First insight into the whole-genome sequence variations in clarithromycin- resistant Helicobacter pylori clinical isolates in Russia

First insight into the whole-genome sequence variations in clarithromycin- resistant Helicobacter pylori clinical isolates in Russia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article First insight into the whole-genome sequence variations in clarithromycin- resistant Helicobacter pylori clinical isolates in Russia Daria Starkova, Nikita Gladyshev, Dmitrii Polev, Alina Saitova, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3900522/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted 14 You are reading this latest preprint version Abstract Clarithromycin (CLR) is a key drug to treat Helicobacter pylori infection, however the data on CLR resistance patterns in Russia are missing. Here, we applied next-generation sequencing (NGS)-based approach to H. pylori clinical isolates from Russia to comprehensively investigate sequence variations, identify putative markers of CLR resistance and correlate them with phenotypic drug susceptibility testing (DST). The phenotypic susceptibility of 44 H. pylori isolates (2014–2022) to CLR was determined by disc diffusion method. All isolates were undergone whole-genome sequencing and submitted to GenBank. Twenty-three H. pylori isolates were phenotypically CLR-resistant and 21- were CLR-susceptible. Based on complete sequence analysis, we showed that among all genetic variants, the combination of mutations A2146G/A2147G in the 23S rRNA gene is the most reliable for prediction of phenotypic DST. For the first time, the average number of nucleotide variants in 108 virulence-associated genes between resistant and susceptible groups were compared. Moreover, this study presents the first WGS insight into genetic diversity of H. pylori in Russia with a particular focus on the molecular basis of drug resistance: the novel mutations were described as potential markers for the resistance development. Of these, the most prominent was frameshift deletion in HP0820 coding region, which is a good candidate for further investigation. Biological sciences/Molecular biology Biological sciences/Genetics/Microbial genetics Biological sciences/Genetics/Mutation Figures Figure 1 Figure 2 Figure 3 Introduction Helicobacter pylori infection is recognized as an important public health concern in more than 50% of the world's population and may be associated with such gastrointestinal diseases as chronic gastritis, gastric/duodenal ulcers, MALT-lymphoma, stomach cancer [ 1 ]. Various regimens for H. pylori eradication therapy are used worldwide, while the choice of appropriate eradication therapy is determined by the local resistance patterns [ 2 ]. Conventional triple therapy (clarithromycin (CLR), proton pump inhibitor (PPI), metronidazole/amoxicillin) is recommended as first-line treatment in countries with less than 15% clarithromycin resistance, while quadruple therapy (bismuth, PPI, metronidazole/tetracycline/amoxicillin/CLR/tinidazole) - where CLR resistance is over 15% [ 2 – 4 ]. According to the newly published Maastricht VI guidelines there are several proposals to improve the effectiveness of the existing eradication therapy regimens: extending the treatment length of triple therapy to 14 days, the usage of a higher dosage of PPI or vonoprazan and the usage of a four-drug regimen supplemented with probiotics [ 3 , 5 ]. To date, Russia belongs to countries with a high prevalence of H. pylori infection (65–90% depending on the region) [ 6 ]. According to a meta-analysis that summarized studies of H. pylori antibiotic resistance in various regions of Russia over 10 years (2011–2020), the resistance rate to CLR reached 10.39%. That indicates a low resistance of the pathogen (< 15%) and allows to consider the triple scheme of treatment as the first-line empirical therapy (based on the absence drug susceptibility testing (DST) data) in our country [ 7 ]. However, the empirical therapy frequently results in treatment failure, which in turn leads to increase and development of antimicrobial resistance of the pathogen. On the other hand, the phenotypic DST approach is hardly feasible in clinical practice due to special laboratory requirements, high cost and labor intensity, let alone difficulties in isolating and growing H. pylori from biopsy specimens. During the last decade, many molecular approaches (such as polymerase chain reaction (PCR), Sanger sequencing, next-generation sequencing (NGS)) were used to identify known and novel markers for prompt prediction of the phenotypic antimicrobial resistance. It is generally accepted that point mutations A2142G and A2143G (in accordance with Taylor et al. numbering) in V domain of the 23S rRNA gene are mainly responsible for H. pylori resistance to CLR [ 8 ]. At the same time, point mutations outside these positions (T1942C, G1939A, C2147G, G2172T, T2182C, A2116G, A2144G/T, A2115G, G2111A, T2117C, G2141A, T2182C, T2717C, T2289C, G2224A, C2245T) vary geographically and their role in CLR resistance is not yet clear [ 9 , 10 ]. Based on whole genome sequencing (WGS) results, some sequence changes in rpl22 (encoding a ribosomal protein that interacts with the 23S rRNA domains) and infB (encoding a translation initiation factor, IF-2) genes in CLR-resistant H. pylori strains were described [ 11 ]. In addition, four gene clusters of efflux pump systems (hp0605-hp0607 ( hefABC ), hp0971-hp0969 ( hefDEF ), hp1327-hp1329 ( hefGHI ), and hp1489-hp1487) were identified as resistance-nodulation-cell division (RND) family and can be also involved in development of resistance to CLR. Furthermore, some authors note that the number of single nucleotide variants (SNVs) in the RND family is significantly higher in resistant than susceptible H. pylori strains [ 8 , 12 , 13 ]. Due to the lack of data on clarithromycin resistance patterns in Russian H. pylori clinical isolates, we aimed to comprehensively investigate sequence variations by NGS- based approach to detect putative markers of clarithromycin resistance in H. pylori clinical isolates from Russia and evaluate the correlation between genotypic and phenotypic DST. Results Phenotypic DST of study cohort For our analysis we selected two types of isolates: resistant (inhibition zone diameter 41 mm). Isolates with intermediate susceptibility were not included in our investigation. Of 44 H. pylori clinical isolates, 27 were obtained from newly diagnosed patients and 17 - from previously treated patients after eradication failure. As a result, 23 out of 44 H. pylori isolates were phenotypically determined as clarithromycin resistant (CLR-resistant), while 21 isolates showed susceptibility to CLR. All isolates from previously treated patients were CLR-resistant by phenotypic DST. Prior to identify a putative markers of CLR resistance in a novel regions of the genome, we examined genetic variations in the full-length 23S rRNA gene (two copies), infB (HP1048), rpl22 (HP1314), as well as four clusters of efflux pump genes (HP0605-HP0607 ( hefABC ), HP0969-HP0971 ( hefDEF ), HP1327-HP1329 ( hefGHI ) and HP1487-HP1489), presumably associated with CLR resistance in H. pylori clinical isolates. Plasmid Detection in H. pylori isolates Among 44 H. pylori genomes analyzed in our study, no putative plasmids encoding resistance genes were found. Single Nucleotide Polymorphisms in 23S rRNA Genes Among 44 H. pylori isolates, 21 nucleotide substitutions were identified in 23S rRNA gene, however, only one of them- A2147G (aka A2143G)- was significantly associated with phenotypic resistance of H. pylori to CLR: of the 23 CLR-resistant isolates, 13 (56.5%) carried the 2147G alleles (p = 0.003). However, A2147 allele was related to CLR susceptibility in 95.2% (20/21) of cases, since one strain phenotypically determined as CLR-susceptible possessed A2147G mutation. The other point mutation – A2146G (aka A2142G) – was found in 13.0% (3/23) of CLR-resistant isolates (p = 0.33). The A2146C allele was not found in our study. All CLR-susceptible isolates (100%; 21/21) possessed A2146 allele. None of the CLR-resistant isolates carried two A2146G/A2147G mutations simultaneously: all 2147G isolates carried the A2146 allele and, vice versa, all 2146G isolates carried the A2147 allele. Seven CLR-resistant isolates had neither A2146G nor A2147G mutations. The agreement between genotypic and phenotypic susceptibility to CLR based on both A2146G/A2147G mutations was 69.6%. Out of 17 H. pylori isolates from previously treated patients, 13 (76,5%) possessed either A2146G or A2147G mutations. This observation is entirely consistent with the general agreement on increasing the resistance to CLR during treatment regimen. Since the H. pylori genome contains two copies of the 23S rRNA gene, we assessed the presence of putative allelic variants due to mutational differences in each copy. A survey of the depth of mapped reads showed that 2146G and 2147G alleles were present in 100% reads of 15/16 CLR-resistant isolates which indicates that mutations has occurred in both copies of 23S rRNA. However, one CLR-resistant isolate (HP220) demonstrated a heterogeneity between two copies of 23S rRNA : the 2147G allele was detected in only 50% reads of one copy, while the second showed 100% reads of A2147 allele. The remaining mutations outside 2146/2147 positions in 23S rRNA gene were found in H. pylori isolates regardless of phenotypic susceptibility (Table S1 ). Nucleotide Variants in rpl22, infB and RND family genes We investigated presence of 12 genes belonging to RND family ( hefABCDEFGHI , HP1487-HP1489) in CLR-resistant and susceptible groups. In addition, we analysed the sequence variations in rpl22 and infB genes, and evaluated the number of variations between the two phenotypic groups, as well as the mutant (A2146G/A2147G) isolates and the wild type. As a result, we found that 12 efflux pump genes (HP0605-HP0607, HP0969-HP0971, HP1327-HP1329, HP1487-HP1489) were presented in all 44 isolates, however, we did not reveal any significant differences in either individual SNVs or total SNVs number between CLR-resistant and susceptible H. pylori isolates. Analysis of sequence variations in infB gene (HP1048) did not reveal any of C718T, A1899G, G160A, G60A mutations which were considered in previous studies as factors hypothetically involved in increasing of antibiotic resistance. Among all types of SNVs, only one missense substitution C2763A was significantly predominated in CLS-susceptible isolates [p = 0.03; OR: 4.6743]. The other synonymous variant G2118A was found specifically in CLR-resistant isolates (26.1%; 6/23), however, involvement of silent mutations in phenotypic consequences is least likely (p = 0.021). We did not find significant differences in total number of missense substitutions neither between CLR-resistant and -susceptible groups nor CLR-resistant mutant isolates (A2146G/A2147G) and the wild type. The most interesting finding among missense variations in the rpl22 gene (HP1314) was the absence of two types of mutations − 3bp (GTG) deletion and 9 bp (TTCCATGTA) insertion - which have been detected in numerous studies. Instead, we identified three missense variants, none of which were associated with phenotypic resistance-susceptibility status (Table S1 ). Novel Sequence Variations in Coding Regions of Russian H. pylori Isolates To detect new putative markers of phenotypic resistance/susceptibility to CLR we conducted a global gene analysis of all H. pylori isolates compared to the H. pylori 26695 reference genome. We investigated nucleotide substitutions rated to high impact on biological sequence in annotation tools and evaluated their association with antibiotic phenotype. Among all genes included in our study, the majority of genomic variants were shared between resistant and susceptible groups of isolates. However, we were able to detect six novel genomic variants harbored exclusively by CLR-resistant isolates and eight variants that were predominated in either resistant or susceptible phenotypic group. These findings were summarized in Table S1 . Among OMP (Outer Membrane Protein) family genes the most interesting findings were related to omp32 , omp5 and omp25 genes. Missense SNV -A2C -resulting in loss of start-codon in the omp32 gene, conservative inframe insertion 7:insTTTTGA in the omp5 gene, and nsSNV C1400T in the omp25 gene, were detected in 21.7% (5/23) CLR-resistant isolates per each (p = 0.049). None of these mutations were found in CLR-susceptible isolates (Table 1 ). Table 1 Resistance and susceptibilityassociated sequence variants in CDS of H. pylori clinical isolates compared to the H. pylori 26695 reference genome. Gene (Locus) Nucleotide change Amino acid change CLR-R (n = 23) N (%) CLR-S (n = 21) N (%) p-Value 23S rRNA A2146G - 3 (13.0) 0 0.23414 A2147G - 13 (56.5) 1 (4.8) 0.00025 infB (HP1048) G2763T Asp921Glu 2 (8.7) 8 (38.1) 0.03099 omp5 (HP0227) 7:insTTTTGA 3:insIleLys 5 (21.7) 0 0.04972 omp32 (HP1501) T2G start codon lost 5 (21.7) 0 0.04972 omp25 (HP1156) C1400T Ala467Val 5 (21.7) 0 0.04972 HP0820 252:delCGGGT / 262:delGG or GGG insTTAGCACA delGly85-fs / delGly88-fs 7 (30.4) 0 0.00943 flhF (HP1035) G376A Ala126Thr 7 (30.4) 2 (9.5) 0.13681 flaB (HP0115) G783T Glu261Asp 6 (26.1) 0 0.02198 flaB (HP0115) C854T Thr285Ile 4 (17.4) 0 0.10931 flaB (HP0115) G964A Ala322Thr 6 (26.1) 1 (4.8) 0.09732 fliD (HP0752) C1997G Pro666Arg 1 (4.3) 7 (33.3) 0.01900 spaB (HP0600) G98A Thr33Ile 12 (52.2) 4 (19.0) 0.03069 spaB (HP0600) G400A Ala134Thr 6 (26.1) 1 (4.8) 0.09732 res2 (HP1521) G1A Met1Val 6 (26.1) 1 (4.8) 0.09732 recA (HP0153) 360TGATACA-366insAAGCACG Asp121Ser 8 (34.8) 2 (9.5) 0.07249 rpl10 (HP1200) C257T Gly86Asp 8 (34.8) 2 (9.5) 0.07249 rpl10 (HP1200) C487A stop gained Glu163* 0 2 (9.5) 0.22198 Several unique frameshift (fs) alterations in HP0820 coding region were detected in 30.4% (7/23) CLR-resistant isolates. A fs deletion 252:delCGGGT was linked with another fsIndel in HP0820 onwards: deletion of two (262-263delGG) or three (262-264delGGG) nucleotides and insertion of TTAGCACA fragment. None of these mutations were detected in CLR-susceptible isolates. Among 28 flagellar family genes three nsSNVs in flaB gene were found predominantly in resistant group, however only one of them (G783T) was significantly associated with resistance (p = 0.021). Alternatively, we revealed that the prevalence of nsSNV (C1997) in the fliD gene was significantly higher in the CLR-susceptible rather than CLR-resistant group (33.3 vs. 4.3%, respectively) [p = 0.019, OR: 0.1057741]. Three multidrug resistance protein genes ( hetA, msbA, spaB ) were analysed in present study. As a result, the proportion of CLR-resistant H. pylori isolates carried two missense substitutions (G98A and G400A) in multidrug resistance protein SpaB gene was much higher than that in CLR-susceptible isolates (52.2 vs. 19.0% [p = 0.030; OR: 4.3874] and 26.1 vs. 4.8% [p = 0.097; OR: 6.1535], respectively). Interestingly, using WGS analysis, Liu et al (2022) showed that the spaB was absent in most strains isolated from Shanghai, whereas in our study this gene was present in all 44 clinical isolates. In addition, the same report showed that four variations (S275N, D353N, 120-121delGG and ins116G117) within the integrase/recombinase gene xerD were significantly associated with both clarithromycin and metronidazole resistance [ 27 ]. In the present study, S275N mutation was found without significant differences between resistant and susceptible phenotypes (82.6 vs. 80.9%; p > 0.05) (Table S1 ). Instead of 120-121GG deletion, we identified missense multiple substitutions of four nucleotides (117-124CTAGGATT→TTAATATC) that were linked together and have prevailed in resistance (34.8%) compared to susceptible (19.0%) group, however not significantly [p = 0.32; OR: 2.1937]. Insertion 116G117 and missense variant D353N were not detected in our study. Five point substitutions within recombinational repair protein RecA gene were linked together and were carried predominantly by resistant (34.8%) rather than susceptible (9.5%) isolates [p = 0.072; OR: 4.674359]. The missense variant (G1A) in the type III restriction enzyme gene ( res2 ) leading to the replacement of start-codon Met with alternative Val was detected predominantly in resistant group, however not significantly [p = 0.097; OR: 0.1472963]. It is known that the replacement of standard AUG with the alternative GUG form leads to a several-fold drop in the translation efficiency [ 28 ]. Since rpl are considered to be the genes with the greatest value, we initially conducted the analysis of 49 ribosomal proteins genes. As a result, we found one nsSNV (C257T) within rpl10 gene (encodes ribosomal protein L10/50S) that was predominated in CLR-resistant group, but not significantly [p = 0.072; OR: 4.6743]. Interestingly, in CLR-susceptible group isolates we found another nsSNV (C487A) in the same gene resulting in a stop codon. Unfortunately, only a few isolates possessed it (2/21), that does not allow us to assess the actual role of this mutation in the development of macrolide susceptibility. Further analysis of the distribution of newly detected mutations significantly associated with resistance to CLR in the A2146G/A2147G-mutated H. pylori clinical isolates, revealed that 6/7 (85.7%) HP0820- mutated isolates possessed either 2146G or 2147G alleles (Table 2 ). Of note, more than half (63.4%) of CLR-resistant isolates with nucleotide alterations we detected in six coding regions were A2146G/A2147G-positive. Table 2 The distribution of newly detected genomic variants in A2146G/A2147G-positive and A2146G/A2147G-negative CLR-resistant H. pylori clinical isolates. Gene (Locus) A2146G/A2147G- positive isolates (n = 16), N (%) A2146G/A2147G- negative isolates (n = 7), N (%) Total no. of CLR-R isolates (n = 23), N (%) omp5 (HP0227) 3 (18.8) 2 (28.6) 5 (21.7) omp32 (HP1501) 3 (18.8) 2 (28.6) 5 (21.7) omp25 (HP1156) 3 (18.8) 2 (28.6) 5 (21.7) HP0820 6 (37.5) 1 (14.3) 7 (30.4) flaB (HP0115) 4 (25.0) 2 (28.6) 7 (30.4) spaB (HP0600) 7 (43.8) 5 (71.4) 12 (52.2) Phylogenetic Analysis and Relationship Between Isolate Relatedness and CLR-Resistance Patterns in H. pylori isolates Based on the core SNVs of 44 Russian H. pylori genomes we constructed the phylogenetic tree based on the maximum likelihood estimation with respect to CLR-resistance patterns (Fig. 1 ). As a result, no isolates relatedness associated with the antibiotic phenotype was revealed. Instead, these isolates had distinct resistance profiles regardless of their genetic relatedness. Distributions of virulenceassociated and multidrug-resistance genes in H. pylori clinical isolates To investigate whether the presence or absence of virulenceassociated genes related to phenotypic resistance H. pylori to CLR, we evaluated the presence of 26 cag pathogenicity island ( cag PAI) genes, 28 flagellar genes, vacuolating cytotoxin gene ( vacA ), ulcer-associated restriction endonuclease and adenine specific DNA methyltransferase genes ( iceA1, hpyIM ), urease gene cluster ( ureABCIEFGH ), gamma-glutamyltranspeptidase gene ( ggt ), virulence factor protein ( mviN ), outer membrane protein family ( omp1 - omp32, ompP1 ), toxin-like outer membrane protein and iron-regulated outer membrane proteins genes ( fecA , frpB ), flagellar family genes ( flaA, flaB, fla, flaA1, flgH, fliF, flgI, fliE, flgB, flgC, flgG, flhA, flhF, flhB, fliP, fliR, fliH, fliI, flgE, flgK, fliD, motA, motB, fliG, fliM, flaG, hpaA, fliN,) , heat shock protein family genes ( hslU , hslV , htpX , ibpB ) in two groups of H. pylori isolates. For the first time, we also compared the average number of missense/nonsense/frameshift nucleotide variants in 108 virulence-associated genes between two phenotypic groups of H. pylori isolates (Fig. 2 ). As a result, we found that among all the genes mentioned above, only the presence of cag PAI genes in the CLR-susceptible group was higher than that in the CLR-resistant group, but not significantly (p = 0.42) (Fig. 3 ). Interestingly, that neither of Russian isolates contained cag2 (HP0521) gene and all 44 H. pylori isolates regardless of antimicrobial susceptibility harbored A589G mutation in the cag13 (HP0534) gene leading to the loss of stop codon and splicing with coding region HP0533 (“-“ chain). Liu Y. et al. (2022) found that the average frequency of the iron-regulated OMP genes ( fecA and frpB ) in the CLR-resistant group was significantly lower than that in the CLR-susceptible group (p = 0.01) [ 27 ]. Unfortunately, we did not find any significant differences in either the average frequency of these genes or the average number of mutations in fecA and frpB genes between both phenotypic groups. As for the remaining genes, we found no statistical differences in either the frequency of these genes or the average number of mutations between two phenotypic groups (p > 0.05). Discussion Despite insufficient knowledge and interregional dynamic differences in the resistance of H. pylori to the main antimicrobials, the level of the primary clarithromycin resistance in Russia is low (11.8%) and allows to consider a standard triple eradication regimen as a first-line therapy. Thus, routine H. pylori susceptibility testing usually is not provided in clinical settings and empirical therapy can cause eradication failures and increasing resistance rate. According to the latest data (2020), the prevalence of clarithromycin resistance in Moscow reached 10.87%, Smolensk − 5.74%, Kazan − 10.0% and in St. Petersburg − 22.26% [ 29 , 30 ]. Such difference indicates the wide variation in resistance rates in Russia, highlighting the need to ongoing examination of CLR-resistance in each geographic region. For our study, we used conventional disc diffusion method to detect susceptibility of H. pylori isolates to CLR. Although the EUCAST has recommended the E-test for MIC breakpoints detection, the high cost of E-test strips makes it difficult to use. Recently, Tang et al. (2020) assessed the disk diffusion technique against E-test and estimated that susceptibility agreement between two methods for clarithromycin is 96.0% [ 14 ]. It was demonstrated that disc diffusion is simple and affordable alternative to E-test for in vitro CLR susceptibility testing [ 31 , 32 ]. The mechanism of drug resistance in H. pylori is believed to be due to mutations (missense/ nonsense substitutions, fs insertions/deletions) located in the chromosome and not related to horizontal transfer of plasmids or other mobile elements [ 33 ]. It is generally accepted that H. pylori resistance to clarithromycin is associated with several point mutations A2146C/G and A2147G (in accordance to H. pylori reference strain 26695 numbering) in the V domain of 23S rRNA gene that decreases the affinity of drug binding to the ribosome by disrupting the peptidyl transferase loop conformation [ 6 ]. In the present study, A2147G was the most frequent mutation found in CLR-resistant H. pylori isolates (56.5%) and highly correlated with CLR resistance. However, A2146G mutation has been detected in only 13% CLR-resistant isolates that does not allow us to assess the actual role of this mutation in the resistance development. These findings are consistent with prior studies conducted in Europe that demonstrated that A2146G/A2147G mutations were present in the genome of 60–90% CLR-resistant isolates [ 8 , 33 , 34 ]. The high rate of CLR-resistant isolates without A2146G/A2147G mutations (30.4%) in our study indicates that these isolates may have genetic determinants other than 23S rRNA gene responsible for pathogen resistance development. Notably, one isolate phenotypically determined as CLR-susceptible in our study possessed A2147G mutation. Such case may indicate the presence of mixed CLR-susceptible and -resistant isolates. As shown by our previous study, this isolate was positive for mixed combinations of vacA alleles (s1s2 and i1i2) indicating the coexistence of several genetically different H. pylori isolates in gastric sites as a result of mixed infection [ 6 ]. Furthermore, there are two copies of the 23S rRNA operon within the H. pylori genome. Sequence differences between both copies have rarely been studied and data on whether 2146/2147- mutations in single or both copies of 23S rRNA are necessary for the strain resistance to occur are still controversial [ 35 ]. In this study, 16 CLR-resistant isolates possessed A2146G/A2147G mutations, and only one of them demonstrated a heterogeneity between two copies of 23S rRNA: the 2147G allele was detected in 50% reads of one copy, while the second copy showed 100% reads of A2147 allele. This observation suggested that H. pylori isolates should be considered resistant if they have the mutation in at least one copy of 23S rRNA gene. Another possible mechanism for CLR resistance is multidrug efflux pump systems. It has been reported that four conserved RND families of efflux pump transporters may be responsible for development of macrolide resistance in H. pylori by reducing intracellular antimicrobial concentration [ 8 , 10 ]. As shown by Iwamoto et al. (2014), there is a significant difference in the number of SNVs in hefABC (hp0605-hp0607) cluster between CLR-resistant and susceptible isolates, as well as CLR-resistant isolates are more prone to single-nucleotide variants in all four clusters of efflux genes, with significant differences for cluster hefABC [ 13 ]. Moreover, Chen et al. (2018) revealed that the number of mutations in the RND family was significantly higher in H. pylori isolates harboring the A2147G point mutation in 23S rRNA gene [ 36 ]. In our study, we did not find any significant differences in number of SNVs among 12 genes either between CLR-resistant and susceptible phenotypes or between isolates carried A2147G mutation and a wild type. Noteworthy, among all types of nucleotide substitutions, we found only single-nucleotide variants: no deletions or insertions were detected in four clusters of efflux genes. This result is consistent with the hypothesis that SNVs in efflux pump genes may contribute to CLR resistance through a synergistic effect between 23S rRNA mutations and efflux pumps. Other genes assumed to be involved in CLR resistance are rpl22 and infB . In fact, several point mutations in infB (C718T, A1899G, G160A, G60A) as well as 9-bp insertion and/or 3-bp deletion in rpl22 genes have been described, which, in combination with 23S rRNA mutations, are predicted to increase the resistance