Helicobacter pylori (H. pylori) is a microaerophilic, flagellated bacillus that is Gram-negative and pleomorphic and has been associated with pre-cancerous lesions and gastric cancer (GC) (Ishaq and Nunn, 2015; Baj et al., 2020). It has been estimated that half of the global population is infected with H. pylori, but <1% of them will progress to GC (Uemura et al., 2001). It has been described that the initial infection occurs during childhood from contaminated water or vegetables (Gomes and de Martinis, 2004; Goh et al., 2011).

Many virulence factors of H. pylori have been associated with cancer progression. The most frequent are the pathogenicity island cagPAI and the vacuolating cytotoxic VacA (Denic et al., 2020; Soluri et al., 2020). The pathogenicity island is a locus of 40 kb and has ~31 genes that encode for a type IV secretion system (Baj et al., 2020). The cagA gene encodes one of the oncoproteins that can be inserted inside the host's cells, leading to cell death (Soluri et al., 2020). The vacA gene is one of the most important virulence factors, and its high genetic diversity is associated with gastric wound development (Denic et al., 2020). The s1m1 alleles are the most pathogenic for the host compared to the s2m2 alleles (Atrisco et al., 2010; Román et al., 2017; Keikha et al., 2020).

Colombia has a high incidence of gastric cancer, and eradication of H. pylori from the gastric mucosa is an ideal treatment plan to revert gastric lesions, including peptic ulcers and chronic and atrophic gastritis (Matta et al., 2018). In Colombia, the first-line treatment for H. pylori infection consists of a standard triple therapy that includes a proton inhibitor, metronidazole, and amoxicillin. However, high levels of metronidazole resistance (81%) (Trespalacios et al., 2010) have forced us to change it to clarithromycin.

The therapeutic failure of first-line treatment in Colombia, associated with drug resistance, led to the use of tetracycline, clarithromycin, and levofloxacin as H. pylori rescue treatments (Camargo et al., 2014; Arévalo et al., 2019). Considering the increasing rates of bacterial antibiotic resistance, it is imperative to study mutations conferring drug resistance and its evolution to define novel evidence-based therapeutic schemes for H. pylori eradication in Colombia.

Out of the 166 isolates, the proportion of genes associated with antibiotic resistance was 3.62% (6/166) for clarithromycin, 7.23% (12/166) for tetracycline, about 12.04% (20/166) for levofloxacin, 25.9% (43/166) for amoxicillin, and 99.3% (165/166) for metronidazole in Colombia (Figure 1). Mutations in the 23S rRNA gene related to clarithromycin resistance were the most frequent. We found five isolates exhibiting the transition of adenine by guanine, A2142G, and only one isolate showed the transition of adenine by guanine in the 2,143 position (A2143G).

We observed 12 isolates carrying mutations in the gene 16S rRNA related to tetracycline resistance. Nine isolates showed changes at the 926 position (either A926G or A926T), and three had the mutation at the position A928C. There were 43 mutations in the gene pbp1A associated with amoxicillin resistance. Among these mutations, 18 isolates exhibited transitions at position R649K, 13 isolates showed mutations at positions T593S, T593A, and T593P, eight isolates had mutations at positions R656P and R656H, and four isolates displayed the mutation at T556S. For gyrA, the gene related to levofloxacin resistance, 11 isolates showed the mutations D91G, D91N, and D91Y, and nine had the mutations N87K and N87I among the 20 evaluated isolates. Finally, regarding metronidazole resistance, we found 164 isolates with the mutation D59N, 78 isolates with the mutation R131K, 77 with the mutation R90K, 43 with the mutations H97T, H97Y, and H97I, 41 with the mutations A118T, A118S, and truncations in 34, 35, 50, and 51 positions in 1, 3, 10, and 1 isolates, respectively, in the gene rdxA.

