High-throughput sequencing to understand pathogen carriage and transmission

Genomic sequencing is a powerful technology in the study of pathogen biology and pathogen transmission. In this thesis I have studied the human nasal carriage of *Staphylococcus aureus* and, in response to the SARS-CoV-2 pandemic, I examine the transmission of SARS-CoV-2, two critically important human pathogens. To address this, I have utilised genomic and individual-level epidemiological data, acquired through the CARRIAGE study and the COVID-19 Genomics UK Consortium (COG-UK). I characterised the microbial community structure that facilitates or prevents persistent nasal colonisation by *Staphylococcus aureus* using 16S rRNA gene sequencing of serially collected nasal swabs from 1,180 individuals. I provide evidence, by defining the carriage status of participants, that alpha and beta diversity of the anterior nares significantly differs by carriage status. I demonstrate that persistent carriage in the anterior nares is negatively associated with multiple species, compared with other carriage states. These findings redefine intermittent carriage, which does not appear to be defined by a distinct microbial community. Using a random forest model, I show that *S. aureus* carriage can be predicted from microbiome data with a moderate degree of accuracy, and explore the phylogenetic relationship of *S. aureus* isolates recovered from nasal swabs with their associated microbiome. To facilitate higher throughput sequencing in the CARRIAGE study, I optimized 16S rRNA gene sequencing. Using nasal samples from healthy human participants and a serially diluted mock microbial community, I compared alpha and beta diversity, and compositional abundance where the PCR amplification was conducted in triplicate, duplicate or as a single reaction, and where manually prepared or premixed mastermix was used. I find no requirement for pooling of PCR amplifications or manual preparation of PCR mastermix, resulting in a more efficient 16S rRNA gene PCR protocol. Moreover, I demonstrate the need for sufficient controls to account for contaminants, which are an important source of bias. I used care homes as a model to understand the utility of whole genome sequencing early in the SARS-CoV-2 pandemic. I reviewed all genomic epidemiology studies on SARS-CoV-2 in care-homes that had been published up to 3 November 2020 and identified numerous sources of, and opportunities to mitigate, transmission of SARS-CoV-2 in care homes using genomics. Importantly, I recognised the importance of combining detailed epidemiological data with SARS-CoV-2 genomic data to inform public health decision making and detail sources of possible error introduced through bioinformatic analysis in multiple studies. To identify the transmission dynamics of SARS-CoV-2 in a university population, I used 482 prospectively sequenced SARS-CoV-2 isolates derived from asymptomatic student screening and symptomatic testing of students and staff at the University of Cambridge from 5 October to 6 December 2020. I performed a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. I found that after a limited number of viral introductions into the university, the majority of student cases were linked to a single genetic cluster, likely dispersed across the university following social gatherings at a venue outside the university. I identified considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and dramatically reduced following a national lockdown. I observed that transmission clusters were largely segregated within the university or within the community. To characterise the contribution of imported SARS-CoV-2 to onwards transmission and establishment of SARS-CoV-2, I evaluated the effectiveness of travellers being required to quarantine for 14 days on return to England in Summer 2020. I identified 4,207 travel-related SARS-CoV-2 cases and their 18,885 contacts, along with 888 associated travel-related SARS-CoV-2 genomes in the UK SARS-CoV-2 sequencing dataset. Quarantining was associated with a lower rate of contacts. Fewer genomically-linked cases were observed for index cases who returned from countries with quarantine requirement compared to cases from countries with no quarantine requirement, but this effect was explained when adjusting for the number of importations for each genome. Furthermore, I identified a large travel-related cluster dispersed across England, which was confirmed with contact-tracing data. In conclusion, I have described the microbial basis for *Staphylococcus aureus* colonisation of the anterior nares; importantly I have re-defined carriage and developed an effective model for predicting carriage status. I have identified the key determinants of SARS-CoV-2 transmission and effective interventions in care homes, a large UK university, and at a national scale, highlighting the clear utility of whole-genome sequencing to inform real-time public health policy.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Aggarwal, Dinesh

Advisors dc:contributor.advisor

• Peacock, Sharon
• Harrison, Ewan

• 16S rRNA
• Carriage
• Microbiome
• SARS-CoV-2
• Staphylococcus aureus
• Tranmission
• Whole-genome sequencing

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/83dacd6e-e63c-478a-8027-c9cd3dbfbb72/download
• https://creativecommons.org/licenses/by/4.0/

Language dc:language

eng

Author Identifier

0000-0002-5938-8172

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/374745