level of H. pylori isolates [ 11 , 37 ]. Interestingly, no indels in rpl22 gene were detected in our study: instead, we found 3-bp deletion in infB gene primarily in CLR-susceptible isolates, but not significantly. Notably, we did not find any of point mutations in infB that were detected in other studies. Among all SNVs in infB , no substitutions unique for resistant isolates were identified. Altogether, these data provide further evidence that high genetic diversity of H. pylori populations is the crucial factor in the searching for putative determinants of resistance. Giving the enormous genetic diversity of H. pylori and high heterogeneity of resistance genotypes across geographical regions, the determining of novel resistance patterns and interpretation obtained results appears to be challenging. WGS-based approaches have enabled to provide a comprehensive view of bacterial genotypes and are particularly useful for tracking novel genetic determinants responsible for antimicrobial resistance of the pathogen [ 10 ]. To identify novel mutations potentially contributing to antibiotic resistance in H. pylori isolates we next sought to analyze genetic variants in other gene families presumably associated with virulence and multidrug resistance. Among all the genes examined, the largest number of nucleotide alterations found specifically in CLR-resistant group, was observed among the OMP genes family: omp5 , omp25 (HopI) and omp32 (HopW). It is well known that OMPs are associated with macrolide resistance in Gram-negative bacteria. Lin et al. (2023) suggested that the alteration of outer membrane proteins is a potential mechanism involved in CLR resistance development in H. pylori , but the specific function and mechanism of action of these proteins need to be further studied [ 37 ]. Interestingly, Thompson et al. (2003) showed the close coexpression of the cagA gene with the omp5 and omp29 genes, suggesting these OMPs are involved in the secretion and/or activation of CagA [ 38 ]. Among all identified variants in the OMP family genes, the missense substitution in the anion-selective porin Omp32, leading to the loss of the start codon, had the greatest impact on the gene. It has been reported that expression of the omp32 gene was down-regulated by amoxicillin [ 39 ]. It is unknown whether the accumulation of such mutations may indirectly be involved in the resistance development, hence its hypothetical impact needs to be further investigated. Another interesting finding: two frameshift alterations that were significantly associated with resistance to CLR are in coding region HP0820 ( cj1247c in C. jejuni ), which Porcelli et al. (2013) described as predicted leaderless mRNA in all species containing this gene, which is always upstream of the uvrC DNA repair gene. The genes translated from the leaderless mRNAs encode the proteins predicted to be involved in stress responses, the outer membrane efflux protein and the predicted multidrug efflux pump [ 40 ]. It is well known that antibiotics create conditions that favor selection of beneficial mutants with elevated mutation rates and this is most often linked to the DNA repair system and SOS response, that can lead to increased activity of associated multi-drug efflux pumps. Given the fact that H. pylori has a defective DNA mismatch repair system (lacking genes as mutS , mutL , and mutH implicated in this pathway), as well as lack of the SOS regulon, the antibiotic-induced mutagenesis in various types of regulatory genes increases the genetic diversity of bacterial populations and, as a consequence, enhances the evolution of antibiotic resistance [ 41 , 42 ]. Missense substitutions in three different flagellar genes flaB , flhF and fliD may also be candidates indirectly involved in underlying mechanisms of resistance and/or susceptibility of H. pylori . The pathogenesis of H. pylori infection is thought to be determined in part by flagellar motility, which has a direct impact on colonization, inflammation, and immune evasion. Flagellar filament FlaB and the filament-capping protein FliD are one of the main structural proteins of the H. pylori flagellum and play an important role in bacterial motility [ 43 ]. FlhF controls flagellar patterns in many bacteria and is required for flagellum assembly and localization [ 44 ]. Even though the actual impact of these mutations on resistance development is difficult to assess, the mutational buildup in the genome may be a result of response to antimicrobials and an important predictor of treatment failure. As for relationship between virulence-associated genes and phenotypic resistance, we determined that there is no significant correlation between the presence of main virulence factors and resistance to CLR. On the one hand, our findings are consistent with majority of previous reports [ 27 , 45 , 46 ]. However, it is worth noting that in the CLR-resistant group the number of cagA -negative isolates predominated twofold compared to those in the CLR-susceptible group (10/23 vs. 5/21). Thus, on the other hand, this observation in consistent with other findings showing that clarithromycin resistance was obviously higher in cagA- negative than in cagA -positive isolates [ 47 , 48 ]. The present study has several limitations. First, despite the high level of agreement between disc diffusion method and E-test, the main drawback in our study is the inability to determine MIC values, which does not allow us to reveal a causal relationship between mutations and high/low MIC values of phenotypic resistant isolates. Second, the sample size is rather small and obtained results do not necessarily reflect resistant patterns in general Russian H. pylori population. Third, we did not provide further experimental analysis to confirm the involvement of newly detected mutations in CLR resistance development. Nevertheless, despite these limitations obtained data provided the foundation for the future investigations. Conclusions This study presents the first WGS insight into genetic diversity of H. pylori in Russia with a particular focus on the molecular basis of drug resistance. For the first time, the average number of missense/nonsense/frameshift nucleotide variants in 108 virulence-associated genes between resistant and susceptible groups were compared. It has been shown that among all genetic variants we obtained, the combination of mutations A2146G/A2147G in the 23S rRNA gene is the most reliable for prediction of phenotypic DST. However, sequencing data did not reveal the involvement of nucleotide variants in any efflux pump, infB or rpl22 genes in phenotypic prediction of CLR resistance, emphasizing the enormous genetic diversity of H. pylori populations. The novel mutations in other genes were described in our study as potential markers for the resistance development. Among them, the most prominent mutation is frameshift deletion (252:CGGGT) in HP0820 coding region, which is a good candidate for further investigation. Materials and methods Study Design and Sampling Collection In total, 44 H. pylori clinical isolates collected between 2014 and 2022 were retrieved from the bacterial strain collection of the St. Petersburg Pasteur Institute, Russia. All strains were isolated from gastric biopsy specimens taken during endoscopy from the separate adult patients with chronic gastritis (n = 32), duodenal ulcer (n = 11) and gastric cancer (n = 1). The patients were 18 men (40.9%) and 26 women (59.1%); median age 45.8 ± 4.5 years (range 22-70 years). For this study, H. pylori isolates were deliberately selected based on their CLR-resistance phenotype and hence are not an epidemiologically representative sample of H. pylori primary antibiotic resistance. Endoscopic biopsy specimens were homogenized and used for the culture on H. pylori -selective medium (Columbia agar base with the addition of 5–7% defibrinated horse blood and 1% IsoVitalex solution). The bacterial cultures were incubated at 37°C under microaerophilic conditions (10% CO 2 , 85% N 2 , 5% O 2 , GasPak 100, BD Biosciences, USA). After 7–10 days incubation H. pylori colonies were identified by microscopy of Gram-staining culture smears and biochemical tests (urease, catalase, and oxidase). Final identification was made using MALDI-TOF MS (Autof MS1000, China). The H. pylori cultures were stored at − 80°C for further examination. Ethics Approval The retrospective study was approved by the Independent Ethics Committee of the St. Petersburg Pasteur Institute, Russia (protocol № 50/04-2019, 22.06.2020). All patient-related data were treated anonymously. All methods in our study were conducted in accordance with relevant guidelines and regulations. Phenotypic Drug Susceptibility Testing (DST) The susceptibility of H. pylori isolates to CLR was determined by conventional disc diffusion method. The test was performed by direct suspension of a 72-h cultivated H. pylori strains in Müller-Hinton broth adjusted to a density of 0.5 OD 600 or 1-2x10 8 cells CFU/ml according to the McFarland scale. Bacterial suspension (0.1 ml) was inoculated onto plates with fastidious Mueller Hinton agar medium supplemented with 5% defibrinated horse blood and evenly distributed over the surface with a spatula. Immediately after inoculation, 6-mm-diameter discs containing CLR (15 µg) were placed on the plate surface (1 disk per plate). After incubation at 37°C under microaerophilic conditions for 72 hours, the inhibition zone diameters were measured in millimeters (mm). H. pylori NCTC 12823 strain was used as a control. Using the linear regression analysis, Tang et al. (2021) calculated the inhibition zone diameter breakpoint to be 41 mm for clarithromycin corresponding to the EUCAST MIC cutoff value of 0.5 mg/L. Such inhibition zone diameter, obtained using the 15-µg CLR disks, also correlate well with the MIC by E-test (r = − 0.894) [ 14 ]. DNA extraction and Sequencing The total DNA of H. pylori isolates was extracted using the QIAamp DNA Mini Kit (QIAGEN GmbH, Germany) according to the manufacturer's guidelines. The DNA concentration of each sample was quantified on a Qubit 4.0 fluorometer. Whole-genome shot-gun DNA libraries were prepared using MGIEasy FS DNA Library Prep Set and then sequenced on a DNBSEQ-G50 sequencer (MGI Tech Co. Ltd, Beijing, China) in a 2*100 paired-end (PE) mode. Bioinformatics analysis The raw paired-end reads were initially analyzed using FastQC software (v.0.12.1; Babraham Institute, Cambridge, UK) to assess their acceptability for further analysis. The reads were trimmed to remove adapters and low-quality sequences (Q-score < 20) and filtered by Trim Galore! (version 0.6.7). Bacterial genomes were assembled de novo using SPAdes assembler software (version 3.13.1) [ 15 ], and the results were evaluated with QUAST (version 5.2.0) [ 16 ]. To identify plasmid-encoded resistance genes, trimmed FASTQ files were analyzed by plasmidSPAdes pipeline (Version 3.15.4) [ 17 ]. Next, high-quality reads were merged by PEAR software [ 18 ] installed via Conda environment [ 19 ] and aligned to the H. pylori 26695 reference genome, available at NCBI GenBank under the accession number AE000511.1. To evaluate the genetic variations between H. pylori isolates and reveal potential genotype-to-phenotype correlations, the insertions/deletions (indels) and SNVs were called from alignments using Snippy pipeline v.4.6.0 ( https://github.com/tseemann/snippy ) with a following parameters: minimum base quality score – 30, minimum mapping quality – 60, minimum coverage – 20, proportion for variant evidence − 0.9 [ 20 ]. CLR-resistant isolates were also assessed for any novel point mutations. Putative impact of identified variants was predicted using SnpEff software v.4.3 [ 21 ]. Aligned nucleotide sequences were visually analyzed using UGENE v. 38.1 [ 22 ]. Particular genome features were retrieved from the UniProt platform ( https://www.uniprot.org/ ) [ 23 ]. Mutations clustering analysis and a maximum likelihood tree was constructed by using the RAxML-NG is a phylogenetic tree inference tool based on core genome obtained using Snippy core [ 24 ]. The reliability of the phylogenetic tree branches was evaluated by bootstrapping method with 1000 replications. All genome assemblies were deposited to the NCBI and available under BioProject “Whole-genome sequence variations in Russian Helicobacter pylori isolates” (Accession: PRJNA1011037 ( https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1011037 ). Statistical Analysis All statistics and data visualization were performed in R programming language, Environment version 4.3.1 with the following packages: tidyverse v. 2.0.0., epitools v. 0.5–10.1, psych v. 2.3.6 [ 25 , 26 ]. The association between genotypic and phenotypic groups was screened using Chi-square and Fisher’s exact tests. The significance level of the differences was set at α = 0.05. We used two-by-two tables to calculate the odds ratio (OR). Declarations Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions N. Gladyshev - bioinformatics, phylogenetics and statistical analysis; A. Svarval - material preparation, data collection, culture methods; D. Polev / A. Saitova – bioinformatics; S. Egorova – supervision; D. Starkova – bioinformatics, study design and writing the first draft manuscript; All authors read and approved the final manuscript. Data Availability All data of this study are presented in the article and supplementary material. The datasets analysed during the current study are available in the NCBI repository and available under BioProject “Whole-genome sequence variations in Russian Helicobacter pylori isolates” (Accession: PRJNA1011037 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1011037). