Understanding the survival mechanisms of Campylobacter jejuni in human hosts and in the environment

Laburpena

Campylobacter is a bacterial pathogen that is transmitted mainly through contaminated food causing the so-called campylobacteriosis, the most frequently reported gastrointestinal disease in the European Union since 2005, with more than 127,000 human cases in 2021. Although campylobacteriosis is a self-limiting disease and the use of antibiotics is reserved for the treatment of the most severe cases and immunocompromised patients, in extreme situations, it can trigger complications such as neurological disorders like the Guillain-Barré syndrome or reactive arthritis, and myocarditis, among others. In most reported cases, Campylobacter jejuni is the predominant species, which represents a serious threat to human health, causing a negative impact on the economy of public health systems. Therefore, this Doctoral Thesis aims to study in more detail the molecular mechanisms of pathogenicity and virulence of C. jejuni to resist the harsh environmental conditions existing along the food supply chain and survive the defense mechanisms of the human host with the purpose of improving food safety. The study of the molecular bases of C. jejuni pathogenicity requires Whole-Genome Sequencing (WGS) of the isolates, as well as subsequent genome analysis of the sequenced data. This arises the need to develop a workflow ―CamPype― to automatically analyse WGS data, especially of this genus. CamPype allows the user to customize the WGS analysis to be performed, including quality control of raw reads and read quality filtering, read extension and assembly, bacterial typing, genome annotation, searching for antibiotic resistance and virulence genes, and plasmids, pangenome construction and identification of nucleotide variants. To validate CamPype, 145 Campylobacter spp. genomes, including the species C. jejuni, Campylobacter coli and Campylobacter lari, corresponding to isolates from different stages of the poultry supply chain, as well as clinical strains, from Spain were analysed. The study revealed a wide genetic diversity between and within these species, with a predominance of the lineage Clonal Complex (CC)21, which was identified at all sampling locations. Likewise, several virulence and antibiotic resistance profiles were identified, with a higher frequency of virulence genes found in the species C. jejuni that could explain its greater abundance in the environment. High rates of antibiotic resistance genes were observed, especially against β-lactams, fluoroquinolones and tetracyclines, especially in C. jejuni and C. coli, which justify the threat to global health of increasing antibiotic resistance. On the way to understanding the behaviour of Campylobacter in the environment, two isolates of C. jejuni that had infected two children from the same family at the same time were genomically compared to find the molecular traits that led the boy to suffer from perimyocarditis after the episode of campylobacteriosis. The isolates turned out to be clones of the same bacteria and only differed in 16 Single Nucleotide Polymorphisms (SNPs), which mainly affected the ON/OFF state of hypervariable genes. These differences suggest that, when infection of the human host occurs, the bacteria are able to modulate the expression of certain genes to adapt its behaviour to the environment, which can lead to the development of subsequent complications in the host depending on his/her state. The two previous strains were also phenotypically characterized, together with three different C. jejuni isolates representing different stages of the poultry supply chain, whose genomes were analysed to search for possible genetic markers responsible for the observed phenotypes. Specifically, the behaviour of the isolates was evaluated against typical stress conditions found in the poultry supply chain, such as their ability to tolerate oxygen and oxidative stress, as well as their ability to form biofilm as an adaptive strategy in the food industry, which depends on bacterial motility. The isolates combined different phenotypic behaviours, with special relevance the different swimming ability of the two isolates related to the perimyocarditis case. Furthermore, the comparative analysis of the genomes revealed differences in the pattern of genes associated with the survival strategies evaluated, although the underlying molecular mechanism of each phenotype was mainly the phase variation of hypervariable genes, which was crucial to modulate the diverse survival mechanisms adopted by the bacteria. The different swimming ability of the two isolates related to the perimyocarditis case led to an in-depth study of the motility phenotype in C. jejuni, which again revealed a great diversity in the swimming ability of the bacteria, as well as in the patterns of genes associated with this phenotype and in phasotypes ―combinations of the ON/OFF state of hypervariable genes―. This indicates that motility is a complex phenotype that combines distinct but complementary genetic mechanisms, which determine different swimming abilities. Among these mechanisms, there exists the presence of genes that code for structural proteins of the flagellum, glycosylation proteins of the flagellum that modify its composition, regulatory proteins and chemoreceptors, although the shortening of proteins as a consequence of point mutations or the expression state of those hypervariable genes, which those involved in the glycosylation of the flagellum stand out, are decisive to explain the variability of this phenotype. The previous analysis was complemented with a Genome-Wide Association Study (GWAS) that revealed new findings involved in the motility phenotype from a statistical point of view, considering both the set of the genes and mutations found among all isolates. In particular, up to four regions in the genome seemed to be involved in the greater or lesser swimming ability of the bacteria, regions that code for membrane, transmembrane, periplasmic and capsule proteins, which suggest a fundamental role for the structure and composition of the membrane and capsule in the performance of the bacteria in viscous environments. In addition to the need for correct assembly of the flagellum in a highly ordered process, the phosphate ABC and ATP-binding transporters could be key in the transference of nutrients for the generation of the proton-motive force across the membrane, necessary for the rotation of the flagellum at greater or lesser speed allowing longer or shorter swimming distances of the bacteria. In this way, these last two studies highlight the importance for understanding the motility phenotype in C. jejuni of studying both the genes directly involved in the construction of the flagellum and those genes responsible for starting up the functioning of this filamentous structure. In conclusion, this Doctoral Thesis demonstrates the potential of CamPype to genomically characterize isolates from different species and origins, generating new data useful for subsequent more specific analyses that allow further studies of the molecular bases of C. jejuni pathogenicity. The bacteria are characterized by their ability to efficiently combine diverse and complex genetic mechanisms, among which there are virulence factors involved in multiple processes, several of which are affected by phase variation and that determine different phenotypes, and diverse mechanisms of antibiotic resistance. Although of all these, the phase variation is the mechanism that gives the bacteria the essential adaptive advantage to face stress situations, overcome adverse environmental conditions and adapt to the host, which makes the behaviour of the bacteria to be almost unpredictable and enhances its prevalence in the environment.