Removal of antimicrobial resistance genes from bacterial strains and communities using CRISPR-Cas9

Antimicrobial resistance (AMR) is one of the largest threats facing modern-day healthcare and society in the coming decades. AMR genes are widely disseminated on genetic vehicles called plasmids, leading to resistant bacteria in many environments. Development of new antibiotics is inefficient, and stewardship of existing antibiotics is often ineffective. One promising novel approach to reduce AMR in bacteria is the delivery of genes coding for CRISPR-Cas9, which can specifically cleave a target sequence of choice – and in this way can be utilised to kill bacteria or remove their resistance plasmids. The general concept of such CRISPR delivery tools has been proven to be effective under laboratory conditions, however antibiotic resensitisation is more complex when targeting natural plasmids in mixed microbial communities. In this thesis, I aimed to develop a CRISPR delivery tool that can reach various species of bacteria embedded in microbial communities and resensitise these to antibiotics, allowing successful treatment using existing antibiotic drugs. In the first chapter, I reviewed the role which plasmids play in the AMR crisis by horizontal transfer of resistance genes. I summarised various approaches of counteracting this, with a focus on CRISPR-mediated AMR plasmid removal. In the second chapter, I engineered a broad host-range plasmid pKJK5 to encode CRISPR-Cas9 (pKJK5::Cas). I showed that this plasmid can be used to block target AMR plasmid uptake in Escherichia and Pseudomonas isolates. In the third chapter, I utilised pKJK5::Cas’ conjugative ability to remove a target AMR plasmid from recipient bacteria, which depended on pKJK5::Cas conjugation efficiency and CRISPR targeting efficiency. In the fourth chapter, I investigated removal of the broad host-range conjugative plasmid RP4 by pKJK5::Cas. I found that presence of toxin-antitoxin systems and target plasmid incompatibility can interfere with the use of pKJK5::Cas. In the fifth chapter, I assayed pKJK5::Cas transfer and maintenance in a synthetic bacterial community. Surprisingly, pKJK5::Cas maintenance and fitness of its host was dependent on community context where the plasmid became lost from a Variovorax host strain in presence of Stenotrophomonas growth partners. Finally, I offer concluding remarks on my data where I speculated under which conditions target plasmid removal may be successful in such a community context.Medical Research Council (MRC

CRISPR-Cas antimicrobials: Challenges and future prospects

This is the final version. Available from PLoS via the DOI in this record.Antimicrobial resistance (AMR) poses a serious threat to modern medicine and may render common infections untreatable. The discovery of new antibiotics has come to a relative standstill during the last decade [1], and developing novel approaches to tackle the spread of AMR genes will require significant efforts in the coming years [2]. In 2014, several groups independently demonstrated how CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR–associated), a bacterial immune system now widely used for genome editing, can selectively remove AMR genes from bacterial populations. Here, we discuss the current state of the field of CRISPR-Cas antimicrobials, the challenges ahead, and how they may be overcome.Biotechnology & Biological Sciences Research Council (BBSRC)Medical Research CouncilNatural Environment Research CouncilWellcome TrustEuropean Research CouncilPeople Programme (Marie Curie Actions) of the European Union’s Horizon 202

Interspecific competition can drive plasmid loss from a focal species in a microbial community

This is the final version. Available on open access from Springer Nature via the DOI in this recordData availability: All data generated during this study are included in the supplementary information files.Plasmids are key disseminators of antimicrobial resistance genes and virulence factors, and it is therefore critical to predict and reduce plasmid spread within microbial communities. The cost of plasmid carriage is a key metric that can be used to predict plasmids’ ecological fate, and it is unclear whether plasmid costs are affected by growth partners in a microbial community. We carried out competition experiments and tracked plasmid maintenance using a model system consisting of a synthetic and stable five-species community and a broad host-range plasmid, engineered to carry different payloads. We report that both the cost of plasmid carriage and its long-term maintenance in a focal strain depended on the presence of competitors, and that these interactions were species specific. Addition of growth partners increased the cost of a high-payload plasmid to a focal strain, and accordingly, plasmid loss from the focal species occurred over a shorter time frame. We propose that the destabilising effect of interspecific competition on plasmid maintenance may be leveraged in clinical and natural environments to cure plasmids from focal strains.Medical Research Council (MRC)Biotechnology and Biological Sciences Research Council (BBSRC)Lister Institute for Preventative MedicineBundesministerium für Bildung und Forschun

Removal of AMR plasmids using a mobile, broad host-range CRISPR-Cas9 delivery tool

