Analysis of drug resistance in Helicobacter pylori by complete genome sequencing in Bama County, Guangxi, China

The resistance rate of clinical Helicobacter pylori (Hp) isolates has increased, however, the mechanism of drug resistance is unclear. In this study, we isolated drug-resistant Hp strains isolated from different areas and different populations of China for genomic analysis. The aim of this study was to investigate drug resistance in Hp from Bama County, Guangxi, China. Minimal inhibitory concentrations (MICs) of clarithromycin, metronidazole and levooxacin were determined and complete genome sequencing was performed with annotation. The presence of hp1181 and hp1184 genes was detected by RT-PCR. The relationships between hp1181, hp1184 and clarithromycin resistance were conrmed by gene mutation and drug-resistant strains. Three drug-resistant Hp strains were isolated from patients with gastritis in Bama County. The strains showed a high degree of homology with hp26695 through complete genome detection and identication. Differences in genome sequences, gene quantity and gene characteristics were detected amongst the three strains. Prediction and analysis of the function on drug-resistant genes indicated that the RNA expression of hp1181 and hp1184 increased in the three strains that were the same in the articially induced clarithromycin-resistant bacteria. After gene knockout, the drug sensitivity of the strains increased signicantly. In summary, The expressions of the genes hp1184 and hp1181 were associated with clarithromycin resistance in the Hp from Bama, Guangxi.


Introduction
Helicobacter pylori (Hp) is recognized as an important human pathogen that colonizes the gastric mucus resulting in super cial gastritis, atrophic gastritis and gastric cancer [1][2][3] . Present treatments for Hp infection include proton pump inhibitors or bismuth in combination with amoxicillin or metronidazole and clarithromycin [4][5] . The rate of drug resistance is increasing due to a wide range of use of antibiotics and high resistance rates to clarithromycin, metronidazole and levo oxacin are associated with the failure of Hp eradication [6][7][8] . The World Health Organization (WHO) gave clarithromycin-resistant Hp as a high priority bacterium for antibiotic research and development [9] .
At present, the mechanism of antibiotic resistance of Hp is not completely understood [10][11] . It is widely accepted that the resistance to these antimicrobials is related to mutations in Hp gene: clarithromycin-resistant strains present three-point mutations in the region of domain V of 23S ribosomal RNA (rRNA): A2142G, A2142C and A2143G [12][13] . In addition to the mutations, the e ux pump cluster is also involved in the development of resistance to clarithromycin [14][15] . Moreover, there may be gene mutation sites that are not yet known and so the mechanism of drug resistance warrants further study. We isolated, cultured Hp from the population in Bama County, which is a township known for the longevity of its residents in Guangxi, and randomly selected three strains of multiple drug-resistant Hp with resistance to clarithromycin. Complete genome sequences were analyzed to ascertain the genomic characteristics of the strains and to reveal the underlying mechanism of drug resistance in Hp.

