Application of whole genome sequencing and metagenomics for diagnosis of tuberculosis

Globally, tuberculosis kills more people than any other infectious disease. Control of the epidemic is impeded by poor diagnostic approaches. My original contribution to knowledge presented in this thesis is towards diagnosis of tuberculosis by (meta)genomic approaches. In the work presented here, I established proof-of-principle that tuberculosis can be detected, identified and somewhat characterised using a shotgun metagenomics approach. I developed an approach for DNA extraction directly from sputum followed by metagenomic sequencing that allowed me to detect sequences from the M. tuberculosis complex in all sixteen samples with low coverage of the H37Rv reference genome. This allowed me to assign the lineage of the MTBC species in thirteen of these samples. This was the basis of the first publication to sequencing tuberculosis without prior culture. I determined that the proportion of human reads in the resulting metagenomic data was a major limitation to characterising the MTBC organisms with greater resolution and sought, though unsuccessfully, to determine methods to remedy this. In doing so, I identified a number of considerations that need to be made when designing studies of human DNA depletion from such heterogeneous clinical samples in the future. Addressing some of the other limitations to using genomics in the diagnosis of tuberculosis, I studied the genotype-phenotype association of first-line drug resistance found in patients in Peru and evaluated the performance diagnostic approaches used. This identified novel mutations associated with pyrazinamide resistance, flaws in the MODS method of antibiotic resistance testing and variation in resistance prediction tools

Whole-genome sequencing illuminates the evolution and spread of multidrug-resistant tuberculosis in Southwest Nigeria.

Nigeria has an emerging problem with multidrug-resistant tuberculosis (MDR-TB). Whole-genome sequencing was used to understand the epidemiology of tuberculosis and genetics of multi-drug resistance among patients from two tertiary referral centers in Southwest Nigeria. In line with previous molecular epidemiology studies, most isolates of Mycobacterium tuberculosis from this dataset belonged to the Cameroon clade within the Euro-American lineage. Phylogenetic analysis showed this clade was undergoing clonal expansion in this region, and suggests that it was involved in community transmission of sensitive and multidrug-resistant tuberculosis. Five patients enrolled for retreatment were infected with pre-extensively drug resistant (pre-XDR) due to fluoroquinolone resistance in isolates from the Cameroon clade. In all five cases resistance was conferred through a mutation in the gyrA gene. In some patients, genomic changes occurred in bacterial isolates during the course of treatment that potentially led to decreased drug susceptibility. We conclude that inter-patient transmission of resistant isolates, principally from the Cameroon clade, contributes to the spread of MDR-TB in this setting, underscoring the urgent need to curb the spread of multi-drug resistance in this region

Global emergence of a hypervirulent carbapenem-resistant <i>Escherichia coli </i>ST410 clone

Carbapenem-resistant Escherichia coli (CREC) ST410 has recently emerged as a major global health problem. Here, we report a shift in CREC prevalence in Chinese hospitals between 2017 and 2021 with ST410 becoming the most commonly isolated sequence type. Genomic analysis identifies a hypervirulent CREC ST410 clone, B5/H24RxC, which caused two separate outbreaks in a children's hospital. It may have emerged from the previously characterised B4/H24RxC in 2006 and has been isolated in ten other countries from 2015 to 2021. Compared with B4/H24RxC, B5/H24RxC lacks the blaOXA-181-bearing X3 plasmid, but carries a F-type plasmid containing blaNDM-5. Most of B5/H24RxC also carry a high pathogenicity island and a novel O-antigen gene cluster. We find that B5/H24RxC grew faster in vitro and is more virulent in vivo. The identification of this newly emerged but already globally disseminated hypervirulent CREC clone, highlights the ongoing evolution of ST410 towards increased resistance and virulence. </p

Transmission of Staphylococcus aureus from Humans to Green Monkeys in The Gambia as Revealed by Whole-Genome Sequencing.

