- Email: [email protected]
The history of Helicobacter pylori: from phylogeography to paleomicrobiology
Accepted Manuscript The history of Helicobacter pylori - From phylogeography to paleomicrobiology Prof Francis Mégraud, Philippe Lehours, Filipa F. Vale PII:
S1198-743X(16)30237-3
DOI:
10.1016/j.cmi.2016.07.013
Reference:
CMI 656
To appear in:
Clinical Microbiology and Infection
Received Date: 16 May 2016 Revised Date:
14 June 2016
Accepted Date: 9 July 2016
Please cite this article as: Mégraud F, Lehours P, Vale FF, The history of Helicobacter pylori - From phylogeography to paleomicrobiology, Clinical Microbiology and Infection (2016), doi: 10.1016/ j.cmi.2016.07.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
history
of
Helicobacter
paleomicrobiology.
pylori
-
from
phylogeography
to
M AN U
The
SC
RI PT
ACCEPTED MANUSCRIPT
Francis Mégraud1, Philippe Lehours1, 2, Filipa F. Vale3
1 Université de Bordeaux, Centre National de Référence des Campylobacters et Hélicobacters, Bordeaux, France
TE D
2 INSERM U1053, Bordeaux, France
3 Host-Pathogen Interactions Unit, Research Institute for Medicines (iMed-ULisboa),
AC C
EP
Instituto de Medicina Molecular, Faculdade de Farmácia da Universidade de Lisboa.
Corresponding author: Prof Francis Mégraud, Laboratoire de Bactériologie, Hôpital Pellegrin, 33077 Bordeaux, France
ACCEPTED MANUSCRIPT
Abstract
RI PT
The study of the gastric pathogen Helicobacter pylori brought us interesting data on the history of mankind. Based on multi locus sequence typing, it was possible to trace the migration of Homo sapiens all around the world, and to infer the time when he went Out of Africa. Beside these phylogeographic aspects, paleomicrobiology gave us important elements
SC
on the life at the Neolithic period, following the discovery of Ötzi, the Iceman, who was living in the Tyrolean Alps 5,200 years ago and from whom an Helicobacter pylori genome
M AN U
was sequenced. This review present the data accumulated in these different fields.
Key words
Multi locus sequence typing (MLST), migration, mummies, genetic diversity, genome
AC C
EP
TE D
sequencing
ACCEPTED MANUSCRIPT
Introduction The history of Helicobacter pylori is fascinating. The first surprise was the discovery of this bacterium from high clinical relevance only at the end of the 20th century (1). In fact, these
RI PT
bacteria had been observed at the beginning of the microbiology development when
microscopic observation was essential, but it was then forgotten probably because there was enough to study with bacteria which could easily grow in culture. Later, the bacteria fell
SC
victim to the belief that the stomach was a sterile organ, i.e. no bacteria could grow at such a low pH, and therefore many observers thought that the bacteria observed were simply in
M AN U
transit from the oral cavity rather than gastric residents.
However, over and above the medical aspect which now allows diseases such as peptic ulcer disease to be cured by a 10 to 14 day antibiotic therapy, as well as gastric carcinoma to be prevented, the role of H. pylori as a witness to human migrations via its phylogeography is
TE D
also a fantastic story and paleomicrobiology studies are especially illuminated by the discovery of Ötzi, the iceman, who died 5,200 years ago (2). These aspects will be reviewed
EP
in this article.
AC C
I. Phylogeographic distribution of the Helicobacter pylori population
A. Characterization of H. pylori populations Several techniques have been developed in order to identify the common source of outbreaks which can also be used to determine the population structure of bacterial pathogens and to reconstruct their evolutionary history. For genetically monomorphic bacteria, it turns out that multilocus sequence typing (MLST) is currently the best method which allows an optimal standardization and is truly portable between laboratories (3).
ACCEPTED MANUSCRIPT MLST was proposed in 1998 as a universal method for bacterial characterization using Neisseria meningitidis as an example and since then has been used for a large number of pathogens. A set of six fragments (each approximately 470 bp) of housekeeping genes was chosen. For each locus, alleles were assigned numbers arbitrarily and dendrograms were
RI PT
constructed from the pairwise differences in multilocus allelic profiles by cluster analysis. This method reliably identified the major meningococcal lineages associated with invasive diseases (4).
SC
With any given genetic dataset, it is important to verify whether there is any evidence that the samples in the data are from a structured population. Moreover, understanding the population
M AN U
structure is an important step for evolutionary studies of any species (5). The approaches frequently employed to analyze MLST data rely on phylogenetic trees and STRUCTURE (6) . The increased availability of high throughput sequencing data poses new statistical challenges for the current methods to identify populations considering all genome data. As a result other
TE D
methodologies have recently been developed, namely fineSTRUCURE (7). H. pylori is a strictly human pathogen, currently colonizing the stomachs of approximately half of the world population. In most subjects it does not lead to major diseases while gastritis
EP
lesions are always present, but peptic ulcer disease can arise in 5-10% of them as well as two cancers: gastric adenocarcinoma in approximately 1% and gastric MALT lymphoma in an
AC C
even smaller proportion.
H. pylori strains are extremely diverse and virtually all non-epidemiologically related strains appear to be different. Achtman et al. were the first to apply MLST using the following seven housekeeping genes: atpA, efp, mutY, ppa, trpC, ureI and yphC (instead of six in the princeps article) to a collection of 20 H. pylori strains from several regions of the world also including sequences of 2 virulence-associated genes (cagA, vacA), which are not currently considered in MLST analysis. They found that clonal descent could still be distinguished despite frequent
ACCEPTED MANUSCRIPT recombinations possibly reflecting geographical separation. Then MLST can be used in the panmictic population of H. pylori (8). Indeed, H. pylori presents a high recombination rate,
B. H. pylori as a tool to follow human migrations
RI PT
which has a greater effect than mutation (9).
H. pylori population structure provides strong evidence of ancient ancestry in Africa and subsequent human migrations. Accumulated evidence points to human migration out of
SC
Africa to the Middle East ~60,000-150,000 year ago and then independently to Europe and Asia (10). Then H. pylori and its human host undergo variation driven by founder effects,
M AN U
isolation, drift and host selection. As a result H. pylori became a marker of human migrations (11).
This was confirmed by Falush et al. who sequenced the seven housekeeping genes from 370 H. pylori strains isolated from 27 geographic, ethnic and linguistic human groups. Of the
TE D
3,850 nucleotides sequenced for each isolate, 1,418 were polymorphic and were used to define bacterial populations with the programme STRUCTURE. (Fig 1) (12). The long branch of hpAfrica2 in the population tree indicates prolonged genetic isolation, and
populations (12).
