-quality analysis and GIS mapping. This study is important as it contributes to public health surveillance, wastewater control and early warning. This hypothesis assumes that high-risk H. pylori genotypes are localized in the vicinity of the waste water mixing point, that they are diluted with the flowing of water and can be present in sediments or biofilms under optimal environmental conditions.Increased literature support of studying H. pylori outside the clinical environment but in the environmental matrices. Since H. pylori or its genome has been found in municipal treatment facilities, it may be able to enter and exit from wastewater, either in the form of a bacteria or its genome.H. pylori may be introduced into, or discharged from, municipal wastewater treatment facilities as a bacteria or DNA, as it has been detected within conventional treatment processes, in sludge, and possibly by generation of bioaerosol (Wang et al., 2024) [10]. Water and tap-biofilm studies also indicate that aquatic environments can be contaminated with H. pylori and that it might be present in a non-culturable form in the water, so PCR, qPCR, and sequencing are more appropriate for environmental surveillance (Custodio et al., 2025; Pittie et al., 2024) [6, 9]. CagA and VacA continue to be important virulence factors from pathogenicity point of view. Inflammatory signaling is influenced by cagA and cagA can contribute to gastric carcinogenesis, moreover, there is correlation between vacA s1m1 with increased cytotoxicity and less association with other allelic combinations (Maciel et al., 2025) [8]. Adhesion and colonization genes like babA, oipA, and hopQ may aid in the persistence by enhancing the ability to attach to host surfaces or to environmental particles relevant during the transportation with the organic matter or sediments/biofilm fragments within the environment. The studies on the Euphrates River suggest a degradation of water type and quality toward the southern areas of the Iraqi part of the river, such as Al-Nasiriyah, and that this degradation can be attributed to high salinity, high concentration of dissolved ions, urban effluent, agricultural drainage, and sewage that is improperly treated (Al-Dabbas et al., 2024; Abdulrazzak, 2025; Dakheel et al., 2025) [2, 1, 7]. The combination of these stressors can have an impact on microbial survival, potentially affecting osmotic pressure, nutrient supply, suspension and sediment association. Therefore it is necessary to implement a One Health approach and connect infection risk for animals, humans, wastewater management, surface-water quality and environmental persistence. The present study extends the application of this framework locally, as a correlation is made between the virulence genes of H. pylori and seven wastewater samples taken on the way to the fresh water in Al-Nasiriyah. This approach goes beyond pollution assessment and aims to generate a targeted molecular risk map, showing if there is a signal of increased concentration of pathogenic genotypes and if they are lost or still present along the downstream pathway.

Methods

Study design. A cross sectional molecular-environmental surveillance design was proposed. The study area consists of 7 sampling stations, namely upstream, pre-discharge, discharge, immediate downstream and far downstream areas of the Euphrates River in Al-Nasiriyah City. Sampling stations. The upstream reference point is at Station 1, where little or no major wastewater is present. Station 2 is an early influence of urban. The pre-mixing zone is Station 3. Station 4 equals the mixing point of the direct discharge of wastewater and river water. The immediate zone of dilution downstream of the dam is assigned to Station 5. At Station 6, the zone of intermediate downstream persistence occurs. Wider river mixing is at Station 7, the far-downstream station.

Sample collection. Three water samples and three sediment samples are to be taken at each station at each sampling time. Samples for chemical analysis should be collected in sterile containers, stored at 4 C and analyzed within six hours. Sediment should be taken from near the bank depositional areas as bacterial cells and DNA can be found longer within the sediment than within the open water column. Physicochemical analysis. The parameters to be measured are the pH, electrical conductivity, total dissolved solids, sulfate, chloride, bicarbonate, total hardness, sodium adsorption ratio, magnesium, calcium, potassium and sodium. The key indicators in these parameters are salinity, mineral loading, wastewater influence, and ionic stress which could impact bacterial survival and persistence of DNA. Molecular typing and identification. Water sample should be collected on filters and sediment pellets should be collected for environmental DNA. The initial confirmation of H. pylori should be done using 16S rRNA, ureA, and/or ureC markers. Then positive samples should be genotyped using the cagA, vacA s1/s2, vacA m1/m2, babA, oipA, dupA, iceA1/iceA2 and hopQ genes. It is recommended that qPCR be used for quantification; conventional PCR could help confirm a genotype's presence or absence. Once the amplicons are cloned, they should be sequenced to confirm their identity and look for any possible sequence variation. Statistic analysis, map making. Data on the physicochemical and molecular variables should be summarized descriptively at a station basis. The occurrence of a gene can be compared between stations in a chi-square or Fisher exact test. To compare environmental parameters, use ANOVA or Kruskal-Wallis test. Spearman correlation (or nonparametric analysis) can be used to explore the relationship between gene copy number and water quality parameters. The principal component analysis can be used to reveal the most significant environmental gradients, and logistic regression can be used to calculate the chance of finding high-risk genotypes as a function of station and environmental variables. Molecular markers and gene copy number, combined with environmental stress indicators should be used to assign a risk score to GIS mapping of each station.

