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Application of next-generation sequencing to characterize novel mutations in clarithromycin-susceptible Helicobacter pylori strains with A2143G of 23S rRNA gene

  • Jiaoe Chen
  • Liping Ye
  • Liangmin Jin
  • Xuehua Xu
  • Peisong Xu
  • Xianjun Wang
  • Hongzhang Li
Open Access
Research

Abstract

Background

Clarithromycin (CLR) resistance has become a predominant factor for treatment failure of Helicobacter pylori eradication. Although the molecular mechanism of CLR resistance has been clearly understood in H. pylori, it is lack of evidence of other genes involved in drug resistance. Furthermore, the molecular mechanism of phenotype susceptible to CLR while genotype of 23S rRNA is mutant with A2143G is unclear. Here, we characterized the mutations of CLR-resistant and -susceptible H. pylori strains to explore bacterial resistance.

Methods

In the present study, the whole genomes of twelve clinical isolated H. pylori strains were sequenced, including two CLR-susceptible strains with mutation of A2143G. Single nucleotide variants (SNVs) were extracted and analyzed from multidrug efflux transporter genes.

Results

We did not find mutations associated with known CLR-resistant sites except for controversial T2182C outside of A2143G in the 23S rRNA gene. Although total SNVs of multidrug efflux transporter gene and the SNVs of HP0605 were significant differences (P ≤ 0.05) between phenotype resistant and susceptible strains. There is no significant difference in SNVs of RND or MFS (HP1181) family. However, the number of mutations in the RND family was significantly higher in the mutant strain (A2143G) than in the wild type. In addition, three special variations from two membrane proteins of mtrC and hefD were identified in both CLR-susceptible strains with A2143G.

Conclusions

Next-generation sequencing is a practical strategy for analyzing genomic variation associated with antibiotic resistance in H. pylori. The variations of membrane proteins of the RND family may be able to participate in the regulation of clinical isolated H. pylori susceptibility profiles.

Keywords

Helicobacter pylori Clarithromycin Susceptible strains 23S rRNA SNVs RND family 

Abbreviations

SNVs

single nucleotide variants

CLR

clarithromycin

RND

resistance-nodulation-cell division

NGS

next-generation sequencing

MFS

major facilitator superfamily

InDels

insertions or deletions

Background

Helicobacter pylori (H. pylori), a Gram-negative and microaerophilic bacterium, has been recognized an important human pathogen that infects approximately 50% of world’s population, and is responsible for the development of upper gastrointestinal disorders, including chronic gastritis, peptic ulcer disease, gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma [1, 2, 3]. In the past few decades, triple therapy regimen consist of a proton pump inhibitor in combination with two antibiotics, such as clarithromycin (CLR) and amoxicillin (AMX) or metronidazole (MTZ), which has been recommended as first-line treatment regimen for H. pylori infection [4]. However, with the increase of H. pylori CLR-resistant strains, this traditional treatment regimen is being replaced by quadruple therapy or precise medical, especially in the area of CLR resistance is higher than 15% [5, 6, 7]. Many reports have indicated that CLR resistance has become a predominant factor for treatment failure in which containing CLR [5, 8].

The majority of H. pylori CLR-resistant strains present three point mutations in the region of domain V of 23S ribosomal RNA (rRNA): A2142G, A2142C and A2143G. Simultaneously, the studies also suggest that some other point mutations may be involved in CLR resistance at position 2115G, G2141A, T2117C, T2182C, T2717C [9, 10, 11]. Another mechanism of resistance to CLR has been reported that five conserved families of multidrug efflux pump transporter contribute to bacterial antibiotic resistance. One of these, the resistance-nodulation-cell division (RND) family, was consisted of an inner membrane efflux protein, a membrane fusion protein and an outer membrane protein. Currently, four gene clusters (HP0605–HP0607, HP0969–HP0971, HP1327–HP1329, HP1487–HP1489) [12, 13, 14, 15] have been established as RND family candidates in H. pylori. Hirata et al. reported that the MIC of CLR-resistant strains was decreased by using efflux pump inhibitor (EPI), indicating that in addition to the point mutation of 23S rRNA gene, the efflux pump cluster is also involved in the development of resistance to CLR [16].

Although the molecular mechanism of CLR resistance has been relatively clearly understood in H. pylori, it is unclear whether other gene mutations associated with CLR resistance outside 23S rRNA and RND family. With increase of the CLR-resistant strains, several studies have suggested that other genetic factors could be involved in the increased antibiotic resistance. Recently, Binh et al. [17] revealed that mutations of insertion or deletion in rpl22 and guanine to adenine point mutation at position 60 in infB gene could be related to CLR resistance using whole-genome sequencing of induced CLR-resistant strains in vitro.