Isolation and culture of Hp
This study had received a strict medical ethics review from Youjiang Medical University for Nationalities. Written informed consent was obtained from each patient. Gastric mucosa tissue samples were collected from the People's Hospital of Bama Yao Autonomous County in patients' gastric body and pylorus with gastritis or gastric ulcers. Isolation and culture of Hp were performed at the Prevention and treatment Research Center for Resistance Microbiological, Youjiang Medical University for Nationalities. Patients investigated had not taken any antibiotics for at least four weeks before examination. The isolation and identi cation of Hp were performed as previously described [16][17] . The culture conditions used Columbia agar plates containing 5% fresh de brinated sheep blood. The microaerophilic conditions included 5% O2, genomic DNA, bacterial arti cial chromosomes or long-length PCR products were sheared into smaller fragments by CovarisS/E210 or using a Bioruptor. The overhangs resulting from fragmentation were converted into blunt ends using T4 DNA polymerase, Klenow Fragment and T4 Polynucleotide Kinase. After adding an 'A' base to the 3' end of the blunt phosphorylated DNA fragments, adapters were ligated to the ends of the DNA fragments. The desired fragments were puri ed though gel-electrophoresis, selectively enriched and ampli ed by PCR. The index tag was introduced into the adapter at the PCR stage as appropriate and a library quality test was performed. Finally, the quali ed BSlibrary was used for sequencing. Genomic component and gene function analyses were performed including gene prediction, tRNA, sRNA, genes annotation and the predicted open reading frames (ORFs) by GO.
Drug resistance gene detection Drug resistance genes were predicted based on the results of the complete genome sequence analysis and selected for detection by RT-PCR.
The reaction for cDNA synthesis was held at 25 ℃ for 10 min, 42 ℃ for 60 min and 99 ℃ for 5 min. The reaction consisted of 32 cycles with each cycle composed of 1 min at 95 ℃, 4 min at 56 ℃ and 7 min at 70 ℃. After a nal extension of 15 min at 72 ℃, the RT-PCR products were visualized by electrophoresis on 1% agarose gel and 15% acrylamide gel with a 200-bp ladder size marker.
Knockout of mutant genes HP1181 and HP 1184 knockout mutants were constructed by insertion of the KAN resistance cassette. Double-knockout mutants were made by natural transformation of the KAN resistance cassette with pBSII KS (as presented by Bi Hongkai, Laboratory of Nanjing Medical University) containing an internal fragment interrupted with a cat cassette from pAV35, with selection for both KAN-and CHL-resistant colonies. Insertion of the KAN and cat resistance cassette at the desired locations in the Hp putative e ux genes was con rmed by PCR.

Induction of drug resistance
The MIC of clarithromycin to Hp26695 was detected. Drug resistance was induced by 1/4MIC. The culture medium was changed every two days and MIC was detected every four days. The concentration of induced drug was changed with MIC.

Bacterial resistance
Three drug-resistant strains isolated and identi ed by Gram stain, urease, oxidase, catalase activity testing, and urease gene PCR testing: the drug resistance information of these strains is summarized in Table 1.

Bacterial sequence information
Based on the valid data from the previous sequencing platform, the CleanData could be assembled for each sample and the optimal assembly results were obtained after multiple adjustments. The assembly sequence was analyzed by correcting single base, circular judgment and plasmid comparison. The results of the genome assembly statistics of each sample are shown in Table 2. These three strains have been uploaded to the NCBI Biosample database: Hpbs1(https://www.ncbi.nlm.nih.gov/biosample/?term=SAMN10461767) Hpbs2(https://www.ncbi.nlm.nih.gov/biosample/?term=SAMN10663081), and Hpbs3(https://www.ncbi.nlm.nih.gov/biosample/? term=SAMN10663175),

Gene information
Gene prediction was applied to determine gene composition. The statistics are shown in Table 3 below.

Analysis of drug resistance gene database
The drug resistance gene numbers of three strains were different in the CARD drug resistance database, which are 14, 13 and 15 genes, respectively. However, after sorting, it was found that some genes were repetitive. The speci c numbers and characteristics of genes are shown in the Tables 6 and 7. NP_207975.1 and NP_207972.1 were e ux pump genes of 26695 strain, i.e. hp1181 and hp1184 genes. Their drug resistance was veri ed by RT-PCR as illustrated in Fig. 2. After knocking out the drug resistance gene, drug sensitivity was signi cantly improved as shown in Fig. 3.

Identi cation of 23S rRNA gene mutations
Three strains were resistant to clarithromycin, so we analyzed and identi ed the sites of clarithromycin-resistant mutations. We found that three strains had mutations in A2142G, A2143G, G2144T, and some had mutations in other sites, as shown in Table 8.

