Genotyping analysis of Helicobacter pylori using multiple-locus variable-number tandem-repeats analysis in five regions of China and Japan
- Chunliang Guo†1, 2, 3,
- Yaling Liao†1,
- Yan Li4,
- Jun Duan5,
- Ying Guo1,
- Yuqian Wu5,
- Yujun Cui6,
- Hongwu Sun1,
- Jinyong Zhang1,
- Bing Chen2Email author,
- Quanming Zou7Email author and
- Gang Guo1Email author
© Guo et al; licensee BioMed Central Ltd. 2011
Received: 20 March 2011
Accepted: 3 September 2011
Published: 3 September 2011
H. pylori (Helicobacter pylori) is the major causative agent of chronic active gastritis. The population of H. pylori shows a high genomic variability among isolates. And the polymorphism of repeat-units of genomics had participated the important process of evolution. Its long term colonization of the stomach caused different clinical outcomes, which may relate to the high degree of genetic variation of H. pylori. A variety of molecular typing tools have been developed to access genetic relatedness in H. pylori isolates. However, there is still no standard genotyping system of this bacterium. The MLVA (Multi-locus of variable number of tandem repeat analysis) method is useful for performing phylogenetic analysis and is widely used in bacteria genotyping; however, there's little application in H. pylori analysis. This article is the first application of the MLVA method to investigate H. pylori from different districts and ethnic groups of China.
MLVA of 12 VNTR loci with high discrimination power based on 30 candidates were performed on a collection of 202 strains of H. pylori which originated from five regions of China and Japan. Phylogenetic tree was constructed using MLVA profiles. 12 VNTR loci presented with high various polymorphisms, and the results demonstrated very close relationships between genotypes and ethnic groups.
This study used MLVA methodology providing a new perspective on the ethnic groups and distribution characteristics of H. pylori.
Helicobacter pylori (H. pylori) is a spiral-shaped, Gram-negative bacterium that infects half the world's population and is the major cause of chronic gastritis, peptic ulcers and gastric malignancies, including gastric non-cardia adenocarcinoma and mucosal-associated lymphoid tissue lymphoma [1, 2]. Most infected individuals present with no clinical symptoms, but approximately 10~20% will develop peptic ulcers and 1% will develop gastric cancer [3, 4], which could be associated with the diversity of H. pylori.
H. pylori exhibits exceptionally high rates of DNA point mutations and intra- and inter-genomic recombination. Recently, many molecular typing tools have been developed to investigate genetic relatedness among H. pylori isolates. However, these methods have limitations including lower discrimination power, or preventing results from different labs being compared [5, 6].
In 1999, MLVA analysis was proposed as a general approach to providing accurate, portable data that were appropriate for the epidemiological investigation of bacterial pathogens [7–11]. However, there's little information concerning populations of H. pylori species using MLVA. Whether this method is available for the H. pylori population is still uncertain.
H. pylori infections in China are common and extensively distributed, with an average infection rate of about 58%. In this study, 12 VNTR loci of the H. pylori genome were identified and used to analyze 202 strains of H. pylori which originated from different regions of China and Japan.
Multi-VNTR loci for H. pylori genome
PCR products amplified from the reference strains 26695, HPAG1 and J99 were identical to the published sequences sizes. Of the locus VNTR-2576 and VNTR-614, the PCR products sequencing were consistence with our electrophoresis results. The exact number of tandem repeats at each locus could be determined from the sizes of the PCR products.
Characteristics of the 12 VNTR loci in the reference H.pylori strains
Position in the reference strains (bp)
Number of repeat times
Repeat unit size (bp)
Related gene in 26695
16605. . 16643
17912. . 17932
16761. . 16778
42061. . 42115
43125. . 43167
42199. . 42252
129983. . 130389
125875. . 126119
1238315. . 1238474
120659. . 120675
118007. . 118023
116640. . 116673
129957. . 130396
1189474. . 1189690
1238289. . 1238481
485276. . 485316
452649. . 452673
448197. . 448261
580530. . 580546
546643. . 546659
544199. . 544227
665196. . 665241
628875. . 628996
625968. . 626121
696789. . 697001
1067559. . 1067708
1112077. . 1112164
1382502. . 1382594
1314612. . 1314776
1360215. . 1360348
1439274. . 1439284
1368268. . 1368279
1412390. . 1412413
1512724. . 1512751
1419518. . 1419531
1464638. . 1464651
Description of 12 VNTR loci analyzing with 202 H.pylori clinical isolates
Forward and Reverse primer (F/R)
Annealing temperature (°C)
Expected product length in 26695 (bp)
Product size range
Allele size range(unites)
Nei's diversity index
F:GAATTGATTATGATTTTCTTGGCAAT R: GCTGAAAACGCTAGGGATAGAGC
1, 2, 5-21, 23
Clustering trend of the strains from different regions and ethnic groups
Correlation between H. pylori MTs and the related diseases
Among the 202 samples, 14.9%, 55.9%, 25.2% and 4.0% of patients presented with non-ulcer dyspepsia (NUD), gastritis (G), peptic ulcer (PU) and gastric cancer (GC), respectively. And in our study there's no significant relationship between the H. pylori MTs and the related diseases.
