Thomas Schott

Comparative genomics of Helicobacter pylori and the human-derived Helicobacter bizzozeronii CIII-1 strain reveal the molecular basis of the zoonotic nature of non-pylori gastric Helicobacter infections in humans.

BackgroundThe canine Gram-negative Helicobacter bizzozeronii is one of seven species in Helicobacter heilmannii sensu lato that are detected in 0.17-2.3% of the gastric biopsies of human patients with gastric symptoms. At the present, H. bizzozeronii is the only non-pylori gastric Helicobacter sp. cultivated from human patients and is therefore a good alternative model of human gastric Helicobacter disease. We recently sequenced the genome of the H. bizzozeronii human strain CIII-1, isolated in 2008 from a 47-year old Finnish woman suffering from severe dyspeptic symptoms. In this study, we performed a detailed comparative genome analysis with H. pylori, providing new insights into non-pylori Helicobacter infections and the mechanisms of transmission between the primary animal host and humans.ResultsH. bizzozeronii possesses all the genes necessary for its specialised life in the stomach. However, H. bizzozeronii differs from H. pylori by having a wider metabolic flexibility in terms of its energy sources and electron transport chain. Moreover, H. bizzozeronii harbours a higher number of methyl-accepting chemotaxis proteins, allowing it to respond to a wider spectrum of environmental signals. In this study, H. bizzozeronii has been shown to have high level of genome plasticity. We were able to identify a total of 43 contingency genes, 5 insertion sequences (ISs), 22 mini-IS elements, 1 genomic island and a putative prophage. Although H. bizzozeronii lacks homologues of some of the major H. pylori virulence genes, other candidate virulence factors are present. In particular, we identified a polysaccharide lyase (HBZC1_15820) as a potential new virulence factor of H. bizzozeronii.ConclusionsThe comparative genome analysis performed in this study increased the knowledge of the biology of gastric Helicobacter species. In particular, we propose the hypothesis that the high metabolic versatility and the ability to react to a range of environmental signals, factors which differentiate H. bizzozeronii as well as H. felis and H. suis from H. pylori, are the molecular basis of the of the zoonotic nature of H. heilmannii sensu lato infection in humans.

Genomic Variation between Campylobacter jejuni Isolates Associated with Milk-Borne-Disease Outbreaks

ABSTRACT Bacterial genome sequencing has led to the development of new approaches for the analysis of food-borne epidemics and the exploration of the relatedness of outbreak-associated isolates and their separation from nonassociated isolates. Using Illumina technology, we sequenced a total of six isolates (two from patients, two from raw bulk milk, and two from dairy cattle) associated with a milk-borne Campylobacter jejuni outbreak in a farming family and compared their genomes. These isolates had identical pulsed-field gel electrophoresis (PFGE) types, and their multilocus sequence typing (MLST) type was ST-50. We used the Ma_1 isolate (milk) as the reference, and its genome was assembled and tentatively ordered using the C. jejuni NCTC 11168 genome as the scaffold. Using whole-genome MLST (wgMLST), we identified a total of three single-nucleotide polymorphisms (SNPs) and differences in poly(G or C) or poly(A or T) tracts in 12 loci among the isolates. Several new alleles not present in the database were detected. In contrast, the sequences of the unassociated C. jejuni strains P14 and 1–12S (both ST-50) differed by 420 to 454 alleles from the epidemic-associated isolates. We found that the fecal contamination of bulk tank milk occurred by highly related sequence variants of C. jejuni, which are reflected as SNPs and differences in the length of the poly(A or T) tracts. Poly(G or C) tracts are reversibly variable and are thus unstable markers for comparison. Further, unrelated strains of ST-50 were clearly separated from the outbreak-associated isolates, indicating that wgMLST is an excellent tool for analysis. In addition, other useful data related to the genes and genetic systems of the isolates were obtained.

Comparative genomics of unintrogressed Campylobacter coli clades 2 and 3

BackgroundCampylobacter jejuni and C. coli share a multitude of risk factors associated with human gastrointestinal disease, yet their phylogeny differs significantly. C. jejuni is scattered into several lineages, with no apparent linkage, whereas C. coli clusters into three distinct phylogenetic groups (clades) of which clade 1 has shown extensive genome-wide introgression with C. jejuni, yet the other two clades (2 and 3) have less than 2% of C. jejuni ancestry. We characterized a C. coli strain (76339) with four novel multilocus sequence type alleles (ST-5088) and having the capability to express gamma-glutamyltranspeptidase (GGT); an accessory feature in C. jejuni. Our aim was to further characterize unintrogressed C. coli clades 2 and 3, using comparative genomics and with additional genome sequences available, to investigate the impact of horizontal gene transfer in shaping the accessory and core gene pools in unintrogressed C. coli.ResultsHere, we present the first fully closed C. coli clade 3 genome (76339). The phylogenomic analysis of strain 76339, revealed that it belonged to clade 3 of unintrogressed C. coli. A more extensive respiratory metabolism among unintrogressed C. coli strains was found compared to introgressed C. coli (clade 1). We also identified other genes, such as serine proteases and an active sialyltransferase in the lipooligosaccharide locus, not present in C. coli clade 1 and we further propose a unique scenario for the evolution of Campylobacter ggt.ConclusionsWe propose new insights into the evolution of the accessory genome of C. coli clade 3 and C. jejuni. Also, in silico analysis of the gene content revealed that C. coli clades 2 and 3 have genes associated with infection, suggesting they are a potent human pathogen, and may currently be underreported in human infections due to niche separation.