However, almost all of the researches were focus on exploring potential antibiotic resistance genes. Until now, to the best of our knowledge, there is very little research on antibiotic susceptibility gene. In our previous research, we isolated two strains that were identified adenine to guanine mutations at position 2143 in 23S rRNA, while the outcome of antibiotic susceptibility testing were susceptibility to CLR or lower CLR resistance, suggesting that there were some other genes participated in the regulation of antibiotic resistance or susceptibility to CLR.

Compared with traditional DNA sequencing, next-generation sequencing (NGS) is a revolution of sequencing technology that can be sequencing millions of DNA molecules massively parallel in less time and at lower cost [18, 19]. To date, more than three thousand microbial genome sequences have been completed and published by using NGS according to the report [20, 21]. Now, NGS has also been used to identify bacterial single nucleotide polymorphisms (SNP) or mutation associated with antibiotic resistance [22, 23, 24]. Although we didn’t identify all of sequencing information that is like a needle in a haystack because a large number of redundant data is generated by the NGS, with the development of sequencing and biological information, the genetic code will be eventually cracked one by one.

In this study, to characterize the multidrug efflux transporter gene variants in the CLR genotype-resistant while phenotype-susceptible strains, we applied Sanger sequencing to detect the genotype of 23S rRNA and NGS to analysis of genomic variation in clinical isolated H. pylori strains. CLR susceptibility testing was performed by E-test and agar dilution. Ten HP strains meeting our requirements were applied to whole-genome sequencing, including four CLR-resistant and six CLR-susceptible strains.

Methods

Isolation and culture of H. pylori

Gastric mucosa tissue samples were collected from patients with upper gastrointestinal disease during endoscopy at Sanmen People’s Hospital and Zhejiang Taizhou Hospital. Isolation and culture of H. pylori were performed at the laboratory of Hangzhou Zhiyuan Medical Inspection Institute. Patients were investigated to have not taken any antibiotics for at least 4 weeks before examination. This study had received a strict medical ethics review, and written informed consent was obtained from every patient.

The isolation and identification of H. pylori were performed as described in previous studies [25, 26]. Gastric mucosa homogenate tissue was transferred onto a Columbia agar plates containing 5% fresh defibrinated sheep blood and cultured under microaerophilic conditions (5% O2, 10% CO2 and 85% N2) at 37 °C for 3–7 days. Suspicious colonies were confirmed by Gram stain, urease, oxidase, and catalase activity testing.

Antibiotic susceptibility testing

The antibiotic resistance of H. pylori to CLR was performed by E-test and agar dilution methods according to the protocols of the Clinical and Laboratory Standards Institute (Wayne, PA, USA) [27]. Briefly, the concentration of H. pylori was regulated with saline to a 2.0 McFarland standard, and the suspensions were inoculated onto Mueller–Hinton agar plate supplemented with 5% sheep blood. The CLR E test strip was attached on the plate and incubated at 37 °C for 3–5 days under microaerophilic conditions. Agar dilution was performed by serial twofold dilutions of CLR. The breakpoint of CLR resistance was ≥ 1 mg/l. ATCC43504 (NCTC11637) was used as the control strain and all tests were performed by Hangzhou Zhiyuan Medical Inspection Institute.

Direct sequencing characterized the mutations of 23S rRNA

According to the reference sequence of HP U27270, HP-23S forward primer (5′-ATGAATGGCGTAACGAGATG-3′) and HP-23S reverse primer (5′-ACACTCAACTTGCGATTTCC-3′) were employed to detect 23S rRNA gene mutations at positions of 2142 and 2143. The PCR reaction was performed in 25-µl reactions containing 2.5 µl of 10× LA Taq Buffer, 4 µl of dNTP mixture (2.5 mM each), 0.5 µl each 10 µM primer, 2 µl template DNA and 0.25 µl of TaKaRa LA Taq™ (5 units/µl). The parameters of PCR were carried out at 94 °C for 5 min, followed by 25 cycles of denaturing at 94 °C for 30 s, annealing at 58 °C for 30 s, and extending at 72 °C for 3 min, with a final extension for 10 min at 72 °C. 1.2% agarose gel electrophoresis was utilized for verifying the PCR products size at 360 bp. To validate the mutations of 23S rRNA, Sanger sequencing was performed with an ABI 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA) using BigDye® Terminator V3.1 according to the manufacturer’s instructions.