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3900522","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":272596803,"identity":"ab88ec6e-9992-40fa-b8ed-b8749432ebc9","order_by":0,"name":"Daria Starkova","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYDCCA2AEBDwgwqBGDiz4gLAWA6iWgmPGYMEEAloYEFo+MCc2gGh8WviOn3144MefP/LmPYePSfwwYEufH3b4IdAWOzndBuxaJM+kGxzsbTMwnHO2LU2yx0Amd+PtNAOglmRjswPYtRgcSGM4wNtgwDiDn8fYgMeALXfj7ASQlgOJ23BpOf+M4eCfPwb2IC2GfwyY0w1np3/Ar+VGGsNhHjaDxBm8PYaPeQyYE+Slc/DbInnjGcNh2Tbj5Bk8xxIfyxgcM9wgnVNwIMEAt1/4zqcxf3zzR852Bk/ygYNv/tTIy89O3/zhQ4WdHC4t2AIETBKrHATkG0hRPQpGwSgYBSMBAAALNWW5KFSGDwAAAABJRU5ErkJggg==","orcid":"","institution":"St. Petersburg Pasteur institute","correspondingAuthor":true,"prefix":"","firstName":"Daria","middleName":"","lastName":"Starkova","suffix":""},{"id":272596804,"identity":"c4a4576c-518e-4d04-9d9d-c6432801acb0","order_by":1,"name":"Nikita Gladyshev","email":"","orcid":"","institution":"St. Petersburg Pasteur institute","correspondingAuthor":false,"prefix":"","firstName":"Nikita","middleName":"","lastName":"Gladyshev","suffix":""},{"id":272596805,"identity":"1ef8c265-5f48-499f-b623-1d44bd943e5b","order_by":2,"name":"Dmitrii Polev","email":"","orcid":"","institution":"St. Petersburg Pasteur institute","correspondingAuthor":false,"prefix":"","firstName":"Dmitrii","middleName":"","lastName":"Polev","suffix":""},{"id":272596806,"identity":"75f37598-f8d5-41a6-9dda-852906ec06b8","order_by":3,"name":"Alina Saitova","email":"","orcid":"","institution":"St. Petersburg Pasteur institute","correspondingAuthor":false,"prefix":"","firstName":"Alina","middleName":"","lastName":"Saitova","suffix":""},{"id":272596807,"identity":"6d3655cf-18f9-4107-9cdb-444a2abfd562","order_by":4,"name":"Svetlana Egorova","email":"","orcid":"","institution":"St. Petersburg Pasteur institute","correspondingAuthor":false,"prefix":"","firstName":"Svetlana","middleName":"","lastName":"Egorova","suffix":""},{"id":272596808,"identity":"887d0f89-117b-4d34-88f1-b0621bb90f5f","order_by":5,"name":"Alena Svarval","email":"","orcid":"","institution":"St. Petersburg Pasteur institute","correspondingAuthor":false,"prefix":"","firstName":"Alena","middleName":"","lastName":"Svarval","suffix":""}],"badges":[],"createdAt":"2024-01-26 16:35:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3900522/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3900522/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-70977-4","type":"published","date":"2024-08-29T15:57:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51114462,"identity":"5e1b7336-dbc5-4dca-8153-dd62b163a6c4","added_by":"auto","created_at":"2024-02-14 10:58:14","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":495452,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of 44 Russian \u003cem\u003eH. pylori\u003c/em\u003e genomes with respect to CLR-resistance patterns. Maximum likelihood tree based on core SNVs alignment from draft genome mapping against \u003cem\u003eH. pylori\u003c/em\u003e 26695 reference strain and resistance profile corresponding to specific genetic determinants that differ between CLR-susceptible and CLR-resistant groups of \u003cem\u003eH. pylori\u003c/em\u003e isolates. The susceptible and resistant patterns are denoted by blue and red rectangles; the presence and absence of the specific loci are denoted by green and grey rectangles, respectively.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3900522/v1/9a401ef7ff9e74b5458dbe67.jpeg"},{"id":51114460,"identity":"5a31536b-0506-4ff9-9b7a-b80b24f9222c","added_by":"auto","created_at":"2024-02-14 10:58:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":25976,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of average number of missense/nonsense/fs nucleotide variants in virulence-associated genes among CLR-resistant and CLR-susceptible groups of \u003cem\u003eH. pylori\u003c/em\u003e isolates.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3900522/v1/ed67f1c98bd2d9a4bef7a11b.png"},{"id":51114459,"identity":"99e7d151-7680-42c9-976c-2680b489383e","added_by":"auto","created_at":"2024-02-14 10:58:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10132,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the presence of \u003cem\u003ecag\u003c/em\u003e pathogenicity island (\u003cem\u003ecag\u003c/em\u003ePAI) between resistant and susceptible groups of \u003cem\u003eH. pylori \u003c/em\u003eisolates.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3900522/v1/bb1192dffd104399da4f1887.png"},{"id":63820963,"identity":"70f46330-b5e6-4bb3-b438-059fa86106cc","added_by":"auto","created_at":"2024-09-02 16:10:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1357408,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3900522/v1/641ab8d5-934c-4cb6-bb7c-f4be91a4cc20.pdf"},{"id":51114461,"identity":"134adb97-a84f-497a-a131-f14f61bd36b8","added_by":"auto","created_at":"2024-02-14 10:58:14","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26598,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3900522/v1/799f50649a7e1248b5785d40.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"First insight into the whole-genome sequence variations in clarithromycin- resistant Helicobacter pylori clinical isolates in Russia","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eHelicobacter pylori\u003c/em\u003e infection is recognized as an important public health concern in more than 50% of the world's population and may be associated with such gastrointestinal diseases as chronic gastritis, gastric/duodenal ulcers, MALT-lymphoma, stomach cancer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVarious regimens for \u003cem\u003eH. pylori\u003c/em\u003e eradication therapy are used worldwide, while the choice of appropriate eradication therapy is determined by the local resistance patterns [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Conventional triple therapy (clarithromycin (CLR), proton pump inhibitor (PPI), metronidazole/amoxicillin) is recommended as first-line treatment in countries with less than 15% clarithromycin resistance, while quadruple therapy (bismuth, PPI, metronidazole/tetracycline/amoxicillin/CLR/tinidazole) - where CLR resistance is over 15% [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. According to the newly published Maastricht VI guidelines there are several proposals to improve the effectiveness of the existing eradication therapy regimens: extending the treatment length of triple therapy to 14 days, the usage of a higher dosage of PPI or vonoprazan and the usage of a four-drug regimen supplemented with probiotics [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo date, Russia belongs to countries with a high prevalence of \u003cem\u003eH. pylori\u003c/em\u003e infection (65\u0026ndash;90% depending on the region) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. According to a meta-analysis that summarized studies of \u003cem\u003eH. pylori\u003c/em\u003e antibiotic resistance in various regions of Russia over 10 years (2011\u0026ndash;2020), the resistance rate to CLR reached 10.39%. That indicates a low resistance of the pathogen (\u0026lt;\u0026thinsp;15%) and allows to consider the triple scheme of treatment as the first-line empirical therapy (based on the absence drug susceptibility testing (DST) data) in our country [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, the empirical therapy frequently results in treatment failure, which in turn leads to increase and development of antimicrobial resistance of the pathogen. On the other hand, the phenotypic DST approach is hardly feasible in clinical practice due to special laboratory requirements, high cost and labor intensity, let alone difficulties in isolating and growing \u003cem\u003eH. pylori\u003c/em\u003e from biopsy specimens.\u003c/p\u003e \u003cp\u003eDuring the last decade, many molecular approaches (such as polymerase chain reaction (PCR), Sanger sequencing, next-generation sequencing (NGS)) were used to identify known and novel markers for prompt prediction of the phenotypic antimicrobial resistance. It is generally accepted that point mutations A2142G and A2143G (in accordance with Taylor et al. numbering) in V domain of the 23S rRNA gene are mainly responsible for \u003cem\u003eH. pylori\u003c/em\u003e resistance to CLR [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. At the same time, point mutations outside these positions (T1942C, G1939A, C2147G, G2172T, T2182C, A2116G, A2144G/T, A2115G, G2111A, T2117C, G2141A, T2182C, T2717C, T2289C, G2224A, C2245T) vary geographically and their role in CLR resistance is not yet clear [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Based on whole genome sequencing (WGS) results, some sequence changes in \u003cem\u003erpl22\u003c/em\u003e (encoding a ribosomal protein that interacts with the 23S rRNA domains) and \u003cem\u003einfB\u003c/em\u003e (encoding a translation initiation factor, IF-2) genes in CLR-resistant \u003cem\u003eH. pylori\u003c/em\u003e strains were described [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, four gene clusters of efflux pump systems (hp0605-hp0607 (\u003cem\u003ehefABC\u003c/em\u003e), hp0971-hp0969 (\u003cem\u003ehefDEF\u003c/em\u003e), hp1327-hp1329 (\u003cem\u003ehefGHI\u003c/em\u003e), and hp1489-hp1487) were identified as resistance-nodulation-cell division (RND) family and can be also involved in development of resistance to CLR. Furthermore, some authors note that the number of single nucleotide variants (SNVs) in the RND family is significantly higher in resistant than susceptible \u003cem\u003eH. pylori\u003c/em\u003e strains [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDue to the lack of data on clarithromycin resistance patterns in Russian \u003cem\u003eH. pylori\u003c/em\u003e clinical isolates, we aimed to comprehensively investigate sequence variations by NGS- based approach to detect putative markers of clarithromycin resistance in \u003cem\u003eH. pylori\u003c/em\u003e clinical isolates from Russia and evaluate the correlation between genotypic and phenotypic DST.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic DST of study cohort\u003c/h2\u003e \u003cp\u003eFor our analysis we selected two types of isolates: resistant (inhibition zone diameter\u0026thinsp;\u0026lt;\u0026thinsp;10 mm) and susceptible (\u0026gt;\u0026thinsp;41 mm). Isolates with intermediate susceptibility were not included in our investigation. Of 44 \u003cem\u003eH. pylori\u003c/em\u003e clinical isolates, 27 were obtained from newly diagnosed patients and 17 - from previously treated patients after eradication failure.\u003c/p\u003e \u003cp\u003eAs a result, 23 out of 44 \u003cem\u003eH. pylori\u003c/em\u003e isolates were phenotypically determined as clarithromycin resistant (CLR-resistant), while 21 isolates showed susceptibility to CLR. All isolates from previously treated patients were CLR-resistant by phenotypic DST.\u003c/p\u003e \u003cp\u003ePrior to identify a putative markers of CLR resistance in a novel regions of the genome, we examined genetic variations in the full-length 23S rRNA gene (two copies), \u003cem\u003einfB\u003c/em\u003e (HP1048), \u003cem\u003erpl22\u003c/em\u003e (HP1314), as well as four clusters of efflux pump genes (HP0605-HP0607 (\u003cem\u003ehefABC\u003c/em\u003e), HP0969-HP0971 (\u003cem\u003ehefDEF\u003c/em\u003e), HP1327-HP1329 (\u003cem\u003ehefGHI\u003c/em\u003e) and HP1487-HP1489), presumably associated with CLR resistance in \u003cem\u003eH. pylori\u003c/em\u003e clinical isolates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid Detection in H. pylori isolates\u003c/h2\u003e \u003cp\u003eAmong 44 \u003cem\u003eH. pylori\u003c/em\u003e genomes analyzed in our study, no putative plasmids encoding resistance genes were found.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSingle Nucleotide Polymorphisms in 23S rRNA Genes\u003c/h2\u003e \u003cp\u003eAmong 44 \u003cem\u003eH. pylori\u003c/em\u003e isolates, 21 nucleotide substitutions were identified in 23S rRNA gene, however, only one of them- A2147G (aka A2143G)- was significantly associated with phenotypic resistance of \u003cem\u003eH. pylori\u003c/em\u003e to CLR: of the 23 CLR-resistant isolates, 13 (56.5%) carried the 2147G alleles (p\u0026thinsp;=\u0026thinsp;0.003). However, A2147 allele was related to CLR susceptibility in 95.2% (20/21) of cases, since one strain phenotypically determined as CLR-susceptible possessed A2147G mutation.