Gnotobiotic piglets were probably the first animals to undergo successful infection with H. pylori (Lambert et al., 1987; Krakowka et al., 1987; Eaton et al, 1989). While these animals have been instrumental in studying the role of urease and motility in the mediation of H. pylori infection induced pathology, key immune features observed in this model can vary significantly from humans. Despite its early applications, researchers have moved away from using this model, likely due to economic limitations and the need for specialized breeding and experimental facilities.

Beagle dogs, both gnotobiotic pups and conventional animals, have been investigated for experimental H. pylori infection. Gnotobiotic pups, infected at 7 days of age, showed successful H. pylori colonization for at least a month post-infection, albeit at a lower level compared to humans (Radin et al., 1990). Gastric lesions were observed upon gross examination with microscopic evidence of immune cell infiltration into the gastric lamina propria. Antibodies specific to H. pylori were developed in conventional beagle dogs that were infected between four and six months of age and observed for up to 24 weeks (Rossi et al., 1999). Animals with an acute infection experienced vomiting and diarrhea. This was followed by polymorphonuclear cell infiltration and subsequent development of gastritis with epithelial changes linked to the development of MALT lymphoma in humans. Even though the pathophysiology of H. pylori infection in dogs closely resembles that of humans, this model has not been adopted more widely. Similarly, there are not many reported studies on feline infections. The initial descriptions of feline H. pylori infection demonstrated the development of lymphofollicular gastritis (Fox et al., 1995; Handt et al., 1995). According to a vaccination study, cats that received the urease antigen orally were protected against the H. pylori challenge (Handt et al., 1995). However, there have not been wider studies with this host species other than reports of isolation and characterization of non-H. pylori Helicobacter isolates.

Insects can be employed to quickly determine in vivo toxicity and efficacy of novel antimicrobial compounds, which makes it possible to conduct more targeted mammalian testing. Galleria mellonella is a wax moth present throughout the world and its larvae have been used as animal model in several research studies for over two decades (Tsai et al, 2016). The larvae of G. mellonella are being utilized more frequently as miniature hosts to study the pathogenesis and virulence components of many bacterial and fungal human pathogens from humans despite the fact that they do not recapitulate all elements of mammalian infection. This has the following benefits: i) adaptation to the human physiological temperature (37°C), ii) presence of a well-characterized phagocytic system, and iii) availability of a comprehensive transcriptome and immune gene repertoire. Several pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, and Campylobacter jejuni, have been shown to be pathogenic in G. mellonella larvae (Tsai et al, 2016). Due to their susceptibility to H. pylori infection and relatively lower thresholds of legal or ethical considerations, G. mellonella larvae have been proposed as a simple in vivo model for the study of H. pylori virulence factors and pathogenic pathways (Giannouli et al., 2014). The experimental paradigm that has been developed can be helpful for screening a relatively large number of clinical H. pylori strains and for correlating the disease state of patients with the virulence of these strains (Giannouli et al., 2014). Recently this model was utilized to demonstrate biofilm formation by multidrug resistant H. pylori strain during its exposure to stress caused by clarithromycin (Krzyżek et al., 2025). The G. mellonella larval infection model recapitulates important aspects of H. pylori pathophysiology and is cost-effective in comparison to mammalian infection models. While it could be useful for the initial evaluation of the impact of H. pylori virulence parameters factors on particular cellular functions, it is unable to completely substitute the well-established and more physiologically relevant in vivo models in the analysis of the complex pathogenic mechanisms underlying H. pylori-related human disease (Giannouli et al., 2014; Ochoa et al., 2021) and the efficacy of therapeutics (Almeida Furquim de Camargo et al., 2020). In summary, the G. mellonella model may lessen reliance on mammalian infection models based on its ability to differentiate between virulent and non-virulent H. pylori isolates, determine putative genes associated with virulence through genome-wide association studies and identify novel molecular targets for antimicrobial therapy.

