Helicobacter pylori Virulence Factors and Clarithromycin Resistance-Associated Mutations in Mexican Patients

Persistent infection with Helicobacter pylori (H. pylori) is an important factor in gastric diseases. The vacA and cagA virulence factors of H. pylori contribute to the development of these diseases. Triple therapy containing clarithromycin has been used to eradicate this infection. Unfortunately, resistance to this antibiotic is the primary cause of treatment failure. This study aimed to determine the prevalence of clarithromycin resistance-associated mutations and to assess the relationship between virulence factors and Mexican patients infected with H. pylori. The cagA and vacA genotypes were determined by multiplex PCR. Furthermore, a qPCR was used to identify mutations of the 23S rRNA gene. This study reported a prevalence of 84.3% of H. pylori among patients with gastric diseases, and the vacA s1m1/cagA+ genotype was the most frequent (44.8%) in antrum and corpus. Analysis of the 23S rRNA gene revealed a 19.8% prevalence of clarithromycin resistance-associated mutations. The most prevalent mutations were A2143G (56%) and A2142C (25%). A significant association (p < 0.05) between the A2142G and the vacA s1m1/cagA+ genotype was detected. In conclusion, we report a high prevalence (>15%) of clarithromycin resistance-associated mutations, and we found an association between the genotypes of virulence factors and a mutation in the 23S rRNA gene.


Introduction
Helicobacter pylori (H. pylori) is a widely distributed bacterium; according to a 2018 meta-analysis, the global prevalence was 44.3% [1,2]. This infection is common in gastrointestinal diseases, from gastritis to gastric cancer [3]. H. pylori has a set of factors that facilitate its persistence in the stomach, ensure its survival, and induce interactions with host cells. Virulence factors are associated with an increased risk of peptic ulcer, gastric adenocarcinoma, or MALT-type lymphoma. In addition, other factors (host inflammatory response, host genetic diversity, and environmental factors) are associated with the development of gastric disease [4][5][6]. The vacA virulence factor is a pore forming toxin and its gene has variable structures in the signal region (s), s1 or s2; intermediate (i), i1 or i2; and the middle region (m), m1 or m2. The s1 and m1 genotype has been subclassified into three subtypes s1a, s1b, and s1c and m1a, m1b, and m1c, respectively [3]. Recently, two additional regions have been identified, the deletion region (d) and the c region (c). The d region is located between the i and m regions. It is classified as a d1 or d2 genotype (with a deletion of 69-81 bp). The c region includes the deletion of 15 bp located at the 3 end of the vacA and is divided into c1 (with deletion) and c2 (without deletion) [7]. Another important virulence factor is cagA; the strains that express cagA are associated with an increased risk of gastric cancer [3,8].
Eradicating this infection markedly reduces the progression or recurrence of these gastric diseases. The first line of treatment for H. pylori elimination consists of a triple therapy containing clarithromycin and a proton pump inhibitor (PPI). Unfortunately, the resistance to clarithromycin has increased [9]. Furthermore, clarithromycin resistance varies depending on the geographical region [10]. In Asia, such as South Korea (60%), China (52%), Japan (31%), and southern Europe (25%), the prevalence is higher, while in northern Europe (7%) and Latin America (12%), the prevalence is lower [11][12][13].
In regions where clarithromycin resistance is >15%, the treatment guidelines recommend the use of bismuth quadruple therapy (PPI, bismuth, tetracycline, and metronidazole); levofloxacin quadruple therapy (PPI, levofloxacin, amoxicillin, and bismuth); or non-bismuth therapy, which includes sequential, concomitant, and hybrid therapies; among these, concomitant therapy (PPI, amoxicillin, clarithromycin, and metronidazole prescribed at the same time) for 14 days is preferred. In recent years, the use of dual (amoxicillin and vonoprazan) or triple (amoxicillin, clarithromycin, and vonoprazan) therapies has been introduced [12,14].
Studies concerning the association between H. pylori vacA/cagA genotypes and clarithromycin resistance are insufficient and remain controversial. Karabiber et al. and Agudo et al. demonstrated that vacA s1c and vacA s2m2 genotypes were more likely to lead to clarithromycin resistance [20,21], and Elviss et al. reported that the vacA s1m2 genotype is more susceptible but not with either vacA s1m1 or vacA s2m2 [22]. It is important to note that in these studies, the association between specific mutations in the 23s rRNA gene and the vacA/cagA genotypes of H. pylori was not reported.
In Mexico, clarithromycin is still widely used and there are very few reports of resistance to clarithromycin and its associated mutations. This study aimed to determine the prevalence of infection, the 23S rRNA domain V mutations associated with clarithromycin resistance, and the relation between virulence factors in H. pylori-infected Mexican patients. Our data showed a high prevalence of H. pylori infection among patients with gastric diseases, with a high frequency of mutations associated with the resistance to clarithromycin, and the mutation A2142G was associated with the vacA s1m1/cagA+ genotype.