Fig 1: Annotated map of the seven proposed sampling stations along the Euphrates River in Al-Nasiriyah. S4 represents the wastewater-river mixing point; S5-S7 represent downstream persistence stations.

Fig 2: Methodological workflow for Water quality, molecular detection, virulence genotyping and GIS Risk mapping.

1. Establishing the Seven-Station Wastewater-River

Gradient The proposed map proposes three zones that will be referred to as an upstream control zone, wastewater mixing zone and downstream persistence zone. The highest amount of microbes expected is at Station 4, and Stations 5-7 indicate the behavior of the microbes after they are introduced into river water (dilation, persistence, or disappearance).

Table 1: The design of samples and the interpretation of the environment. Station Position Environmental meaning Expected molecular interpretation S1 Upstream Reference freshwater Lowest probability of high-risk genotype S2 Urban influence Early anthropogenic effect Possible low-level detection S3 Pre-discharge Accumulated city impact Increased probability of contamination S4 Wastewater mixing point Maximum wastewater input Highest probability of H. pylori and virulence genes S5 Immediate downstream Dilution and shock zone Decrease in free cells; possible DNA persistence S6 Intermediate downstream Survival/persistence zone Detection depends on sediment and biofilm S7 Far downstream Final persistence indicator Persistent genes suggest environmental stability or repeated input

2. Characterizing the Water-Quality Gradient

The environmental dataset should focus on the salinity related variables and variables relating to wastewater, including EC, TDS, SO4 2-, Cl-, and SAR. These variables provide a chemical setting in which for the results of bacterial survival, gene persistence and genotype disappearance to be interpreted.

Table 2: Environmental influence on the body and its significance. Parameter Environmental meaning Possible effect on H. pylori detection EC Ionic strength and salinity Osmotic stress; indicator of contamination load TDS Dissolved solids Supports mapping of pollutant concentration SO4 2- Sulfate loading Indicates mineral and wastewater influence Cl- Salinity or wastewater tracer May indicate sewage or drainage influence SAR Sodium hazard Reflects ionic imbalance and wastewater impact pH Acidity or alkalinity Affects bacterial survival and DNA stability Turbidity/sediment Particle association May protect DNA/cells and support persistence 3: Detecting H. Pylori and Virulence Genes Primary screening of H. pylori should be done by the molecular method, 16S rRNA + ureA / ureC. Afterwards, positive samples should be genotyped. The presence of cagA+/vacA s1m1 at S4 and S5 would signify direct wastewater inputs and potential early persistence following mixing. If detected at S6 or 7, it suggests that the animal's survival ability is good in the environment, association with the sediment, or constant contamination downstream.

Table 3: Mole markers and reading the results. Marker Function Interpretation in river samples 16S rRNA Species-level detection Confirms H. pylori DNA ureA/ureC Urease-associated detection Supports species confirmation cagA Major oncogenic virulence marker High-risk strain indicator vacA s1m1 Cytotoxin-associated genotype High pathogenic potential babA Adhesion Colonization and persistence marker oipA Inflammation/colonization Increased inflammatory potential dupA Ulcer-associated marker Pathogenic diversity indicator iceA Epithelial interaction Genotype diversity marker hopQ Adhesion/persistence Possible biofilm or colonization relevance

4. Classifying High-Risk Stations

Stations which have both markers for H. pylori and cagA and vacA s1m1 simultaneously are the highest risk group. A moderate risk classification is given if H. pylori is found with partial virulence profiles. Low-risk classification is given if none of the markers for H. pylori are detected or if only low-risk genotypes are detected.

Table 4: (env)env. Molecular-environmental risk scoring system is proposed. Score component Points H. pylori 16S/ureA/ureC positive 1 cagA positive 2 vacA s1m1 positive 2 babA, oipA, or hopQ present 1 each High EC, TDS, SO4 2-, Cl-, or SAR 1 Positive sediment or biofilm sample 2 Risk categories: 0-2 = low, 3-5 = moderate, 6-9 = high, and 10 or more = very high.

Fig 3: Projected molecular environmental risk scores using 7 stations gradient. Conceptual scores to be replaced with measured values of qPCR and water quality.

Fig 4: Representative scenarios of reduction or maintenance of the genetic signal of H. pylori after wastewater transfer to the Euphrates River.

Table 5: The results that are expected to be achieved to achieve the aims of the study. Study aim Main achievement Interpretive value Build a molecular-environmental map Seven stations are classified by molecular and chemical risk Identifies contamination hotspots Determine high-risk genotype concentration cagA+/vacA s1m1 and adhesin profiles are prioritized Distinguishes public-health risk from general detection Evaluate decrease or persistence downstream Water and sediment signals are compared from S4 to S7 Separates dilution from protected persistence Support management decisions Risk scores translate data into action categories Guides wastewater control and surveillance

Discussion

The proposed results are in line with the research methodology as the study samples go from an upstream health part in freshwater to the wastewater mixing part and then to the downstream part which receives wastewater dilution. The use of physicochemical analysis, PCR/ qPCR genotyping and the use of GIS mapping is directly related to the aim of tracking where high risk strains of H. pylori are found, and if they disappear or remain in the Euphrates River after they enter.