EP
probably reflects a long period of independent evolution prior to the splits into the other
AC C
The hpAfrica1 group could be divided into hspSAfrica and hspWAfrica. The hspWAfrica were found at a high frequency in West Africa and America. The hpEurope were found in Europe but also in America, Australia and West Asia, reflecting more recent human migrations. Almost all strains from East Asia were assigned to the hpEastAsia population from which the subpopulations hspEAsia, hspAmerind, isolated from Amerindians and Inuits, and hspMaori, found in Oceania, were derived (13). From this current population of H. pylori, using a linkage model it was possible to infer
ACCEPTED MANUSCRIPT ancestral populations: Africa1, Africa2, East Asia, Europe 1 (AE1) and Europe 2 (AE2) (Fig 1). The high diversity in hpEurope would come from the mixture between AE1 and AE2 which probably reached Europe at different times and from different sources: AE1 was indeed
RI PT
present throughout Central Asia in addition to Europe and AE2 came from North Africa and Middle East.
Interestingly, in a recent study, Vale et al. were able to clearly differentiate between two
SC
European populations, present mainly in Northern Europe (presumably closer to AE1) and in Southern Europe (presumably closer to AE2) using a phage sequence typing based on 2
M AN U
prophage genes of H. pylori (integrase and holin) but present in only a minority of strains (14).
Further subgroups were identified using fineSTRUCTURE for the analysis of 29 H. pylori genome wide single nucleotide polymorphisms (SNPs) by Yahara et al. The hpEurope were
TE D
divided in two subgroups (and two singletons) and highest difference was found for hpEastAsia. In this population hspAmerind was divided into 4 subgroups and hspEAsia into another four groups, although a limited number of samples that do not represent H. pylori
EP
diversity were used (15). The population structure of H. pylori was inferred in another group of 60 H. pylori genomes using three approaches, including STRUCTURE run on a subset of
AC C
100 kb for 29242 SNPs. This approach identified fewer populations than previously, which included Africa2, Africa1, EurAsia (Europe and Asia), EastAsia and America, but using a limited sample size with little geographic diversity. For instance all strains forming the population America came from the same country (Peru) (16). The populations identified in different studies are in agreement with each other, and in a way show how H. pylori populations are organized in a more general (fewer populations) or detailed (larger number of populations) perspective. However, understanding the detailed structure of H. pylori
ACCEPTED MANUSCRIPT population is an exciting field, that novel analyzed sequences and high throughput sequencing data starts to reveal. The phylogeographic distribution of H. pylori is therefore an additional important tool to
mitochondrial DNA (mtDNA) and languages.
RI PT
investigate human evolution and migration which can be added to the traditional tools, e.g.
Similar to the situation in humans, genetic diversity in H. pylori decreases with geographic distance from East Africa where Homo sapiens originated. It was then possible to hypothesize
SC
that H. pylori spread from East Africa around 58,000 ± 3,500 years ago and that modern humans were already infected by H. pylori when their migrations out of Africa began (Fig. 2)
M AN U
(17).
A comparable proportion of the genetic diversity of humans based on autosomal microsatellites was accounted to geographic distance from East Africa (77% of the variance vs 72% for H. pylori) also indicating a migration between 50,000 and 70,000 years ago (18).
TE D
Several articles have reported more specific migrations of H. pylori (and humans) around the world. An interesting one is the peopling of the Pacific region. Indeed, 40,000 to 50,000 years ago Australia, New Guinea and Tasmania were connected in a continent called Sahul which
EP
apparently was colonized only once by humans. The study of diverse aboriginal people from these regions allowed the formation of a distinct biogeographic group called hpSahul, its split
AC C
dating back 31,000-37,000 years ago. In contrast, hspMaori is a subgroup of hpEastAsia which is considered to be a marker for the entire Austronesian expansion and the origin would be some tribes of aboriginal Taiwanese, these results being in agreement with the data coming from language studies. This group corresponds to a second wave of migration into the Pacific (13). Breurec et al. also studied the complex peopling of Southeast Asia using H. pylori MLST. They were able to find at least three human migrations to this area: 1) a migration from India
ACCEPTED MANUSCRIPT introduced hpEurope in Thailand, Cambodia and Malaysia, 2) a migration into Vietnam and Cambodia carrying hspEAsia, and 3) a migration from Southern China to Thailand carrying hpAsia 2 (19). Interestingly, the distribution of these haplotypes shape the incidence of gastric cancer in these countries, given that hspEAsia has a stronger association with this disease, as
origins with their European counterparts (20).
RI PT
is observed in Vietnam. Devi et al. confirmed that H. pylori strains in India share ancestral
The peopling of America can also be traced via H. pylori. Strains from indigenous people of
SC
this continent (Columbia, Alaska) possessed novel vacA and cagA gene structures most closely related to East Asian H. pylori (21). It was also possible to show East Asian loci in
M AN U
strains from Amerindian subjects from Amazonia which were not present in the Mestizo population (22). These data were confirmed by Falush et al. who showed that the hspAmerind haplotype was derived from hpEastAsia and was probably introduced by humans migrating from Asia via the Bering Strait 11,000 years ago (12).
TE D
Another study was carried out to answer how long humans were infected by H. pylori prior to the out-of-Africa event. It included strains obtained from the KhoiSan people of South Africa, a unique relic of hunter-gatherer lineages which diverged 90,000 to 150,000 years ago
EP
according to mtDNA data (23). It was found that almost half of the San population H. pylori haplotypes belong to hpAfrica2. When hpAfrica2 was compared to hpAfrica1 and
AC C
Helicobacter acinonychis - a Helicobacter species which is the closest to H. pylori and found in large felines (cheetah, tiger) - hpAfrica2 and H. acinonychis clustered together, and separately from hpAfrica1. Interestingly H. acinonychis is thought to have arisen from a host jump from humans which occurred 43,000 to 45,000 years ago while the estimated minimum age of the association of H. pylori with humans is 100,000 years (range 88,000-116,000). The Bantus who originate from Central Africa (Nigeria, Cameroon) expanded around 5,000 years ago into the Southern part of Africa and appear to have acquired some hpAfrica2 haplotypes
ACCEPTED MANUSCRIPT from the San, while the San acquired hpAfrica1 from them (Fig 2) (24).
II. Paleomicrobiology and H. pylori There are limited data concerning the direct detection of H. pylori in ancient populations. H.
RI PT
pylori lives in an organ, the stomach, which does not allow a good chance of preservation over numerous years, in contrast, for example, to bacteria protected from the environment in teeth or bones. Nevertheless, bodies which were mummified either voluntarily or due to
SC
freezing in an appropriate environment have offered us a few examples of paleomicrobiology. A. Pre-Columbian mummies
M AN U
Mummification is defined as the preservation of soft tissues from the enzymatic process of post mortem putrefaction. For this, special conditions are required, in particular natural dryness which can be the case in funeral caves. However, as part of funeral rituals the bodies may be wrapped in blankets woven from absorbent vegetable fibers. The internal organs
seldomly conserved.