\u003c/p\u003e \u003cp\u003eThe other point mutation \u0026ndash; A2146G (aka A2142G) \u0026ndash; was found in 13.0% (3/23) of CLR-resistant isolates (p\u0026thinsp;=\u0026thinsp;0.33). The A2146C allele was not found in our study. All CLR-susceptible isolates (100%; 21/21) possessed A2146 allele. None of the CLR-resistant isolates carried two A2146G/A2147G mutations simultaneously: all 2147G isolates carried the A2146 allele and, vice versa, all 2146G isolates carried the A2147 allele. Seven CLR-resistant isolates had neither A2146G nor A2147G mutations. The agreement between genotypic and phenotypic susceptibility to CLR based on both A2146G/A2147G mutations was 69.6%.\u003c/p\u003e \u003cp\u003eOut of 17 \u003cem\u003eH. pylori\u003c/em\u003e isolates from previously treated patients, 13 (76,5%) possessed either A2146G or A2147G mutations. This observation is entirely consistent with the general agreement on increasing the resistance to CLR during treatment regimen.\u003c/p\u003e \u003cp\u003eSince the \u003cem\u003eH. pylori\u003c/em\u003e genome contains two copies of the 23S rRNA gene, we assessed the presence of putative allelic variants due to mutational differences in each copy. A survey of the depth of mapped reads showed that 2146G and 2147G alleles were present in 100% reads of 15/16 CLR-resistant isolates which indicates that mutations has occurred in both copies of 23S rRNA. However, one CLR-resistant isolate (HP220) demonstrated a heterogeneity between two copies of 23S \u003cem\u003erRNA\u003c/em\u003e: the 2147G allele was detected in only 50% reads of one copy, while the second showed 100% reads of A2147 allele.\u003c/p\u003e \u003cp\u003eThe remaining mutations outside 2146/2147 positions in 23S rRNA gene were found in \u003cem\u003eH. pylori\u003c/em\u003e isolates regardless of phenotypic susceptibility (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eNucleotide Variants in rpl22, infB and RND family genes\u003c/h2\u003e \u003cp\u003eWe investigated presence of 12 genes belonging to RND family (\u003cem\u003ehefABCDEFGHI\u003c/em\u003e, HP1487-HP1489) in CLR-resistant and susceptible groups. In addition, we analysed the sequence variations in \u003cem\u003erpl22\u003c/em\u003e and \u003cem\u003einfB\u003c/em\u003e genes, and evaluated the number of variations between the two phenotypic groups, as well as the mutant (A2146G/A2147G) isolates and the wild type.\u003c/p\u003e \u003cp\u003eAs a result, we found that 12 efflux pump genes (HP0605-HP0607, HP0969-HP0971, HP1327-HP1329, HP1487-HP1489) were presented in all 44 isolates, however, we did not reveal any significant differences in either individual SNVs or total SNVs number between CLR-resistant and susceptible \u003cem\u003eH. pylori\u003c/em\u003e isolates.\u003c/p\u003e \u003cp\u003eAnalysis of sequence variations in \u003cem\u003einfB\u003c/em\u003e gene (HP1048) did not reveal any of C718T, A1899G, G160A, G60A mutations which were considered in previous studies as factors hypothetically involved in increasing of antibiotic resistance. Among all types of SNVs, only one missense substitution C2763A was significantly predominated in CLS-susceptible isolates [p\u0026thinsp;=\u0026thinsp;0.03; OR: 4.6743]. The other synonymous variant G2118A was found specifically in CLR-resistant isolates (26.1%; 6/23), however, involvement of silent mutations in phenotypic consequences is least likely (p\u0026thinsp;=\u0026thinsp;0.021). We did not find significant differences in total number of missense substitutions neither between CLR-resistant and -susceptible groups nor CLR-resistant mutant isolates (A2146G/A2147G) and the wild type.\u003c/p\u003e \u003cp\u003eThe most interesting finding among missense variations in the \u003cem\u003erpl22\u003c/em\u003e gene (HP1314) was the absence of two types of mutations \u0026minus;\u0026thinsp;3bp (GTG) deletion and 9 bp (TTCCATGTA) insertion - which have been detected in numerous studies. Instead, we identified three missense variants, none of which were associated with phenotypic resistance-susceptibility status (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eNovel Sequence Variations in Coding Regions of Russian H. pylori Isolates\u003c/h2\u003e \u003cp\u003eTo detect new putative markers of phenotypic resistance/susceptibility to CLR we conducted a global gene analysis of all \u003cem\u003eH. pylori\u003c/em\u003e isolates compared to the \u003cem\u003eH. pylori\u003c/em\u003e 26695 reference genome. We investigated nucleotide substitutions rated to high impact on biological sequence in annotation tools and evaluated their association with antibiotic phenotype. Among all genes included in our study, the majority of genomic variants were shared between resistant and susceptible groups of isolates. However, we were able to detect six novel genomic variants harbored exclusively by CLR-resistant isolates and eight variants that were predominated in either resistant or susceptible phenotypic group. These findings were summarized in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAmong OMP (Outer Membrane Protein) family genes the most interesting findings were related to \u003cem\u003eomp32\u003c/em\u003e, \u003cem\u003eomp5\u003c/em\u003e and \u003cem\u003eomp25\u003c/em\u003e genes. Missense SNV -A2C -resulting in loss of start-codon in the \u003cem\u003eomp32\u003c/em\u003e gene, conservative inframe insertion 7:insTTTTGA in the \u003cem\u003eomp5\u003c/em\u003e gene, and nsSNV C1400T in the \u003cem\u003eomp25\u003c/em\u003e gene, were detected in 21.7% (5/23) CLR-resistant isolates per each (p\u0026thinsp;=\u0026thinsp;0.049). None of these mutations were found in CLR-susceptible isolates (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResistance and susceptibilityassociated sequence variants in CDS of \u003cem\u003eH. pylori\u003c/em\u003e clinical isolates compared to the \u003cem\u003eH. pylori\u003c/em\u003e 26695 reference genome.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene (Locus)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNucleotide change\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmino acid change\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCLR-R (n\u0026thinsp;=\u0026thinsp;23)\u003c/p\u003e \u003cp\u003eN (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCLR-S (n\u0026thinsp;=\u0026thinsp;21)\u003c/p\u003e \u003cp\u003eN (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep-Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23S rRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA2146G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (13.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.23414\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA2147G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13 (56.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (4.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.00025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003einfB\u003c/em\u003e (HP1048)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG2763T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAsp921Glu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (8.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (38.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.03099\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eomp5\u003c/em\u003e (HP0227)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7:insTTTTGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3:insIleLys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (21.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.04972\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eomp32\u003c/em\u003e (HP1501)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT2G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003estart codon lost\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (21.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.04972\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eomp25\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(HP1156)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC1400T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAla467Val\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (21.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.04972\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHP0820\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e252:delCGGGT /\u003c/p\u003e \u003cp\u003e262:delGG or GGG insTTAGCACA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edelGly85-fs / delGly88-fs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7 (30.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.00943\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eflhF\u003c/em\u003e (HP1035)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG376A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAla126Thr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7 (30.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (9.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.13681\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eflaB\u003c/em\u003e (HP0115)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG783T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGlu261Asp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (26.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.02198\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eflaB\u003c/em\u003e (HP0115)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC854T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThr285Ile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (17.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.10931\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eflaB\u003c/em\u003e (HP0115)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG964A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAla322Thr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (26.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (4.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.09732\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003efliD\u003c/em\u003e (HP0752)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC1997G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePro666Arg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (4.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7 (33.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.01900\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003espaB\u003c/em\u003e (HP0600)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG98A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThr33Ile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12 (52.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 (19.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.03069\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003espaB\u003c/em\u003e (HP0600)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG400A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAla134Thr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (26.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (4.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.09732\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eres2\u003c/em\u003e (HP1521)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG1A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMet1Val\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (26.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (4.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.09732\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erecA\u003c/em\u003e (HP0153)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e360TGATACA-366insAAGCACG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAsp121Ser\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (34.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (9.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.07249\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpl10\u003c/em\u003e (HP1200)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC257T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGly86Asp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (34.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (9.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.07249\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpl10\u003c/em\u003e (HP1200)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC487A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003estop gained Glu163*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (9.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.22198\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSeveral unique frameshift (fs) alterations in HP0820 coding region were detected in 30.4% (7/23) CLR-resistant isolates. A fs deletion 252:delCGGGT was linked with another fsIndel in HP0820 onwards: deletion of two (262-263delGG) or three (262-264delGGG) nucleotides and insertion of TTAGCACA fragment. None of these mutations were detected in CLR-susceptible isolates.\u003c/p\u003e \u003cp\u003eAmong 28 flagellar family genes three nsSNVs in \u003cem\u003eflaB\u003c/em\u003e gene were found predominantly in resistant group, however only one of them (G783T) was significantly associated with resistance (p\u0026thinsp;=\u0026thinsp;0.021). Alternatively, we revealed that the prevalence of nsSNV (C1997) in the \u003cem\u003efliD\u003c/em\u003e gene was significantly higher in the CLR-susceptible rather than CLR-resistant group (33.3 vs. 4.3%, respectively) [p\u0026thinsp;=\u0026thinsp;0.019, OR: 0.1057741].