Genetic affinities of Helicobacter pylori isolates from ethnic Arabs in Kuwait
© Albert et al; licensee BioMed Central Ltd. 2010
Received: 21 June 2010
Accepted: 5 July 2010
Published: 5 July 2010
Helicobacter pylori is one of the most genetically diverse of bacterial species, and since the 5'-end of cagA gene and the middle allele of vacA gene of H. pylori from different populations exhibit considerable polymorphisms, these sequence diversities were used to gain insights into the genetic affinities of this gastric pathogen from different populations. Because the genetic affinity of Arab strains from the Arabian Gulf is not known, we carried out genetic analysis based on sequence diversities of the cagA and the vacA genes of H. pylori from 9 ethnic Arabs in Kuwait. The analysis showed that the Kuwaiti isolates are closely related to the Indo-European group of strains, although some strains have a tendency to form a separate cluster close to the Indo- European group, but clearly distinct from East Asian strains. However, these results need to be confirmed by analyses of neutral markers (house-keeping genes in a multi-locus sequence typing [MLST]) platform. The profiling of virulence-associated genes may have resulted from ecologically distinct populations due to human migration and geographical separation over long periods of time.
Helicobacter pylori, the Gram-negative, spiral, microaerobic bacterium, infects more than half of the world's population . It is a major cause of gastritis and peptic ulcer disease, and a risk factor for gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma . There is extraordinary diversity among H. pylori strains as evident by DNA fingerprinting . This diversity has been enhanced by extensive interstrain gene transfer and recombination [4, 5]. In contrast, much stronger clonality, with the predominance of relatively fewer clones, is seen in populations of several other much-studied bacterial species [6, 7]. The great diversity among H. pylori strains implies a striking lack of selection for just one or a few genotypes that might be best adapted for all humans.
The virulence associated cagA, and vacA genes provide examples in which evolutionary dynamics are likely to have been shaped by local differences in host physiology. CagA and VacA proteins each enter target cells and affect several normal cellular signal transduction pathways, with strengths and specificities that vary geographically.
The sequence diversities at the 5'-end of the cagA gene and the mid-region alleles of the vacA gene have been used to deduce the genetic relationships among strains from East Asia, South Asia, South America, Africa, Europe and USA [8, 9]. However, there are no data on the genetic affinity of Arab strains from the Arabian Gulf Region including those from Kuwait, although some data exist on their genotypes [10, 11]. In the current study, we analysed the genetic relationship of Kuwaiti strains with strains from other ethnic world populations. In order to investigate the gene pool diversity of Kuwaiti isolates of H. pylori, we sequenced putative virulence genes cagA and vacA, whose allele frequency have been shown to vary by ethnic group .
H. pylori isolates were cultured from the gastric biopsies of dyspeptic ethnic Arab Kuwaiti patients in a previous study on antibiotic susceptibility conducted during 2003-2005 . The isolates were stored in brain heart infusion broth (Difco Laboratories, Detroit, Michigan, USA) with 15% glycerol at -70°C. The isolates were grown microaerobically on brain heart infusion agar supplemented with sheep blood, IsoVitalex and Dent supplement as described previously . Chromosomal DNA was extracted by the CTAB (hexadecyl-trimethyl ammonium bromide) method  from culture derived from a single colony. A 219 bp fragment from the 5'-end of the cagA was amplified by PCR with the primers, cagA5 and cagA2 . PCR was carried out in a 50-μl volume using 10 ng DNA, 25 μl of HotStarTaq Master Mix (catalogue number 203445, Qiagen, Valencia, California, USA), 10 pmol of each primer, for 40 cycles using the following conditions: 95°C for 60 s, 55°C for 60 s, and 72°C for 90 s. A 700 bp middle region of the vacA gene was amplified with the primers, VAm-F and VAm-R . The concentrations of various PCR components were the same as in the cagA PCR, but amplification was done in 32 cycles with the following conditions: 94°C for 40 s, 55°C for 40 s, and 72°C for 70 s. The m1 (567 bp) or m2 (642 bp) allele of the middle region of the vacA gene was determined by PCR with the primers, VAG-F and VAG-R . The concentration of PCR ingredients were the same as the other two PCRs above, but amplification was done in 35 cycles with the following conditions: 94°C for 60 s, 55°C for 60 s, and 72°C for 60 s. The PCR products were visualised by agarose gel electrophoresis after staining with ethidium bromide and examination under ultraviolet light. The PCR products were purified by High Pure PCR product purification kit (Roche Diagnostics, Indianapolis, Indiana, USA) according to manufacturer's instructions and directly sequenced. DNA sequencing was performed by the dideoxynucleotide chain termination method with an ABI PRISM BigDye Termination Cycle Sequencing Ready Reaction kit (Perkin-Elmer-Applied Biosystems, Forster City, California, USA) in an ABI PRISM automated sequencer. The DNA sequences were manually edited and compared with the selected sequences in the public database http://www.ncbi.nlm.nih.gov/nucleotide/ with the PHYLIP programme of J. Felsenstein http://evolution.genetics.washington.edu/phylip.html. The genetic trees were generated using DDBJ programme http://www.ddbj.nig.ac.jp and visualised using Treeview programme (version1.61; http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). The lengths of the cagA gene and the vacA gene used in the analyses were 209 bp and 620 bp (from the 700 bp amplified region) respectively.
