Curr Microbiol (2009) 59:123129

DOI 10.1007/s00284-009-9407-x

Core Genome Haplotype Diversity and vacA Allelic Heterogeneity

of Chinese Helicobacter pylori Strains
Y. L. Liao G. Guo X. H. Mao Q. H. Xie
W. J. Zhang X. F. Liu Q. M. Zou

Received: 13 January 2009 / Accepted: 27 March 2009 / Published online: 19 May 2009
Springer Science+Business Media, LLC 2009

Abstract The human gastric pathogen, Helicobacter

pylori, has co-evolved with its host and established itself in
the human stomach possibly millions of years ago.
Therefore, the diversity of this bacterium is important in its
clinical manifestations. Our aim has been to evaluate the
genetic diversity of 40 H. pylori clinical isolates from four
different parts of China. The methods of multi-locus
sequence typing and vacA allele genotyping were used to
assess their genetic diversity. To discriminate MLST, the
vacA genotype method was used to identify strains.
Patients from the northern, eastern, southern, and southwestern parts of China were recruited randomly from the
cities of Beijing, Shanghai, Guangzhou, and Chongqing,
respectively. Most of the sequence types are new and have
never been reported in the database of the H. pylori multilocus sequence typing system. The most prevalent vacA
genotype in patients was s1a/m2 (80.0%), followed by s1b/
m2 (17.5%). In contrast, the s1a/m1 genotype was scarcely
represented (2.5%). The vacA genotype varied for each ST.
These results showed that the MLST method offers high
resolution of the H. pylori isolates in China when compared
to vacA genotyping. The vacA allelic s1a has been correlated with the peptic ulcer. Because of the paucity of data
on human isolates due to the absence of systematic
investigations of H. pylori in China, the data provide useful

Y. L. Liao
G. Guo
X. H. Mao
Q. H. Xie

W. J. Zhang
X. F. Liu
Q. M. Zou (&)
Department of Clinical Microbiology and Immunology, Faculty
of Medical Laboratory Science, Third Military Medical
University, Chongqing, China
e-mail: qmzou@mail.tmmu.com.cn
Y. L. Liao
e-mail: yaya_lynn@126.com

information for understanding the epidemiology of

H. pylori in China from the viewpoint of nucleotide
sequence databases.

Introduction
Helicobacter pylori is the major causative agent of chronic
active gastritis, persistently colonizing the gastric mucosa
of human hosts for decades or a whole life-time [1]. This
organism is probably one of the most prevalent human
bacterial pathogens, and its colonization of the stomach is
an important etiological factor in the pathogenesis of gastric cancer [26]. However, most people infected with
H. pylori remain asymptomatic and 1020% of the patients
go on to develop peptic ulcer or gastric cancer. One possible explanation for this is that H. pylori has exceptional
genetic variability and interspecies diversity [7].
The expression of disease is associated with bacterial
factors. Several potential virulence factors have been suggested to play a prominent role in pathogenesis caused by
H. pylori [810]. Numerous epidemiological studies on
H. pylori have focused on the vacA gene. This virulence
factor of the gene exists in almost all the H. pylori strains,
while the cagA gene is present in *60% of strains, and
both are associated in particular with the differences in
disease risk. The vacA signal sequence (s sequence) located
in the region encoding the N-terminal signal r has two
major genotypes, s1 and s2, with two variations of s1, viz.
s1a and s1b. And the mid-region of vacA (m allele) has two
major genotypes, m1 and m2 [1113]. Moreover, particular
vacA genotypes vary in their geographic prevalence and
may serve as markers for the ancestry of the H. pylori
isolates [1417].