\u003c/p\u003e \u003cp\u003eThree multidrug resistance protein genes (\u003cem\u003ehetA, msbA, spaB\u003c/em\u003e) were analysed in present study. As a result, the proportion of CLR-resistant \u003cem\u003eH. pylori\u003c/em\u003e isolates carried two missense substitutions (G98A and G400A) in multidrug resistance protein SpaB gene was much higher than that in CLR-susceptible isolates (52.2 vs. 19.0% [p\u0026thinsp;=\u0026thinsp;0.030; OR: 4.3874] and 26.1 vs. 4.8% [p\u0026thinsp;=\u0026thinsp;0.097; OR: 6.1535], respectively). Interestingly, using WGS analysis, Liu et al (2022) showed that the \u003cem\u003espaB\u003c/em\u003e was absent in most strains isolated from Shanghai, whereas in our study this gene was present in all 44 clinical isolates. In addition, the same report showed that four variations (S275N, D353N, 120-121delGG and ins116G117) within the integrase/recombinase gene \u003cem\u003exerD\u003c/em\u003e were significantly associated with both clarithromycin and metronidazole resistance [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the present study, S275N mutation was found without significant differences between resistant and susceptible phenotypes (82.6 vs. 80.9%; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Instead of 120-121GG deletion, we identified missense multiple substitutions of four nucleotides (117-124CTAGGATT\u0026rarr;TTAATATC) that were linked together and have prevailed in resistance (34.8%) compared to susceptible (19.0%) group, however not significantly [p\u0026thinsp;=\u0026thinsp;0.32; OR: 2.1937]. Insertion 116G117 and missense variant D353N were not detected in our study.\u003c/p\u003e \u003cp\u003eFive point substitutions within recombinational repair protein RecA gene were linked together and were carried predominantly by resistant (34.8%) rather than susceptible (9.5%) isolates [p\u0026thinsp;=\u0026thinsp;0.072; OR: 4.674359]. The missense variant (G1A) in the type III restriction enzyme gene (\u003cem\u003eres2\u003c/em\u003e) leading to the replacement of start-codon Met with alternative Val was detected predominantly in resistant group, however not significantly [p\u0026thinsp;=\u0026thinsp;0.097; OR: 0.1472963]. It is known that the replacement of standard AUG with the alternative GUG form leads to a several-fold drop in the translation efficiency [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince \u003cem\u003erpl\u003c/em\u003e are considered to be the genes with the greatest value, we initially conducted the analysis of 49 ribosomal proteins genes. As a result, we found one nsSNV (C257T) within \u003cem\u003erpl10\u003c/em\u003e gene (encodes ribosomal protein L10/50S) that was predominated in CLR-resistant group, but not significantly [p\u0026thinsp;=\u0026thinsp;0.072; OR: 4.6743]. Interestingly, in CLR-susceptible group isolates we found another nsSNV (C487A) in the same gene resulting in a stop codon. Unfortunately, only a few isolates possessed it (2/21), that does not allow us to assess the actual role of this mutation in the development of macrolide susceptibility.\u003c/p\u003e \u003cp\u003eFurther analysis of the distribution of newly detected mutations significantly associated with resistance to CLR in the A2146G/A2147G-mutated \u003cem\u003eH. pylori\u003c/em\u003e clinical isolates, revealed that 6/7 (85.7%) HP0820- mutated isolates possessed either 2146G or 2147G alleles (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Of note, more than half (63.4%) of CLR-resistant isolates with nucleotide alterations we detected in six coding regions were A2146G/A2147G-positive.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe distribution of newly detected genomic variants in A2146G/A2147G-positive and A2146G/A2147G-negative CLR-resistant \u003cem\u003eH. pylori\u003c/em\u003e clinical isolates.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene (Locus)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA2146G/A2147G- positive isolates (n\u0026thinsp;=\u0026thinsp;16), N (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA2146G/A2147G- negative isolates (n\u0026thinsp;=\u0026thinsp;7), N (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal no. of CLR-R isolates (n\u0026thinsp;=\u0026thinsp;23), N (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eomp5\u003c/em\u003e (HP0227)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3 (18.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2 (28.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5 (21.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eomp32\u003c/em\u003e (HP1501)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3 (18.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2 (28.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5 (21.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eomp25\u003c/em\u003e (HP1156)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3 (18.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2 (28.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5 (21.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHP0820\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6 (37.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1 (14.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7 (30.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eflaB\u003c/em\u003e (HP0115)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4 (25.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2 (28.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7 (30.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003espaB\u003c/em\u003e (HP0600)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7 (43.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5 (71.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12 (52.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003ePhylogenetic Analysis and Relationship Between Isolate Relatedness and CLR-Resistance Patterns in H. pylori isolates\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eBased on the core SNVs of 44 Russian \u003cem\u003eH. pylori\u003c/em\u003e genomes we constructed the phylogenetic tree based on the maximum likelihood estimation with respect to CLR-resistance patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As a result, no isolates relatedness associated with the antibiotic phenotype was revealed. Instead, these isolates had distinct resistance profiles regardless of their genetic relatedness.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDistributions of virulenceassociated and multidrug-resistance genes in H. pylori clinical isolates\u003c/h2\u003e \u003cp\u003eTo investigate whether the presence or absence of virulenceassociated genes related to phenotypic resistance \u003cem\u003eH. pylori\u003c/em\u003e to CLR, we evaluated the presence of 26 cag pathogenicity island (\u003cem\u003ecag\u003c/em\u003ePAI) genes, 28 flagellar genes, vacuolating cytotoxin gene (\u003cem\u003evacA\u003c/em\u003e), ulcer-associated restriction endonuclease and adenine specific DNA methyltransferase genes (\u003cem\u003eiceA1, hpyIM\u003c/em\u003e), urease gene cluster (\u003cem\u003eureABCIEFGH\u003c/em\u003e), gamma-glutamyltranspeptidase gene (\u003cem\u003eggt\u003c/em\u003e), virulence factor protein (\u003cem\u003emviN\u003c/em\u003e), outer membrane protein family (\u003cem\u003eomp1\u003c/em\u003e-\u003cem\u003eomp32, ompP1\u003c/em\u003e), toxin-like outer membrane protein and iron-regulated outer membrane proteins genes (\u003cem\u003efecA\u003c/em\u003e, \u003cem\u003efrpB\u003c/em\u003e), flagellar family genes (\u003cem\u003eflaA, flaB, fla, flaA1, flgH, fliF, flgI, fliE, flgB, flgC, flgG, flhA, flhF, flhB, fliP, fliR, fliH, fliI, flgE, flgK, fliD, motA, motB, fliG, fliM, flaG, hpaA, fliN,)\u003c/em\u003e, heat shock protein family genes (\u003cem\u003ehslU\u003c/em\u003e, \u003cem\u003ehslV\u003c/em\u003e, \u003cem\u003ehtpX\u003c/em\u003e, \u003cem\u003eibpB\u003c/em\u003e) in two groups of \u003cem\u003eH. pylori\u003c/em\u003e isolates. For the first time, we also compared the average number of missense/nonsense/frameshift nucleotide variants in 108 virulence-associated genes between two phenotypic groups of \u003cem\u003eH. pylori\u003c/em\u003e isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs a result, we found that among all the genes mentioned above, only the presence of \u003cem\u003ecag\u003c/em\u003ePAI genes in the CLR-susceptible group was higher than that in the CLR-resistant group, but not significantly (p\u0026thinsp;=\u0026thinsp;0.42) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Interestingly, that neither of Russian isolates contained \u003cem\u003ecag2\u003c/em\u003e (HP0521) gene and all 44 \u003cem\u003eH. pylori\u003c/em\u003e isolates regardless of antimicrobial susceptibility harbored A589G mutation in the \u003cem\u003ecag13\u003c/em\u003e (HP0534) gene leading to the loss of stop codon and splicing with coding region HP0533 (\u0026ldquo;-\u0026ldquo; chain).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLiu Y. et al. (2022) found that the average frequency of the iron-regulated OMP genes (\u003cem\u003efecA\u003c/em\u003e and \u003cem\u003efrpB\u003c/em\u003e) in the CLR-resistant group was significantly lower than that in the CLR-susceptible group (p\u0026thinsp;=\u0026thinsp;0.01) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Unfortunately, we did not find any significant differences in either the average frequency of these genes or the average number of mutations in \u003cem\u003efecA\u003c/em\u003e and \u003cem\u003efrpB\u003c/em\u003e genes between both phenotypic groups.\u003c/p\u003e \u003cp\u003eAs for the remaining genes, we found no statistical differences in either the frequency of these genes or the average number of mutations between two phenotypic groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite insufficient knowledge and interregional dynamic differences in the resistance of \u003cem\u003eH. pylori\u003c/em\u003e to the main antimicrobials, the level of the primary clarithromycin resistance in Russia is low (11.8%) and allows to consider a standard triple eradication regimen as a first-line therapy. Thus, routine \u003cem\u003eH. pylori\u003c/em\u003e susceptibility testing usually is not provided in clinical settings and empirical therapy can cause eradication failures and increasing resistance rate. According to the latest data (2020), the prevalence of clarithromycin resistance in Moscow reached 10.87%, Smolensk \u0026minus;\u0026thinsp;5.74%, Kazan \u0026minus;\u0026thinsp;10.0% and in St. Petersburg \u0026minus;\u0026thinsp;22.26% [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Such difference indicates the wide variation in resistance rates in Russia, highlighting the need to ongoing examination of CLR-resistance in each geographic region.\u003c/p\u003e \u003cp\u003eFor our study, we used conventional disc diffusion method to detect susceptibility of \u003cem\u003eH. pylori\u003c/em\u003e isolates to CLR. Although the EUCAST has recommended the E-test for MIC breakpoints detection, the high cost of E-test strips makes it difficult to use. Recently, Tang et al. (2020) assessed the disk diffusion technique against E-test and estimated that susceptibility agreement between two methods for clarithromycin is 96.0% [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. It was demonstrated that disc diffusion is simple and affordable alternative to E-test for \u003cem\u003ein vitro\u003c/em\u003e CLR susceptibility testing [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mechanism of drug resistance in \u003cem\u003eH. pylori\u003c/em\u003e is believed to be due to mutations (missense/ nonsense substitutions, fs insertions/deletions) located in the chromosome and not related to horizontal transfer of plasmids or other mobile elements [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. It is generally accepted that \u003cem\u003eH. pylori\u003c/em\u003e resistance to clarithromycin is associated with several point mutations A2146C/G and A2147G (in accordance to \u003cem\u003eH. pylori\u003c/em\u003e reference strain 26695 numbering) in the V domain of 23S rRNA gene that decreases the affinity of drug binding to the ribosome by disrupting the peptidyl transferase loop conformation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In the present study, A2147G was the most frequent mutation found in CLR-resistant \u003cem\u003eH. pylori\u003c/em\u003e isolates (56.5%) and highly correlated with CLR resistance. However, A2146G mutation has been detected in only 13% CLR-resistant isolates that does not allow us to assess the actual role of this mutation in the resistance development. These findings are consistent with prior studies conducted in Europe that demonstrated that A2146G/A2147G mutations were present in the genome of 60\u0026ndash;90% CLR-resistant isolates [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The high rate of CLR-resistant isolates without A2146G/A2147G mutations (30.4%) in our study indicates that these isolates may have genetic determinants other than 23S rRNA gene responsible for pathogen resistance development.