UNLABELLED: Staphylococcus aureus is an important pathogen of humans and animals. We genome sequenced 90 S. aureus isolates from The Gambia: 46 isolates from invasive disease in humans, 13 human carriage isolates, and 31 monkey carriage isolates. We inferred multiple anthroponotic transmissions of S. aureus from humans to green monkeys (Chlorocebus sabaeus) in The Gambia over different time scales. We report a novel monkey-associated clade of S. aureus that emerged from a human-to-monkey switch estimated to have occurred 2,700 years ago. Adaptation of this lineage to the monkey host is accompanied by the loss of phage-carrying genes that are known to play an important role in human colonization. We also report recent anthroponotic transmission of the well-characterized human lineages sequence type 6 (ST6) and ST15 to monkeys, probably because of steadily increasing encroachment of humans into the monkeys' habitat. Although we have found no evidence of transmission of S. aureus from monkeys to humans, as the two species come into ever-closer contact, there might be an increased risk of additional interspecies exchanges of potential pathogens. IMPORTANCE: The population structures of Staphylococcus aureus in humans and monkeys in sub-Saharan Africa have been previously described using multilocus sequence typing (MLST). However, these data lack the power to accurately infer details regarding the origin and maintenance of new adaptive lineages. Here, we describe the use of whole-genome sequencing to detect transmission of S. aureus between humans and nonhuman primates and to document the genetic changes accompanying host adaptation. We note that human-to-monkey switches tend to be more common than the reverse and that a novel monkey-associated clade is likely to have emerged from such a switch approximately 2,700 years ago. Moreover, analysis of the accessory genome provides important clues as to the genetic changes underpinning host adaptation and, in particular, shows that human-to-monkey switches tend to be associated with the loss of genes known to confer adaptation to the human host

Estimating the time-varying reproduction number of SARS-CoV-2 using national and subnational case counts

Background: Assessing temporal variations in transmission in different countries is essential for monitoring the epidemic, evaluating the effectiveness of public health interventions and estimating the impact of changes in policy. Methods: We use case and death notification data to generate daily estimates of the time-varying reproduction number globally, regionally, nationally, and subnationally over a 12-week rolling window. Our modelling framework, based on open source tooling, accounts for uncertainty in reporting delays, so that the reproduction number is estimated based on underlying latent infections. Results: Estimates of the reproduction number, trajectories of infections, and forecasts are displayed on a dedicated website as both maps and time series, and made available to download in tabular form. Conclusions:  This decision-support tool can be used to assess changes in virus transmission both globally, regionally, nationally, and subnationally. This allows public health officials and policymakers to track the progress of the outbreak in near real-time using an epidemiologically valid measure. As well as providing regular updates on our website, we also provide an open source tool-set so that our approach can be used directly by researchers and policymakers on confidential data-sets. We hope that our tool will be used to support decisions in countries worldwide throughout the ongoing COVID-19 pandemic.</ns4:p

Alpaca Field Behaviour When Cohabitating with Lambing Ewes

A common strategy to reduce predator attack on livestock is the deployment of guardian alpacas. However, little research has been conducted on the behaviour of this species while housed with other livestock. This study monitored two male alpacas cohabitating with 180 lambing ewes in order to quantify field behaviour in two phases. Phase one assessed diurnal patterns of alpacas and lambing ewes using Global Navigation Satellite System (GNSS) collars recording data over 41 days, in combination with observational recordings. Phase two developed an alpaca behavioural ethogram through continuous observations from 05:30 to 19:30 h over a 3-day period. The two alpacas shared similar behaviours with commonality of distance travelled, and both species exhibited an increase in activity level based on speed between the times of 05:00 and 17:00 h. The GNSS data indicated that the alpacas flocked with the ewes at night sharing the same resting location, however, would spend time during the day on the outskirts of the paddock. Alpacas were observed to spend the majority of the observation period in two behavioural states: grazing (57%) and resting (27%). As a result of this study we were able to catalogue a range and frequency of field behaviours which alpacas exhibit while cohabitating with lambing ewes. However, further research is needed to determine in more detail how these behaviours correspond with the effectiveness of this species as a livestock guardian

