**3. Methods for Detection of** *H. pylori* **Antibiotic Resistance**

The methods used to detect antibiotic resistance are divided into phenotypic and molecular genetic. Phenotypic methods include: diffusion method (disk diffusion), serial dilution method (in agar, in broth) and combined E-test [23–28].

The method of serial dilutions (limiting dilutions) is based on the determination of a quantitative indicator characterizing the microbiological activity of an antibiotic–the minimum inhibitory concentration (MIC). To determine the MIC, concentrations of the drug prepared in advance are added to the nutrient medium, followed by inoculation and incubation of nutrient medium with the antibiotic. After incubation, bacterial growth is assessed and the active dose of the antibiotic is determined [26,27]. Depending on the concentrations to which *H. pylori* strains are sensitive, they can be divided into sensitive, moderately resistant and resistant. The dilution method using liquid nutrient media is quite convenient and it can be automated, however, due to the difficulties of cultivating *H. pylori*, this method is not convenient for large-scale application.

The disk-diffusion method is based on the diffusion of the drug from the carrier into a dense nutrient medium and suppression of the growth of the studied culture in the zone where the antibiotic concentration exceeds the minimum inhibitory concentration [28]. A paper disk is used as an antibiotic carrier in the disk diffusion method. The effect of an antibiotic is estimated by the diameter size of the growth suppression zone. The growth inhibition diameter, the lower the MIC of the antibiotic and the more active it is against the microorganism under study. Due to the presence of a sufficiently long period of time between the preparation of the medium and the start of this test, the result may be distorted due to a change in the redox potential of the medium, which makes it impossible to use this method for the study of metronidazole. It should be noted that there are no clear criteria for interpreting the results obtained from this method; therefore, at present, the disk diffusion method is not practically used.

The E-test is a type of the disk-diffusion method, where a polymer strip is used as an antibiotic carrier, on which a drug concentration gradient is applied. The activity of the antibiotic is also assessed by the inhibition zone of the microorganisms' growth–the drop-shaped zone of growth inhibition. The MIC value is assessed in the place where the zone of growth suppression is closely adjacent to the drug carrier [23–25].

Criteria for assessing the sensitivity of *H. pylori* to antimicrobial agents are shown in Table 1.


**Table 1.** Criteria for assessing the sensitivity of *H. pylori* to antimicrobial agents. (in accordance with EUCAST).

Molecular genetic methods for detecting antibiotic resistance include real-time polymerase chain reaction (RT-PCR), hybridization with oligonucleotide probes, analysis of restriction fragment length polymorphism (RFLP), Sanger sequencing and NGS sequencing. These methods facilitate the identification of specific mutations that lead to antibiotic resistance.

The PCR-RFLP method is based on amplification of DNA gene regions and selective cleavage of PCR products using restriction endonucleases that recognize mutation sites.

Real-time PCR is the most sensitive and specific method for the detection of infectious agents in comparison with traditional methods (phenotypic, immunological). Real-time polymerase chain reaction is a repetitive cycle of target DNA synthesis. In each cycle, the number of copies of the amplified region is doubled, which makes it possible to generate a DNA fragment bounded by a pair of selected primers in an amount sufficient for its detection using fluorescent probes in 35 cycles. By the end of the procedure, at least 1012 fragments should accumulate. The real-time PCR method allows both to determine the presence of a pathogen and its quantification it. Since the kinetics of amplicon accumulation directly depends on the number of the evaluated matrix copies, which allows quantitative measurements of DNA and RNA of infectious agents. To date, real-time polymerase chain reaction method is characterized by one of the lowest error rates [29]. A group of scientists led by M. Pichon conducted a study of *H. pylori* resistance to clarithromycin in stool samples using real-time PCR. Sensitivity and specificity for detecting *H. pylori* were 96.3% (95% CI, 92–98%) and 98.7% (95% CI, 97–99%), respectively [30].

Fluorescence in situ hybridization (FISH) is used to detect *H. pylori* antibiotic resistance in biopsy specimens. The method is applicable to histological preparations. The interpretation of the results is carried out using a fluorescent microscope.

The Sanger sequencing method is the analysis of the studied DNA section's nucleotide sequence of the, namely, the determination of the exact order of the nucleotides in the DNA

molecule. With its help, in one working cycle, it is possible to "read" sequences up to 1000 base pairs long with a high accuracy of 98% [31].

NGS–next generation sequencing. The main advantage of the method is high performance and accuracy of the method. Modern sequencers have a capacity of more than 15 billion base pairs per run, maximum read length of more than 600 base pairs and ability to analyze up to 96 samples per run. However, this method is very expensive and requires highly qualified personnel. Therefore, its implementation into clinical practice remains problematic. It is possible that in the future, with the reduction in the cost of analysis, NGS would be implemented into routine clinical practice [32].

A significant advantage of molecular genetic methods in comparison to phenotypic methods is their automation, lower labor input, and high accuracy of results.

Hulten K.G. with a group of scientists compared molecular genetic (NGS) and phenotypic methods for determining the sensitivity of *H. pylori* to amoxicillin, clarithromycin, metronidazole, levofloxacin, tetracycline and rifabutin. The NGS analysis was aimed at studying the already known antimicrobial resistance genes *23SrRNA*, *gyrA, 16SrRNA*, *pbp1*, *rpoB*, and *rdxA*. The study was carried out on tissue samples of the gastric mucosa fixed in formalin. The results showed that compared to the phenotypic method, the NGS method determined resistance to clarithromycin, levofloxacin, rifabutin and tetracycline with higher accuracy. However, the results for amoxicillin and metronidazole were noted to be less accurate. This fact might be attributed to the poorly understood molecular characteristics of this antibiotic resistance type. There is a possibility that genetic changes in other genes may also be associated with resistance to metronidazole and amoxicillin [33].

The results of recent study regarding the evaluation of *H. pylori* resistance to antibiotics demonstrate a close correlation between the results of NGS analysis of stool samples and gastric biopsy samples from the same patients, which indicates the suitability of feces as a biological material for molecular genetic identification of *H. pylori* antibiotic resistance [34]. Comparative characteristics of *H. pylori* antibiotic resistance research methods are presented in Table 2.


**Table 2.** Comparison of methods for determining the antibiotic resistance of *H. pylori*.

Interesting findings have been reported in a review paper by Francesca Celiberto et al. on a molecular genetic study of *H. pylori* antibiotic resistance in stool samples. The authors analyzed scientific publications for the period from 1996. The results of these studies showed high sensitivity, and majority of them–high specificity, of the molecular method for determining *H. pylori* antibiotic resistance in stool samples compared to phenotypic methods and RT-PCR of gastric biopsies. Interestingly, the method proved to be more reliable in diagnosing the infection in comparison with commonly used non-invasive diagnostic methods: 13C-urease breath test and fecal antigen determination [35].
