Molecular characterization of biofilm production and whole genome sequencing of selected Campylobacter concisus oral and clinical strains

Huq, M 2016, Molecular characterization of biofilm production and whole genome sequencing of selected Campylobacter concisus oral and clinical strains, Doctor of Philosophy (PhD), Applied Science, RMIT University.

Document type: Thesis
Collection: Theses

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Title Molecular characterization of biofilm production and whole genome sequencing of selected Campylobacter concisus oral and clinical strains
Author(s) Huq, M
Year 2016
Abstract Campylobacter concisus is a fastidious, hydrogen-requiring bacterium normally found in the human oral cavity, and is currently considered as an emerging pathogen. It has been isolated from gingivitis, periodontitis, foot ulcers, gastritis, and from intestinal biopsies of patients with inflammatory bowel disease (IBD). It is a heterogeneous species of phenotypically indistinguishable strains belonging to different genomospecies. C. concisus is also known to produce biofilms on glass, stainless steel, and polystyrene plastic. However, there are little data available on its biofilm properties. The regulation of biofilm formation has been linked to the signalling protein “LuxS” in many oral pathogens. In this study C. concisus biofilms were phenotypically characterized and the role of the luxS gene in biofilm formation was investigated. Comparative genomic analysis of selected oral and intestinal C. concisus strains was also performed to improve the knowledge in the present literature.

Biofilm formation by 14 clinical and 19 oral C. concisus strains was assessed by the crystal violet assay. All tested C. concisus strains were capable of producing biofilms in different levels, with oral strains being the highest producers. The biofilms were phenotypically characterized by phase contrast microscopy, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM). Different morphological stages of biofilm formation (attachment, maturation and dispersion) were observed by phase contrast microscopy. Completely developed biofilms were observed by CLSM on day five of biofilm formation with a mixture of dead and live bacteria within its structure. In addition, the aggregation of the biofilm and the presence of extracellular polymeric substances (EPS) were confirmed by SEM.

A luxS PCR product (309 bp) was amplified from all clinical and oral strains. Furthermore, to assess the role of LuxS in biofilm formation, a luxS mutant was created by inserting a kanamycin cassette within the luxS gene of RMIT-O17, the highest biofilm-forming strain. RMIT-O17 and the luxS-mutant were subjected to different phenotypic tests. A significant reduction (p < 0.05) in the terms of biofilm formation, motility, and invasion of the intestinal epithelial INT 407 cells were observed in the luxS-mutant compared to the wild type. However, no significant difference was found between the adherence properties of the luxS-mutant and the parental strain.

As a heterogenic bacterium C. concisus can be typed into at least two distinct genomospecies (A & B). In this study comparative genomic analysis was performed on the genomes of four C. concisus strains, all belonged to genomospecies A. Those are the genomes of RMIT-O17, along with an oral isolate from an individual with Crohn’s disease (RMIT-JF1) and the two intestinal strains, RCH 26, a faecal strain from a child with gastroenteritis and AUS22-Bd2, a unique strain isolated from the duodenum of an IBD patient. In addition, the genomes of two reference strains C. concisus 13826 and ATCC 33237T, available from the National Center for Biotechnology Information (NCBI), were included in subsequent comparative genomic analyses. The sizes of the sequenced four genomes ranged from 1.83-1.94 Mbp (1,859-2,048 encoded proteins) with the genome of RMIT-JF1 being the largest.

Pairwise comparisons of the genomes of RMIT-O17, RMIT-JF1, RCH 26 and AUS22-Bd2 with C. concisus 13826 and ATCC 33237T were performed using Mauve alignment tools. The comparison with C. concisus 13826 showed a very high level of gene shuffling (new gene combinations for genetic variation), while a high level of similarity and contiguity was observed with ATCC 33237T. Two plasmids, 22 kb and 3.3 kb, were detected in the genome of RCH 26 yet the other three genomes did not contain any plasmid. The 3.3kb plasmid was a unique, high copy number plasmid with no similarity to other C. concisus genome sequences available in the database.

The pan and core genomes of the four sequenced C. concisus strains including C. concisus 13826 and ATCC 33237T consisted of 2,790 and 1,463 protein coding genes, respectively. The clusters of orthologous groups (COG) of protein distribution patterns showed the involvement of more core genes in amino acid metabolism and transport; energy production and conversion and translation; ribosomal structure and biogenesis. Pan genes appeared to be enriched in replication, recombination and repair, in addition to cell wall/membrane/envelope biogenesis related functions. This was also supported by the Blast2GO enrichment analysis, where the enrichment of RNA processes, and metabolic and biosynthetic processes were observed within the core genome of C. concisus. On the other hand, in the pan genome enrichment of defence responses, DNA-related processes such as DNA integration and DNA restriction-modification suggests that several cellular defence mechanisms do exist in these C. concisus strains to probably survive phage attacks. In addition other genes which could be related to phenotypic characteristics such as invasiveness, adherence and motility.were identified in the four sequenced C. concisus genomes.

Phylogenetic trees were generated based on the ribosomal RNA (rrn) operon from 12 C. concisus strains. The regions from each rrn operon produced different phylogenetic trees demonstrating sequence differences between these strains in relation to the 5S rRNA, 16S rRNA, 23S rRNA, and other intergenic regions, which supports the heterogenic nature of this species. Furthermore, C. concisus strains were classified into the two known genomospecies (A & B) based on the indels present within each operon. Within the rrn operon, the 23S rRNA gene was suggested as a reliable region for C. concisus typing.

From the findings of this study it can be concluded that the C. concisus oral strains are higher biofilm producers than the intestinal strains, which could be a survival mechanism in their normal habitat. The LuxS plays a role in biofilm formation and other virulence properties in C. concisus; however, this is yet to be confirmed by genetic or chemical complementation. In addition, genomic analysis of the genomes of oral and intestinal C. concisus strains indicated that there is a significant difference in gene content in these strains depending on the isolation site and the clinical history of the host, rather than the genomospecies.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Applied Science
Subjects Genomics
Clinical Microbiology
Keyword(s) Campylobacter concisus
Comparative genomic analysis
rrn operon
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