DNA extraction and whole-genome sequencing

The total genomic DNAs of H. pylori were extracted by using Invitrogen Purelink Genomic DNA Mini Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. The concentration of each genomic DNA sample was quantified with Qubit dsDNA HS assay kit (Life Technologies). For each sample, 1 μg genomic DNA was randomly uniformly fragmented to < 500 bp by sonication (Diagenode Bioruptor UCD-200) and the library was prepared by using NEB Next® Ultra™ DNA Library Prep Kit for Illumina® following the manufacturer’s protocol. Quality control of the library was identified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) with DNA 1000 chip according to the manufacturer’s instructions. Whole-genome sequencing was performed with the Illumina HiSeq (Illumina, San Diego, CA, USA) platform to generate 2 × 150-bp paired-end reads. Image analysis and base calling were conducted by the HiSeq Control Software (HCS) and GAPipeline-1.6 (Illumina) on the HiSeq instrument.

Genome analysis and single nucleotide variations (SNVs) calling

To reduce the false discovery rate of SNVs, the low-quality bases (the quality of both ends bases < Q20) and raw reads (> 10 N bases and reads lengths < 75 bp) were trimmed by Trimmomatic (version 0.30). The remaining clean reads were mapped against the reference genome with GenBank accession NC_000915 and CP003904 using BWA [28] (version 0.7.12). SNVs and InDel were called using SAMTOOLS’s [29] (version 1.1) Mpileup module and Bcftools with default parameters, and finally a series of mutations were generation.

Variations of multidrug resistance genes

In order to validate the role of multidrug efflux transporter gene in CLR-resistant and CLR-susceptible H. pylori, the variations of RND family, major facilitator superfamily (MFS) and adenosine triphosphate (ATP)-binding cassette (ABC) superfamilies had been analyzed, including HP0605–HP0607, HP0969–HP0971, HP1327–HP1329, HP1487–HP1489, HP1181, HP1184, HP0600, HP0613, HP0759, HP1082, HP1206, HP1220, HP1321 and HP1486.

Statistical analysis

All statistical analyses were conducted with SPSS statistical software package version 19.0 (SPSS Inc., Chicago, IL, USA). The relationships between variations of multidrug resistance genes and CLR resistance/susceptibility were investigated by Student’s t-test. A P value ≤ 0.05 was considered statistically significant.

Results

Genotype of 23S rRNA and phenotype of H. pylori resistant to CLR

For twelve clinical isolated strains, CLR susceptibility testing indicated that the six of strains were susceptible to CLR and six of strains were resistant to CLR. The genotype of 23S rRNA from H. pylori was verified by Sanger sequencing (Fig. 1). Eight of them were mutant with A2143G and four strains were wild type (Table 1). All of phenotype-resistant strains presented mutation A to G at position 2143 of the 23S rRNA. However, two mutant-type (S2, S3) of 23S rRNA gene at position 2143 (A>G) were also detected in six phenotype-susceptible strains.
Fig. 1

The results of Sanger sequencing for genotype of 23S rRNA in H. pylori. a The wild type without mutation at position 2143. b The mutant strain with mutation of A>G at position 2143

Table 1

The results of whole-genome sequencing for each sample

Strain ID

CLR susceptibility testing

Total reads (clean reads)

Mapping to genome reads

Covered length

Coverage (%)

Average depth

Total SNVs

Total InDels

S1

Ra

9,815,508

8,780,946

1,517,430

90.98

545.66

67,219

106

S2

S b

10,573,646

9,813,458

1,534,369

91.99

601.38

68,671

95

S3

S

10,269,810

9,529,590

1,527,013

91.55

565.38

70,135

101

S4

R

9,931,112

8,921,164

1,553,847

93.16

522.64

67,929

132

S5

R

9,965,800

8,012,304

1,522,585

91.29

459.11

67,580

134

S6

S

8,326,490

7,203,062

1,537,013

92.15

528.19

68,032

99

S7

S

8,023,030

7,541,380

1,536,514

92.12

549.71

68,496

112

S8

R

7,687,812

7,171,310

1,522,399

91.28

535.58

69,891

104

S9

S

11,190,968

9805694

1,522,469

91.28

731.93

68,658

67

S10

S

12,167,520

11,643,382

1,539,435

92.3

888.02

70,933

94

S11

R

9,756,502

8,630,028

1,517,874

91.01

532.33

67,285

112

S12

R

6,575,708

6,090,808

1,521,421

91.22

378.89

67,231

177

S2 and S3 (italic values) indicates the phenotype-susceptible strains with mutant in A2143G of 23S rRNA gene

aR indicated the strain was resistant to clarithromycin

bS indicated the strain was susceptible to clarithromycin

Overview of whole-genome sequencing of clinical isolated H. pylori

In the present study, twelve strains of H. pylori genome were successfully figured out by Hiseq sequencer. After trimming the low-quality reads, clean reads ranged from 6.6 to 12.2 million (Table 1). Clean reads were directly mapped to the reference genome. Coverage and average depth ranged from 90.98 to 93.16% and from 378 to 888, respectively. Therefore, the efficient reads were sufficient for subsequent analysis of SNVs. The number of SNVs and InDels ranged from 67,219 to 70,933 and from 67 to 177, respectively. There were no significant differences between CLR-resistant and CLR-susceptible strains in SNVs despite more prevalent in CLR-susceptible strains. However, the number of InDels was significantly decreased in CLR-susceptible strains.