123

124

Y. L. Liao et al.: Core Genome Haplotype Diversity and vacA Allelic Heterogeneity

Table 1 Background information of the H. pylori isolates

Isolation region

Beijing (10)

Shanghai (10)

Guangzhou (10)

Chongqing (10)

Dyspepsia

Chronic gastritis

20

Peptic ulcer

12

The multi-locus sequence typing (MLST) was proposed

in 1998 [18] as a general approach to provide accurate,
portable data that were appropriate for the epidemiological
investigation of bacterial pathogens, and which also
reflected their evolutionary and population biology. This
method uses genetic variations at multiple chromosomal
locations and allows generation of sequence data that may
be available via the Internet for comparison between different laboratories. It applies neutral or slowly accumulating genetic variations in housekeeping genes, the
evolution of which is constrained by their requirement to
encode functional products, and which is not affected by
the rapid evolution detected within genes encoding proteins
that influence survival in a particular niche [1921].
Helicobacter pylori infections in China were common
and extensively distributed, with an average infection rate
of *58%. Significant intra-familial clustering was
observed, which might be due to close contact among
family members. However, most people harboring
H. pylori are asymptomatic, and only a few patients
infected with this bacterium develop peptic ulcer or gastric
cancer. One possible explanation for the different clinical
findings is that patients are infected by different strains of
H. pylori.

Materials and Methods

Patients
Clinical isolates of H. pylori were obtained from 2001 to
2007 in various parts of China. None of the patients had
received non-steroidal anti-inflammatory drugs or antacids,
and none had recently (more than half a year) been prescribed antibiotics. The patient age range was 2580 years,
and the proportion of each gender was 50% (Table 1). The
diagnosis was made by two endoscopists. The study was
performed in accordance with the Declaration of Helsinki.
Informed consent was obtained from each patient prior to
entering the study.
Isolation and Culture of H. pylori Isolates
Three biopsies samples were taken from each patient (two
from the antrum, one from the body of the stomach). The
biopsies were placed in saline solution (NaCl 0.85%) and

123

Total (40)

taken within 4 h to the laboratory for analysis. Specimens

were cultured on brain-heart infusion (BHI) agar containing
5% rabbit blood, incubated for 5 days at 37C under
microaerobic conditions (O2, 5%; CO2, 15%; N2, 80%).
Helicobacter pylori microbes were identified on the basis of
typical colony morphology, characteristic appearance with
Gram staining, and positive testing for urease. Three-day
liquid cultures of H. pylori were obtained by inoculating
some positive colonies from these plates into 20 ml brucella
broth supplemented with 5% rabbit blood. Chromosomal
DNA was extracted from the pellet using TIANGEN
Bacteria DNA Kits and its integrity measured by electrophoresis in 0.8% agarose gel. Reference strains of H. pylori
were obtained in a lyophilized form from the National
Collection of Type Cultures (NCTC): NCTC 11637 (type
strain, Australia); NCTC 11638(type strain, Australia).
MLST
MLST relied on sequences obtained from seven housekeeping genes: atpA, efp, mutY, ppa, trpC, ureI, vacA, and
yphC (Table 2). PCR products were amplified with oligonucleotide primers designed as previously described
(http://pubmlst.org/helicobacter/) [16]. PCR products from
isolates were purified for sequencing using QIAquick gel
extraction kit (QIAGEN, Germany), in accordance with the
manufacturers instructions. The DNA sequences of both
strands were determined with an ABI PRISM 310 DNA
sequencer (Applied Biosystems). Sequences were assembled from the resultant chromatograms by using DNAssist
software package (Gene Codes Corp.). MLST alleles and
sequence types were assigned to the isolates by comparing
the data to the H. pylori MLST database (http://
helicobacterpylori.mlst.net). Each isolate was defined by
an allelic profile consisting of the eight integers, and each
unique allelic profile was assigned as a sequence type (ST).
Sequence type analysis and recombination tests (START)
was used as a useful tool in the analysis of MLST data,
including data summary, lineage assignment, tests for
recombination, and tests for selection [22]. Related STs
were clustered into clonal complexes or lineages using the
eBURST to examine patterns of evolutionary descent [23].
Analysis of fixed differences, polymorphic sites, and the
ratio of non-synonymous substitutions to synonymous
substitutions was performed with the DnaSP software
package (version 4.00) [24]. The index of association (Ia)

Bifunctional indole-3-glycerol
phosphate synthase
Vacuolating
cytotoxin
GTP-binding
protein
ATP synthase
subunit alpha
Gene product or
function