Study Population, Gastric Biopsies, and DNA Extraction
The study's participants were recruited from the endoscopy department at the Hospital Juárez de Mexico in Mexico City. The criteria for selecting patients were as follows: adults (18 to 80 years old) who had not received antibiotic treatment to eradicate H. pylori, PPIs, or gastric pH-neutralizing agents during the 15 days before the endoscopic procedure. Excluded patients from the study were patients with endoscopy contraindications, who recently ingested NAIDS and/or antibiotics, and patients with severe concomitant diseases. Written informed consent was obtained from all participants. Finally, 108 patients were included, and four gastric biopsies (two from the antrum, two from de corpus) were obtained from each patient by an endoscopic procedure. Two biopsies were fixed with 4% PFA for histopathology analysis; the other two were transported in 120 µL of cold sterile 1× PBS; and tissue was recovered from PBS with sterile forceps, which was immediately frozen in liquid nitrogen and finally crushed. Biopsies that were not processed were stored at 70 • C until DNA extraction. According to the manufacturer, nucleic acid extraction was performed using the Quick-DNA Microprep Plus Kit (Zymo Research; Irving, CA, USA). The Research and Bioethics Committee of the Hospital Juárez de México approved this work (ethical approval code: HJM 2260/13-A), and each patient signed an informed consent form.

Detection of Helicobacter Pylori and Virulence Markers by PCR
Helicobacter pylori DNA was detected by PCR that was targeting the 16S rRNA gene. The 25 µL PCR mixture contained 200 ng DNA template, 1.5 mM MgCl 2 , 200 µM dNTPs, 0.2 µM each primer, and 1U Taq DNA polymerase. The thermal cycling conditions were 95 • C for 5 min, 30 cycles of 95 • C for 1 min, 58 • C for 1 min, 72 • C for 1 min, and a final extension at 72 • C for 5 min. The H. pylori virulence markers were determined by multiplex PCR using primers targeting the cagA and vacA s/m regions [23]. Briefly, the master mix included 100-200 ng of DNA, 2.5 pmol of primers to target vacA s1/s2, 25 pmol of primers to target vacA m1/m2, 10 pmol of primers to target cagA, 0.25 mM of each dNTPs, 1 U of Taq DNA polymerase, and 1.5 mM of MgCl 2 . All PCR products were visualized by applying agarose gel (1.5%) electrophoresis and staining with ethidium bromide ( Figure 1). The thermal cycling profiles were the same as those mentioned above. DNA from the H. pylori 43504 (vacA s1m1/cagA+) and Tx30a (vacA s2m2/cagA-) strains (kindly donated by Gloria Fernández-Tilapa) were used as a positive control in all reactions. All primer sequences are described in Table 1.

Determination of Clarithromycin Resistance Mutations by qPCR
A qPCR was performed to detect 23S rRNA gene point mutations associated with clarithromycin resistance. Primers targeting the 23S rRNA gene and TaqMan™ MGB probes (Applied Biosystems, Waltham, MA, USA) were used (Table 1) to identify the mutations A2142G, A2142C, A2143C, A2143G, and A2144G. Briefly, the 25 µL PCR mixture contained 200 ng DNA template, 1.5 mM MgCl2, 200 µM dNTPs, 0.2 µM primers, 0.1 µM wild type VIC-probe, 0.1 µM FAM-modified probe, and 1.5 U Taq DNA polymerase. The assays for each mutation were performed separately. The PCR mixtures were assayed with the following thermal cycling conditions using StepOne™ Real-Time PCR System (Applied Biosystems, Waltham, MA, USA): 95 °C for 5 min, 45 cycles of 95 °C for 30 sec, and 58 °C for 40 sec. Experiments were carried out in duplicates and DNA from 700392 (ATCC 26695) and 43,504 (ATCC 11637) strains of H. pylori were used as the positive controls, sterile deionized water and DNA from W3110 strain of E. coli were used as negative controls. Data were analyzed with StepOne version 2.3 software.