Identification of 23S rRNA gene mutations

To analyze the associations between phenotypic resistance and genotypic resistance, the mutations of the 23S rRNA gene were investigated. Consistent with Sanger sequencing, whole-genome sequencing indicated that six phenotype-resistant strains and two phenotype-susceptible strains had mutation A>G at position 2143. Simultaneously, the mutations outside of 2143 had been extracted (Table 2). Totally, we found fourteen mutations in addition to 2143. In this study, we did not find any mutations associated with known CLR-resistant sites except for T2182C and there is no significant difference between resistant strain and susceptible strain regardless of whether there was mutant with A2143G.
Table 2

SNVs of the 23S rRNA outside of 2143

Nucleotide position

Ref

Mutation

S1

S2

S3

S4

S5

S6

S7

S8

S9

S10

S11

S12

973

G

T

+

+

+

973

G

A

+

+

973

G

C

+

+

+

+

+

1023

G

A

+

+

+

1279

A

T

+

1280

A

G

+

1314

G

A

+

+

+

1513

G

A

+

+

+

+

2173

C

T

+

+

+

2182

T

C

+

+

+

+

+

+

+

+

+

+

+

2302

A

G

+

+

2485

T

C

+

+

2143

A

G

+

+

+

+

+

+

+

+

S2 and S3 (italic values) indicates the phenotype-susceptible strains with mutant in A2143G of 23S rRNA gene

+ Represented that the mutation had been detected and − represented the mutation had not been detected

Identification of multidrug efflux transporter gene mutations

To characterize the mutations of multidrug efflux transporter genes, we focused on the study of the RND family, MFS and ABC superfamilies in H. pylori. Prior to identification of gene mutation in multidrug efflux genes, we removed the synonymous and InDels mutations in the CDS region. All mutations of these genes were presented in Table 3. Regardless of whether the strain is resistant to CLR, gene mutations were detected in all H. pylori of multidrug efflux transporter genes. The gene mutations of membrane fusion proteins (HP0606, HP0970, HP1328, HP1488) were significantly less than inner membrane proteins or outer membrane proteins in RND family.
Table 3

SNVs of multidrug efflux pump transporter genes

Gene

Strain

S1

S2

S3

S4

S5

S6

S7

S8

S9

S10

S11

S12

HP0605

21

18

23

24

23

16

20

27

15

17

29

27

HP0606

5

5

6

1

4

7

5

6

8

5

2

8

HP0607

38

34

32

32

33

30

22

31

32

32

27

36

HP0969

26

23

25

25

24

25

26

23

26

26

24

23

HP0970

7

9

11

7

7

9

8

8

8

9

9

7

HP0971

11

15

12

11

13

12

14

11

8

9

10

11

HP1327

22

22

21

24

22

22

21

22

23

25

23

22

HP1328

15

12

15

14

12

14

13

14

15

14

12

14

HP1329

28

29

36

30

28

25

33

30

26

24

32

30

HP1487

22

21

15

20

21

18

16

18

18

19

17

16

HP1488

7

4

3

6

4

4

5

6

5

4

4

5

HP1489

25

28

23

22

23

25

19

20

24

29

25

19

HP0600

50

3

1

52

48

23

20

46

5

31

47

44

HP0613

9

7

6

9

7

15

3

9

10

15

8

5

HP0759

10

11

10

10

11

11

8

10

11

9

8

11

HP1082

10

13

12

10

11

12

17

11

15

11

12

16

HP1181

10

9

11

9

10

7

11

12

12

9

9

12

HP1184

14

19

14

22

15

14

12

18

13

14

17

15

HP1206

13

16

14

13

14

13

27

16

16

18

18

12

HP1220

3

3

4

3

2

1

4

3

1

1

2

1

HP1321

26

30

29

22

24

29

32

30

24

24

30

28

HP1486

21

18

18

18

18

17

16

17

18

15

17

12

Total

393

349

341

384

374

349

352

388

333

360

382

374

S2 and S3 (italic values) indicates the phenotype-susceptible strains with mutant in A2143G of 23S rRNA gene

We didn’t find significant differences in gene mutations of RND or MFS (HP1181) families between CLR phenotype resistant strains and CLR phenotype susceptible strains. However, the total SNVs of multidrug efflux genes were significant differences between them. Unexpectedly, when we were grouped according to the 23S rRNA genotype, the number of mutations in the RND family was significantly higher in the mutant strain (A2143G) than in the wild type, and the difference was statistically significant. These results suggested the mutations from multidrug efflux genes may play an important role in CLR-resistance, and the mutations of the RND family may change the resistance of H. pylori to CLR with A2143G.

