Pathogen genomics of methicillin resistant staphylococcus aureus and leishmania
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Infectious diseases caused by the single-celled eukaryotic parasite Leishmania and the methicillin-resistant Staphylococcus aureus (MRSA) bacterium are major public health problems in many countries. In this thesis, I use genomics to explore the genomic plasticity of Leishmania and characterise the genomic and transcriptomic responses of MRSA treated with an antibiotic called oxacillin. The Leishmania parasite is transmitted by sandflies and can be maintained in the wild by various animals, as well as in people. It causes leishmaniasis, which is often difficult to treat and can prove fatal. In order to understand the Leishmania spp. infecting wild animals and their relationships to human-infecting Leishmania, we assembled, annotated and analysed the genomes of three Leishmania spp. The first of these was from a rodent in Ethiopia which we identified as Leishmania adleri using a phylogenomic approach. This species is part of the Sauroleishmania subgenera, whose genomes are expected to have 36 chromosomes and can infect reptiles. We found evidence of two novel independent chromosomal fission events in L. adleri using both our genome and an unassembled L. adleri sample isolated from a lizard. This resulted in 38 chromosomes, which was a novel finding because there was no evidence of these fissions in the sole published genome from the same subgenus: L. tarentolae Parrot-TarII. Extensive gene amplifications and aneuploidy were discovered in all three Sauroleishmania samples analysed, in common with previous work on other Leishmania spp., highlighting the lack of differentiation between animal- and human- infecting species. This new L. adleri genome is a high-quality annotated draft suitable for use as a reference, is the first assembled sequence available for L. adleri, and is only the second species in the Sauroleishmania subgenus to have a published genome. The other two Leishmania samples were isolated from dogs with leishmaniasis in Colombia and these were assembled and analysed using the same approach. A control genome was assembled using reads from the L. braziliensis genome so that we could quantify the completeness of our assemblies and identify any problems caused by our assembly approach. We classified our samples as L. naiffi and L. guyanensis, both members of the subgenus Viannia, whose members are only found in the Americas, predominately South America. This is the first report of L. naiffi in Colombia and in dogs illustrating the usefulness of genomics in disease surveillance. These genomes are also the first genomes for these two species. We compared both genomes with multiple other species from this subgenus and identified a 45 kb amplification in many Viannia spp. as well as a minichromosome in L. shawi M8408. Genes with high copy number and those unique to both species and the Viannia subgenus as a whole were also documented, which will aid development of diagnostics for this subgenus. Multiple responses to drug treatment with oxacillin have been investigated in many MRSA lineages. In this thesis, colleagues and I examined the genomic and transcriptomic responses of a community acquired MRSA strain (USA300) in a continuous culture (chemostat) experiment as well as in growth on agar plates. MRSA can exhibit heterogeneous resistance (HeR) which occurs when most cells in a sample are susceptible to low levels of antibiotic and only a few cells are highly resistant. A highly homogenously resistant (HoR) can be selected from a HeR sample using high doses of oxacillin. We discovered a novel tandem amplification of SCCmecIV in a drug resistant sample taken from a chemostat experiment. SCCmecIV is a mobile genetic element that harbours the mecA gene which facilitates resistance to β-lactam antibiotics, such as oxacillin. Multiple SNPs and indels at genes previously implicated in resistance were also identified. HeR isolates treated with oxacillin had low-frequency SNPs at some genes as well as numerous differentially expressed genes, whereas HoR samples had a nonsynonymous SNP at the gdpP gene, but few differentially expressed genes. This demonstrated that HeR cell populations responded to oxacillin by modifying gene expression regulation, whereas HoR ones had a genetic mutation to become resistant. We also found that purine metabolism had a role in oxacillin stress response because it was highly down-regulated at all levels of oxacillin, and SNPs and indels were discovered at two genes in this pathway (apt and guaA). Overall, we have assembled the genomes of three Leishmania spp., discovered novel chromosomal fission events in L. adleri and documented the presence of L. naiffi in a dog in Colombia for the first time. These genomes, coupled with that of L. guyanensis have extended our understanding of genome architecture and plasticity in Leishmania and will facilitate future research by others on these species. We have found a novel amplification of SCCmecIV in response to drug treatment demonstrating the need to search for copy number variation in addition to SNPs and indels, and found multiple responses to various levels of oxacillin, some of which had not been previously reported. These findings have important clinical implications for drug treatment of S. aureus as they demonstrate that amplification of large mobile elements can occur and that these can be maintained on the chromosome with variable copy number in response to drug pressure. Furthermore, commonly mutated genes and pathways in resistant samples show that cells converge on common solutions to survive drug treatment and these genes/pathways could serve as drug targets.
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