We found 55 multidrug-resistant isolates. Five were resistant to clarithromycin and metronidazole; six to tetracycline and metronidazole; 32 to amoxicillin and metronidazole; four isolates were resistant to tetracycline, amoxicillin, and metronidazole; six were resistant to amoxicillin, levofloxacin, and metronidazole; and one was resistant to clarithromycin, levofloxacin, and metronidazole. Finally, we found one isolate with drug resistance-associated mutations to four antibiotics, including tetracycline, amoxicillin, levofloxacin, and metronidazole (Table 2).

In the core genome phylogenomic tree, we observed independent lineages of hpAfrica2, hspWAfrica, hpEAsia, hpEurope, hspAmerindian, and an independent clade of Colombian isolates (hspColombia) (Gutiérrez et al., 2017). We found that isolates clustered in the Native American clades had a close evolutionary relationship with Asian isolates (Kodaman et al., 2014). Moreover, in the cluster formed by Colombian isolates (hspColombia), we observed three hpEuropean and two hspWAfrican isolates. Finally, we found a group of seven isolates that were grouped with the de hpEurope linage, and only two isolates were grouped with hpAfrica2 (Figure 2).

In Colombia, there is a high prevalence of H. pylori infection (~80%) (Bravo et al., 2003) associated with gastric cancer patients, mostly in the Andean region. The genetic evolution of H. pylori, initially introduced in the Americas by the Europeans during colonization, due to adaptation to the Neotropical environmental features and the host genetics, led to the emergence of local strains (hspColombia) with resistant patterns that seem to be adjusted to the local therapy. In Colombia, it has been found that strains of African ancestry have high resistance to antibiotics despite their low virulence. However, strains from the Andean area show higher susceptibility than those from coastal Colombia (Figueroa et al., 2012). This finding may be linked to the strong African background found in the Colombian Pacific area, where H. pylori strains of African origin have coevolved with the human host, thereby allowing a higher contact with antibiotics used in treatments for multiple infections, including parasites and other bacteria, giving H. pylori the opportunity to develop resistance to the antibiotic. A different situation from Colombia has been described in the Andean mountains, where the coevolution of H. pylori-human seems to be disrupted. In these places, there is a high prevalence of new strains with less exposure to antibiotics and a lower resistance rate to antibiotics that are used for eradication, but also a higher incidence of gastric cancer (Figueroa et al., 2012; Matta et al., 2018; Mannion et al., 2021).

To eradicate the H. pylori infection, it is crucial to know the susceptibility and response to antibiotics to obtain the best treatment. In Colombia, the H. pylori treatment scheme is based on the combination of two antibiotics and a proton pump inhibitor such as omeprazole. Recently, the therapeutic efficacy of this triple therapy in Colombia has decreased due to the high resistance rate to antibiotics. For example, in Bogota, the proportion of resistance to metronidazole is ~97.3% (Yepes et al., 2008) (described by the E-test technique), almost the same as in our study, where we found 99.3% of resistance to this antibiotic, taking into account that most of the studied isolates in this study are from the Cundi-Boyacense mountains.

In this study, we found that D59N in rdxA was the most frequent mutation associated with resistance to metronidazole. This finding is in line with one study from the Cauca region that identified the same mutations with a frequency of 78.9% (Acosta et al., 2017). However, the resistance to metronidazole data could vary depending on the region of Colombia. For example, in several microbiological studies, the mean resistance ranges from 66 to 83% (Camargo et al., 2014).

Amoxicillin, a first-line antibiotic used for H. pylori eradication in Colombia, is at a high risk of failure due to the increased spread of resistant isolates ranging from 3.8 to 17% (Camargo et al., 2014; Atehortúa et al., 2020). We found mutations associated with resistance to amoxicillin as high as 25.9%. The highest frequency of amoxicillin resistance (20.5%) in Colombia was found in Tumaco City (Figueroa et al., 2012).