Recently, many bacterial genomes have been fully sequenced, and analysis of the sequenced genomes has revealed the presence of variable proportions of repeats, including tandem repeats. Short repeat motifs undergo frequent variation in the number of repeated units. MLVA is an appropriate method for bacterial typing and identification, for determining genetic diversity, and for the tracing-back of highly mono-morphological species [12–14]. The MLVA typing was reported to have a high-quality species identification capability and a high discriminatory power. The method has been used in the analysis of many bacteria [15–18], but little research has been carried out in H. pylori. Therefore, this study established the H. pylori MLVA system and applied to type clinical strains.
The H. pylori genome has a number of repeat sequences, and their repeat number results in divergence. The 12 loci identified were distributed throughout the genome. These loci had different variations in different isolates and were able to typing H. pylori successfully.
The H. pylori MTs were clustered with ethnic groups, consistent with the previous reports [19, 20]. The Han strains were selected from Southern China and had little relationship to Mongolian strains from Northern China or Tibetan strains from Western China. It may demonstrate an apparent cluster tendency in different regions and ethnic groups, but there were some exceptions, which may because, unlike other Asian countries with relatively homogeneous populations, China has a heterogeneous population from various ethnic groups. Therefore, there may be more opportunity for DNA transfer between strains of different genotypes in China than other countries. While Tibet is a relatively closed region, H. pylori strains from this area have a good cluster.
The H. pylori genome shows a high degree of genetic diversity among strains [21, 22], but weakly clonal groupings of different diseases were detected, and these could be superimposed on a pattern of free recombination. And the relationship between particular H. pylori genotype and related disease has not been sure.
MLVA is a useful molecular tool for epidemiological investigations and recognition of laboratory cross-contamination [23–25]. VNTR analysis thus provides multiple independent characteristics for phylogenetic analysis. Studies have indicated that MLVA is sufficient to resolve closely related isolates. In contrast, combining loci with lower variability values is suitable for establishing clear phylogenetic patterns among strains that have evolved over a longer time period. Theoretically, the greater the number of loci used, the higher the discriminatory power that can be achieved, and subtler phylogenetic relationships among bacterial strains can be established.
At the present time, the MLVA was established and applied to examine the clonal relationships between H. pylori isolates from China and Japan. The loci used in this study provided high discriminatory power and successfully separated isolates of different strains from different geographical areas. And there was a particularly evident of H. pylori from Tibet, a relatively closed region, which showed better cluster than other ethnic groups. The data will aid in the development of a genomic polymorphism database of H. pylori. We have established a preliminary profile of MLVA but more information is required for a comprehensive profile.
China is a large country containing 56 ethnic groups and a large population. Therefore, further studies are required including isolates from more regions and over several more time-frames.
The studies indicated that MLVA method, based on 12 VNTR loci, is sufficient to resolve closely related isolates for the purpose of H. pylori genotyping analysis. This study used MLVA methodology provided a new perspective on the ethnic groups distribution characteristics of H. pylori.
H. pylori strains and DNA preparation
Background information of the 202 H. pylori clinical strains
No. of isolates
Bacteria were separated and cultured in Skirrow medium with 5% fresh sheep blood at 37°C for 24 h~72 h in a micro-aerobic environment. H. pylori genomes were extracted using genomic DNA isolation kits (Omega Biotek Inc). Culture and identification of H. pylori were done by appropriate biochemical tests and amplification of 16S rDNA using species-specific primers
Selection and identification the VNTR loci of H. pylori
VNTR loci were selected from the MLVA database http://minisatellites.u-psud.fr/ASPSamp/base_ms/bact.php by estimating the size of PCR products on agarose gels. The repeat sequence of loci ≥ 10 bp, consistency of repeat unit ≥ 90% and a minimum of two alleles in three reference strains of H. pylori (26695, HPAG1, J99) were selected for this research. The locations, copy numbers, sizes of the loci and the gene(s) involved are also listed in Table 1.