TE D
being the last to dehydrate, they may be susceptible to putrefaction, and therefore are
The first study concerning H. pylori in pre-Columbian mummies was indeed performed by
EP
Correa et al. They took advantage of the availability of a new kit to detect H. pylori antigens by ELISA (H. pylori Stool Antigen Kit, Meridian Diagnostics) to look for H. pylori in
AC C
coprolites from pre Columbian mummies from Chile. There were 7 positive specimens out of the 16 tested but only 2 of them, from the same mummy, were blocked by anti H. pylori IgG confirming the presence of H. pylori antigens. These specimens came from the mummy of a 25-year old male who died of pneumonia. His cardiomegaly indicated his Andean origin and he was mummified in 300 AD, i.e. 1,700 years ago. Another 1,800 year old mummy gave a strong positive result but which was only partly inhibited by the anti H. pylori IgG (25, 26). In another study the natural orifices of two mummies allowed to explore the internal organs
ACCEPTED MANUSCRIPT with a gastroscope (Pentax EG-1840) and to obtain material from their stomach. The histological analysis of the tissue samples did not allow to distinguish a complete gastric mucosa, but by carrying out PCRs it was possible to detect the presence of H. pylori in 2 of the 4 biopsies from the same mummy while other specimens from tongue and brain tissue
RI PT
were negative. The 109 bp DNA fragment of the 16S rRNA gene hybridized with a specific probe and had 99% homology with H. pylori reference strains. In addition, a real-time PCR targeting the ureB gene was positive in one of the samples. This mummy was from a man
the Chihuahua State, northern Mexico (27).
SC
aged 50 to 60 years and dates back to 1000-1400 AD, i.e. 550 to 950 years old, and lived in
M AN U
These data confirm the presence of H. pylori in Amerindians and it can be hypothesized that the strains were from the hspAmerind haplotype as proposed in previous studies (21, 22). B.
Frozen mummies
• - In August 1999, the remains of a male subject were discovered by hunters in a glacier
TE D
located in British Columbia on a First Nation territory and was confirmed to be of First Nation ancestry. Radiocarbon dating suggested that he died approximately between 1670 and 1850 which is a time when contact with the Europeans in this area was possible.
EP
PCRs were performed on gastric tissue extracts targeting vacA, the PCR products were sequenced and compared to reference sequences. vacA is a gene specific to H. pylori and it
AC C
was found in the stomach of this mummified body. The authors looked at the different alleles and showed that mid regions was hybrid m2a/m1d while the signal sequence was s2. The presence of an s2 allele being unusual in Asian strains it suggests that the strain is potentially a hybrid between European strains and Asian strains (28). • - Ötzi, the Tyrolean iceman. In September 1991, a well conserved male frozen body was discovered in the Otzal Alps at an altitude of 3,200 m, in Italy at 90 meters from the Austro-Italian border, who is now called
ACCEPTED MANUSCRIPT Ötzi or the iceman. Surprisingly the frozen body was almost completely mummified and it dated back to 5,200 years, i.e. from the late Neolithic period which makes it the oldest mummy found to date. Numerous studies have been performed on this mummy in order to get insight on the life
technology during these last 25 years (29).
RI PT
conditions at this time, with several rebounds and also new data linked to improved
For example, a big surprise was the discovery of an arrowhead found in his left shoulder
SC
which indicated that he was injured and probably died of this injury, which had not been detected on the first x-rays performed. In the same manner, the Iceman's stomach was
M AN U
discovered during a reappraisal of radiological data and contained the food ingested shortly before his death.
In order to get more information on the mummy, for the 20th anniversary of his discovery, the decision was made to thaw the body which is maintained at a constant temperature of -6°C
TE D
and this was done in October 2010. Among several teams of specialists devoted to different organs, one was in charge of the gastrointestinal tract. The appropriate endoscope was selected in order to perform a gastroscopy; unfortunately, the collapsed oesophagus did not
EP
allow the utilization of this route and a laparoscopy was performed. Material was obtained from the gastric content, the gastric wall and from the length of the small and large intestines.
AC C
This material was dispatched to several laboratories and was tested by various methods: culture, PCR targeting different genes of H. pylori, and sequencing using the Roche 454 apparatus. However, the results for H. pylori detection were negative at this time (2010). Later, a metagenomic approach coupled with targeted genome capture generated 15,350 reads of H. pylori DNA throughout the gastrointestinal tract with an abundance decreasing from the stomach towards the lower intestinal tract, and with negative results for the muscle tissue used as control. When aligned with the H. pylori reference strain 26695, the reads showed damage
ACCEPTED MANUSCRIPT patterns indicative of ancient DNA. The strain was cagA positive and vacA s1a/i1/m1 type as are the more virulent modern H. pylori strains. Stigmates of gastric inflammation, via the presence of high level of calprotectin subunits, were also noted. More details on the Iceman H. pylori strain can be found in the original article and supplementary data (2).
RI PT
It was also possible to compare the MLST haplotype of the Iceman's strain to the database of 1,603 strains using the STRUCTURE programme and the strain was assigned to the modern population of hpAsia2. This information is interesting in light of the uncertainty concerning
SC
the time of recombination between AE1 (derived from hpAsia2) and AE2 (derived from hpNEAfrica) which formed the current hpEurope, as mentioned above.
M AN U
So it appears that 5,200 years ago, AE1 was present in central Europe with a minimum of admixture from AE2 (6.5%, probability interval 1.5-13.5%) which places it close to modern hpAsia2 found in India. These results were confirmed in further analyses. Indeed, the low proportion of AE2 ancestry observed in this strain suggests an introgression into Europe after
TE D
the Copper Age, i.e. later than previously thought, or at least later in Central Europe (Otzal Alps). However, the limit of such a conclusion is that it is based on only one strain.
EP
In conclusion, beyond medical aspects, phylogeography and paleomicrobiology of H. pylori especially with the new imaging and sequencing approaches are able to bring unexpected data
AC C
on the history of humanity.
Acknowledgement: Filipa F. Vale is recipient of a fellowship from FCT (SFRH/BPD/95125/2013).
ACCEPTED MANUSCRIPT Figure legends Fig 1. Relationships between modern populations (A), modern subpopulations (B), and ancestral populations (C) of Helicobacter pylori (Ref 12) Relationships between modern populations (A), modern subpopulations (B), and ancestral populations (C) of H. pylori. The black lines show neighbor-joining population trees as measured byδ̂, the net
RI PT
nucleotide distance between populations. The circle diameters indicate their genetic diversity, measured as the average genetic distance between random pairs of individuals. The larger circles in (A) versus (C) reflect the effects of admixture between ancestral populations. Filled arcs reflect the number of isolates (A and B) or nucleotides (C) in each population. Color coding is consistent in
M AN U
AE1 and AE2 populations. Scales are at lower right.