In the previous study on the antibiotic susceptibility of H. pylori strains conducted in Al-Adan Hospital in Kuwait, cultures were available from 39 ethnic Arab Kuwaiti patients . Of these cultures, 16 were amplified by the cagA gene primers, 21 by the vacA gene primers and 9 by primers for both the genes. Our study attempted to analyze the genomes of the 9 Kuwaiti isolates in defining the evolutionary history of these strains. The isolate numbers are: 7, 16, 21, 46, 202, 220, 43, 25, and 42675. The sequence obtained during this study have been assigned the following accession numbers [GenBank:GU212658 to GenBank:GU212666] (cagA 5'-end), [GenBank:GU212667 to GenBank:GU212669 and GenBank:GU441847] (vacA m1 allele) and [GenBank:GU441844, GenBank:GU441846, GenBank:GU441848 to GenBank:GU441850] (vacA m2 allele).
cagA and vacA genes are virulence genes under selective pressure and hence subjected to sequence variation. Therefore, the data presented in this study need to be confirmed by sequence analyses of neutral genes such as house-keeping genes used in multi-locus sequence typing (MLST). Such analyses have been carried out for H. pylori isolates from various ethnic groups in India  and Iran . These studies showed that isolates in these countries essentially belonged to the population of hpEurope, though isolates from various ethnic groups belonged to distinct subpopulation groups. Also, our conclusion of mixed gene in Kuwaiti isolates should be confirmed by Bayesian statistics.
The Arab population in the Middle East is a distinct ethnic race. Although the region is geographically separate, it established contacts with other regions of the world through colonisation and trade hundreds of years ago. Our study has shown that the strains that infect ethnic Arab Kuwaiti population is closely related to the Indo-European strains although some strains have a tendency to form a separate cluster close to the Indo- European group but far away from East Asian strains. This suggests a common ancestry of strains circulating in Kuwait, India and the European countries. The ancestry of these strains is different from that of the strains circulating in the Far East since they formed a distinct cluster. Human migration and geographical separation over long periods of time may have resulted in ecologically distinct populations of H. pylori infecting individuals in different continents. The profiling of virulence-associated genes like cagA and vacA provide examples in which evolutionary dynamics might have been shaped by local differences in environmental conditions and host physiology and may have resulted in ecologically distinct populations due to human migration and geographical separation over long periods of time. The remarkable differences found to-date between various Asian and European H. pylori populations in at least a few loci along with the present data from Kuwaiti strains should encourage further analyses of strains from relatively understudied geographic areas and human ethnic groups like Middle Eastern regions where human migration and geographical separation over long periods of time are prominent. Such "geographic genomics" may unearth new genes that affect human infection, increase our understanding of bacterium-host interactions in colonisation and disease, and impart new insights into the evolution of this diverse and globally distributed human pathogen.
- Parsonnet J: Incidence of Helicobacter pylori infection. Aliment Pharmacol Ther. 1995, 9: 45-51.PubMedGoogle Scholar
- Dunn BE, Cohen H, Blaser MJ: Helicobacter pylori. Clin Microbiol Rev. 1997, 40: 720-741.Google Scholar
- Akopyanz N, Bukanov NO, Westblom TU, Berg DE: DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res. 1992, 20: 5137-5142. 10.1093/nar/20.19.5137.PubMed CentralView ArticlePubMedGoogle Scholar
- Kersulyte D, Chalkauskas H, Berg DE: Emergence of recombinant strains of Helicobacter pylori during human infection. Mol Microbiol. 1999, 31: 31-43. 10.1046/j.1365-2958.1999.01140.x.View ArticlePubMedGoogle Scholar
- Suerbaum S, Maynard Smith J, Bapumia K, Morelli G, Smith NH, Kuntsmann E, Dyrek I, Achtman M: Free recombination within Helicobacter pylori. Proc Natl Acad Sci USA 1998. 1998, 95: 12619-12624. 10.1073/pnas.95.21.12619.View ArticleGoogle Scholar