Mice have been a crucial model organism to study different human infectious diseases and genetic disorders. The predominantly used wildtype mouse strains in H. pylori infection are C57BL/6 and BALB/c, followed by less commonly used Swiss albino, ICR and Kunming mice (Ansari and Yamaoka, 2022; Wei et al., 2019; Kimura et al., 1998). These models are used to study the effects of H. pylori colonization and to test the efficacy of different antimicrobials, phytochemicals, and probiotics. The bacterial strain used to infect these mice presents another important facet to the infection as mice do not get colonized by H. pylori as readily as humans (Salama et al, 2013). It is interesting to note that before H. pylori infection models were developed, mouse infection models and vaccination trials extensively employed a feline Helicobacter isolate named H. felis. H. felis lacks the cag pathogenicity island and produces severe atrophic gastritis in C57BL/6 mice, but only mild disease in BALB/c mice (Zhang and Moss, 2012). In 1997, Lee et al., introduced the first standardized mouse infection model of H. pylori with the Sydney strain SS1 derived from an individual with duodenal ulcers (Lee et al., 1997). The original strain was cag ⁺ vac ⁺ (PMSS1) but underwent modification during experimental infection to yield the type 4 secretion system (T4SS) deficient SS1 strain widely used in infection studies nowadays. The parental PMSS1 strain is often used to study the effect of a functional cagPAI system (Arnold et al., 2011) although it has been reported that this genetic island is susceptible to genetic rearrangement and disruption leading to variability in bacterial virulence features (Dyer et al., 2018). Overall, H. pylori infection in wildtype mice frequently results in milder gastritis or slowly progressive illnesses, and these models offer less insight into the toxicity of clinical H. pylori strains. Furthermore, while these wildtype mice infected with H. pylori and H. felis develop lymphocytic gastritis, they generally do not advance to more severe conditions such as peptic ulcers or stomach cancer (Zhang and Moss, 2012).

To compensate for the deficiencies of the wildtype mouse models, several knockout and transgenic mice have been developed over the years. These have incrementally paved the way for the identification of critical host factors implicated in H. pylori pathogenesis and the development of gastric cancer. Some examples are reviewed below.

H. pylori infection-induced gastric inflammation is largely mediated by IFN-gamma and double-knockout mice have been demonstrated to allow longer colonization by strains that are unable to infect wildtype mice (Sawai et al., 1999). Chronic infection is typically associated with significantly lower inflammation in this model which has been used for many vaccine studies. Similar observations have been made in mice deficient in TNF-alpha signaling (Yamamoto et al., 2004).

H. pylori infection has a synergistic effect on the development of gastric cancer in individuals with IL-1β gene polymorphisms with the most severe gastric pathology being observed in patients with both host and bacterial high-risk genotypes (El-Omar et al., 2000). IL-1 receptor knockout mice have lower gastritis scores and associated pathology markers such as nitric oxide production (Huang et al., 2013). Gastrokine-2 is an anti-inflammatory protein produced in the gastric epithelium and its deletion drives premalignant gastric inflammation and tumor progression in mice that is accelerated by H. pylori infection (Menheniott et al., 2016). Furthermore, GKN-2 expression is progressively lost during the progression of gastric cancer, and it plays a causal role in its development (Chung Nien Chin et al., 2020). During early H. pylori infection, Fas-antigen mediated apoptosis depletes gastric parietal and chief cells which are then replaced by metaplastic glandular lineages resistant to Fas-apoptosis. This has been modelled in Fas antigen–deficient (lpr) mice that develop invasive stomach lesions post H. pylori infection (Cai et al., 2005).

Transgenic mice are genetically engineered mice that harbor gene insertions from different species. Humanized insulin/Gastrin (INS-GAS) transgenic mice are frequently used to model stomach cancer as they have high circulating levels of pancreatic gastrin (Jiang and Yu, 2017). These mice spontaneously develop atrophic gastritis and intestinal metaplasia which then progress to corpus-centric cancer. PMSS1 infection in IN-GAS mice results in invasive carcinoma whereas SS1 infection causes only dysplasia (Lofgren et al., 2011; Stair et al., 2023), thus demonstrating the importance of bacterial factors in determining disease progression. Transgenic expression of H. pylori CagA in mice has been shown to induce gastrointestinal and hematopoietic neoplasms (Ohnishi et al., 2008).