Elongation
factor P

Inorganic
pyrophosphatase

A/G-specific adenine
glycosylase

Urease accessory
protein

456
444
510
585
627
Fragment length
(bp)

410

398

420

vacA
efp
atpA
Gene locus

Table 2 Information of MLST housekeeping genes

ppa

mutY

ureI

yphC

trpC

Y. L. Liao et al.: Core Genome Haplotype Diversity and vacA Allelic Heterogeneity

125

provided a measure of the linkage disequilibrium between

alleles by the program START.
Detection of vacA Genotype Status
PCR was carried out in a total volume of 50 ll containing
100 ng genomic DNA (Table 3), 200 lM each of dNTP
(Takara, USA), 109 PCR buffer (20 mM TrisHCl, pH
8.4), 2 ll MgCl2, 0.75 mM of each primer, and 0.5 ll Taq
polymerase (2 U/ll). For each batch assayed, distilled
water instead of the genomic DNA templates provided a
negative control. The reaction mixtures were cycled in an
automated thermal cycle under the conditions shown in
Table 1 [25]. After amplification, 10 ll of PCR product
was electrophoresed on 1.5% agarose gel, stained with
ethidium bromide, and examined under a UV illuminator.
Results
The internal fragments of 8t loci were amplified and
sequenced for 40 cultures of H. pylori. We included 12
patients with peptic ulcer, 20 with chronic gastritis, and
eight with dyspepsia. All eight loci appeared to be under
stabilizing selective pressure, as indicated by the dN/dS
ratios being substantially \1 (Table 4).
Table 5 shows that there are 38 STs, most being
unique, and had not previously been described at the
H. pylori MLST database. An UPGMA (unweighted pair
group mean average) cluster analysis of distance matrix
showed that the strains isolated from four different cities,
as well as the two controlled strains, could be clustered in
three groups (Fig. 1). The total Ia was 5.27, and mean trial
variance was 0.014 with max trials variance of 0.115.
There was no evidence for linkage; therefore, genetic
diversity in H. pylori is caused primarily by recombination.
MLST clonal groupings have been defined as sets of bacteria sharing a significant number of identical alleles. Isolates were grouped together where they shared at least five
of the eight MLST loci by eBURST analysis (Fig. 2).
Associations within alleles at housekeeping loci were
quantified by calculating the Ia. Clonal populations to
assess whether genetic diversity in H. pylori was caused
primarily by mutation or recombination, where mutations
predominated in generating genetic variation show significant associations (linkage disequilibrium) within alleles at
different loci. Non-clonal populations, where recombination predominated, show low degrees of association
between alleles at different loci. The total Ia was 0.753, and
there is no evidence of linkage, and therefore genetic
diversity in H. pylori is caused primarily by recombination.
The primers on the allele s1, s2 and m1, m2 enabled the
differentiation and characteristics of allele of s region and
m region in vacA genotype which were isolated from our

123

126

Y. L. Liao et al.: Core Genome Haplotype Diversity and vacA Allelic Heterogeneity

Table 3 Primer sequence and PCR conditions used for amplification of vacA alleles of H. pylori
Amplified region

Primer sequence (50 ? 30 )

Product size (bp)

PCR conditions

vacAs1a

F: GTCAGCATCACACCGCAAC

190

94C, 1 min; 55C, 1 min; 72C,

1 min (35 cycles)

R: CTGCTTGAATGCGCCAAAC
vacAs1b

F: AGCGCCATACCGCAAGAG

187

R: CTGCTTGAATGCGCCAAAC
vacAs2

F: GCTAACACGCCAAATGATCC

199

R: CTGCTTGAATGCGCCAAAC
vacAm1

F: GGTCAAAATGCGGTCATGG

290

R:CCATTGGTACCTGTAGAAAC
vacAm2

F: GGAGCCCCAGGAAACATTG

352

R: CATAACTAGCGCCTTGCAC
F forward primer sequence, R reverse primer sequence
Table 4 The dN/dS results of eight loci
Locus

S (mean)

N (mean)

n (S ? N)