Genome analysis of Campylobacter jejuni strains isolated from a waterborne outbreak

BackgroundWaterborne Campylobacter jejuni outbreaks are common in the Nordic countries, and PFGE (pulsed field gel electrophoresis) remains the genotyping method of choice in outbreak investigations. However, PFGE cannot assess the clonal relationship between isolates, leading to difficulties in molecular epidemiological investigations. Here, we explored the applicability of whole genome sequencing to outbreak investigation by re-analysing three C. jejuni strains (one isolated from water and two from patients) from an earlier resolved Finnish waterborne outbreak from the year 2000.ResultsOne of the patient strains had the same PFGE profile, as well as an identical overall gene synteny and three polymorphisms in comparison with the water strain. However, the other patient isolate, which showed only minor differences in the PFGE pattern relative to the water strain, harboured several polymorphisms as well as rearrangements in the integrated element CJIE2. We reconstructed the genealogy of these strains with ClonalFrame including in the analysis four C. jejuni isolated from chicken in 2012 having the same PFGE profile and sequence type as the outbreak strains. The three outbreak strains exhibited a paraphyletic relationship, implying that the drinking water from 2000 was probably contaminated with at least two different, but related, C. jejuni strains.ConclusionsOur results emphasize the capability of whole genome sequencing to unambiguously resolve the clonal relationship between isolates of C. jejuni in an outbreak situation and evaluate the diversity of the C. jejuni population.

Evolution and comparative genomics of Campylobacter jejuni ST-677 clonal complex

Campylobacter is the most common bacterial cause of gastroenteritis in the European Union with over 200,000 laboratory-confirmed cases reported annually. This is the first study to describe findings related to comparative genomics analyses of the sequence type (ST)-677 clonal complex (CC), a Campylobacter jejuni lineage associated with bacteremia cases in humans. We performed whole-genome sequencing, using Illumina HiSeq sequencing technology, on five related ST-677 CC isolates from two chicken farms to identify microevolution taking place at the farms. Our further aim was to identify novel putative virulence determinants from the ST-677 CC genomes. For this purpose, clinical isolates of the same CC were included in comparative genomic analyses against well-known reference strains of C. jejuni. Overall, the ST-677 CC was recognized as a highly clonal lineage with relatively small differences between the genomes. Among the farm isolates differences were identified mainly in the lengths of the homopolymeric tracts in genes related to the capsule, lipo-oligosaccharide, and flagella. We identified genomic features shared with C. jejuni subsp. doylei, which has also been shown to be associated with bacteremia in humans. These included the degradation of the cytolethal distending toxin operon and similarities between the capsular polysaccharide biosynthesis loci. The phase-variable GDP-mannose 4,6-dehydratase (EC 4.2.1.47) (wcbK, CAMP1649), associated with the capsular polysaccharide biosynthesis locus, may play a central role in ST-677 CC conferring acid and serum resistance during different stages of infection. Homology-based searches revealed several additional novel features and characteristics, including two putative type Vb secretion systems and a novel restriction modification/methyltransferase gene cluster, putatively associated with pathogenesis and niche adaptation.

Genetic heterogeneity of Campylobacter jejuni NCTC 11168 upon human infection

Campylobacter jejuni NCTC 11168 variants before and after accidental human infection were sequenced with Illumina technology and mapped against the isogenic reference genome applying the Breseq pipeline. Only the frequencies of length variations of homopolymeric tracts in the contingency genes Cj0045c, Cj0456c, Cj1139c, Cj1145c, and Cj1306c and a deletion in Cj0184c were significantly different after human passage (p<0.01). Our results highlight differences in the selection of C. jejuni variants after human infection compared with those observed in animal models, emphasizing the genetic diversity of C. jejuni NCTC 11168 and the possible role of the host in the selection of bacterial determinants that might be involved in the adaptation and disease development.

Genome sequence of Helicobacter bizzozeronii strain CIII-1, an isolate from human gastric mucosa

The canine-adapted Helicobacter bizzozeronii is the only nonpylori Helicobacter species isolated from human gastric biopsy tissue. Here we present the genome sequence of strain CIII-1, isolated from a 45-year-old female patient with severe gastric symptoms. This is the first genome sequence of nonpylori gastric Helicobacter isolated from human gastritis.

Complete Genome Sequence of a Variant of Campylobacter jejuni NCTC 11168

Campylobacter jejuni NCTC 11168 is widely used in research, but at least two variants have been reported. The available genome was sequenced from a variant which later showed a different phenotype and gene expression profile. Here we present the complete genome sequence of a second variant of C. jejuni NCTC 11168.

Microevolution of a zoonotic Helicobacter population colonizing the stomach of a human host before and after failed treatment

To investigate the microevolution of Helicobacter bizzozeronii in the human stomach, comparative genomics of antrum-derived populations, obtained 3 months before (T0) and 6 months after (T1) an unsuccessful eradication treatment, was performed. For each time point, the DNA of bacterial mass, representing the population diversity in three biopsies, was mixed in equal amounts and sequenced using Illumina technology. Polymorphic sites (PSs) were detected by mapping the reads against an isogenic reference genome, derived from a corpus isolate obtained at T0. The total numbers of PSs detected in the H. bizzozeronii population at T0 and T1 were 128 and 223, affecting 81 and 134 coding sequences, respectively. At T0 in 91.4% of the PSs the mutation appeared at a frequency of 50% or less. On the contrary, in the majority of the PSs observed in T1 (71.3%) the mutation had a frequency >75%. Although only a minority of mutations were fixed in the antrum-derived population at T0, a certain level of allelic variability, compared with the corpus-derived reference genome, was present and most likely arose as consequence of the long-term colonization of the patient. The treatment probably induced a sudden decrease of population size, selecting a subpopulation, which acted as founder for the new population at T1 characterized by a higher number of fixed mutations. These data demonstrate that genome plasticity is an important common prerequisite among gastric Helicobacter species for adaptation to the stomach environment allowing the bacterium to evolve rapidly once a selective pressure is applied.