Mongolian gerbils are small rodents that show similar symptoms to humans, such as appetite and weight loss, and recount many attributes of H. pylori-induced gastric colonization, inflammation, ulcers, and cancers (Noto et al., 2016). After it was discovered that Mongolian gerbils can mirror several characteristics of H. pylori-induced human stomach inflammation and cancer, these rodents captured considerable interest and attracted a lot of attention, particularly for vaccine studies (Jeremy et al., 2006; Guo et al., 2019; Lv et al., 2014; Noto et al., 2016; Wu et al., 2008). However, the timeline for cancer development varies greatly between studies and appears to be contingent on the infecting H. pylori strain. Gerbil infection by a modified H. pylori strain with an inducible T4SS has demonstrated that while an infectious trigger can be instrumental for cancer development, cancer progression does not necessarily depend on the persistent presence of the infectious agent (Lin et al., 2020). Comparative genomic studies in this model have shown that the genomes of three strains recovered from infected gerbil stomachs showed mutations in babA, tlpB, and gltS genes, all of which are linked to host adaptation (Suzuki et al., 2019).

This model has also been used in the study of probiotics in H. pylori eradication (Kuo et al., 2013; Zheng et al., 2024). Probiotics have been shown to inhibit H. pylori growth, adhesion, and the production of virulence factors in vitro. In the gerbil infection model, probiotics have been demonstrated to prevent the colonization of H. pylori (Zheng et al., 2024; Kuo et al., 2013; Isobe et al., 2012). This model has also demonstrated that the presence of H. pylori and related inflammation can alter microbiome composition in the non-inflamed regions of the gastrointestinal tract (Heimesaat et al., 2014). While the Mongolian gerbil infection model has been instrumental in dissecting the critical determinants mediating host-pathogen interactions during H. pylori infection, the lack of molecular resources has stymied its wider application within the research community.

The guinea pig stomach structure is akin to that of humans with a dietary requirement for vitamin C like humans and other primates. H. pylori infection in this model, first described in the late 1990’s, produces an inflammatory response driven by IL-8 secretion by gastric epithelial cells (Sturegård et al., 1998; Kleanthous et al, 1998). Infections with either the rodent adapted SS1 strain or clinical H. pylori isolates from gastric biopsies were able to establish lasting colonization and induce seroconversion in infected animals. Chronic infection was associated with the development of antral gastritis and lymphoid follicles like that of H. pylori-infected humans. Epithelial cell proliferation as evidenced by the presence of a large population of Ki67-positive gastric cells, is readily observed in this model (Gonciarz et al., 2019). This model has been used in a recent study delineating the role of H. pylori in driving metabolic syndrome (Tomaszewska et al., 2024). Despite the similarities with human gastric anatomy and H. pylori related pathophysiology, this model has not been widely used.

Non-human primates, such as macaques, can be a suitable model for H. pylori infections due to their physiological and anatomical similarities with humans. Rhesus macaques (Macaca mulatta) acquire H. pylori in the stomach mucosa and experience chronic gastritis (Solnick et al., 2003, Solnick et al., 2001) and glandular atrophy, the precursor to stomach cancer (Dubois et al., 1999). However, it remains unclear if macaques naturally harbor H. pylori and act as a reservoir in wildlife or whether they acquire the bacteria upon contact with humans, after capture (Solnick et al., 2003, Solnick et al., 2006). Non-human primates are mostly used in testing vaccines against H. pylori (Lee, 2001). The genetic and physiological parallels between primates and humans make this model appealing and practical for research on H. pylori-related gastritis, but the high cost, time and labor requirements along with ethical considerations prevent its widespread use in Helicobacter research (Barnhill et al, 2016).