\u003c/p\u003e \u003cp\u003eNotably, one isolate phenotypically determined as CLR-susceptible in our study possessed A2147G mutation. Such case may indicate the presence of mixed CLR-susceptible and -resistant isolates. As shown by our previous study, this isolate was positive for mixed combinations of \u003cem\u003evacA\u003c/em\u003e alleles (s1s2 and i1i2) indicating the coexistence of several genetically different \u003cem\u003eH. pylori\u003c/em\u003e isolates in gastric sites as a result of mixed infection [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, there are two copies of the 23S rRNA operon within the \u003cem\u003eH. pylori\u003c/em\u003e genome. Sequence differences between both copies have rarely been studied and data on whether 2146/2147- mutations in single or both copies of 23S rRNA are necessary for the strain resistance to occur are still controversial [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this study, 16 CLR-resistant isolates possessed A2146G/A2147G mutations, and only one of them demonstrated a heterogeneity between two copies of 23S rRNA: the 2147G allele was detected in 50% reads of one copy, while the second copy showed 100% reads of A2147 allele. This observation suggested that \u003cem\u003eH. pylori\u003c/em\u003e isolates should be considered resistant if they have the mutation in at least one copy of 23S rRNA gene.\u003c/p\u003e \u003cp\u003eAnother possible mechanism for CLR resistance is multidrug efflux pump systems. It has been reported that four conserved RND families of efflux pump transporters may be responsible for development of macrolide resistance in \u003cem\u003eH. pylori\u003c/em\u003e by reducing intracellular antimicrobial concentration [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. As shown by Iwamoto et al. (2014), there is a significant difference in the number of SNVs in \u003cem\u003ehefABC\u003c/em\u003e (hp0605-hp0607) cluster between CLR-resistant and susceptible isolates, as well as CLR-resistant isolates are more prone to single-nucleotide variants in all four clusters of efflux genes, with significant differences for cluster \u003cem\u003ehefABC\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Moreover, Chen et al. (2018) revealed that the number of mutations in the RND family was significantly higher in \u003cem\u003eH. pylori\u003c/em\u003e isolates harboring the A2147G point mutation in 23S rRNA gene [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In our study, we did not find any significant differences in number of SNVs among 12 genes either between CLR-resistant and susceptible phenotypes or between isolates carried A2147G mutation and a wild type. Noteworthy, among all types of nucleotide substitutions, we found only single-nucleotide variants: no deletions or insertions were detected in four clusters of efflux genes. This result is consistent with the hypothesis that SNVs in efflux pump genes may contribute to CLR resistance through a synergistic effect between 23S rRNA mutations and efflux pumps.\u003c/p\u003e \u003cp\u003eOther genes assumed to be involved in CLR resistance are \u003cem\u003erpl22\u003c/em\u003e and \u003cem\u003einfB\u003c/em\u003e. In fact, several point mutations in \u003cem\u003einfB\u003c/em\u003e (C718T, A1899G, G160A, G60A) as well as 9-bp insertion and/or 3-bp deletion in \u003cem\u003erpl22\u003c/em\u003e genes have been described, which, in combination with 23S rRNA mutations, are predicted to increase the resistance level of \u003cem\u003eH. pylori\u003c/em\u003e isolates [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Interestingly, no indels in \u003cem\u003erpl22\u003c/em\u003e gene were detected in our study: instead, we found 3-bp deletion in \u003cem\u003einfB\u003c/em\u003e gene primarily in CLR-susceptible isolates, but not significantly. Notably, we did not find any of point mutations in \u003cem\u003einfB\u003c/em\u003e that were detected in other studies. Among all SNVs in \u003cem\u003einfB\u003c/em\u003e, no substitutions unique for resistant isolates were identified. Altogether, these data provide further evidence that high genetic diversity of \u003cem\u003eH. pylori\u003c/em\u003e populations is the crucial factor in the searching for putative determinants of resistance.\u003c/p\u003e \u003cp\u003eGiving the enormous genetic diversity of \u003cem\u003eH. pylori\u003c/em\u003e and high heterogeneity of resistance genotypes across geographical regions, the determining of novel resistance patterns and interpretation obtained results appears to be challenging. WGS-based approaches have enabled to provide a comprehensive view of bacterial genotypes and are particularly useful for tracking novel genetic determinants responsible for antimicrobial resistance of the pathogen [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo identify novel mutations potentially contributing to antibiotic resistance in \u003cem\u003eH. pylori\u003c/em\u003e isolates we next sought to analyze genetic variants in other gene families presumably associated with virulence and multidrug resistance. Among all the genes examined, the largest number of nucleotide alterations found specifically in CLR-resistant group, was observed among the OMP genes family: \u003cem\u003eomp5\u003c/em\u003e, \u003cem\u003eomp25\u003c/em\u003e (HopI) and \u003cem\u003eomp32\u003c/em\u003e (HopW). It is well known that OMPs are associated with macrolide resistance in Gram-negative bacteria. Lin et al. (2023) suggested that the alteration of outer membrane proteins is a potential mechanism involved in CLR resistance development in \u003cem\u003eH. pylori\u003c/em\u003e, but the specific function and mechanism of action of these proteins need to be further studied [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Interestingly, Thompson et al. (2003) showed the close coexpression of the \u003cem\u003ecagA\u003c/em\u003e gene with the \u003cem\u003eomp5\u003c/em\u003e and \u003cem\u003eomp29\u003c/em\u003e genes, suggesting these OMPs are involved in the secretion and/or activation of CagA [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Among all identified variants in the OMP family genes, the missense substitution in the anion-selective porin Omp32, leading to the loss of the start codon, had the greatest impact on the gene. It has been reported that expression of the \u003cem\u003eomp32\u003c/em\u003e gene was down-regulated by amoxicillin [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. It is unknown whether the accumulation of such mutations may indirectly be involved in the resistance development, hence its hypothetical impact needs to be further investigated.\u003c/p\u003e \u003cp\u003eAnother interesting finding: two frameshift alterations that were significantly associated with resistance to CLR are in coding region HP0820 (\u003cem\u003ecj1247c\u003c/em\u003e in \u003cem\u003eC. jejuni\u003c/em\u003e), which Porcelli et al. (2013) described as predicted leaderless mRNA in all species containing this gene, which is always upstream of the \u003cem\u003euvrC\u003c/em\u003e DNA repair gene. The genes translated from the leaderless mRNAs encode the proteins predicted to be involved in stress responses, the outer membrane efflux protein and the predicted multidrug efflux pump [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. It is well known that antibiotics create conditions that favor selection of beneficial mutants with elevated mutation rates and this is most often linked to the DNA repair system and SOS response, that can lead to increased activity of associated multi-drug efflux pumps. Given the fact that \u003cem\u003eH. pylori\u003c/em\u003e has a defective DNA mismatch repair system (lacking genes as \u003cem\u003emutS\u003c/em\u003e, \u003cem\u003emutL\u003c/em\u003e, and \u003cem\u003emutH\u003c/em\u003e implicated in this pathway), as well as lack of the SOS regulon, the antibiotic-induced mutagenesis in various types of regulatory genes increases the genetic diversity of bacterial populations and, as a consequence, enhances the evolution of antibiotic resistance [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMissense substitutions in three different flagellar genes \u003cem\u003eflaB\u003c/em\u003e, \u003cem\u003eflhF\u003c/em\u003e and \u003cem\u003efliD\u003c/em\u003e may also be candidates indirectly involved in underlying mechanisms of resistance and/or susceptibility of \u003cem\u003eH. pylori\u003c/em\u003e. The pathogenesis of \u003cem\u003eH. pylori\u003c/em\u003e infection is thought to be determined in part by flagellar motility, which has a direct impact on colonization, inflammation, and immune evasion. Flagellar filament FlaB and the filament-capping protein FliD are one of the main structural proteins of the \u003cem\u003eH. pylori\u003c/em\u003e flagellum and play an important role in bacterial motility [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. FlhF controls flagellar patterns in many bacteria and is required for flagellum assembly and localization [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Even though the actual impact of these mutations on resistance development is difficult to assess, the mutational buildup in the genome may be a result of response to antimicrobials and an important predictor of treatment failure.\u003c/p\u003e \u003cp\u003eAs for relationship between virulence-associated genes and phenotypic resistance, we determined that there is no significant correlation between the presence of main virulence factors and resistance to CLR. On the one hand, our findings are consistent with majority of previous reports [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, it is worth noting that in the CLR-resistant group the number of \u003cem\u003ecagA\u003c/em\u003e-negative isolates predominated twofold compared to those in the CLR-susceptible group (10/23 vs. 5/21). Thus, on the other hand, this observation in consistent with other findings showing that clarithromycin resistance was obviously higher in \u003cem\u003ecagA-\u003c/em\u003enegative than in \u003cem\u003ecagA\u003c/em\u003e-positive isolates [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present study has several limitations. First, despite the high level of agreement between disc diffusion method and E-test, the main drawback in our study is the inability to determine MIC values, which does not allow us to reveal a causal relationship between mutations and high/low MIC values of phenotypic resistant isolates. Second, the sample size is rather small and obtained results do not necessarily reflect resistant patterns in general Russian \u003cem\u003eH. pylori\u003c/em\u003e population. Third, we did not provide further experimental analysis to confirm the involvement of newly detected mutations in CLR resistance development. Nevertheless, despite these limitations obtained data provided the foundation for the future investigations.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study presents the first WGS insight into genetic diversity of \u003cem\u003eH. pylori\u003c/em\u003e in Russia with a particular focus on the molecular basis of drug resistance. For the first time, the average number of missense/nonsense/frameshift nucleotide variants in 108 virulence-associated genes between resistant and susceptible groups were compared. It has been shown that among all genetic variants we obtained, the combination of mutations A2146G/A2147G in the 23S rRNA gene is the most reliable for prediction of phenotypic DST. However, sequencing data did not reveal the involvement of nucleotide variants in any efflux pump, \u003cem\u003einfB\u003c/em\u003e or \u003cem\u003erpl22\u003c/em\u003e genes in phenotypic prediction of CLR resistance, emphasizing the enormous genetic diversity of \u003cem\u003eH. pylori\u003c/em\u003e populations. The novel mutations in other genes were described in our study as potential markers for the resistance development. Among them, the most prominent mutation is frameshift deletion (252:CGGGT) in HP0820 coding region, which is a good candidate for further investigation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design and Sampling Collection\u003c/h2\u003e \u003cp\u003eIn total, 44 \u003cem\u003eH. pylori\u003c/em\u003e clinical isolates collected between 2014 and 2022 were retrieved from the bacterial strain collection of the St. Petersburg Pasteur Institute, Russia. All strains were isolated from gastric biopsy specimens taken during endoscopy from the separate adult patients with chronic gastritis (n\u0026thinsp;=\u0026thinsp;32), duodenal ulcer (n\u0026thinsp;=\u0026thinsp;11) and gastric cancer (n\u0026thinsp;=\u0026thinsp;1). The patients were 18 men (40.9%) and 26 women (59.1%); median age 45.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 years (range 22-70 years). For this study, \u003cem\u003eH. pylori\u003c/em\u003e isolates were deliberately selected based on their CLR-resistance phenotype and hence are not an epidemiologically representative sample of \u003cem\u003eH. pylori\u003c/em\u003e primary antibiotic resistance.