Targeted Sequencing Technology for the Characterisation of Microbes and Antibiotic Resistance Markers from Low-biomass Environmental Samples

Yang, Qing (2024). Targeted Sequencing Technology for the Characterisation of Microbes and Antibiotic Resistance Markers from Low-biomass Environmental Samples. University of Birmingham. Ph.D.

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Abstract

The healthcare environment is host to diverse communities of pathogenic bacteria with varying levels of antibiotic resistance. Within these communities, the exchange of genetic material, such as through horizontal gene transfer, further amplifies the spread of antibiotic resistance posing a threat to patients. Surveilling and managing this diversity through genetic characterisation are crucial for effective infection control. By analysing the genetic material of pathogenic bacteria, i.e. their DNA genome, valuable insights in genetic variation within a particular pathogen species can be gained. However, it remains difficult to effectively sequence genes and genomes from trace amounts of target DNA in hospital environmental samples (such as potable water, surfaces, and air) due to low-input or degraded DNA.

In this thesis I implemented a comprehensive approach to detect and analyse pathogenic bacteria and antibiotic resistance genes in low-biomass environmental samples including tap water, p-trap water, wastewater, air dust and surface by combining multiple culture-free targeted sequencing technologies and methodologies. By utilising 16S rRNA amplicon sequencing and high-throughput-quantitative PCR approaches, the dynamic patterns of pan-pathogenic bacteria, antibiotic resistance genes, and mobile genetic elements in nosocomial environments were sensitively identified, correlated and traced.

Of particular importance, this thesis establishes two novel multiplex-PCR-based panels that enabled rapid and accurate diagnostic typing and investigation of antibiotic resistance in Legionella pneumophila and Pseudomonas aeruginosa, with a sensitivity down to as few as three bacterial cells using Oxford Nanopore MinION sequencing. The prevalence of sequence type 36 (ST36) and a novel ST of L. pneumophila was confirmed from the common water-source during the winter and summer seasons, respectively. Additionally, ST549 of P. aeruginosa was found to be associated with various antibiotic resistance traits, including fluoroquinolone, carbapenem, and aminoglycoside resistance, through the occurrence of antibiotic resistance genes and genetic polymorphisms of gyrase and efflux-pump regulator genes linked to antibiotic resistance.

Overall, this thesis demonstrates the utility of novel targeted enrichment technologies for the comprehensive detection and analysis of pathogens and ARGs, particularly in hard-to-sequence environmental samples containing low starting input. These findings contribute to the understanding of epidemic tracing and outbreak assessment, as well as antibiotic resistance dynamics, thereby facilitating the development of effective surveillance and management strategies.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):