Induced pluripotent stem cells (iPSC) are derived from adult somatic cells that undergo genetic reprogramming to attain a state akin to embryonic stem cells, achieved through the enforced expression of specific genes and factors crucial for maintaining pluripotency (Ye et al, 2013). Organoid models, a relatively recent advancement in three-dimensional (3D) cell cultivation systems, can be derived from iPSCs by controlled differentiation steps (Pellegrino and Gutierrez, 2021; Seidlitz et al, 2021). Gastric organoids can effectively mimic the cellular diversity and architectural complexity of the stomach, encompassing various epithelial cell types like mucous-secreting cells, chief cells, parietal cells, and enteroendocrine cells (Seidlitz et al, 2021). Given that the stomach serves as the primary site for H. pylori colonization and infection in humans, gastric organoids have emerged as invaluable tools for modeling infection and related gastric diseases such as cancer or ulcers (Ku et al., 2022). Compared to immortalized gastric cancer cell lines, organoid cultures offer a closer recapitulation of in vivo conditions, particularly in studying interactions between H. pylori and the apical-junctional complex (Uotani et al., 2019).

Intestinal organoids, also known as enteroids or colonoids, are derived from intestinal stem cells and mimic the cellular composition and architecture of the intestine (Lee et al., 2018; Mahe et al., 2013; Ohki et al., 2020). Although H. pylori primarily infects the stomach, studies suggest that it can transiently colonize the duodenum and colon (Fujimori, 2021). A model of mouse intestinal organoids generated from isolated intestinal crypts demonstrated the role of gastric hormones in inflammation and repair thus showing that endocrine cells within these organoids closely resemble those in the gut (Ohki et al., 2020). Given the roles played by gastric hormones in H. pylori-associated disease manifestation, intestinal organoids can be infected with H. pylori to study its interaction with the intestinal epithelium and investigate potential extra-gastric effects of the infection (Becher et al., 2010; Walduck and Becher, 2012) including alterations in gut microbiota composition and gastrointestinal symptoms such as diarrhea and irritable bowel syndrome (Tan and Goh, 2012; Tohumcu et al., 2024; Cui et al., 2025).

To recapitulate the host immune response to H. pylori infection, researchers can co-culture organoids with immune cells such as macrophages, dendritic cells, and T cells. Co-culture systems grow stomach organoids with immune cells from the same host. This helps us learn more about the roles that innate and adaptive immune cells play in H. pylori infection (Idowu et al., 2022). Sebrell et al., 2019, investigated the recruitment of dendritic cells during H. pylori infection using human gastric organoids co-cultured with monocyte-derived dendritic cells and cataloged the various chemokines that were expressed in response to infection (Sebrell et al., 2019). The effect of H. pylori infection on cytokine production by innate immune cells using co-cultured gastric organoids and macrophages from infected mice has been studied as well (Suarez et al., 2019). These studies revealed a remarkable increase in cytokine production in Nod1-deficient cells, particularly when both macrophages and organoids lacked Nod1, suggesting that functional Nod1 suppresses cytokine production (Suarez et al., 2019).

Additionally, changes in mucin and antimicrobial peptide expression resulting from activation of innate and adaptive immunity have been investigated in these models. The bactericidal activity of mucus in a two-dimensional mucosoid culture model was demonstrated where it also acts as a physical barrier against H. pylori attachment (Boccellato et al., 2019). A co-culture of mucosoid organoids offers the potential to explore the effects of CD4+ T cell subsets and innate lymphoid cells on epithelial antimicrobial activity and offers opportunities to generate vaccinated organoids to test specific cytokines or hormone’s roles in protective responses as well as its associated mechanisms. Research employing co-culture organoids containing immune cells from the same host sheds light on the interaction between H. pylori and the host immune system during infection and advances our knowledge of the immunopathogenesis of diseases brought on by H. pylori.