Special variations in CLR-susceptible strains with A2143G

In the present study, we found two H. pylori strains were susceptible to CLR, while the genotypes of 23S rRNA were mutant with A2143G. To understand the cause of this discrepancy, analysis of the special variations in both strains was performed. The special variations were extracted by removing the mutations present in other CLR resistant strains with mutation of A2143G. Totally, 320 non-synonymous variations were obtained, and we selected 16 variations in 14 genes as putative susceptible gene (Table 4).
Table 4

The special variations were identified in CLA-resistant strain with A2143G

Gene

Position of mutationa

Ref

Mutation

Amino acid

Annotation

Mean depth

HP0254

265

G

A

G89S

Outer membrane protein HopG

465.5

rpsA

482

G

A

G161D

30S ribosomal protein S1

543.5

mtrC

542

C

T

T181I

Membrane fusion protein

525.5

HP0498

744

T

G

F248L

Sodium and chloride dependent transporter

597.5

proWX

1650

A

C

R550S

Osmoprotectant ABC transporter permease

642

HP0853

943

C

A

R315S

ABC transporter ATP-binding protein

441.5

hypD

474

G

A (stop)

W158X

Hydrogenase maturation factor

512

hefD

13

G

T

G5C

Outer membrane protein

583.5

hefD

82

A

G

M28V

Outer membrane protein

346

tolB

210

T

G

D70E

Translocation protein

705.5

rpsP

226

G

A

A76T

30S ribosomal protein S16

650

HP1181

826

A

G

I276V

Multidrug transporter

506

rplA

464

G

A

S155N

50S ribosomal protein L16

561.5

HP1220

661

G

A

A221T

ABC transporter ATP-binding protein

628

HP1220

662

C

T

A221V

ABC transporter ATP-binding protein

624.5

moaA

76

C

T (stop)

Q26X

Molybdenum cofactor biosynthesis protein A

528.5

aNucleotide position was determined by the respective reference sequence from NC_000915

Variations were mainly concentrated in membrane proteins and ABC transport ATP-binding proteins. Membrane proteins of the RND family, including mtrC and hefD, were identified variations in H. pylori CLR-susceptible strains with mutation of A2143G. The variations of major facilitator superfamily of HP1181 and ABC superfamilies were found in both strains. Simultaneously, two terminate mutations were observed in Hydrogenase maturation factor (hypD) and molybdenum cofactor biosynthesis protein A (moaA).

Discussion

CLR has been widely used as a first-line drug for H. pylori eradication therapy, and has achieved remarkable achievements for the past few decades [4, 30]. Although the mechanism of CLR resistance has been well illustrated, it is difficult to explain the bacterial antibiotic resistance of some strains with different genotype and phenotype. However, researchers were mainly committed to exploring the genes associated with CLR resistance [11, 17]. To our best knowledge, there is little attention on CLR susceptible gene. In this study, to characterize the variations of multidrug efflux transporter genes, we first complete the whole-genome sequencing of CLR-susceptible H. pylori with mutation of A2143G in the 23S rRNA.

In the present study, we applied HiSeq 2500 platform to generate sufficient reads for analyzing the whole genome sequences of H. pylori. The clean short overlapping reads after quality control were directly mapped against reference genome without assembly [31]. Although the coverage was not very high, the multidrug efflux transporter genes and 23S rRNA gene were well covered with depth of at least 300-fold. Because of genomic gaps, we didn’t choice to analysis of insertions or deletions (InDels).

To elucidate the differences in genotype and phenotype, the analysis of 23S rRNA gene mutation was carried out, and the point mutations at position 2143 were consistent with NGS. Consequently, NGS was a precise method to distinguish the point mutation in genome [22, 23, 24]. Overall, mutations of 23S rRNA were disorganized and unregulated. There were no significant differences between CLR phenotype-resistant strains and CLR phenotype-susceptible strains, and nor between CLR genotype-resistant strains and CLR genotype-susceptible strains. On the contrary, the mutation of T2182C was detected in all strains except for S7. Although gene mutation of T2182 was identified with low resistance level in previous study [32], this mutation was detected in most of the strains in China and the result of this discrepancy may be contribute to geographical and genetic factors [2].