Pairwise comparisons

dN

r dN

dS

r dS

dN/dS

atpA

151.2

475.8

627

630

0.0013

0.0013

0.0922

0.0304

0.0138

efp

89.2

318.8

408

666

0.0023

0.0027

0.1345

0.0482

0.0174

mutY

93.8

326.2

420

595

0.0153

0.0082

0.1549

0.0555

0.0988

ppa
trpC

85.5
100.1

310.5
355.9

396
456

666
630

0.0039
0.0193

0.0032
0.0154

0.0858
0.1688

0.0417
0.0815

0.0455
0.1146

ureI

135.7

449.3

585

666

0.0042

0.0034

0.0779

0.0358

0.0533

vacA

94.3

349.7

444

630

0.0089

0.0086

0.1264

0.0422

0.0701

yphC

111.9

398.1

510

630

0.0104

0.0056

0.1025

0.0558

0.1014

H. pylori strains. Table 6 shows that the most frequently

observed H. pylori vacA allele genotype is the s1a/m2
allele (80%), followed by s1b/m2 (17.5%). In contrast, the
s1a/m1 genotype is scarcely represented (2.5%). The vacA
s1a genotype showed significant difference between the
peptic ulcer group and the other two groups, suggesting
that the s1a genotype is associated with peptic ulcer. While
m2 genotype is the main subtype of the vacA m allele,
there was no difference among three groups. Of the 40
isolates, the distribution of STs was diverse with the vacA
genotypes. Figure 3 presents the results of electrophoretic
examination of PCR products for H. pylori gene vacA
signal region and middle region in DNA extracted from 40
patients in accordance with their isolating regions.
Of the 40 isolates, the distribution of STs was diverse
among the vacA genotypes. So far, there is no clear association between vacA genotypes and STs.

Discussion
This is a novel in which the MLST approach has been used
to examine the clonal relationships of H. pylori isolates of
China. The MLST method provides a scalable typing

123

system that reflects the population and evolutionary biology of the bacterium, and makes possible valid comparison
of results between different laboratories. Although the
number of isolates was relatively small, the diversity of the
isolates was established by identification of their different
genotypes.
Most of new STs have never been found among the
database of H. pylori MLST systems. China is a country
with 1.2 billion people, where people eat their meals
together, which probably increases the risk of H. pylori
infection, and lead to a more genetic diversity of this
bacterium. Helicobacter pylori and man seem to have an
historic association and its clonal relationships is only
apparent in the very short term, for example, during
transmission from one host to another [26, 27]. Consistent
with our study, Falush et al. [16] had proposed that
H. pyloris non-clonal population structure may be the
result of the frequent recombination between distinct
strains during mixed colonization. We also found that most
alleles from independent strains of H. pylori are unique.
Because of the high levels of recombination among the
isolates of H. pylori, it seemed that almost every isolate had
a different ST number. Although recombination may occur
on a global scale for most genes studied, two weakly clonal

Y. L. Liao et al.: Core Genome Haplotype Diversity and vacA Allelic Heterogeneity
Table 5 Sequence type of H. pylori isolates
Number of H. pylori
isolates

Region

Year

ST

Beijing

20072009

413

421

422

430

436

20042007

437

7
8

438
417

400

10
11

401
Shanghai

20072004

420

12

426

13

423

14

411

15

20022011

412

16

414

17

424

18

20012004

19
20
21

406
408
427

Guangzhou

20062011

402

22
23

404
405

24

402

25

416

26

20072004

431

27

435

28

433

29

418

30
31

419
Chongqing

20072004

410

32

410

33

407

34

434

35

425

36

428

37

409

38
39

429
432

40

403

NCTC11637

415

NCTC11638

30

complexes were noted, which suggests that, with time,

more clonal groupings will be recognized if larger samplings are taken.
The incidence of gastric carcinoma of China is among
the highest in the world. There are also indications of