\u003c/p\u003e \u003cp\u003eEndoscopic biopsy specimens were homogenized and used for the culture on \u003cem\u003eH. pylori\u003c/em\u003e-selective medium (Columbia agar base with the addition of 5\u0026ndash;7% defibrinated horse blood and 1% IsoVitalex solution). The bacterial cultures were incubated at 37\u0026deg;C under microaerophilic conditions (10% CO\u003csub\u003e2\u003c/sub\u003e, 85% N\u003csub\u003e2\u003c/sub\u003e, 5% O\u003csub\u003e2\u003c/sub\u003e, GasPak 100, BD Biosciences, USA). After 7\u0026ndash;10 days incubation \u003cem\u003eH. pylori\u003c/em\u003e colonies were identified by microscopy of Gram-staining culture smears and biochemical tests (urease, catalase, and oxidase). Final identification was made using MALDI-TOF MS (Autof MS1000, China). The \u003cem\u003eH. pylori\u003c/em\u003e cultures were stored at \u0026minus;\u0026thinsp;80\u0026deg;C for further examination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEthics Approval\u003c/h2\u003e \u003cp\u003eThe retrospective study was approved by the Independent Ethics Committee of the St. Petersburg Pasteur Institute, Russia (protocol № 50/04-2019, 22.06.2020). All patient-related data were treated anonymously. All methods in our study were conducted in accordance with relevant guidelines and regulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic Drug Susceptibility Testing (DST)\u003c/h2\u003e \u003cp\u003eThe susceptibility of \u003cem\u003eH. pylori\u003c/em\u003e isolates to CLR was determined by conventional disc diffusion method. The test was performed by direct suspension of a 72-h cultivated \u003cem\u003eH. pylori\u003c/em\u003e strains in M\u0026uuml;ller-Hinton broth adjusted to a density of 0.5 OD\u003csub\u003e600\u003c/sub\u003e or 1-2x10\u003csup\u003e8\u003c/sup\u003e cells CFU/ml according to the McFarland scale. Bacterial suspension (0.1 ml) was inoculated onto plates with fastidious Mueller Hinton agar medium supplemented with 5% defibrinated horse blood and evenly distributed over the surface with a spatula. Immediately after inoculation, 6-mm-diameter discs containing CLR (15 \u0026micro;g) were placed on the plate surface (1 disk per plate). After incubation at 37\u0026deg;C under microaerophilic conditions for 72 hours, the inhibition zone diameters were measured in millimeters (mm). \u003cem\u003eH. pylori\u003c/em\u003e NCTC 12823 strain was used as a control. Using the linear regression analysis, Tang et al. (2021) calculated the inhibition zone diameter breakpoint to be 41 mm for clarithromycin corresponding to the EUCAST MIC cutoff value of 0.5 mg/L. Such inhibition zone diameter, obtained using the 15-\u0026micro;g CLR disks, also correlate well with the MIC by E-test (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.894) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and Sequencing\u003c/h2\u003e \u003cp\u003eThe total DNA of \u003cem\u003eH. pylori\u003c/em\u003e isolates was extracted using the QIAamp DNA Mini Kit (QIAGEN GmbH, Germany) according to the manufacturer's guidelines. The DNA concentration of each sample was quantified on a Qubit 4.0 fluorometer. Whole-genome shot-gun DNA libraries were prepared using MGIEasy FS DNA Library Prep Set and then sequenced on a DNBSEQ-G50 sequencer (MGI Tech Co. Ltd, Beijing, China) in a 2*100 paired-end (PE) mode.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics analysis\u003c/h2\u003e \u003cp\u003eThe raw paired-end reads were initially analyzed using FastQC software (v.0.12.1; Babraham Institute, Cambridge, UK) to assess their acceptability for further analysis. The reads were trimmed to remove adapters and low-quality sequences (Q-score\u0026thinsp;\u0026lt;\u0026thinsp;20) and filtered by Trim Galore! (version 0.6.7). Bacterial genomes were assembled de novo using SPAdes assembler software (version 3.13.1) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and the results were evaluated with QUAST (version 5.2.0) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo identify plasmid-encoded resistance genes, trimmed FASTQ files were analyzed by plasmidSPAdes pipeline (Version 3.15.4) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Next, high-quality reads were merged by PEAR software [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] installed via Conda environment [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and aligned to the \u003cem\u003eH. pylori\u003c/em\u003e 26695 reference genome, available at NCBI GenBank under the accession number AE000511.1. To evaluate the genetic variations between \u003cem\u003eH. pylori\u003c/em\u003e isolates and reveal potential genotype-to-phenotype correlations, the insertions/deletions (indels) and SNVs were called from alignments using Snippy pipeline v.4.6.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/tseemann/snippy\u003c/span\u003e\u003cspan address=\"https://github.com/tseemann/snippy\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with a following parameters: minimum base quality score \u0026ndash; 30, minimum mapping quality \u0026ndash; 60, minimum coverage \u0026ndash; 20, proportion for variant evidence \u0026minus;\u0026thinsp;0.9 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. CLR-resistant isolates were also assessed for any novel point mutations. Putative impact of identified variants was predicted using SnpEff software v.4.3 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Aligned nucleotide sequences were visually analyzed using UGENE v. 38.1 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Particular genome features were retrieved from the UniProt platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Mutations clustering analysis and a maximum likelihood tree was constructed by using the RAxML-NG is a phylogenetic tree inference tool based on core genome obtained using Snippy core [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The reliability of the phylogenetic tree branches was evaluated by bootstrapping method with 1000 replications.\u003c/p\u003e \u003cp\u003eAll genome assemblies were deposited to the NCBI and available under BioProject \u0026ldquo;Whole-genome sequence variations in Russian \u003cem\u003eHelicobacter pylori\u003c/em\u003e isolates\u0026rdquo; (Accession: PRJNA1011037 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/bioproject/PRJNA1011037\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1011037\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistics and data visualization were performed in R programming language, Environment version 4.3.1 with the following packages: tidyverse v. 2.0.0., epitools v. 0.5\u0026ndash;10.1, psych v. 2.3.6 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe association between genotypic and phenotypic groups was screened using Chi-square and Fisher\u0026rsquo;s exact tests. The significance level of the differences was set at α\u0026thinsp;=\u0026thinsp;0.05. We used two-by-two tables to calculate the odds ratio (OR).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting Interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eN. Gladyshev - bioinformatics, phylogenetics and statistical analysis;\u003c/p\u003e\n\u003cp\u003eA. Svarval - material preparation, data collection, culture methods;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD. Polev / A. Saitova \u0026ndash; bioinformatics;\u003c/p\u003e\n\u003cp\u003eS. Egorova \u0026ndash; supervision;\u003c/p\u003e\n\u003cp\u003eD. Starkova \u0026ndash; bioinformatics, study design and writing the first draft manuscript;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData Availability\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll data of this study are presented in the article and supplementary material.\u003c/p\u003e\n\u003cp\u003eThe datasets analysed during the current study are available in the NCBI repository and available under BioProject \u0026ldquo;Whole-genome sequence variations in Russian \u003cem\u003eHelicobacter pylori\u003c/em\u003e isolates\u0026rdquo; (Accession: PRJNA1011037 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1011037).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMannion A, Dzink-Fox J, Shen Z, Piazuelo MB, Wilson KT, Correa P, Peek RM Jr, Camargo MC, Fox JG (2021) Helicobacter pylori Antimicrobial Resistance and Gene Variants in High- and Low-Gastric-Cancer-Risk Populations. 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Rev Inst Med Trop Sao Paulo 60:e25. doi: 10.1590/s1678-9946201860025\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eOktem-Okullu S, Cekic-Kipritci Z, Kilic E, Seymen N, Mansur-Ozen N, Sezerman U, Gurol Y (2020) Analysis of Correlation between the Seven Important Helicobacter pylori Virulence Factors and Drug Resistance in Patients with Gastritis. Gastroenterol Res Pract 2020:3956838. doi: 10.1155/2020/3956838\u003c/li\u003e\n \u003cli\u003eHaddadi MH, Negahdari B, Asadolahi R, Bazargani A (2020) Helicobacter pylori antibiotic resistance and correlation with cagA motifs and homB gene. Postgrad Med 132(6):512-520. doi: 10.1080/00325481.2020.1753406\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBrennan DE, Dowd C, O\u0026apos;Morain C, McNamara D, Smith SM (2018) Can bacterial virulence factors predict antibiotic resistant Helicobacter pylori infection? World J Gastroenterol 24(9):971-981. doi: 10.3748/wjg.v24.i9.971\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3900522/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3900522/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eClarithromycin (CLR) is a key drug to treat \u003cem\u003eHelicobacter pylori\u003c/em\u003e infection, however the data on CLR resistance patterns in Russia are missing. Here, we applied next-generation sequencing (NGS)-based approach to \u003cem\u003eH. pylori\u003c/em\u003e clinical isolates from Russia to comprehensively investigate sequence variations, identify putative markers of CLR resistance and correlate them with phenotypic drug susceptibility testing (DST). The phenotypic susceptibility of 44 \u003cem\u003eH. pylori\u003c/em\u003e isolates (2014\u0026ndash;2022) to CLR was determined by disc diffusion method. All isolates were undergone whole-genome sequencing and submitted to GenBank. Twenty-three \u003cem\u003eH. pylori\u003c/em\u003e isolates were phenotypically CLR-resistant and 21- were CLR-susceptible. Based on complete sequence analysis, we showed that among all genetic variants, the combination of mutations A2146G/A2147G in the 23S rRNA gene is the most reliable for prediction of phenotypic DST. For the first time, the average number of nucleotide variants in 108 virulence-associated genes between resistant and susceptible groups were compared. Moreover, this study presents the first WGS insight into genetic diversity of \u003cem\u003eH. pylori\u003c/em\u003e in Russia with a particular focus on the molecular basis of drug resistance: the novel mutations were described as potential markers for the resistance development. Of these, the most prominent was frameshift deletion in HP0820 coding region, which is a good candidate for further investigation.\u003c/p\u003e","manuscriptTitle":"First insight into the whole-genome sequence variations in clarithromycin- resistant Helicobacter pylori clinical isolates in Russia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-14 10:58:09","doi":"10.21203/rs.3.rs-3900522/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-12T04:00:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-11T15:46:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-10T11:22:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-08T13:28:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-07T00:03:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"09aca2bd-e96e-474c-bbb3-b7e7ed28811b","date":"2024-03-26T09:40:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5a7b14a2-3e07-44c5-a878-f092fd9e4e0c","date":"2024-03-26T07:43:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"05324a42-014f-4ff1-948e-da9bcf44f817","date":"2024-03-26T07:42:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"723dffd5-aad5-47fa-8213-5c766ccbf633","date":"2024-03-26T07:11:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-26T07:05:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-19T11:06:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-02-13T09:03:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-13T06:38:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-01-26T16:32:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"af48f3f4-8bf7-4b0f-a2cd-b5ac1c3ae5bf","owner":[],"postedDate":"February 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28730986,"name":"Biological sciences/Molecular biology"},{"id":28730987,"name":"Biological sciences/Genetics/Microbial genetics"},{"id":28730988,"name":"Biological sciences/Genetics/Mutation"}],"tags":[],"updatedAt":"2024-09-02T16:02:06+00:00","versionOfRecord":{"articleIdentity":"rs-3900522","link":"https://doi.org/10.1038/s41598-024-70977-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-08-29 15:57:37","publishedOnDateReadable":"August 29th, 2024"},"versionCreatedAt":"2024-02-14 10:58:09","video":"","vorDoi":"10.1038/s41598-024-70977-4","vorDoiUrl":"https://doi.org/10.1038/s41598-024-70977-4","workflowStages":[]},"version":"v1","identity":"rs-3900522","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3900522","identity":"rs-3900522","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}