This is the final version. Available on open access from the Microbiology Society via the DOI in this recordAntimicrobial resistance (AMR) genes are widely disseminated on plasmids. Therefore, interventions aimed at blocking plasmid uptake and transfer may curb the spread of AMR. Previous studies have used CRISPR-Cas-based technology to remove plasmids encoding AMR genes from target bacteria, using either phage- or plasmid-based delivery vehicles that typically have narrow host ranges. To make this technology feasible for removal of AMR plasmids from multiple members of complex microbial communities, an efficient, broad host-range delivery vehicle is needed. We engineered the broad host-range IncP1-plasmid pKJK5 to encode cas9 programmed to target an AMR gene. We demonstrate that the resulting plasmid pKJK5::csg has the ability to block the uptake of AMR plasmids and to remove resident plasmids from Escherichia coli. Furthermore, due to its broad host range, pKJK5::csg successfully blocked AMR plasmid uptake in a range of environmental, pig- and human-associated coliform isolates, as well as in isolates of two species of Pseudomonas. This study firmly establishes pKJK5::csg as a promising broad host-range CRISPR-Cas9 delivery tool for AMR plasmid removal, which has the potential to be applied in complex microbial communities to remove AMR genes from a broad range of bacterial species.Medical Research Council (MRC)Biotechnology and Biological Sciences Research Council (BBSRC)Lister Institute for Preventative MedicineJPI-AMR HARISSA programme (MISTAR)Bundesministerium für Bildung und ForschungEuropean Research Council (ERC)College of Life and Environmental Sciences, University of Exete

Emotional design and human-robot interaction

Recent years have shown an increase in the importance of emotions applied to the Design field - Emotional Design. In this sense, the emotional design aims to elicit (e.g., pleasure) or prevent (e.g., displeasure) determined emotions, during human product interaction. That is, the emotional design regulates the emotional interaction between the individual and the product (e.g., robot). Robot design has been a growing area whereby robots are interacting directly with humans in which emotions are essential in the interaction. Therefore, this paper aims, through a non-systematic literature review, to explore the application of emotional design, particularly on Human-Robot Interaction. Robot design features (e.g., appearance, expressing emotions and spatial distance) that affect emotional design are introduced. The chapter ends with a discussion and a conclusion.info:eu-repo/semantics/acceptedVersio

Localization with random time-periodic quantum circuits

We consider a random time evolution operator composed of a circuit of random unitaries coupling even and odd neighboring spins on a chain in turn. In spirit of Floquet evolution, the circuit is time-periodic; each timestep is repeated with the same random instances. We obtain analytical results for arbitrary local Hilbert space dimension d: On a single site, average time evolution acts as a depolarising channel. In the spin 1/2 (d=2) case, this is further quantified numerically. For that, we develop a new numerical method that reduces complexity by an exponential factor. Haar-distributed unitaries lead to full depolarization after many timesteps, i.e. local thermalization. A unitary probability distribution with tunable coupling strength allows us to observe a many-body localization transition. In addition to a spin chain under a unitary circuit, we consider the analogous problem with Gaussian circuits. We can make stronger statements about the entire covariance matrix instead of single sites only, and find that the dynamics is localising. For a random time evolution operator homogeneous in space, however, the system delocalizes

Probabilistic object detection: Definition and evaluation

We introduce Probabilistic Object Detection, the task of detecting objects in images and accurately quantifying the spatial and semantic uncertainties of the detections. Given the lack of methods capable of assessing such probabilistic object detections, we present the new Probability-based Detection Quality measure (PDQ). Unlike AP-based measures, PDQ has no arbitrary thresholds and rewards spatial and label quality, and foreground/background separation quality while explicitly penalising false positive and false negative detections. We contrast PDQ with existing mAP and moLRP measures by evaluating state-of-the-art detectors and a Bayesian object detector based on Monte Carlo Dropout. Our experiments indicate that conventional object detectors tend to be spatially overconfident and thus perform poorly on the task of probabilistic object detection. Our paper aims to encourage the development of new object detection approaches that provide detections with accurately estimated spatial and label uncertainties and are of critical importance for deployment on robots and embodied AI systems in the real world

LunaRoo: A proposal for the Google Lunar XPrize payload opportunity with the part time scientists team

This proposal describes the innovative and competitive lunar payload solution developed at the Queensland University of Technology (QUT)–the LunaRoo: a hopping robot designed to exploit the Moon's lower gravity to leap up to 20m above the surface. It is compact enough to fit within a 10cm cube, whilst providing unique observation and mission capabilities by creating imagery during the hop. This first section is deliberately kept short and concise for web submission; additional information can be found in the second chapter