A PCR reaction mixture (30 ml) containing 10 ng of DNA template, 0.5 mM of each primer, 1 unit of Taq DNA polymerase, 200 mM of dNTPs and 10 × PCR buffer (500 mM KCl, 100 mM TrisHCl (pH 8.3) 25 mM MgCl2) was utilized. Amplification was carried out in a DNA thermocycler (MJ Research PTC-225) with denaturation at 94°C for 8 min, followed by 30 cycles of denaturation at 94°C for 45 s, annealing at 52°C for 45 s and elongation at 72°C for 1 min . A 10-min elongation at 72°C was performed after the last cycle to ensure complete extension of the amplicons.
Five μl of the PCR products were run on standard 3% agarose gels in 0.56TBE buffer at 8-10 V/cm. Gel lengths of 10 to 40 cm were used according to PCR product size and repeat unit size. Strains in which alleles had been precisely measured by re-sequencing or by direct comparison with a sequenced reference strain were used (In this study DNA from 26695, HPAG1 and J99 were used for this purpose). Multiple interspersed negative controls containing no DNA were included each time PCR was performed.
PCR products of 202 strains on VNTR-2576 and VNTR-614 sites were sequenced directly with a Taq Dye Deoxy Terminator Cycle Sequencing Kit on an ABI 377 sequencer (Applied Biosystems).
The number of repeat units in 12 VNTR loci were analyzed and inputted into BioNumerics version 5.1 software (Applied-Maths, Sint-Martens-Latem, Belgium), and gel images were obtained using the BioNumerics software package version 6.0 (Applied-Maths, Sint-Martens-Latem, Belgium) or using UVB gel image analysis. The number of repeat units in each locus was deduced by the amplicon size, flanking sequence length and repeat unit size. Data from agarose gel electrophoresis and UVB gel image analysis, obtained by capillary electrophoresis machines, were imported into BioNumerics by creating a virtual gel image. Gel image data were converted into characteristics data sets. Cluster analysis of Neighbor-joining tree (N-J) was carried out using the categorical similarity coefficient and the Ward method. A minimum spanning tree was inferred using characteristic data from cluster analysis. The polymorphism of each locus was represented by Nei's diversity index , calculated as DI = 1-∑(allelic frequency)2.
Reproducibility and stability of 12 VNTR loci via in-vitro passage
Twenty clinical strain genomes from China and Japan were amplified and multiple DNA samples from each strain yielded PCR products with identical sizes at all loci. Chongqing26 and Tibet36 each yielded no product at one locus, possibly because of mutations or poor quality DNA samples.
The stabilities of the VNTR loci were investigated in a long-term experiment in which the 20 test H. pylori isolates used were sub-cultured into fresh Skirrow medium 30 times by serial passages at two or three day intervals. The DNA from the strains cultivated in each passage was extracted and subjected to MLVA analysis. The results of the VNTR analysis demonstrated no difference in tandem repeat numbers (data not shown).
List of abbreviations
variable-number tandem repeat
multiple-locus VNTR analysis
We thank Prof Chihiro Sasakawa of Institute of Medical Science University for providing 15 H. pylori strains of Tokyo. This work was supported by the fund of Chinese National Natural Science Foundation project (No. 31070445); Major State Basic Research Development Program of China (973 Program) (No. 2009CB522606).