In present study, to analyze the discrepancy between CLR-resistant strains and CLR-susceptible strains, the mutations of twenty-two multidrug efflux transporter genes were extracted. HP0605 knockout mutant presents more susceptibility to novobiocin and sodium deoxycholate [12]. Agreement with previous study, we found that the total SNVs of multidrug efflux transporter gene and the SNV of HP0605 were significant differences (P ≤ 0.05) between phenotype resistant and susceptible strains, while not been found in other genes [33]. Thus, we speculate the SNVs of HP0605 probably have an effect on H. pylori resistance to CLR. Unexpectedly, the SNVs of the RND family were significantly higher in mutant strain (A2143G) than in wild type. The different results mainly contributed to the two strains with different genotype and phenotype. We speculated that the 23S rRNA mutant strains with susceptible to CLR were initially resistant to CLR. Nonetheless, with the living environment and genetic changes, the phenotype of CLR has transformed. This can illustrate that these two strains exhibit more tendency to other strains with consistent genotype and phenotype in genetics.

Efflux pump systems have been identified in bacteria to be associated with antibiotic resistance [14, 15, 16]. The RND family, commonly used as a Gram-negative bacteria antibiotic study, is also used for H. pylori. Amsterdam et al. [12] revealed that more susceptible to metronidazole (MTZ) for HP0605 and HP0971 double-knockout mutant H. pylori strain. The expression of membrane proteins of the RND family has been a hot spot in the study of bacterial resistance. It has been proven that TolC and its homologues play an important role in molecules efflux, virulence and drug resistance [34]. hefD (HP0971) and mtrC (HP0606) are an outer membrane protein (TolC) and a membrane fusion protein (AcrA) of the RND family, respectively. The result of NGS has shown that there have three special mutations present in two CLR-susceptible strains with genotype of A2143G, and not in others. And also, this phenomenon has been found in outer membrane protein hopG (HP0254). Therefore, we attempt to speculate that the variations of outer membrane protein are associated with bacterial antibiotic resistance in H. pylori. Although the exact molecular mechanism of H. pylori with genotype of A2143G susceptible to CLR is unclear, our findings have indicated that the variations of membrane proteins possibly contribute to influence H. pylori resistance to CLR.

Of course, our research also has some limitations due to the problem of coverage and we did not analyze the impact of InDels on the experimental results. We empirically selected some genes that were associated with drug resistance for SNVs analysis. Indeed, we have characterized some special variations of HP0606 and HP0971 in CLR-susceptible strains with mutation of A2143G, and we believed that there have some other genes that regulate the susceptibility of bacteria to antibiotics. Undeniably, whole-genome sequencing of H. pylori provides a new way to solve the problem of bacteria antibiotic resistance.

Conclusion

In this study, we successfully isolated two CLR-susceptible H. pylori strains with mutation A2143G of 23S rRNA gene. Genome variations were analyzed by whole-genome sequencing between twelve H. pylori strains with different CLR resistances. The data of sequencing suggested that the next-generation sequencing of clinical isolated H. pylori is a useful method for identifying genome variations. Analysis of multidrug efflux transporter gene mutation results indicated that membrane proteins of RND family possibly play an indispensable role in resistance to CLR. Further studies of H. pylori genomic variation should be paid more attention to disentomb potential gene associated with antibiotic resistance or susceptibility.

Notes

Authors’ contributions

HL conceived and designed the study. JC, LY, LJ, XX and XW contributed to collected samples. PX performed the laboratory tests. HL, JC, LY and PX participated in analyzing data and writing the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank Fei Meng for excellent technical assistance and much appreciate Yunhui Liu for proofreading of manuscript.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The strains and sequencing data are available from the corresponding author on reasonable request.

Consent for publication

Not applicable.

Ethics approval and consent to participate

This study had received a strict medical ethics review. Written informed consent was obtained from the patients.

Funding

This study was financially supported by Medical and health plan of Zhejiang Province (2015DTA020), Public Technology Application Research of Zhejiang Province Science and Technology Hall (2017C33192 and 2016C33232) and Natural Science Foundation of Zhejiang Province (LY13H190003).

