UZ Gent, medische microbiologie
Abstract 2 advanced bachelor of bioinformatics 2019-2020: Comparative genome analysis of Gardnerella species with a focus on taxonomy
Bacterial vaginosis is a disturbance of the healthy vaginal microbiome where the lactobacilli are replaced with anaerobes such as Gardnerella vaginalis. This increases the acidity in the vagina. Under normal circumstances, the lactic acid-producing lactobacilli ensure a fairly high acidity in the natural environment of the vagina. Bacterial vaginosis is associated with premature birth and increased incidence of sexually transmitted infections including HIV.
Gardnerella vaginalis, the key pathogen in BV, has recently been shown to actually comprise 13 different species, based on the comparison of 81 genomes. Of these 13 species, some were suggested to be more virulant than others. My task in at the traineeship was to update this taxonomy. For this all the genomes of Gardnerella vaginalis from NCBI were downloaded. From this information different kinds of data were extracted with the use of python to in the first place control if the sequences were indeed of Gardnerella vaginalis. This was done by using the 16S ribosomal RNA region of the genome and if this was not present in the data other genes were used for this quality control. Control gene was used in a blast, if the top hit was Gardnerella vaginalis the genome passed the quality control else the genome could not be classified as Gardnerella vaginalis. The second part of the traineeship was the update of the figure where the ANI (average nucleotide identity) and the DDH (DNA-DNA-hybridization) are placed next to each other for proper subdivision of the different subspecies of Gardnerella vaginalis.
Both the average nucleotide identity and DNA-DNA-hybridization used the genomes provided from NCBI. The results were gathered and placed in excel and filtered.
The process of gathering the 16S ribosomal RNA, getting the genome files and using the ANI tool were atomized using python. Here the only input needed is the csv file from NCBI that can be downloaded when going to the genome page of a bacteria. This script is thus also usable for researching other bacteria than Gardnerella vaginalis. The DDH and the quality control and the filtering of the results to get the wanted structure are done manually due to a lack of time but will be atomized by the next trainee.
When looking at this figure different groups are visible which separate the different genomes in subspecies. This can be important for the next step when looking at the virulent genes, the virulent genes can differ in these groups and thus the activity of the specific made bacteriophage can differ between these groups.
The last phase of the internship was to use the virulent genes found in publications and provided from the internship to determine which genes are present on the genomes of these bacteria and which genes to use in the phage therapy to target these bacteria without interacting with the other bacteria present in the microbiome. In this way specific bacteria species can be eradicated from a microbiome to restore balance.
Abstract Bachelor Project FBT 2018-2019: Development and optimization of ddPCR for detection and qualification of a SNP linked to anthelmintic resistance in Trichuris trichiura
Soil-transmitted helminths (Ascaris lumbricoides, Trichuris trichiura and the hookworms Ancylostoma duodenale and Necator americanus) are a group of intestinal parasitic nematodes that infect one fifth of the world’s population and cause significant morbidity. Preventive chemotherapy with benzimidazoles (BZ, i.e. albendazole or mebendazole) is implemented as the main strategy for controlling Soil-transmitted helminthiasis. However, there is grounded fear that drug resistance may develop as a result of (1) the high degree of drug pressure, (2) the suboptimal doses and (3) the reliance on only two drugs that have a common mode of action.
Experience from the veterinary field has further ratified these concerns. In veterinary nematodes, BZ-resistance is caused by single nucleotide polymorphisms (SNPs) located in the β-tubulin isotype 1 gene at codon position 167, 198 and/or 200. Therefore, the development of a system to monitor the emergence and spread of anthelmintic resistance is crucial. In this study, a novel competitive digital droplet PCR (ddPCR) assay was designed for the detection of both the wildtype and the SNP 200 in the β-tubulin isotype 1 gene of T. trichiura. This species was chosen based on data that indicate that BZ efficacy against T. trichiura is unsatisfactory, hence likely the biggest chance of finding resistance. The newly developed ddPCR assays were first optimized using quantitative Polymerase Chain Reaction (qPCR). The results indicate that the final assay allows specific detection and quantification of mutant and wild-type targets.
Further optimization is necessary to make the assay even more efficient, as it may be valuable for the detection and monitoring of anthelminthic resistance in the future.
C. Heymanslaan 10