- Ouakaa-Kchaou A, Elloumi H, Gargouri D, Kharrat J, Ghorbel A: Helicobacter pylori and gastric cancer. Tunis Med. 2010, 88 (7): 459-461.PubMedGoogle Scholar
- Shin CM, Kim N, Yang HJ, Cho SI, Lee HS, Kim JS, Jung HC, Song IS: Stomach cancer risk in gastric cancer relatives: interaction between Helicobacter pylori infection and family history of gastric cancer for the risk of stomach cancer. J Clin Gastroenterol. 2010, 44 (2): e34-39. 10.1097/MCG.0b013e3181a159c4.PubMedView ArticleGoogle Scholar
- Abdullah M, Ohtsuka H, Rani AA, Sato T, Syam AF, Fujino MA: Helicobacter pylori infection and gastropathy: A comparison between Indonesian and Japanese patients. World J Gastroentero. 2009, 15 (39): 4928-4931. 10.3748/wjg.15.4928.View ArticleGoogle Scholar
- Ernst PB, Peura DA, Crowe SE: The translation of Helicobacter pylori basic research to patient care. Gastroenterology. 2006, 130 (1): 188-206. 10.1053/j.gastro.2005.06.032. quiz 212-183PubMedView ArticleGoogle Scholar
- Ben-Darif E, De Pinna E, Threlfall EJ, Bolton FJ, Upton M, Fox AJ: Comparison of a semi-automated rep-PCR system and multilocus sequence typing for differentiation of Salmonella enterica isolates. J Microbiol Methods. 2010, 81 (1): 11-16. 10.1016/j.mimet.2010.01.013.PubMedView ArticleGoogle Scholar
- Do T, Gilbert SC, Clark D, Ali F, Fatturi Parolo CC, Maltz M, Russell RR, Holbrook P, Wade WG, Beighton D: Generation of diversity in Streptococcus mutans genes demonstrated by MLST. PLoS One. 2010, 5 (2): e9073-10.1371/journal.pone.0009073.PubMedPubMed CentralView ArticleGoogle Scholar
- de Haan CP, Kivisto RI, Hakkinen M, Corander J, Hanninen ML: Multilocus sequence types of Finnish bovine Campylobacter jejuni isolates and their attribution to human infections. BMC Microbiol. 2010, 10: 200-10.1186/1471-2180-10-200.PubMedPubMed CentralView ArticleGoogle Scholar
- Olsen JS, Aarskaug T, Skogan G, Fykse EM, Ellingsen AB, Blatny JM: Evaluation of a highly discriminating multiplex multi-locus variable-number of tandem-repeats (MLVA) analysis for Vibrio cholerae. J Microbiol Methods. 2009, 78 (3): 271-285. 10.1016/j.mimet.2009.06.011.PubMedView ArticleGoogle Scholar
- Pourcel C, Andre-Mazeaud F, Neubauer H, Ramisse F, Vergnaud G: Tandem repeats analysis for the high resolution phylogenetic analysis of Yersinia pestis. BMC Microbiol. 2004, 4: 22-10.1186/1471-2180-4-22.PubMedPubMed CentralView ArticleGoogle Scholar
- Smith KL, De Vos V, Bryden HB, Hugh-Jones ME, Klevytska A, Price LB, Keim P, Scholl DT: Meso-scale ecology of anthrax in southern Africa: a pilot study of diversity and clustering. J Appl Microbiol. 1999, 87 (2): 204-207. 10.1046/j.1365-2672.1999.00871.x.PubMedView ArticleGoogle Scholar
- Swaminathan B, Gerner-Smidt P, Ng LK, Lukinmaa S, Kam KM, Rolando S, Gutierrez EP, Binsztein N: Building PulseNet International: an interconnected system of laboratory networks to facilitate timely public health recognition and response to foodborne disease outbreaks and emerging foodborne diseases. Foodborne Pathog Dis. 2006, 3 (1): 36-50. 10.1089/fpd.2006.3.36.PubMedView ArticleGoogle Scholar
- Her M, Kang SI, Cho DH, Cho YS, Hwang IY, Heo YR, Jung SC, Yoo HS: Application and evaluation of the MLVA typing assay for the Brucella abortus strains isolated in Korea. BMC Microbiol. 2009, 9: 230-10.1186/1471-2180-9-230.PubMedPubMed CentralView ArticleGoogle Scholar
- Wang YW, Watanabe H, Phung DC, Tung SK, Lee YS, Terajima J, Liang SY, Chiou CS: Multilocus variable-number tandem repeat analysis for molecular typing and phylogenetic analysis of Shigella flexneri. BMC Microbiol. 2009, 9: 278-10.1186/1471-2180-9-278.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhang X, Hai R, Wei J, Cui Z, Zhang E, Song Z, Yu D: MLVA distribution characteristics of Yersinia pestis in China and the correlation analysis. BMC Microbiol. 2009, 9: 205-10.1186/1471-2180-9-205.PubMedPubMed CentralView ArticleGoogle Scholar