Statistical Analysis
Quantitative data from the statistical analysis were presented as mean, standard de-

Determination of Clarithromycin Resistance Mutations by qPCR
A qPCR was performed to detect 23S rRNA gene point mutations associated with clarithromycin resistance. Primers targeting the 23S rRNA gene and TaqMan™ MGB probes (Applied Biosystems, Waltham, MA, USA) were used (Table 1)

Statistical Analysis
Quantitative data from the statistical analysis were presented as mean, standard deviation (SD), range, or percentages. The association between H. pylori resistance to clarithromycin and virulence genotypes was analyzed using Fisher's exact test using ggplot2 software. A p < 0.05 was considered statistically significant.
An analysis of the 23S rRNA gene revealed a prevalence of 19.8% (18/91) of clarithromycin resistance-associated mutations (Clr-ram). According to the biopsy region, for 56.2% (9/16) of patients, H. pylori with the A2143G mutation was detected in the antrum and 50% (8/16) was detected in corpus. A2142G represented 25% (4/16) in both the antrum and corpus, and 6.2% (1/16) of the A2142C mutation were represented only in an antrum biopsy. Interestingly, mixed mutations A2143G/A2142G (6.2%) in the both antrum and corpus, A2143C/A2144G (6.2%) in the corpus, and A2143G/A2142G/A2142C (12.5%) in the antrum, were also detected ( Figure 2 and Table 5).     The association between the Clr-ram and the cagA/vacA genotypes was also analyzed. The A2143G mutation was present in most combined vacA/cagA genotypes detected in this study. The vacA s1m1/cagA+ genotype exhibited A2143G, A2142G, and mixed mutations. The A2142G mutation was associated with this genotype in both the antrum and the corpus (p = 0.019 and p = 0.003, by Fisher's exact test, respectively). Furthermore, the A2143G mutation was detected in the vacA s2m2/cagA− genotype (Table 5).