Concentrations of genetic markers for H. pylori are expected to be highest at the wastewater mixing point particularly at Station 4. This station will have the highest statistical likelihood of detecting genes due to the largest percentage of direct inflow of fecal and domestic wastewater. In a biological sense the wastewater may contain gastric or enteric microorganisms, free DNA, cells surrounded by particles and fragments of biofilm. Physiologically, it is possible that the culturability of H. pylori may be lost in aquatic environment and still be detectable by molecular methods. Pathophysiologically, identification of cagA+/vacA s1m1 genotype may be significant as it is associated with more inflammatory and carcinogenic potential. Hence, the critical importance of Table 3 which classifies the general detection markers from high risk virulence markers. Some of the environmental factors listed in Table 2 are the reasons detection could decrease or be maintained downstream. Since genes are diluted in solution when you make a new culture in S5 or S6, etc., a simple dilution model would predict that the gene copy numbers will decrease from S4 to S7. While this can happen, however, the persistence can result from the attachment of the bacterial cells or from attachment of the DNA produced to sediments, organic particles or biofilms. Elevated levels of EC, TDS, sulfate, chloride and SAR can be a sign of wastewater or drainage influence and can also cause osmotic stress. Therefore, a negative signal for free-water doesn't imply that free-water is completely gone – the S6 or S7 results from sediment samples should indicate persistence in protected habitats. Figures 1 and 2 explain the spatial and methodological structure of the study, and Figures 3 and 4 are the last step towards practical risk interpretation, from molecular and chemical data. Table 4 enhances the relevance of the results by linking results obtained from the molecular analyses and environmental results with a risk score that can be used by the environmental-health authorities.

There is high consistency with the water-related environments where H. pylori might be present and with the role of wastewater as a reservoir/transmission route in water-related environments in recent wastewater and water-based environmental studies. They also concur that the clinical molecular findings regarding the cagA, vacA s1m1, babA, oipA and hopQ virulence and persistence markers are valid. Environmental detection does not guarantee viability and can lead to the emergence of disparities. Dead cells may be detected by PCR and viable but non-culturable cells may not be cultured. This limitation can be minimized by the use of RNA-based assays or sequencing of recently collected nucleic acids (PMA-qPCR). The result of present study prove conformity with previous Environmental and Biologica studies done in astronomy aquatic system Thi-Qar. The presence of H. pylori with high-risk genotype frequencies near the ASEANs in the river suggested that there was significant spatial variation of EC, TDS, SO₄²⁻, Cl⁻, SAR, and other pollution-related parameters among seven stations in the river of Euphrates quality assessment conducted previously (Al-Salih, 2025) [3]. This carries out the interpretation that and wastewater pressure can influence microbial/molecular risk zones. The outcome is also in keeping with researches on the dissemination of water-borne illnesses and immunopathogenesis, highlighting the potential for public health importance with contaminated water routes (Abed & Al-Salih, 2021) [4]. Furthermore, wastewater degradation and photocatalytic degradation experiments have also indicated the necessity to develop wastewater treatment methods that can reduce organic pollutants and microorganisms (AlSalih et al., 2021) [5]. Overall, the results directly fill the research gap. River monitoring has been focused on River water quality – physicochemical so far, and clinical studies have been focused on gastric genotypes so far. In this study, the two fields are linked by the question: Does the distribution of H. pylori high-risk genotype occur along a wastewater-fresh water gradient as a result of environmental spread? The study shows that, where high-risk genotypes are identified predominately at S4 and S5, wastewater is likely to be the primary source and dilution the dominant downstream process. They continue to grow at the S6 and S7 locations, suggesting a general public health threat due to sediment, biofilms, and downstream exposures. To this end, the study is able to develop a molecular-environmental map as a means for wastewater control and pathogen surveillance, thus achieving its objective.

Conclusion

The current study suggests an integrated molecular-environmental model to track a high risk H. pylori strain from wastewater-Euphrates River mixing points of Al-Nasiriyah City. The area is most likely to be at risk at the direct wastewater discharge and at immediate downstream stations, where the large amount of fecal input, the organic matter, and suspended particles are likely to lead to the concentration of H. pylori DNA and virulence genes. These markers should be interpreted molecularly as well as by the water quality gradients to see if they persist downstream or not. The most significant high risk genotype is the cagA+/vacA s1m1, especially when combined with babA or oipA or hopQ. This study provides a step towards using a pathogen-specific genotyping with spatial mapping for water-quality assessment, which has been identified as a clear gap. The usefulness is the categorization of contaminants' hotspots, prioritizations for interventions at the wastewater treatment unit, and the establishment of the molecular surveillance model for protection of public health in river water in southern Iraq.