127

significant geographic differences among H. pylori strains

[4, 16]. Unlike some countries, such as Japan or Korea,
where a relatively homogeneous population exist, China has
had a mixture of people from different ethnic groups
throughout its history. Therefore, the opportunity for the
transfer of DNA between strains of different genotypes may
be higher than in these other countries. The phylogenetic
tree generated from allelic profile data gives evolutionary
information about the isolates, as seen from UPGMA and
split decomposition analyses on the allelic profile data.
Although the H. pylori genome shows a high degree of
genetic diversity among strains [2, 28] weakly clonal
groupings were detected, which could be superimposed on a
pattern of free recombinationan outcome that provides a
basis for further analyses on population genetics.
And VacA protein plays a role in its pathogenicity,
which induces the formation of intracellular vacuoles in
eukaryotic cells in vitro. The most frequently observed
H. pylori vacA allele genotype is the s1a/m2 allele (80%),
followed by s1b/m2 (17.5%), while s1a/m1 genotype is
scarcely represented (2.5%). Production of vacuolating
cytotoxin is related to the mosaic structure of vacA, with
the distribution of vacA genotypes showing a difference
between the four parts of China. The s1a strains are considered to be more cytotoxic, and were frequently detected
in patients with peptic ulcer. However, the reason for no
distinct differences being found within the middle region of
vacA gene in different chronic gastrosia remains unclear.
There are several reports on the relationship between
MLST and H. pyloris virulence factor cagA gene [17, 29].
We therefore inspected the MLST and vacA genetype,
which is also well associated with the differences in disease
risk as the cagA gene. The vacA genotyping was used as a
traditional method to evaluate the discrimination of the
MLST. The MLST method gave better discrimination. In
general, isolates that had the same vacA genotype had variable ST genotypes, suggesting that there was no obvious link
between vacA genotype and ST genotypes from on our data.
In conclusion, this study demonstrates that the adaptation of MLST for strain characterization of H. pylori
directly from clinical specimens has an important role in
the epidemiological and population studies of this panmictic pathogen. It provides a high discrimination tool for
identifying H. pylori strains. The application of MLST
explored the population structure of H. pylori where
recombination plays an important role in Chinese. We have
shown that the vacA s1a strains can be considered more
cytotoxic, as frequently found in patients with peptic ulcer.
However, China is a big country with many people, so
further studies are needed to improve our understanding of
the population biology and structure of H. pylori, including
isolates from more regions and over several more timeframes.

123

128

Y. L. Liao et al.: Core Genome Haplotype Diversity and vacA Allelic Heterogeneity

Fig. 1 An UPGMA
dendrogram of the mean
normalized pair-wise
differences between alleles for
eight gene fragments. The
number of nucleotide
differences between each pair of
alleles was normalized to a
maximal value of 1.0 before
averaging over the eight gene
fragments. The resulting matrix
was used for a cluster analysis
using the unweighted pair-group
mean average clustering method

16 - ST-414 (69,64,146,73,68,69,234,212)
27 - ST-435 (213,198,272,245,234,324,406,339)
13 - ST-423 (185,177,222,185,188,213,342,268)
22 - ST-404 (12,9,18,13,12,16,16,13)
30 - ST-419 (122,72,161,146,72,146,287,234)
34 - ST-434 (216,205,292,256,274,349,405,334)
23 - ST-405 (13,11,20,16,13,18,17,20)
40 - ST-403 (11,6,12,12,11,12,13,11)
8 - ST-417 (124,145,179,156,146,161,293,237)
11 - ST-420 (146,164,185,161,153,185,301,272)
33 - ST-407 (22,25,25,22,20,26,28,28)
4 -ST-430 (190,192,266,205,225,293,401,301)
14 - ST-411 (28,28,42,28,26,52,69,146)
37 - ST-409 (25,26,26,25,21,28,43,64)
26 - ST-431 (189,188,228,192,198,234,356,293)
3 - ST-422 (179,153,198,179,185,197,336,291)
15 - ST-412 (43,43,63,43,28,55,146,161)
32 - ST-410 (26,27,28,26,22,43,55,69)
31 - ST-410 (26,27,28,26,22,43,55,69)
35 - ST-425 (64,55,69,188,55,67,179,189)
39 - ST-432 (18,20,22,20,41,357,20,22)
19 - ST-408 (20,21,23,21,18,22,22,25)
24 - ST-402 (8,2,11,11,6,11,12,8)
21 - ST-402 (8,2,11,11,6,11,12,8)
17 - ST-424 (62,50,68,64,43,64,161,179)
29 - ST-418 (105,69,153,98,69,72,245,228)
18 - ST-406 (16,18,21,18,16,20,18,21)
25 - ST-416 (16,18,21,18,16,179,18,268)
20 - ST-427 (192,190,228,216,220,255,365,296)
2 - ST-421 (161,161,216,168,161,190,335,289)
28 - ST-433 (222,201,291,253,245,346,404,328)
12 - ST-426 (188,179,224,189,189,230,347,292)
38 - ST-429 (209,197,256,235,228,306,403,317)
36 - ST-428 (198,92,234,222,226,301,402,305)
10 - ST-401 (385,58,395,396,397,397,400,400)
9 - ST-400 (395,395,394,395,396,396,399,399)
6 - ST-437 (226,60,295,284,291,60,407,344)
5 - ST-436 (60,209,293,272,291,365,68,343)
7 - ST-438 (184,215,296,285,289,366,190,190)
42 - ST-30 (30,30,30,30,30,30,30,30)
41 - ST-415 (30,68,30,30,30,30,30,220)
1 - ST-413 (68,56,72,72,64,68,198,192)
0.01