Discussion
The present study aimed to determine the frequency and type of mutations in the 23S rRNA gene of Helicobacter pylori detected in Mexican patients. Furthermore, we investigated the association between vacA/cagA genotypes and 23S rRNA mutations.
We found a high prevalence of H. pylori (84.3%) in patients with gastric disease. This result is in agreement with other studies: in southern Mexico, 76.5% (150/196) of H. pylori was reported in gastric biopsies [26]; furthermore, in an age-adjusted analysis, 80% of adults 25 years and older were infected [2]. In general, the prevalence of the H. pylori infection is high in Latin American adults: in Mexico, it ranges from 70% to 90%; in Guatemala, 65%; in Chile, 70-90%; and in Brazil, 82% [27].
H. pylori has different virulence factors, of which cagA and vacA are the most studied. Several studies have reported the heterogeneity of the H. pylori cagA/vacA genotype [28,29]. In this study, the most frequent genotype was vacA s1m1/cagA+, in both the antrum and corpus. Other studies performed in southern Mexico reported 71.1% and 69.7% [30,31]; furthermore, by gastric pathology, the prevalence of this genotype was 70.7% in chronic gastritis, 57.9% in gastric ulcers, and 81.3% in gastric cancer [32]. These findings suggest that the vacA s1m1/cagA+ genotype predominates in the Mexican population. It is essential to note that the percentages of the vacA/cagA genotype combinations can vary by geographical region. For example, in Wenzhou, China, the genotype vacA s1m1/cagA+ was 90.9% [33]; in southern Vietnam, it was 51.5% [34], and while in northern Spain, it was found in 20.6% and 54.5%, due to mild and severe disease, respectively [35]. Similarly, when the combination of the s and m alleles of vacA is analyzed, the prevalence also differs. The vacA m1 allele is common in North Asian countries, including Japan and South Korea, whereas the m2 allele is predominant in Southeast Asia, including Taiwan, China, and Vietnam [36]. Interestingly, in this study, the vacA s1m1/s2m2/cagA+ genotype in both the antrum and corpus was detected in one patient, and the vacA s1m1/s1m2/cagA− genotype was detected in the corpus in two patients. Mixed genotypes have already been reported [37,38], but this event is occasional. On the other hand, we did not detect the s or m allele in some cases, as reported in other Mexican populations [39].
The detection of antimicrobial resistance to H. pylori is usually based on culture approaches (E test or agar dilution method) [40]. However, the special conditions required for specimen transport, growth, and time-consuming processes make them challenging to implement in research laboratories in developing countries. Therefore, it has been necessary to implement molecular tests. PCR-based approaches have been used as alternative assays. They are fast to perform, accurate, and are used directly on different biological samples [41,42]. Some studies have compared the agreement between the culture and the qPCR. For example, Monno et al. reported an 80.6% concordance between the qPCR and the E-test for detecting clarithromycin resistance in gastric biopsies, and Bimaeil et al. reported a significant concordance between these methods (Kappa = 0.85) [43,44]. The Maastricht V/Florence consensus report has already recognized that detecting genes and mutations associated with antibiotic resistance using molecular technologies is a valuable tool for detecting gastric biopsy specimens directly [14]. Therefore, we decided to determine the Clr-ram in the domain V of the 23S rRNA gene in H. pylori using a qPCR assay.
Mutations conferring resistance to clarithromycin were detected in 19.8% (18/91) of patients harboring H. pylori, and A2143G (56.2%) and A2142G (25%) were the most frequent. These results differ slightly from those reported by Alarcón-Millán et al. They reported 12.5% of the A2143G mutation in the southern Mexico population [30]. Furthermore, in a different region of Mexico, A2143G (57.1%) and A2142G (14.3%) were reported [45]. On the other hand, the prevalence of mutations in other regions of the world is also different. For example, the prevalence of mutations was 37.7% in the Korean population, with A2143G in 90.3% of cases, A2142G in 8.0% and A2142G/A2143G (mixed mutations) represented in 1.7% of cases [46]. In China, the prevalence of A2143G varies between 10-14% [47]; in Malaysia, it was 90.5% [48]; in Iran, A2143G fluctuates between 47.1-68.7% and A2142G 33.3-5.6% [49][50][51]; and in Brazil, the A2143G mutation was the most prevalent (77.8%) [52]. Despite the heterogeneity in the prevalence of 23S rRNA gene mutations of H. pylori in different regions, the A2143G mutation remains the most common. In this study, we also detected mixed mutations in three patients; similar results have been reported in other studies [53,54]. Probably in these patients, the presence of heteroresistant strains of H. pylori is suggested; therefore, the efficacy of clarithromycin therapy could be diminished.
Some authors have analyzed the virulence genes and clarithromycin resistance, and it has been suggested that H. pylori vacA and cagA genotypes affect the eradication rates of bacteria [55,56]. Our study reported that the vacA s1m1/cagA+ genotype was associated with the A2142G mutation. This result is not in agreement with Agudo et al., who reported that clarithromycin resistance in H. pylori isolates was strongly associated with the vacA s2/m2 genotype; however, no association with 23S rRNA gene mutations and vacA/cagA genotypes is reported [21]. Otherwise, different studies have not found an association between vacA genotypes and antibiotic resistance [57,58]. Therefore, multicenter studies are needed to study the relationship between clarithromycin resistance-associated mutations and vacA/cagA genotypes of H. pylori.
There are some limitations to this study. First, no culture-based clarithromycin susceptibility tests were conducted. Second, only vacA/cagA genotypes and 23s rRNA classical point mutations were determined, and the mutations were not corroborated by sequencing; third, small sample size.
In conclusion, our data show a high prevalence of H. pylori infection among patients with gastric diseases, with a high frequency of mutations associated with resistance to clarithromycin. The A2143G mutation was the most frequent and the A2142G mutation was associated with the vacA s1m1/cagA+ genotype. These results suggest a change from clarithromycin-based therapy.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/pathogens12020234/s1, Table S1: Distribution of different vacA and cagA genotypes according to anatomical site and gastric disease.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.