Publisher’s Note

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References

  1. 1.
    McColl KE. Clinical practice. Helicobacter pylori infection. N Engl J Med. 2010;362:1597–604.CrossRefPubMedGoogle Scholar
  2. 2.
    Fock KM, Ang TL. Epidemiology of Helicobacter pylori infection and gastric cancer in Asia. J Gastroenterol Hepatol. 2010;25:479–86.CrossRefPubMedGoogle Scholar
  3. 3.
    Park JB, Koo JS. Helicobacter pylori infection in gastric mucosa-associated lymphoid tissue lymphoma. World J Gastroenterol. 2014;20:2751–9.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Malfertheiner P, Megraud F, O’Morain C, Bazzoli F, El-Omar E, Graham D, Hunt R, Rokkas T, Vakil N, Kuipers EJ. Current concepts in the management of Helicobacter pylori infection: the Maastricht III Consensus Report. Gut. 2007;56:772–81.CrossRefPubMedGoogle Scholar
  5. 5.
    Malfertheiner P, Megraud F, O’Morain CA, Atherton J, Axon AT, Bazzoli F, Gensini GF, Gisbert JP, Graham DY, Rokkas T, et al. Management of Helicobacter pylori infection—the Maastricht IV/Florence Consensus Report. Gut. 2012;61:646–64.CrossRefPubMedGoogle Scholar
  6. 6.
    Katelaris PH, Forbes GM, Talley NJ, Crotty B. A randomized comparison of quadruple and triple therapies for Helicobacter pylori eradication: the QUADRATE Study. Gastroenterology. 2002;123:1763–9.CrossRefPubMedGoogle Scholar
  7. 7.
    Mokhtare M, Hosseini V, Tirgar Fakheri H, Maleki I, Taghvaei T, Valizadeh SM, Sardarian H, Agah S, Khalilian A. Comparison of quadruple and triple Furazolidone containing regimens on eradication of Helicobacter pylori. Med J Islam Repub Iran. 2015;29:195.PubMedPubMedCentralGoogle Scholar
  8. 8.
    Pilotto A, Leandro G, Franceschi M, Rassu M, Bozzola L, Furlan F, Di Mario F, Valerio G. The effect of antibiotic resistance on the outcome of three 1-week triple therapies against Helicobacter pylori. Aliment Pharmacol Ther. 1999;13:667–73.CrossRefPubMedGoogle Scholar
  9. 9.
    Versalovic J, Shortridge D, Kibler K, Griffy MV, Beyer J, Flamm RK, Tanaka SK, Graham DY, Go MF. Mutations in 23S rRNA are associated with clarithromycin resistance in Helicobacter pylori. Antimicrob Agents Chemother. 1996;40:477–80.PubMedPubMedCentralGoogle Scholar
  10. 10.
    Fontana C, Favaro M, Minelli S, Criscuolo AA, Pietroiusti A, Galante A, Favalli C. New site of modification of 23S rRNA associated with clarithromycin resistance of Helicobacter pylori clinical isolates. Antimicrob Agents Chemother. 2002;46:3765–9.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Francesco VD, Zullo A, Hassan C, Giorgio F, Rosania R, Ierardi E. Mechanisms of Helicobacter pylori antibiotic resistance: an updated appraisal. World J Gastrointest Pathophysiol. 2011;2:35–41.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    van Amsterdam K, Bart A, van der Ende A. A Helicobacter pylori TolC efflux pump confers resistance to metronidazole. Antimicrob Agents Chemother. 2005;49:1477–82.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Tsugawa H, Suzuki H, Muraoka H, Ikeda F, Hirata K, Matsuzaki J, Saito Y, Hibi T. Enhanced bacterial efflux system is the first step to the development of metronidazole resistance in Helicobacter pylori. Biochem Biophys Res Commun. 2011;404:656–60.CrossRefPubMedGoogle Scholar
  14. 14.
    Mehrabadi JF, Sirous M, Daryani NE, Eshraghi S, Akbari B, Shirazi MH. Assessing the role of the RND efflux pump in metronidazole resistance of Helicobacter pylori by RT-PCR assay. J Infect Dev Ctries. 2011;5:88–93.CrossRefPubMedGoogle Scholar
  15. 15.
    Bina JE, Alm RA, Uria-Nickelsen M, Thomas SR, Trust TJ, Hancock RE. Helicobacter pylori uptake and efflux: basis for intrinsic susceptibility to antibiotics in vitro. Antimicrob Agents Chemother. 2000;44:248–54.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Hirata K, Suzuki H, Nishizawa T, Tsugawa H, Muraoka H, Saito Y, Matsuzaki J, Hibi T. Contribution of efflux pumps to clarithromycin resistance in Helicobacter pylori. J Gastroenterol Hepatol. 2010;25(Suppl 1):S75–9.CrossRefPubMedGoogle Scholar
  17. 17.
    Binh TT, Shiota S, Suzuki R, Matsuda M, Trang TT, Kwon DH, Iwatani S, Yamaoka Y. Discovery of novel mutations for clarithromycin resistance in Helicobacter pylori by using next-generation sequencing. J Antimicrob Chemother. 2014;69:1796–803.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Metzker ML. Sequencing technologies—the next generation. Nat Rev Genet. 2010;11:31–46.CrossRefPubMedGoogle Scholar
  19. 19.