Fig. 2 Using BURST program

to perform split decomposition
analysis on allelic profile data.
This is a clustering algorithm
designed for use with MLST
data, as a way of displaying the
relationships between closelyrelated isolates of a bacterial
species or population. Most of
the STs are singleton ST, which
may show the high diversity of
H. pylori stains. And BURST
analysis shows the relationship
between ST416 and ST406, and
they come from the same
potential ancestral stain. ST415
and ST30 are type strain from a
Australian patients

123

Y. L. Liao et al.: Core Genome Haplotype Diversity and vacA Allelic Heterogeneity

129

Table 6 Relationship of the subtypes of vacA gene and different chronic gastrosia
Group

Number of isolates

vacAs1a

vacAs1b

vacAm2

vacAm1

Chronic gastritis

20

15

20

Peptic ulcer

12

12

11

33 (82.5%)

7 (17.5%)

39 (97.5%)

1 (2.5%)

Dyspepsia
Total

40

100%
80%

m2

m2

60%
40%

s1b

20%

s1a

0%

Beijing

m2

m2

s1b

s1b

s1a

s1a

Shanghai

Guangzhou

m2

13.

s1b
s1a

s1b

14.

s1a
Chongqing

15.

Fig. 3 The frequency of alleles of the vacA genotypes in four

different cities of China
Acknowledgments This work was supported by a grant-in-aid for
the Major State Basic Research Development Program of China (973
Program) (No. 2009CB522606). We thank Yiqi Du, Jide Wang, and
Fukun Wang for technical assistance.

16.

17.
18.

References
1. Wirth T, Wang X, Linz B et al (2004) Distinguishing human
ethnic groups by means of sequences from Helicobacter pylori:
lessons from Ladakh. Proc Natl Acad Sci USA 101:47464751
2. Suerbaum S, Michetti P (2002) Helicobacter pylori infection.
N Engl J Med 347:11751186
3. Hentschel E, Brandstatter G, Dragosics B et al (1993) Effect of
ranitidine and amoxicillin plus metronidazole on the eradication
of Helicobacter pylori and the recurrence of duodenal ulcer.
N Engl J Med 328:308312
4. Covacci A, Telford JL, Parsonnet J et al (1999) Helicobacter
pylori virulence and genetic geography. Science 284:13281333
5. The EUROGAST Study Group (1993) An international association between Helicobacter pylori infection, gastric cancer. Lancet
341:13591362
6. Ernst PB, Gold BD (2000) The disease spectrum of Helicobacter
pylori: the immunopathogenesis of gastroduodenal ulcer and
gastric cancer. Annu Rev Microbiol 54:615640
7. Achtman M, Azuma T, Berg DE et al (1999) Recombination and
clonal groupings within Helicobacter pylori from different geographical regions. Mol Microbiol 32:459470
8. Carroll IM, Khan AA, Ahmed N (2004) Revisiting the pestilence
of Helicobacter pylori: insights into geographical genomics and
pathogen evolution. Infect Genet Evol 4:8190
9. Hurtado A, Chahal B, Owen RJ et al (1994) Genetic diversity of
the Helicobacter pylori hemagglutinin/protease (hap) gene.
FEMS Microbiol Lett 123:173178
10. Blaser MJ, Berg DE (2001) Helicobacter pylori genetic diversity
and risk of human disease. J Clin Invest 107:767773
11. Atherton JC, Tummuru MK, Blaser MJ et al (1995) Mosaicism in
vacuolating cytotoxin alleles of Helicobacter pylori. Association
of specific vacA types with cytotoxin production and peptic
ulceration. J Biol Chem 270:1777117777
12. McClain MS, Cao P, Iwamoto H et al (2001) A 12-amino-acid
segment, present in type s2 but not type s1 Helicobacter pylori