SC
different parts of the figure, except for modern hpEurope, which is an admixture between the ancestral
Fig 2. Chronological reconstruction of the major population events occurring during the intimate human-Helicobacter pylori association. (Ref 24)
Black lines indicate undifferentiated populations and all other lines are color-coded according to population. The sequence of events is as follows: 1) Initial acquisition of H. pylori by a
TE D
human ancestor; 2) Divergence of H. pylori into two super-lineages; 3) First successful migration of modern humans Out of Africa via the southern route; 4) H. pylori divergence into hpAfrica1 and hpNEAfrica with migration eastwards (hpNEAfrica) and westwards
EP
(hpAfrica1); 5) Divergence of H. pylori out of Africa into hpSahul and 6) hpAsia2 and hpEastAsia; 7) Host jump from San to large felines giving rise to H. acinonychis. 8) Southward migration of San carrying the ancestor of hpAfrica2; 9) Second successful
AC C
migration Out of Africa via the Levant; 10) Hybridization of AE1 from central and south-west Asia and AE2 from north-east Africa in the Middle East or western Asia resulting in hpEurope; 11) Spread of hpEurope bacteria to Europe; 12) Back migration from the Middle East, and Spain spreading hpEurope into North Africa. Dates in italics represent estimates obtained from sources other than H. pylori.
ACCEPTED MANUSCRIPT References
1. Warren JR, Marshall BJ. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. The Lancet. 1983;321:1273–5. 2. Maixner F, Krause-Kyora B, Turaev D, Herbig A, Hoopmann MR, Hallows JL, et al. The 5300-year-old Helicobacter pylori genome of the Iceman. Science. 2016;351:162-5.
RI PT
3. Achtman M. Evolution, population structure, and phylogeography of genetically monomorphic bacterial pathogens. Annu. Rev. Microbiol. 2008;62:53-70.
4. Maiden MC, Bygraves JA, Feil E, Morelli G, Russell JE, Urwin R, et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Nat. Acad. Sci. U. S. A. 1998;95:3140-5.
SC
5. Patterson N, Price AL, Reich D. Population structure and eigenanalysis. PLoS Gen. 2006;2:e190.
M AN U
6. Falush D, Stephens M, Pritchard JK. Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics. 2003;164:1567-87. 7. Lawson DJ, Hellenthal G, Myers S, Falush D. Inference of population structure using dense haplotype data. PLoS Gen. 2012;8:e1002453. 8. Achtman M, Azuma T, Berg DE, Ito Y, Morelli G, Pan ZJ, et al. Recombination and clonal groupings within Helicobacter pylori from different geographical regions. Mol. Microbiol. 1999 ;32:459-70.
TE D
9. Morelli G, Didelot X, Kusecek B, Schwarz S, Bahlawane C, Falush D, et al. Microevolution of Helicobacter pylori during prolonged infection of single hosts and within families. PLoS Gen. 2010;6:e1001036.
EP
10. Cavalli-Sforza L, Menozzi P, Piazza A, editors. The history and geography of human genes: Princeton University Press, New Jersey; 1996.
AC C
11. Dominguez-Bello MG, Menozzi P, Piazza A. The human microbiota as a marker for migrations of individuals and populations. Annu. Rev. Anthropol.. 2011;40:451-74. 12. Falush D, Wirth T, Linz B, Pritchard JK, Stephens M, Kidd M, et al. Traces of human migrations in Helicobacter pylori populations. Science. 2003;299:1582-5. 13. Moodley Y, Linz B, Yamaoka Y, Windsor HM, Breurec S, Wu JY, et al. The peopling of the Pacific from a bacterial perspective. Science. 2009;323:527-30. 14. Vale FF, Vadivelu J, Oleastro M, Breurec S, Engstrand L, Perets TT, et al. Dormant phages of Helicobacter pylori reveal distinct populations in Europe. Sci Reports. 2015;5:14333. 15. Yahara K, Furuta Y, Oshima K, Yoshida M, Azuma T, Hattori M, et al. Chromosome painting in silico in a bacterial species reveals fine population structure. Mol. Biol. Evol. 2013 ;30:1454-64.
ACCEPTED MANUSCRIPT 16. Montano V, Didelot X, Foll M, Linz B, Reinhardt R, Suerbaum S, et al. Worldwide Population Structure, Long-Term Demography, and Local Adaptation of Helicobacter pylori. Genetics. 2015;200:947-63. 17. Linz B, Balloux F, Moodley Y, Manica A, Liu H, Roumagnac P, et al. An African origin for the intimate association between humans and Helicobacter pylori. Nature.2007;445:915-8.
RI PT
18. Zhivotovsky LA, Underhill PA, Cinnioglu C, Kayser M, Morar B, Kivisild T, et al. The effective mutation rate at Y chromosome short tandem repeats, with application to human population-divergence time. Am. J. Hum Gen. 2004;74:50-61.
SC
19. Breurec S, Guillard B, Hem S, Brisse S, Dieye FB, Huerre M, et al. Evolutionary history of Helicobacter pylori sequences reflect past human migrations in Southeast Asia. PloS One. 2011;6:e22058. 20. Devi SM, Ahmed I, Francalacci P, Hussain MA, Akhter Y, Alvi A, et al. Ancestral European roots of Helicobacter pylori in India. BMC Genomics. 2007;8:184.
M AN U
21. Yamaoka Y, Orito E, Mizokami M, Gutierrez O, Saitou N, Kodama T, et al. Helicobacter pylori in North and South America before Columbus. FEBS letters 2002;517:180-4. 22. Ghose C, Perez-Perez GI, Dominguez-Bello MG, Pride DT, Bravi CM, Blaser MJ. East Asian genotypes of Helicobacter pylori strains in Amerindians provide evidence for its ancient human carriage. Proc. Nat. Acad. Sci. U. S. A. 2002;99:15107-11.
TE D
23. Behar DM, Villems R, Soodyall H, Blue-Smith J, Pereira L, Metspalu E, et al. The dawn of human matrilineal diversity. Am. J. Hum Gen. 2008;82:1130-40. 24. Moodley Y, Linz B, Bond RP, Nieuwoudt M, Soodyall H, Schlebusch CM, et al. Age of the association between Helicobacter pylori and man. PLoS Pathogens. 2012;8:e1002693.
EP
25. Correa P, Willis D, Allison MJ, Gerszten E. Helicobacter pylori in pre-Columbian mummies. Gastroenterology. 1998;114:A956-A
AC C
26. Allison MJ, Bergman T, Gerszten E. Further studies on fecal parasites in antiquity. Am. J.Clin. Pathol. 1999;112:605-9. 27. Castillo-Rojas G, Cerbon MA, Lopez-Vidal Y. Presence of Helicobacter pylori in a Mexican Pre-Columbian Mummy. BMC Microbiol. 2008;8:119. 28. Swanston T, Haakensen M, Deneer H, Walker EG. The characterization of Helicobacter pylori DNA associated with ancient human remains recovered from a Canadian glacier. PloS One. 2011;6:e16864. 29. Cano RJ, Tiefenbrunner F, Ubaldi M, Del Cueto C, Luciani S, Cox T, et al. Sequence analysis of bacterial DNA in the colon and stomach of the Tyrolean Iceman. Am. J. Phys Anthropol. 2000 ;112:297-309.
