Epidemiology and taxogenomics of the genus arcobacter

Resumen

The genus Arcobacter belongs to the family Campylobacteraceae and some of the species of the genus had been related with human and animal infections. Arcobacter butzleri is the most prevalent species in humans, followed by A. cryaerophilus, A. skirrowii and A. thereius. However, the routes followed by the bacteria to produce infections are still poorly known. Moreover, despite several tools have been described for the epidemiological studies in bacteria, as the Multilocus Sequence Typing (MLST), a few studies have been developed about their epidemiology and genotypes. On the other hand, no specific antibiotics have been proposed for the treatment of Arcobacter infections. The absence of a specific treatment along with the increasing resistance to some antibiotics indicates the need for performing studies on the Arcobacter antibiotic resistance in order to ensure the correct treatment and the control of resistance mechanisms. Secondly, regarding the taxonomy of Arcobacter, the number of species of the genus has increased exponentially in the recent years. This increment has been produced by different factors, as the use of new molecular tools, new methodologies for bacteria isolation or the sampling of new environments. Due to these factors, potential new Arcobacter species had been also isolated in our laboratory. Additionally, these new species showed a wide range of similarities of the 16S rRNA gene (91.2-99.6%), that is the classical tool used in taxonomy to differentiate genera at values <95%. The observed 16S rRNA gene similarities evidences that the taxonomy of the genus must be analysed in order to ensure that all the species are indeed belonging to the same genus. Another taxonomic aspect of Arcobacter is the one related with the species A. cryaerophilus, that has been classified in two different sub groups since its description. However, the use of genomic information may allow to determine the correct classification of this A. cryaerophilus subgroups. The aim of this thesis is i) to characterize the epidemiological relationship of clinical strains isolated from the faeces of patients from three Spanish hospitals using the MLST approach and to screen them for their antibiotic susceptibility and for the presence of virulence genes; ii) to determine, using a polyphasic taxonomic approach, including genomic information, if seven Arcobacter isolates obtained from different sources can be considered new species; iii) to perform a polyphasic re-evaluation of the taxonomic diversity of Arcobacter cryaerophilus including genomic analyses; iv) to re-assess the taxonomy of the genus Arcobacter using phylogenetic and genomic analyses including several genomic indexes and a phenotypic characterization. To achieve these goals, several methodologies were used. The strains were isolated using different protocols in blood agar or marine agar and incubated at 30ºC for 24-72 hours, depending on their requirements. For the first objective of this thesis, the clinical strains were identified on the basis of the phylogenetic analysis of the sequences of the rpoB housekeeping gene. Then, their epidemiological relationships were determined using the MLST scheme described for Arcobacter. The presence of virulence genes was tested using a previously described PCR method. The antibiotic resistance profiles of each strain was performed using the disc diffusion method following recommendations of the CLSI for Campylobacter. For the second objective, the identification of the strains was attempted using at first three previously described multiplex-PCRs and the 16S rDNA-RFLP method. The final identification at species level was performed, as indicated previously, with the rpoB gene. For the strains that represented potential new taxa, a phylogenetic analysis using the 16S rRNA gene and the concatenated sequences of five housekeeping genes (atpA, gyrA, gyrB, hsp60 and rpoB) was performed. Additionally, the genome of the potential new species and those of other known species of Arcobacter were sequenced and compared. Comparison with the closest species was performed using the Average Nucleotide Identity (ANI) and the in silico DNA-DNA-hybridization (isDDH) and their cut-off values >96% and >70%, respectively for genomes belonging to the same species. The description of the potential new species included also the phenotypic characterization of the strains and the study of the morphology and the presence of flagella with the Transmission Electron Microscope. For the third objective, a total of 52 strains were analysed, isolated from different countries in different years from a wide range of sources. The phylogenetic analysis of these strains was performed with the concatenated sequences of four housekeeping genes (atpA, gyrB, hsp60 and rpoB). Additionally, the genomes of seven representative strains of Arcobacter cryaerophilus were sequenced and compared, along with six genomes obtained from GenBank. These genomes were annotated with RAST and PATRIC servers. The genomic comparison included the analysis of the virulence and resistance genes using several databases (an in-house database, VFDB, Victors Database, PATRIC_VF, ARDB, ARG-ANNOT and CARD), the analysis of metabolic pathways and functional characteristics, and the phylogenetic analysis of the core genome. The fourth objective involved the analysis of 55 Arcobacter sequenced genomes and of 11 genomes obtained from the public databases. The genomes were annotated with Prokka and the coding sequences were annotated with RAST. Phylogenetic analyses were performed with the 16S and 23S rRNA genes, with 13 housekeeping genes and with the core genome. Additionally, five genomic indexes i.e. ANI, isDDH, Average Aminoacid Identity (AAI), the Percentage Of Conserved Proteins (POCP) and the Relative Synonymous Codon Usage (RSCU), were calculated to use them as indexes for species and genera differentiation. For the complete characterization of the genus, a phenotypic analysis was performed and the metabolic inference from the genomes was analysed using Traitar software. The main conclusions of this thesis are summarized in this 8 points: 1) The fact that epidemiological unrelated A. butzleri strains show the same Sequence Type indicates that other techniques with higher resolution should be developed to effectively recognize the infection source; 2) The demonstrated resistance of 10.7% of the strains of A. butzleri to ciprofloxacin, one of the antibiotics recommended for the treatment of the intestinal infections produced by this bacteria, indicates the importance of selecting in each case the most effective treatment; 3) Four new of Arcobacter spp. named A. canalis, A. lacus, A. caeni and A. miroungae are proposed; 4) Genomic analyses evidenced the existence of 10 potential new species that are pending to be completely described; 5) The genomic and polyphasic analysis of the species A. cryaerophilus evidenced the existence of four cryptic genomovars within this species for which the names A. cryaerophilus gv. pseudocryaerophilus, A. cryaerophilus gv. crypticus, A. cryaerophilus gv. cryaerophilus, and A. cryaerophilus gv. occultus are proposed; 6) The taxogenomic analysis of the genus Arcobacter along with the phenotypical characterization allowed the division of the genus in at least six different genera, for which the names Aliiarcobacter gen. nov., Pseudoarcobacter gen. nov., Haloarcobacter gen. nov., Malacoarcobacter gen. nov., Poseidoniarcobacter gen. nov., and Arcomarinus gen. nov., are proposed. Those genera embrace all the previously defined Arcobacter species, with the exception of A. nitrofigilis.; 7) Values of 75.2-81.8% for ANI, 19.5-24.8% for isDDH, 67.6-80.3% for AAI, and 67.0-75.4% for POCP enabled to delineate the above mentioned 6 new genera for the family Arcobacteraceae. The study of the RSCU using a Principal Component Analysis clustered the species within the 7 genera.