Clarithromycin is an antibiotic from the macrolide family that inhibits the protein synthesis of the HPrrnB region in the 23S rRNA component. The frequency of mutations associated with resistance to this antibiotic was 3.62%, the lowest found compared to the other studied antibiotics. Colombia has been described as having a low resistance rate for this antibiotic, between 2.2 and 4% (Álvarez et al., 2009; Acosta et al., 2014). However, high resistance rates have been reported for this antibiotic, such as 15, 17.72, 18.8, 19.8, and 63.1% (Yepes et al., 2008; Henao et al., 2009; Trespalacios et al., 2010; Roldán et al., 2019), depending on the country regions. We observed a resistance rate of 7.23% for tetracycline harboring mutations associated with this antibiotic resistance. The most frequent changes found were in positions 926 (A926G and A926T) and A928C. However, in Colombia, the reported resistance rate to this antibiotic has been high, according to a previous study that reported a resistance rate of 85.7% (Yepes et al., 2008). Finally, levofloxacin is an antibiotic that has been used as a rescue therapy for unsuccessful first-line treatment. The main mutations in the gene gyrA have been associated with therapeutic failure to eradicate H. pylori. In Colombia, a resistance rate between 11.8 and 27.3% has been identified (Trespalacios et al., 2016). In this study, we found a 12.04% resistance rate, similar to previous studies.

The finding of strains with multi-resistance genotypes to two, three, and four antibiotics makes the hypothesis more correct about why the antibiotics fail in patients. Despite having assisted in controlling and treating the symptoms with the specialist, the symptoms and the H. pylori infection persist. In addition, we found multi-resistant isolates, especially one in the Nariño region, showing resistant genotypes to four antibiotics, which is why the World Health Organization (WHO) has declared H. pylori a priority in the search for new and effective antibiotics (World Health Organization, 2017).

Commercial kits have been developed based on real-time PCR to detect mutations in specific genes such as 23S rRNA (A2142C, A2142G, and A2143G) and gyrA (N87K, D91G, D91N, and D91Y) to identify genotypic resistance from gastric tissue obtained by biopsies due to the time consuming and difficult in vitro culturing of H. pylori (Cambau et al., 2009; Scaletsky et al., 2011; Redondo et al., 2018), which could be applied as a new diagnosis and treatment strategy in Colombia.

However, one of our limitations is the possibility of other existing mutations in different genes at multiple sites that could affect the grade of resistance. For example, it is known that the gene pbp1A has an important role in amoxicillin resistance. This could be affected or regulated by other genes such as pbp2, pbp3, hopC, hofH, and the presence of the betalactamase TEM-1 in the bacteria (Domanovich-Asor et al., 2021).

Developing Next Generation Sequencing (NGS) technology will help find and suggest new H. pylori treatment models to prevent gastric pathogenesis through pathogen identification and genetic characterization like virulence genes, the evolutive lineage, and in the host, gastric cancer susceptibility genes (Malfertheiner et al., 2022). This study offers a preamble to antibiotic resistance mutation screening. However, it is necessary to conduct more studies that allow us to delve deeper into the H. pylori genome and the emergence of resistance (Malfertheiner et al., 2022).

The determination of antibiotic resistance-associated mutations by H. pylori in Colombia is of major importance in the treatment and eradication of bacterial infection. This study is the first to employ innovative diagnostic methods such as NGS, which offer significant advantages over traditional culture methods. These new methods enable faster and more accurate detection of mutations, which can positively impact the selection of effective treatments. Moreover, the successful application of NGS in Colombia can serve as a model for other countries and regions, accelerating global progress in diagnosing and treating antibiotic resistance in H. pylori.

The phylogenomic tree showed that the H. pylori isolate belongs to an independent lineage, hspColombia, allowing the identification of a circulating strain in the Andean region from Colombia. We observed a high H. pylori resistance rate to metronidazole. It was suggested not to use this antibiotic in eradication therapies as a first-line treatment.

Clarithromycin and tetracycline are antibiotics that do not pass the established resistance rate threshold in the consensus for Colombia. Levofloxacin is an antibiotic whose resistance is increasing in Colombia. We suggest continuing surveillance of the resistance rates for this antibiotic.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://pubmlst.org/organisms?title=Helicobacter+pylori.

Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements.

KG and AP designed and coordinated the study, acquired and analyzed data, and interpreted the data. KG, LM, and AP wrote the manuscript and approved the final version of the article. All authors contributed to the article and approved the submitted version.