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Figure 1
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Figure 2
S1198-743X(16)30237-3
DOI:
10.1016/j.cmi.2016.07.013
Reference:
CMI 656
To appear in:
Clinical Microbiology and Infection
Received Date: 16 May 2016 Revised Date:
14 June 2016
Accepted Date: 9 July 2016
Please cite this article as: Mégraud F, Lehours P, Vale FF, The history of Helicobacter pylori - From phylogeography to paleomicrobiology, Clinical Microbiology and Infection (2016), doi: 10.1016/ j.cmi.2016.07.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
history
of
Helicobacter
paleomicrobiology.
pylori
-
from
phylogeography
to
M AN U
The
SC
RI PT
ACCEPTED MANUSCRIPT
Francis Mégraud1, Philippe Lehours1, 2, Filipa F. Vale3
1 Université de Bordeaux, Centre National de Référence des Campylobacters et Hélicobacters, Bordeaux, France
TE D
2 INSERM U1053, Bordeaux, France
3 Host-Pathogen Interactions Unit, Research Institute for Medicines (iMed-ULisboa),
AC C
EP
Instituto de Medicina Molecular, Faculdade de Farmácia da Universidade de Lisboa.
Corresponding author: Prof Francis Mégraud, Laboratoire de Bactériologie, Hôpital Pellegrin, 33077 Bordeaux, France
ACCEPTED MANUSCRIPT
Abstract
RI PT
The study of the gastric pathogen Helicobacter pylori brought us interesting data on the history of mankind. Based on multi locus sequence typing, it was possible to trace the migration of Homo sapiens all around the world, and to infer the time when he went Out of Africa. Beside these phylogeographic aspects, paleomicrobiology gave us important elements
SC
on the life at the Neolithic period, following the discovery of Ötzi, the Iceman, who was living in the Tyrolean Alps 5,200 years ago and from whom an Helicobacter pylori genome
M AN U
was sequenced. This review present the data accumulated in these different fields.
Key words
Multi locus sequence typing (MLST), migration, mummies, genetic diversity, genome
AC C
EP
TE D
sequencing
ACCEPTED MANUSCRIPT
Introduction The history of Helicobacter pylori is fascinating. The first surprise was the discovery of this bacterium from high clinical relevance only at the end of the 20th century (1). In fact, these
RI PT
bacteria had been observed at the beginning of the microbiology development when
microscopic observation was essential, but it was then forgotten probably because there was enough to study with bacteria which could easily grow in culture. Later, the bacteria fell
SC
victim to the belief that the stomach was a sterile organ, i.e. no bacteria could grow at such a low pH, and therefore many observers thought that the bacteria observed were simply in
M AN U
transit from the oral cavity rather than gastric residents.
However, over and above the medical aspect which now allows diseases such as peptic ulcer disease to be cured by a 10 to 14 day antibiotic therapy, as well as gastric carcinoma to be prevented, the role of H. pylori as a witness to human migrations via its phylogeography is
TE D
also a fantastic story and paleomicrobiology studies are especially illuminated by the discovery of Ötzi, the iceman, who died 5,200 years ago (2). These aspects will be reviewed
EP
in this article.
AC C
I. Phylogeographic distribution of the Helicobacter pylori population
A. Characterization of H. pylori populations Several techniques have been developed in order to identify the common source of outbreaks which can also be used to determine the population structure of bacterial pathogens and to reconstruct their evolutionary history. For genetically monomorphic bacteria, it turns out that multilocus sequence typing (MLST) is currently the best method which allows an optimal standardization and is truly portable between laboratories (3).
ACCEPTED MANUSCRIPT MLST was proposed in 1998 as a universal method for bacterial characterization using Neisseria meningitidis as an example and since then has been used for a large number of pathogens. A set of six fragments (each approximately 470 bp) of housekeeping genes was chosen. For each locus, alleles were assigned numbers arbitrarily and dendrograms were
RI PT
constructed from the pairwise differences in multilocus allelic profiles by cluster analysis. This method reliably identified the major meningococcal lineages associated with invasive diseases (4).
SC
With any given genetic dataset, it is important to verify whether there is any evidence that the samples in the data are from a structured population. Moreover, understanding the population
M AN U
structure is an important step for evolutionary studies of any species (5). The approaches frequently employed to analyze MLST data rely on phylogenetic trees and STRUCTURE (6) . The increased availability of high throughput sequencing data poses new statistical challenges for the current methods to identify populations considering all genome data. As a result other
TE D
methodologies have recently been developed, namely fineSTRUCURE (7). H. pylori is a strictly human pathogen, currently colonizing the stomachs of approximately half of the world population. In most subjects it does not lead to major diseases while gastritis
EP
lesions are always present, but peptic ulcer disease can arise in 5-10% of them as well as two cancers: gastric adenocarcinoma in approximately 1% and gastric MALT lymphoma in an
AC C
even smaller proportion.
H. pylori strains are extremely diverse and virtually all non-epidemiologically related strains appear to be different. Achtman et al. were the first to apply MLST using the following seven housekeeping genes: atpA, efp, mutY, ppa, trpC, ureI and yphC (instead of six in the princeps article) to a collection of 20 H. pylori strains from several regions of the world also including sequences of 2 virulence-associated genes (cagA, vacA), which are not currently considered in MLST analysis. They found that clonal descent could still be distinguished despite frequent
ACCEPTED MANUSCRIPT recombinations possibly reflecting geographical separation. Then MLST can be used in the panmictic population of H. pylori (8). Indeed, H. pylori presents a high recombination rate,
B. H. pylori as a tool to follow human migrations
RI PT
which has a greater effect than mutation (9).
H. pylori population structure provides strong evidence of ancient ancestry in Africa and subsequent human migrations. Accumulated evidence points to human migration out of
SC
Africa to the Middle East ~60,000-150,000 year ago and then independently to Europe and Asia (10). Then H. pylori and its human host undergo variation driven by founder effects,
M AN U
isolation, drift and host selection. As a result H. pylori became a marker of human migrations (11).
This was confirmed by Falush et al. who sequenced the seven housekeeping genes from 370 H. pylori strains isolated from 27 geographic, ethnic and linguistic human groups. Of the
TE D
3,850 nucleotides sequenced for each isolate, 1,418 were polymorphic and were used to define bacterial populations with the programme STRUCTURE. (Fig 1) (12). The long branch of hpAfrica2 in the population tree indicates prolonged genetic isolation, and
populations (12).