    Mardis ER. Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:387–402.CrossRefPubMedGoogle Scholar
  20. 20.
    Kyrpides NC, Hugenholtz P, Eisen JA, Woyke T, Goker M, Parker CT, Amann R, Beck BJ, Chain PS, Chun J, et al. Genomic encyclopedia of bacteria and archaea: sequencing a myriad of type strains. PLoS Biol. 2014;12:e1001920.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Punina NV, Makridakis NM, Remnev MA, Topunov AF. Whole-genome sequencing targets drug-resistant bacterial infections. Hum Genomics. 2015;9:19.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Chen Y, Mukherjee S, Hoffmann M, Kotewicz ML, Young S, Abbott J, Luo Y, Davidson MK, Allard M, McDermott P, Zhao S. Whole-genome sequencing of gentamicin-resistant Campylobacter coli isolated from US retail meats reveals novel plasmid-mediated aminoglycoside resistance genes. Antimicrob Agents Chemother. 2013;57:5398–405.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Daum LT, Rodriguez JD, Worthy SA, Ismail NA, Omar SV, Dreyer AW, Fourie PB, Hoosen AA, Chambers JP, Fischer GW. Next-generation ion torrent sequencing of drug resistance mutations in Mycobacterium tuberculosis strains. J Clin Microbiol. 2012;50:3831–7.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Girault G, Blouin Y, Vergnaud G, Derzelle S. High-throughput sequencing of Bacillus anthracis in France: investigating genome diversity and population structure using whole-genome SNP discovery. BMC Genomics. 2014;15:288.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Su P, Li Y, Li H, Zhang J, Lin L, Wang Q, Guo F, Ji Z, Mao J, Tang W, et al. Antibiotic resistance of Helicobacter pylori isolated in the Southeast Coastal Region of China. Helicobacter. 2013;18:274–9.CrossRefPubMedGoogle Scholar
  26. 26.
    Tong YF, Lv J, Ying LY, Xu F, Qin B, Chen MT, Meng F, Tu MY, Yang NM, Li YM, Zhang JZ. Seven-day triple therapy is a better choice for Helicobacter pylori eradication in regions with low antibiotic resistance. World J Gastroenterol. 2015;21:13073–9.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: seventeenth informational supplement M100-S17. Wayne: CLSI; 2007.Google Scholar
  28. 28.
    Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25:1754–60.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R. Genome project data processing S: the sequence alignment/map format and SAMtools. Bioinformatics. 2009;25:2078–9.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Zagari RM, Bianchi-Porro G, Fiocca R, Gasbarrini G, Roda E, Bazzoli F. Comparison of 1 and 2 weeks of omeprazole, amoxicillin and clarithromycin treatment for Helicobacter pylori eradication: the HYPER Study. Gut. 2007;56:475–9.CrossRefPubMedGoogle Scholar
  31. 31.
    Cao Q, Didelot X, Wu Z, Li Z, He L, Li Y, Ni M, You Y, Lin X, Li Z, et al. Progressive genomic convergence of two Helicobacter pylori strains during mixed infection of a patient with chronic gastritis. Gut. 2015;64:554–61.CrossRefPubMedGoogle Scholar
  32. 32.
    Kim KS, Kang JO, Eun CS, Han DS, Choi TY. Mutations in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance. J Korean Med Sci. 2002;17:599–603.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Iwamoto A, Tanahashi T, Okada R, Yoshida Y, Kikuchi K, Keida Y, Murakami Y, Yang L, Yamamoto K, Nishiumi S, et al. Whole-genome sequencing of clarithromycin resistant Helicobacter pylori characterizes unidentified variants of multidrug resistant efflux pump genes. Gut Pathog. 2014;6:27.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Sharff A, Fanutti C, Shi J, Calladine C, Luisi B. The role of the TolC family in protein transport and multidrug efflux. From stereochemical certainty to mechanistic hypothesis. Eur J Biochem. 2001;268:5011–26.CrossRefPubMedGoogle Scholar

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© The Author(s) 2018

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  • Jiaoe Chen
    • 1
  • Liping Ye
    • 2
  • Liangmin Jin
    • 1
  • Xuehua Xu
    • 1
  • Peisong Xu
    • 3
  • Xianjun Wang
    • 4
  • Hongzhang Li
    • 1
  1. 1.Department of GastroenterologySanmen People’s HospitalSanmenPeople’s Republic of China
  2. 2.Department of GastroenterologyZhejiang Taizhou HospitalTaizhouPeople’s Republic of China
  3. 3.Department of Research ServiceZhiyuan Inspection Medical InstituteHangzhouPeople’s Republic of China
  4. 4.Clinical LaboratoryHangzhou First People’s HospitalHangzhouPeople’s Republic of China

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