The use of animal models has greatly advanced our understanding of the critical determinants of H. pylori pathology and factors involved in mediating host response to the infection, particularly the many drivers of the different phases leading to the development of gastric cancer (Ansari and Yamaoka, 2022). These models have also enabled the testing of many antimicrobials and vaccines over the years. Although early research on H. pylori infection was conducted mostly on non-rodent models, the isolation and generation of the mouse-adapted H. pylori strain has made the mouse model the cornerstone of H. pylori infection studies. However, there are several limitations to using mice as a surrogate for human pathophysiology and anatomy. Mouse metabolic processes are much faster compared to humans, and they can tolerate higher doses of most administered substances due to a quicker rate of kidney filtration and excretion (Sharma and McNeill, 2009). Furthermore, the mouse stomach is quite distinct from the human organ. Mice have a squamous glandular epithelium rather than the oxyntic type found in humans and typically display gastritis features within the stomach corpus as opposed to the antral gastritis observed more typically in humans, following H. pylori infection. Stomach cancer is also not easily modeled in this host without interfering with the expression of specific genes. Mongolian gerbils and guinea pigs have been suggested as alternatives, but they have not had the same success as the mouse models, perhaps due to the lack of molecular resources and relevant expertise.

In recent years, the complexity of non-animal models, particularly 3D organoids, has increased tremendously, putting these at par with traditional animal models. In some instances, human organoids are superlative to animal models with the added benefit of being more efficient and cost-effective. The 3Rs’ (replace, reduce and refine) objectives that underpin the ethical frameworks governing animal research in most countries today can be complemented by an increased reliance on non-animal models in the coming years. These models can further encompass in silico simulations, which can inform subsequent in vitro or animal studies, as well as advanced 2D/3D cell cultures and ex vivo tissue explant cultures. This review focuses specifically on the emerging popularity and application of 3D organoid models within the H. pylori research community.

Organoids derived from genetically modified stem cells or patient-derived iPSCs allow for the investigation of host genetic factors influencing susceptibility to H. pylori infection and disease outcomes. Current iPSC derived gastric organoids closely resemble the cellular composition and architecture of the stomach. Technological advances have made it possible to generate adult stem cell-derived organoids from tissues obtained from a healthy or diseased donor (Pellegrino and Gutierrez, 2021). Donor-matched organoids have diverse applications in the development of precision medicine, particularly for gastric cancer, which continues to have a poor five-year survival rate. Furthermore, gastric organoids can be adapted for high-throughput screening assays to identify novel therapeutics targeting H. pylori infection and associated diseases (Du et al., 2020; Lukonin et al, 2021; Li et al., 2022). Organoid-based screening platforms enable the evaluation of drug efficacy, toxicity, and mode of action in a scalable and cost-effective manner. The added dimension of immune cell co-culture allows for the investigation of both innate and adaptive immune responses to H. pylori infection, providing a more detailed understanding of host-pathogen interactions. This approach permits the study of immune cell recruitment, activation, and function within the context of the gastric or intestinal epithelium during H. pylori infection and provides opportunities to study vaccine-induced immune responses against H. pylori.

Obvious limitations of these models are related to the availability and accessibility to the organoid model of interest besides the difficulties pertaining to the optimization of culture conditions and the reproducibility of experimental results (Eicher et al, 2018; Pang et al., 2022). Adult stem cells and iPSCs from genetically and phenotypically well-characterized donor pools may be difficult to source. Culture media for any organoid must be carefully optimized to support the growth and differentiation of stem cells while maintaining the physiological relevance of the model (Sato et al., 2009). Achieving the appropriate balance of growth factors, signaling molecules, and nutrients is crucial for the long-term maintenance of organoid cultures, while also avoiding the common pitfall of batch variation in media components. The composition and stiffness of the extracellular matrix (ECM) used for embedding gastric organoids can influence their growth, differentiation, and functionality. Optimization of ECM components, such as Matrigel^® or synthetic hydrogels, is necessary to better mimic the native gastric microenvironment (Pang et al., 2022). Furthermore, most organoids lack vasculature which can lead to necrosis of underlying cells and hypoxia-induced stress responses unrelated to infection pathophysiology.