GR13-type plasmids in Acinetobacter potentiate the accumulation and horizontal transfer of diverse accessory genes

Carbapenem and other antibiotic resistance genes (ARGs) can be found in plasmids in Acinetobacter , but many plasmid types in this genus have not been well-characterized. Here we describe the distribution, diversity and evolutionary capacity of rep group 13 (GR13) plasmids that are found in Acinetobacter species from diverse environments. Our investigation was prompted by the discovery of two GR13 plasmids in A. baumannii isolated in an intensive care unit (ICU). The plasmids harbour distinct accessory genes: pDETAB5 contains bla (NDM-1) and genes that confer resistance to four further antibiotic classes, while pDETAB13 carries putative alcohol tolerance determinants. Both plasmids contain multiple dif modules, which are flanked by pdif sites recognized by XerC/XerD tyrosine recombinases. The ARG-containing dif modules in pDETAB5 are almost identical to those found in pDETAB2, a GR34 plasmid from an unrelated A. baumannii isolated in the same ICU a month prior. Examination of a further 41 complete, publicly available plasmid sequences revealed that the GR13 pangenome consists of just four core but 1186 accessory genes, 123 in the shell and 1063 in the cloud, reflecting substantial capacity for diversification. The GR13 core genome includes genes for replication and partitioning, and for a putative tyrosine recombinase. Accessory segments encode proteins with diverse putative functions, including for metabolism, antibiotic/heavy metal/alcohol tolerance, restriction-modification, an anti-phage system and multiple toxin–antitoxin systems. The movement of dif modules and actions of insertion sequences play an important role in generating diversity in GR13 plasmids. Discrete GR13 plasmid lineages are internationally disseminated and found in multiple Acinetobacter species, which suggests they are important platforms for the accumulation, horizontal transmission and persistence of accessory genes in this genus

Endemicity and diversification of carbapenem-resistant Acinetobacter baumannii in an intensive care unit.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major public health concern globally. Often studied in the context of hospital outbreaks, little is known about the persistence and evolutionary dynamics of endemic CRAB populations. METHODS: A three-month cross-sectional observational study was conducted in a 28-bed intensive care unit (ICU) in Hangzhou, China. A total of 5068 samples were collected from the hospital environment (n = 3985), patients (n = 964) and staff (n = 119). CRAB isolates were obtained from 10.5% of these samples (n = 532). All of these isolates, plus an additional 19 from clinical infections, were characterised through whole-genome sequencing. FINDINGS: The ICU CRAB population was dominated by OXA-23-producing global clone 2 isolates (99.3% of all isolates) that could be divided into 20 distinct clusters, defined through genome sequencing. CRAB was persistently present in the ICU, driven by regular introductions of distinct clusters. The hospital environment was heavily contaminated, with CRAB isolated from bed units on 183/335 (54.6%) sampling occasions but from patients on only 72/299 (24.1%) occasions. CRAB was spread to adjacent bed units and rooms, and following re-location of patients within the ICU. We also observed three horizontal gene transfer events between CRAB strains in the ICU, involving three different plasmids. INTERPRETATION: The epidemiology of CRAB in this setting contrasted with previously described clonal outbreaks in high-income countries, highlighting the importance of environmental CRAB reservoirs in ICU epidemiology and the unique challenges in containing the spread of CRAB in ICUs where this important multidrug-resistant pathogen is endemic. FUNDING: This work was undertaken as part of the DETECTIVE research project funded by the Medical Research Council (MR/S013660/1), National Natural Science Foundation of China (81861138054, 32011530116, 31970128, 31770142), Zhejiang Province Medical Platform Backbone Talent Plan (2020RC075), and the National Key Research and Development Program of China grant (2018YFE0102100). W.v.S was also supported by a Wolfson Research Merit Award (WM160092)