- Smith JM, Smith NH, O'Rourke M, Spratt BG: How clonal are bacteria?. Proc Natl Acad Sci USA. 1993, 90: 4384-4388. 10.1073/pnas.90.10.4384.PubMed CentralView ArticlePubMedGoogle Scholar
- Feil EJ, Maiden MC, Achtman M, Spratt BG: The relative contributions of recombination and mutation to the divergence of clones of Neisseria meningitidis. Mol Biol Evol. 1999, 16: 1496-1502.View ArticlePubMedGoogle Scholar
- Mukhopadhyay AK, Kersulyte D, Jeong JY, Datta S, Ito Y, Chowdhury S, Santra A, Bhattacharya SK, Azuma T, Nair GB, Berg DE: Distinctiveness of genotypes of Helicobacter pylori in Calcutta, India. J Bacteriol. 2000, 182: 3219-3227. 10.1128/JB.182.11.3219-3227.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Rahman M, Mukhopadhyay AK, Nahar S, Datta S, Ahmad MM, Sarker S, Masud IM, Engstrand L, Albert MJ, Nair GB, Berg DE: DNA-level characterization of Helicobacter pylori strains from patients with overt disease and with benign infections in Bangladesh. J Clin Microbiol. 2003, 41: 2008-2014. 10.1128/JCM.41.5.2008-2014.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Al Qabandi A, Mustafa AS, Siddique I, Khajah AK, Madda JP, Junaid TA: Distribution of vacA and cagA genotypes of Helicobacter pylori in Kuwait. Acta Trop. 2005, 93: 283-288. 10.1016/j.actatropica.2005.01.004.View ArticlePubMedGoogle Scholar
- Momenah AM, Tayeb MT: Helicobacter pylori cagA and iceA genotypes status and risk of peptic ulcer in Saudi patients. Saudi Med J. 2007, 28: 382-385.PubMedGoogle Scholar
- Kersulyte D, Mukhopadhyay AK, Velapatin OB, Su W, Pan Z, Garcia C, Hernandez V, Valdez Y, Mistry RS, Gilman RH, Yuan Y, Gao H, Alarco´n T, Lo´pez-Brea M, Nair GB, Chowdhury A, Datta S, Shirai M, Nakazawa T, Ally R, Segal I, Wong BCY, Lam SK, Olfat FO, Bore´n T, Engstrand L, Torres O, Schneider R, Thomas JE, Czinn S, Berg DE: Differences in genotypes of Helicobacter pylori from different human populations. J Bacteriol. 2000, 182: 3210-3218. 10.1128/JB.182.11.3210-3218.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Albert MJ, Al-Mekhaizeem K, Neil L, Dhar R, Dhar PM, Al-Ali M, Al-Abkal HM, Haridas S: High prevalence and level of resistance to metronidazole, but lack of resistance to other antimicrobials in Helicobacter pylori, isolated from a multiracial population in Kuwait. Aliment Pharmacol Ther. 2006, 24: 1359-1366. 10.1111/j.1365-2036.2006.03144.x.View ArticleGoogle Scholar
- Chattopadhyay S, Patra R, Ramamurthy T, Chowdhury A, Santra A, Dhali GK, Bhattacharya SK, Berg DE, Nair GB, Mukhopadhyay AK: Multiplex PCR assay for rapid detection and genotyping of Helicobacter pylori directly from biopsy specimens. J Clin Microbiol. 2004, 42: 2821-2824. 10.1128/JCM.42.6.2821-2824.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Tomb JF, White O, Kerlavage AR, Clayton RA, Sutton GG, Fleischmann RD, Ketchum KA, Klenk HP, Gill S, Dougherty BA, Nelson K, Quackenbush J, Zhou L, Kirkness EF, Peterson S, Loftus B, Richardson D, Dodson R, Khalak HG, Glodek A, McKenney K, Fitzegerald LM, Lee N, Adams MD, Hickey EK, Berg DE, Gocayne JD, Utterback TR, Peterson JD, Kelley JM, Cotton MD, Weidman JM, Fujii C, Bowman C, Watthey L, Wallin E, Hayes WS, Borodovsky M, Karp PD, Smith HO, Fraser CM, Venter JC: The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature. 1997, 388: 539-547. 10.1038/41483.View ArticlePubMedGoogle Scholar
- van der Ende A, Pan ZJ, Bart A, van der Hulst RW, Feller M, Xiao SD, Tytgat GN, Dankert J: cagA-positive Helicobacter pylori populations in China and The Netherlands are distinct. Infect Immun. 1998, 66: 1822-1826.PubMed CentralPubMedGoogle Scholar
- Devi SM, Ahmed I, Francalacci P, Hussain MA, Akhter Y, Alvi A, Sechi LA, Megraud F, Ahmed N: Ancestral European roots of Helicobacter pylori in India. BMC Genomics. 2007, 8: 184-10.1186/1471-2164-8-184.PubMed CentralView ArticlePubMedGoogle Scholar
- Latifi-Navid S, Ghorashi SA, Siavoshi F, Linz B, Massarrat S, Khegay T, Salmanian A-H, Shayesteh AA, Masoodi M, Ghanadi K, Ganji A, Suerbaum S, Achtman M, Malekzadeh R, Falush D: Ethnic and geographic differentiation of Helicobacter pylori within Iran. PLoS ONE. 2010, 5: e9645-10.1371/journal.pone.0009645.PubMed CentralView ArticlePubMedGoogle Scholar
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