19.

20.
21.
22.

23.

24.

25.

26.

27.

28.

29.

VacA proteins, abolishes cytotoxin activity and alters membrane

channel formation. J Bacteriol 183:64996508
Telford JL, Comanducci M, Burroni D et al (1994) Gene structure
of the Helicobacter pylori cytotoxin and evidence of its key role
in gastric disease. J Exp Med 179:16531658
Van Doorn LJ, Figueiredo C, Megraud F et al (1999) Geographic
distribution of vacA allelic types of Helicobacter pylori. Gastroenterology 116:823830
Ghose C, Pride DT, Bravi CM et al (2002) East Asian genotypes
of Helicobacter pylori strains in Amerindians provide evidence
for its ancient human carriage. Proc Natl Acad Sci USA 99:
1510715111
Falush D, Wirth T, Linz B et al (2003) Traces of human
migrations in Helicobacter pylori populations. Science 299:
15821585
Devi SM, Ahmed I, Francalacci P et al (2007) Ancestral European roots of Helicobacter pylori in India. BMC Genomics 8:184
Maiden MC, Bygraves JA, Feil E et al (1998) Multilocus
sequence typing: a portable approach to the identification of
clones within populations of pathogenic microorganisms. Proc
Natl Acad Sci USA 95:31403145
Selander RK, Caugant DA, Ochman H et al (1986) Methods of
multi-locus enzyme electrophoresis for bacterial population
genetics and systematics. Appl Environ Microbiol 51:873884
Urwin R, Maiden MC (2003) Multi-locus sequence typing: a tool
for global epidemiology. Trends Microbiol 11:479487
Cooper JE, Feil EJ (2004) Multi-locus sequence typingwhat is
resolved? Trends Microbiol 12:373377
Jolley KA, Feil EJ, Chan MS et al (2001) Sequence type analysis
and recombinational tests (START). Bioinformatics 17:1230
1231
Feil EJ, Li BC, Aanensen DM et al (2004) eBURST: inferring
patterns of evolutionary descent among clusters of related bacterial genotypes from multi-locus sequence typing data. J Bacteriol 186:15181530
Rozas J, Sanchez-DelBarrio JC, Messeguer X et al (2003) DnaSP,
DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19:24962497
Koehler CI, Mues MB, Dienes HP et al (2003) Helicobacter
pylori genotyping in gastric adenocarcinoma and MALT lymphoma by multiplex PCR analyses of paraffin wax embedded
tissues. Mol Pathol 56:3642
Kraft C, Stack A, Josenhans C et al (2006) Genomic changes
during chronic Helicobacter pylori infection. J Bacteriol 188:
249254
Suerbaum S, Smith JM, Bapumia K et al (1998) Free recombination within Helicobacter pylori. Proc Natl Acad Sci USA
95:1261912624
Taylor DE, Eaton M, Chang N et al (1992) Construction of a
Helicobacter pylori genome map and demonstration of diversity
at the genome level. J Bacteriol 174:68006806
Devi SM, Ahmed I, Khan AA et al (2006) Genomes of Helicobacter pylori from native Peruvians suggest admixture of
ancestral and modern lineages and reveal a western type cagpathogenicity island. BMC Genomics 7:191

123