Gene mutation induced in drug-resistant strains
After induction with clarithromycin, Hp26695 drug resistance was enhanced on the 12th day, reached the highest level on day 16 and increased to 8μg/ml on the 24th day. The expressions of hp1181 and hp1184 were also increased with increasing clarithromycin resistance, especially hp1184, as shown in Fig. 4. Only A2142G and A 2143G mutations were detected in 23S RNA, with no other mutation sites being found as shown in Table 9. These data indicated that these two genes may be involved early in the regulation of clarithromycin resistance.

Discussion
The treatment of Hp remains based on bismuth tetralogy at present. Hp is eradicated clinically using common antibiotics including clarithromycin, amoxicillin, metronidazole, tetracycline and levo oxacin star. However, in recent years, the growing rate of antibiotic resistance has resulted in the failure of Hp eradication [19][20] . The most serious resistance has developed to drugs including metronidazole, clarithromycin, and levo oxacin star. The common mechanisms of bacterial resistance are the production of inactivated enzymes, change in the target position of antibacterial drugs, change in the permeability of bacterial outer membrane, effects on the active out ow system, formation of bacterial bio lm and cross resistance [21][22][23] . There are some differences in the mechanisms of drug resistance of each kind of bacteria, however, the same kind of bacteria still have different resistances to the same antibiotic in different areas [24] . The mechanism of drug resistance of Hp remains unclear and needs further study.
We selected drug-resistant strains using metronidazole, clarithromycin and levo oxacin for genome sequencing analysis. We found that there were no signi cant differences from the number of drug-resistant genes in the CARD database. This may be because two kinds of antibiotic resistance can develop and the drug-resistant genes in Hp are mainly hp1181 and hp1184. hp1181 is putative NDA translocase, related to the major facilitator superfamily, and is an integral membrane protein; hp1184 belongs to the MATE family, another translocase, resulting in the aforementioned susceptibility. These can contribute to resistance via a multi-drug resistance e ux protein, active-e ux of antibiotics and other e ux pump genes, such as HefA. After knockout of these two genes, the MICs of the drugs were signi cantly decreased and the sensitivity increased. It is noteworthy that in addition to these two genes, the GE2270A gene of Enterococcus and Mura gene of E. coli also show a correlation. It is likely that the drug-resistant plasmids of other strains invade Hp through transformation or other mechanisms. Bacteria other than Hp in the gastric mucosa of patients indirectly con rm this view. The main reason for this may be long-term acid resistant treatment, gastric erosion, or intestinal bacterial re ux. This will lead to drug resistance becoming more di cult to prevent and control, so we must attach great importance thereto. In addition, all three strains have clarithromycin resistance. The mechanism of resistance to clarithromycin is mainly re ected in the mutations A2142G, A2143G, G2144T. In addition, it is common that there are several mutations in the same strain. hp1181 and hp1184 are related to multi-drug resistance and also to clarithromycin resistance, which has been previously reported in the literature [25][26] . The RNA expressions of hp1181 and hp1184 were increased with the emergence of clarithromycin resistance with hp1184 showing the fastest increase. Therefore, these genes are also involved in the regulation of drug resistance and may be one of the mechanisms of Hp resistance to clarithromycin. Compared with the clinical isolates, 23S RNA mutation sites of Hp were less frequent in arti cially induced strains that had only A2142G and A2143G mutations. These may be due to the single factor of arti cial induction that is not as complex as human stomach environment. More importantly, hp1184 mutation may be the earliest and most persistent response to clarithromycin resistance, and it may be the main target gene for the prevention and treatment of clarithromycin resistance.
The genetic characteristics of multi-drug-resistant strains in this area were preliminarily identi ed: the relationship between hp1181, hp1184 and clarithromycin resistance was demonstrated through genome sequencing analysis and gene function identi cation to drug-resistant Hp from Bama County, Guangxi Province. Our study further provided an improved experimental basis for the prevention and treatment of drug   Table 2 Sequence information of three drug-resistant strains Table 3 The gene information of three drug-resistant strains Table 4 Gene annotation statistics A   Figure 1 The circular genome analysis of three resistant strains. A. Hpbs1; B. Hpbs2; C. Hpbs3. Figure 2