EP
probably reflects a long period of independent evolution prior to the splits into the other
AC C
The hpAfrica1 group could be divided into hspSAfrica and hspWAfrica. The hspWAfrica were found at a high frequency in West Africa and America. The hpEurope were found in Europe but also in America, Australia and West Asia, reflecting more recent human migrations. Almost all strains from East Asia were assigned to the hpEastAsia population from which the subpopulations hspEAsia, hspAmerind, isolated from Amerindians and Inuits, and hspMaori, found in Oceania, were derived (13). From this current population of H. pylori, using a linkage model it was possible to infer
ACCEPTED MANUSCRIPT ancestral populations: Africa1, Africa2, East Asia, Europe 1 (AE1) and Europe 2 (AE2) (Fig 1). The high diversity in hpEurope would come from the mixture between AE1 and AE2 which probably reached Europe at different times and from different sources: AE1 was indeed
RI PT
present throughout Central Asia in addition to Europe and AE2 came from North Africa and Middle East.
Interestingly, in a recent study, Vale et al. were able to clearly differentiate between two
SC
European populations, present mainly in Northern Europe (presumably closer to AE1) and in Southern Europe (presumably closer to AE2) using a phage sequence typing based on 2
M AN U
prophage genes of H. pylori (integrase and holin) but present in only a minority of strains (14).
Further subgroups were identified using fineSTRUCTURE for the analysis of 29 H. pylori genome wide single nucleotide polymorphisms (SNPs) by Yahara et al. The hpEurope were
TE D
divided in two subgroups (and two singletons) and highest difference was found for hpEastAsia. In this population hspAmerind was divided into 4 subgroups and hspEAsia into another four groups, although a limited number of samples that do not represent H. pylori
EP
diversity were used (15). The population structure of H. pylori was inferred in another group of 60 H. pylori genomes using three approaches, including STRUCTURE run on a subset of
AC C
100 kb for 29242 SNPs. This approach identified fewer populations than previously, which included Africa2, Africa1, EurAsia (Europe and Asia), EastAsia and America, but using a limited sample size with little geographic diversity. For instance all strains forming the population America came from the same country (Peru) (16). The populations identified in different studies are in agreement with each other, and in a way show how H. pylori populations are organized in a more general (fewer populations) or detailed (larger number of populations) perspective. However, understanding the detailed structure of H. pylori
ACCEPTED MANUSCRIPT population is an exciting field, that novel analyzed sequences and high throughput sequencing data starts to reveal. The phylogeographic distribution of H. pylori is therefore an additional important tool to
mitochondrial DNA (mtDNA) and languages.
RI PT
investigate human evolution and migration which can be added to the traditional tools, e.g.
Similar to the situation in humans, genetic diversity in H. pylori decreases with geographic distance from East Africa where Homo sapiens originated. It was then possible to hypothesize
SC
that H. pylori spread from East Africa around 58,000 ± 3,500 years ago and that modern humans were already infected by H. pylori when their migrations out of Africa began (Fig. 2)
M AN U
(17).
A comparable proportion of the genetic diversity of humans based on autosomal microsatellites was accounted to geographic distance from East Africa (77% of the variance vs 72% for H. pylori) also indicating a migration between 50,000 and 70,000 years ago (18).
TE D
Several articles have reported more specific migrations of H. pylori (and humans) around the world. An interesting one is the peopling of the Pacific region. Indeed, 40,000 to 50,000 years ago Australia, New Guinea and Tasmania were connected in a continent called Sahul which
EP
apparently was colonized only once by humans. The study of diverse aboriginal people from these regions allowed the formation of a distinct biogeographic group called hpSahul, its split
AC C
dating back 31,000-37,000 years ago. In contrast, hspMaori is a subgroup of hpEastAsia which is considered to be a marker for the entire Austronesian expansion and the origin would be some tribes of aboriginal Taiwanese, these results being in agreement with the data coming from language studies. This group corresponds to a second wave of migration into the Pacific (13). Breurec et al. also studied the complex peopling of Southeast Asia using H. pylori MLST. They were able to find at least three human migrations to this area: 1) a migration from India
ACCEPTED MANUSCRIPT introduced hpEurope in Thailand, Cambodia and Malaysia, 2) a migration into Vietnam and Cambodia carrying hspEAsia, and 3) a migration from Southern China to Thailand carrying hpAsia 2 (19). Interestingly, the distribution of these haplotypes shape the incidence of gastric cancer in these countries, given that hspEAsia has a stronger association with this disease, as
origins with their European counterparts (20).
RI PT
is observed in Vietnam. Devi et al. confirmed that H. pylori strains in India share ancestral
The peopling of America can also be traced via H. pylori. Strains from indigenous people of
SC
this continent (Columbia, Alaska) possessed novel vacA and cagA gene structures most closely related to East Asian H. pylori (21). It was also possible to show East Asian loci in
M AN U
strains from Amerindian subjects from Amazonia which were not present in the Mestizo population (22). These data were confirmed by Falush et al. who showed that the hspAmerind haplotype was derived from hpEastAsia and was probably introduced by humans migrating from Asia via the Bering Strait 11,000 years ago (12).
TE D
Another study was carried out to answer how long humans were infected by H. pylori prior to the out-of-Africa event. It included strains obtained from the KhoiSan people of South Africa, a unique relic of hunter-gatherer lineages which diverged 90,000 to 150,000 years ago
EP
according to mtDNA data (23). It was found that almost half of the San population H. pylori haplotypes belong to hpAfrica2. When hpAfrica2 was compared to hpAfrica1 and
AC C
Helicobacter acinonychis - a Helicobacter species which is the closest to H. pylori and found in large felines (cheetah, tiger) - hpAfrica2 and H. acinonychis clustered together, and separately from hpAfrica1. Interestingly H. acinonychis is thought to have arisen from a host jump from humans which occurred 43,000 to 45,000 years ago while the estimated minimum age of the association of H. pylori with humans is 100,000 years (range 88,000-116,000). The Bantus who originate from Central Africa (Nigeria, Cameroon) expanded around 5,000 years ago into the Southern part of Africa and appear to have acquired some hpAfrica2 haplotypes
ACCEPTED MANUSCRIPT from the San, while the San acquired hpAfrica1 from them (Fig 2) (24).
II. Paleomicrobiology and H. pylori There are limited data concerning the direct detection of H. pylori in ancient populations. H.
RI PT
pylori lives in an organ, the stomach, which does not allow a good chance of preservation over numerous years, in contrast, for example, to bacteria protected from the environment in teeth or bones. Nevertheless, bodies which were mummified either voluntarily or due to
SC
freezing in an appropriate environment have offered us a few examples of paleomicrobiology. A. Pre-Columbian mummies
M AN U
Mummification is defined as the preservation of soft tissues from the enzymatic process of post mortem putrefaction. For this, special conditions are required, in particular natural dryness which can be the case in funeral caves. However, as part of funeral rituals the bodies may be wrapped in blankets woven from absorbent vegetable fibers. The internal organs
seldomly conserved.
