Detection of Helicobacter pylori Microevolution and Multiple Infection, and Genomic Analysis of Helicobacter pylori Strains

Abstract

In the past decades, Helicobacter pylori has received the attention of many researchers because of its known relation with gastric cancer. Although many studies have tried to decipher the exact relation between the bacteria and cancer state, and several virulence factors have been discovered, an exact answer has not been found yet. Further work should be made in order to study more accurately the genome of this bacterium and to understand its precise involvement. The bacterium is characterised for a highly genetic diversity, meaning it is continuously changing in order to adapt itself to its hostile niche, the human stomach. Infection by H. pylori is estimated to affect half of the world’s population, being more extended in developing countries than in developed ones, possibly due to the high consumption of antibiotics and the increased level of sanitation in the latest. It has been demonstrated that the gastric lumen can be colonized by more than just one strain of the bacterium, sometimes these strains could have evolved from the same ‘mother’ strain, or they could come from unrelated strains. The study of these situations is important in order to elucidate if there is just one strain who is responsible for starting the pathogenic cascade, and what are the specific differences between the different strains that inhabit the human stomach. On the first work of this thesis, our group studied the usefulness of six housekeeping genes for the detection of H. pylori infection and the characterization of various strains isolated from gastric isolates, studying as well their phylogeny. In some cases, the distance value between the strains was high, indicating and event of multiple infection. In other cases, small differences were found between clones, suggesting events of microevolution rather than multiple infection. This work was further extended with the study of the usefulness of amplicon sequencing of these housekeeping genes in the detection of microevolution and mixed infections from gastric biopsies of patients with dyspeptic symptoms and different histopathological findings (from atrophy to adenocarcinoma). Five gastric biopsies from four patients infected by H. pylori were involved in this study. We detected in all the analyzed gastric biopsies multiple H. pylori infections with a predominant strain. These results suggest that H. pylori colonizes the human stomach through diverse infection circumstances that lead to a gastric multi-infection with a predominant strain together alongside other minority strains. Furthermore, it was shown that mixed infections are the main status in the colonization of the human gastric mucosa. The last part of this thesis started with a preliminary study of 51 complete sequenced

H. pylori genomes and further focused on three genomes obtained from the same patient in order to analyse and compare them. Particularly, these isolates were sampled at the same time from a stomach with adenocarcinoma, one strain was from the non- tumoral tissue, and the other two were isolated from the tumoral tissue. They all lacked from the most noticeable virulence factor, the cag pathogenicity island; one of the most studied and the main factor related to the malignancy of the bacterium. On the other hand, we found differences in the genotype of the vacuolating cytotoxin gene (vacA) and in genes related with urease, the outer membrane and flagella. Despite the contributions made in this thesis, further studies are needed to find better genetic markers of H. pylori related to virulence and progression to gastric cancer.

Helicobacter pylori és el focus d’atenció de molts estudis a causa de la seva coneguda relació amb el càncer gàstric. Encara que molts estudis han tractat de desxifrar la relació exacta entre el bacteri i la progressió del càncer, i s'han descobert diversos factors de virulència, no s'ha trobat una resposta exacta. En la primera part d'aquesta tesi, es va estudiar la utilitat de sis gens conservats per la detecció de la infecció per H. pylori i la caracterització de diverses soques aïllades de biòpsies gàstriques, estudiant-se també la seva filogènia. En alguns casos, el valor de la distància entre les soques era alt, fet indicatiu d’un esdeveniment d'infecció múltiple. En altres casos, es van trobar petites diferències entre els clons, el que suggereix esdeveniments de microevolució en lloc d’infecció múltiple. Aquesta tesi es va ampliar amb l'estudi de la utilitat de la seqüenciació d'amplicons d'aquests gens conservats en la detecció de microevolució i infeccions múltiples en biòpsies gàstriques de pacients amb símptomes dispèptics. En aquest estudi es van analitzar cinc biòpsies gàstriques de quatre pacients infectats per H. pylori. En totes les biòpsies gàstriques analitzades es van detectar infeccions múltiples per H. pylori amb una soca predominant. Aquests resultats suggereixen que H. pylori colonitza l'estómac humà mitjançant diverses circumstàncies d'infecció que condueixen a una infecció múltiple gàstrica amb una soca predominant juntament amb altres soques minoritàries. L'última part d'aquesta tesi va començar amb l’estudi preliminar de 51 genomes complets d’H. pylori i es va centrar després en l’estudi i comparació de tres genomes obtinguts del mateix pacient. Específicament, aquestes soques van ser aïllades alhora d’un estómac amb adenocarcinoma, on una soca provenia del teixit no tumoral i les altres dues del teixit tumoral. Totes mancaven del factor de virulència més destacat, l'illa de patogenicitat cag; un dels factors més estudiats i el més relacionat amb la malignitat del bacteri. Per una altra banda, vam trobar diferències en el genotip del gen vacA i en gens relacionats amb la ureasa, la membrana externa i els flagels.