- Beranek A, Mikula C, Rabold P, Arnhold D, Berghold C, Lederer I, Allerberger F, Kornschober C: Multiple-locus variable-number tandem repeat analysis for subtyping of Salmonella enterica subsp. enterica serovar Enteritidis. Int J Med Microbiol. 2009, 299 (1): 43-51. 10.1016/j.ijmm.2008.06.002.PubMedView ArticleGoogle Scholar
- Ghebremichael S, Groenheit R, Pennhag A, Koivula T, Andersson E, Bruchfeld J, Hoffner S, Romanus V, Kallenius G: Drug resistant Mycobacterium tuberculosis of the Beijing genotype does not spread in Sweden. PLoS One. 2010, 5 (5): e10893-10.1371/journal.pone.0010893.PubMedPubMed CentralView ArticleGoogle Scholar
- Klevytska AM, Price LB, Schupp JM, Worsham PL, Wong J, Keim P: Identification and characterization of variable-number tandem repeats in the Yersinia pestis genome. J Clin Microbiol. 2001, 39 (9): 3179-3185. 10.1128/JCM.39.9.3179-3185.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Schouls LM, Spalburg EC, van Luit M, Huijsdens XW, Pluister GN, van Santen-Verheuvel MG, van der Heide HG, Grundmann H, Heck ME, de Neeling AJ: Multiple-locus variable number tandem repeat analysis of Staphylococcus aureus: comparison with pulsed-field gel electrophoresis and spa-typing. PLoS One. 2009, 4 (4): e5082-10.1371/journal.pone.0005082.PubMedPubMed CentralView ArticleGoogle Scholar
- Falush D, Wirth T, Linz B, Pritchard JK, Stephens M, Kidd M, Blaser MJ, Graham DY, Vacher S, Perez-Perez GI, et al: Traces of human migrations in Helicobacter pylori populations. Science. 2003, 299 (5612): 1582-1585. 10.1126/science.1080857.PubMedView ArticleGoogle Scholar
- Linz B, Balloux F, Moodley Y, Manica A, Liu H, Roumagnac P, Falush D, Stamer C, Prugnolle F, van der Merwe SW, et al: An African origin for the intimate association between humans and Helicobacter pylori. Nature. 2007, 445 (7130): 915-918. 10.1038/nature05562.PubMedPubMed CentralView ArticleGoogle Scholar
- Hovey JG, Watson EL, Langford ML, Hildebrandt E, Bathala S, Bolland JR, Spadafora D, Mendz GL, McGee DJ: Genetic microheterogeneity and phenotypic variation of Helicobacter pylori arginase in clinical isolates. Bmc Microbiology. 2007, 7:Google Scholar
- Suerbaum S, Kraft C, Dewhirst FE, Fox JG: Helicobacter nemestrinae ATCC 49396T is a strain of Helicobacter pylori (Marshall et al. 1985) Goodwin et al. 1989, and Helicobacter nemestrinae Bronsdon et al. 1991 is therefore a junior heterotypic synonym of Helicobacter pylori. Int J Syst Evol Microbiol. 2002, 52 (Pt 2): 437-439.PubMedView ArticleGoogle Scholar
- Hidalgo A, Carvajal A, La T, Naharro G, Rubio P, Phillips ND, Hampson DJ: Multiple-locus variable-number tandem-repeat analysis of the swine dysentery pathogen, Brachyspira hyodysenteriae. J Clin Microbiol. 2010, 48 (8): 2859-2865. 10.1128/JCM.00348-10.PubMedPubMed CentralView ArticleGoogle Scholar
- Litrup E, Christensen H, Nordentoft S, Nielsen EM, Davies RH, Helmuth R, Bisgaard M: Use of multiple-locus variable-number tandem-repeats analysis (MLVA) typing to characterize Salmonella Typhimurium DT41 broiler breeder infections. J Appl Microbiol. 2010Google Scholar
- Weniger T, Krawczyk J, Supply P, Niemann S, Harmsen D: MIRU-VNTRplus: a web tool for polyphasic genotyping of Mycobacterium tuberculosis complex bacteria. Nucleic Acids Res. 2010, 38 (Suppl): W326-331.PubMedPubMed CentralView ArticleGoogle Scholar
- Li Y, Cui Y, Hauck Y, Platonov ME, Dai E, Song Y, Guo Z, Pourcel C, Dentovskaya SV, Anisimov AP, et al: Genotyping and phylogenetic analysis of Yersinia pestis by MLVA: insights into the worldwide expansion of Central Asia plague foci. PLoS One. 2009, 4 (6): e6000-10.1371/journal.pone.0006000.PubMedPubMed CentralView ArticleGoogle Scholar
- Hunter PR, Gaston MA: Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J Clin Microbiol. 1988, 26 (11): 2465-2466.PubMedPubMed CentralGoogle Scholar
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