TE D
being the last to dehydrate, they may be susceptible to putrefaction, and therefore are
The first study concerning H. pylori in pre-Columbian mummies was indeed performed by
EP
Correa et al. They took advantage of the availability of a new kit to detect H. pylori antigens by ELISA (H. pylori Stool Antigen Kit, Meridian Diagnostics) to look for H. pylori in
AC C
coprolites from pre Columbian mummies from Chile. There were 7 positive specimens out of the 16 tested but only 2 of them, from the same mummy, were blocked by anti H. pylori IgG confirming the presence of H. pylori antigens. These specimens came from the mummy of a 25-year old male who died of pneumonia. His cardiomegaly indicated his Andean origin and he was mummified in 300 AD, i.e. 1,700 years ago. Another 1,800 year old mummy gave a strong positive result but which was only partly inhibited by the anti H. pylori IgG (25, 26). In another study the natural orifices of two mummies allowed to explore the internal organs
ACCEPTED MANUSCRIPT with a gastroscope (Pentax EG-1840) and to obtain material from their stomach. The histological analysis of the tissue samples did not allow to distinguish a complete gastric mucosa, but by carrying out PCRs it was possible to detect the presence of H. pylori in 2 of the 4 biopsies from the same mummy while other specimens from tongue and brain tissue
RI PT
were negative. The 109 bp DNA fragment of the 16S rRNA gene hybridized with a specific probe and had 99% homology with H. pylori reference strains. In addition, a real-time PCR targeting the ureB gene was positive in one of the samples. This mummy was from a man
the Chihuahua State, northern Mexico (27).
SC
aged 50 to 60 years and dates back to 1000-1400 AD, i.e. 550 to 950 years old, and lived in
M AN U
These data confirm the presence of H. pylori in Amerindians and it can be hypothesized that the strains were from the hspAmerind haplotype as proposed in previous studies (21, 22). B.
Frozen mummies
• - In August 1999, the remains of a male subject were discovered by hunters in a glacier
TE D
located in British Columbia on a First Nation territory and was confirmed to be of First Nation ancestry. Radiocarbon dating suggested that he died approximately between 1670 and 1850 which is a time when contact with the Europeans in this area was possible.
EP
PCRs were performed on gastric tissue extracts targeting vacA, the PCR products were sequenced and compared to reference sequences. vacA is a gene specific to H. pylori and it
AC C
was found in the stomach of this mummified body. The authors looked at the different alleles and showed that mid regions was hybrid m2a/m1d while the signal sequence was s2. The presence of an s2 allele being unusual in Asian strains it suggests that the strain is potentially a hybrid between European strains and Asian strains (28). • - Ötzi, the Tyrolean iceman. In September 1991, a well conserved male frozen body was discovered in the Otzal Alps at an altitude of 3,200 m, in Italy at 90 meters from the Austro-Italian border, who is now called
ACCEPTED MANUSCRIPT Ötzi or the iceman. Surprisingly the frozen body was almost completely mummified and it dated back to 5,200 years, i.e. from the late Neolithic period which makes it the oldest mummy found to date. Numerous studies have been performed on this mummy in order to get insight on the life
technology during these last 25 years (29).
RI PT
conditions at this time, with several rebounds and also new data linked to improved
For example, a big surprise was the discovery of an arrowhead found in his left shoulder
SC
which indicated that he was injured and probably died of this injury, which had not been detected on the first x-rays performed. In the same manner, the Iceman's stomach was
M AN U
discovered during a reappraisal of radiological data and contained the food ingested shortly before his death.
In order to get more information on the mummy, for the 20th anniversary of his discovery, the decision was made to thaw the body which is maintained at a constant temperature of -6°C
TE D
and this was done in October 2010. Among several teams of specialists devoted to different organs, one was in charge of the gastrointestinal tract. The appropriate endoscope was selected in order to perform a gastroscopy; unfortunately, the collapsed oesophagus did not
EP
allow the utilization of this route and a laparoscopy was performed. Material was obtained from the gastric content, the gastric wall and from the length of the small and large intestines.
AC C
This material was dispatched to several laboratories and was tested by various methods: culture, PCR targeting different genes of H. pylori, and sequencing using the Roche 454 apparatus. However, the results for H. pylori detection were negative at this time (2010). Later, a metagenomic approach coupled with targeted genome capture generated 15,350 reads of H. pylori DNA throughout the gastrointestinal tract with an abundance decreasing from the stomach towards the lower intestinal tract, and with negative results for the muscle tissue used as control. When aligned with the H. pylori reference strain 26695, the reads showed damage
ACCEPTED MANUSCRIPT patterns indicative of ancient DNA. The strain was cagA positive and vacA s1a/i1/m1 type as are the more virulent modern H. pylori strains. Stigmates of gastric inflammation, via the presence of high level of calprotectin subunits, were also noted. More details on the Iceman H. pylori strain can be found in the original article and supplementary data (2).
RI PT
It was also possible to compare the MLST haplotype of the Iceman's strain to the database of 1,603 strains using the STRUCTURE programme and the strain was assigned to the modern population of hpAsia2. This information is interesting in light of the uncertainty concerning
SC
the time of recombination between AE1 (derived from hpAsia2) and AE2 (derived from hpNEAfrica) which formed the current hpEurope, as mentioned above.
M AN U
So it appears that 5,200 years ago, AE1 was present in central Europe with a minimum of admixture from AE2 (6.5%, probability interval 1.5-13.5%) which places it close to modern hpAsia2 found in India. These results were confirmed in further analyses. Indeed, the low proportion of AE2 ancestry observed in this strain suggests an introgression into Europe after
TE D
the Copper Age, i.e. later than previously thought, or at least later in Central Europe (Otzal Alps). However, the limit of such a conclusion is that it is based on only one strain.
EP
In conclusion, beyond medical aspects, phylogeography and paleomicrobiology of H. pylori especially with the new imaging and sequencing approaches are able to bring unexpected data
AC C
on the history of humanity.
Acknowledgement: Filipa F. Vale is recipient of a fellowship from FCT (SFRH/BPD/95125/2013).
ACCEPTED MANUSCRIPT Figure legends Fig 1. Relationships between modern populations (A), modern subpopulations (B), and ancestral populations (C) of Helicobacter pylori (Ref 12) Relationships between modern populations (A), modern subpopulations (B), and ancestral populations (C) of H. pylori. The black lines show neighbor-joining population trees as measured byδ̂, the net
RI PT
nucleotide distance between populations. The circle diameters indicate their genetic diversity, measured as the average genetic distance between random pairs of individuals. The larger circles in (A) versus (C) reflect the effects of admixture between ancestral populations. Filled arcs reflect the number of isolates (A and B) or nucleotides (C) in each population. Color coding is consistent in
M AN U
AE1 and AE2 populations. Scales are at lower right.
SC
different parts of the figure, except for modern hpEurope, which is an admixture between the ancestral
Fig 2. Chronological reconstruction of the major population events occurring during the intimate human-Helicobacter pylori association. (Ref 24)
Black lines indicate undifferentiated populations and all other lines are color-coded according to population. The sequence of events is as follows: 1) Initial acquisition of H. pylori by a
TE D
human ancestor; 2) Divergence of H. pylori into two super-lineages; 3) First successful migration of modern humans Out of Africa via the southern route; 4) H. pylori divergence into hpAfrica1 and hpNEAfrica with migration eastwards (hpNEAfrica) and westwards
EP
(hpAfrica1); 5) Divergence of H. pylori out of Africa into hpSahul and 6) hpAsia2 and hpEastAsia; 7) Host jump from San to large felines giving rise to H. acinonychis. 8) Southward migration of San carrying the ancestor of hpAfrica2; 9) Second successful
AC C
migration Out of Africa via the Levant; 10) Hybridization of AE1 from central and south-west Asia and AE2 from north-east Africa in the Middle East or western Asia resulting in hpEurope; 11) Spread of hpEurope bacteria to Europe; 12) Back migration from the Middle East, and Spain spreading hpEurope into North Africa. Dates in italics represent estimates obtained from sources other than H. pylori.
ACCEPTED MANUSCRIPT References
1. Warren JR, Marshall BJ. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. The Lancet. 1983;321:1273–5. 2. Maixner F, Krause-Kyora B, Turaev D, Herbig A, Hoopmann MR, Hallows JL, et al. The 5300-year-old Helicobacter pylori genome of the Iceman. Science. 2016;351:162-5.
RI PT
3. Achtman M. Evolution, population structure, and phylogeography of genetically monomorphic bacterial pathogens. Annu. Rev. Microbiol. 2008;62:53-70.
4. Maiden MC, Bygraves JA, Feil E, Morelli G, Russell JE, Urwin R, et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Nat. Acad. Sci. U. S. A. 1998;95:3140-5.
SC
5. Patterson N, Price AL, Reich D. Population structure and eigenanalysis. PLoS Gen. 2006;2:e190.
M AN U
6. Falush D, Stephens M, Pritchard JK. Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics. 2003;164:1567-87. 7. Lawson DJ, Hellenthal G, Myers S, Falush D. Inference of population structure using dense haplotype data. PLoS Gen. 2012;8:e1002453. 8. Achtman M, Azuma T, Berg DE, Ito Y, Morelli G, Pan ZJ, et al. Recombination and clonal groupings within Helicobacter pylori from different geographical regions. Mol. Microbiol. 1999 ;32:459-70.
TE D
9. Morelli G, Didelot X, Kusecek B, Schwarz S, Bahlawane C, Falush D, et al. Microevolution of Helicobacter pylori during prolonged infection of single hosts and within families. PLoS Gen. 2010;6:e1001036.
EP
10. Cavalli-Sforza L, Menozzi P, Piazza A, editors. The history and geography of human genes: Princeton University Press, New Jersey; 1996.
AC C
11. Dominguez-Bello MG, Menozzi P, Piazza A. The human microbiota as a marker for migrations of individuals and populations. Annu. Rev. Anthropol.. 2011;40:451-74. 12. Falush D, Wirth T, Linz B, Pritchard JK, Stephens M, Kidd M, et al. Traces of human migrations in Helicobacter pylori populations. Science. 2003;299:1582-5. 13. Moodley Y, Linz B, Yamaoka Y, Windsor HM, Breurec S, Wu JY, et al. The peopling of the Pacific from a bacterial perspective. Science. 2009;323:527-30. 14. Vale FF, Vadivelu J, Oleastro M, Breurec S, Engstrand L, Perets TT, et al. Dormant phages of Helicobacter pylori reveal distinct populations in Europe. Sci Reports. 2015;5:14333. 15. Yahara K, Furuta Y, Oshima K, Yoshida M, Azuma T, Hattori M, et al. Chromosome painting in silico in a bacterial species reveals fine population structure. Mol. Biol. Evol. 2013 ;30:1454-64.
ACCEPTED MANUSCRIPT 16. Montano V, Didelot X, Foll M, Linz B, Reinhardt R, Suerbaum S, et al. Worldwide Population Structure, Long-Term Demography, and Local Adaptation of Helicobacter pylori. Genetics. 2015;200:947-63. 17. Linz B, Balloux F, Moodley Y, Manica A, Liu H, Roumagnac P, et al. An African origin for the intimate association between humans and Helicobacter pylori. Nature.2007;445:915-8.
RI PT
18. Zhivotovsky LA, Underhill PA, Cinnioglu C, Kayser M, Morar B, Kivisild T, et al. The effective mutation rate at Y chromosome short tandem repeats, with application to human population-divergence time. Am. J. Hum Gen. 2004;74:50-61.
SC
19. Breurec S, Guillard B, Hem S, Brisse S, Dieye FB, Huerre M, et al. Evolutionary history of Helicobacter pylori sequences reflect past human migrations in Southeast Asia. PloS One. 2011;6:e22058. 20. Devi SM, Ahmed I, Francalacci P, Hussain MA, Akhter Y, Alvi A, et al. Ancestral European roots of Helicobacter pylori in India. BMC Genomics. 2007;8:184.
M AN U
21. Yamaoka Y, Orito E, Mizokami M, Gutierrez O, Saitou N, Kodama T, et al. Helicobacter pylori in North and South America before Columbus. FEBS letters 2002;517:180-4. 22. Ghose C, Perez-Perez GI, Dominguez-Bello MG, Pride DT, Bravi CM, Blaser MJ. East Asian genotypes of Helicobacter pylori strains in Amerindians provide evidence for its ancient human carriage. Proc. Nat. Acad. Sci. U. S. A. 2002;99:15107-11.
TE D
23. Behar DM, Villems R, Soodyall H, Blue-Smith J, Pereira L, Metspalu E, et al. The dawn of human matrilineal diversity. Am. J. Hum Gen. 2008;82:1130-40. 24. Moodley Y, Linz B, Bond RP, Nieuwoudt M, Soodyall H, Schlebusch CM, et al. Age of the association between Helicobacter pylori and man. PLoS Pathogens. 2012;8:e1002693.
EP
25. Correa P, Willis D, Allison MJ, Gerszten E. Helicobacter pylori in pre-Columbian mummies. Gastroenterology. 1998;114:A956-A
AC C
26. Allison MJ, Bergman T, Gerszten E. Further studies on fecal parasites in antiquity. Am. J.Clin. Pathol. 1999;112:605-9. 27. Castillo-Rojas G, Cerbon MA, Lopez-Vidal Y. Presence of Helicobacter pylori in a Mexican Pre-Columbian Mummy. BMC Microbiol. 2008;8:119. 28. Swanston T, Haakensen M, Deneer H, Walker EG. The characterization of Helicobacter pylori DNA associated with ancient human remains recovered from a Canadian glacier. PloS One. 2011;6:e16864. 29. Cano RJ, Tiefenbrunner F, Ubaldi M, Del Cueto C, Luciani S, Cox T, et al. Sequence analysis of bacterial DNA in the colon and stomach of the Tyrolean Iceman. Am. J. Phys Anthropol. 2000 ;112:297-309.
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Figure 1
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Figure 2