The rates and routes of transmission of multidrug resistant Klebsiella clones and genes into the clinic from environmental sources.

Lead Research Organisation: University of Bath
Department Name: Biology and Biochemistry

Abstract

Recent years have seen a dramatic global increase of strains of bacteria that pose a high risk in health care settings due to broad resistance to a wide range of antibiotics.

Our research project will concentrate on Klebsiella pneumoniae (Klebsiella), a leading cause of multidrug resistant hospital-acquired infections globally, particularly among neonates, the elderly and immunosuppressed patients, with reported mortality rates upwards of 50%. It is also responsible for an increasing burden due to community-acquired invasive infections, which can lead to pyogenic liver abscess, pneumonia, and meningitis. In addition to causing disease, Klebsiella is an ecological generalist and can be carried asymptomatically in the intestinal tract, skin, nose, and throat of healthy individuals, and can thrive in a range of arthropod, bird or plant hosts (including crops and trees) and environmental niches such as water and soil. It can also cause invasive disease in several animal species, and it is a common cause of mastitis in dairy herds.

In order to control the spread of Klebsiella through targeted surveillance and intervention policies it is necessary to identify the sources of emergent community and health-care associated infection from the interlinked and varied niches encompassing "the environment". Environmental prevalence data provides a critical baseline, but is not always sufficient to inform on public health policy. For instance, the observation that gulls frequently harbour Klebsiella is, by itself, insufficient to guide policy on managing gull populations, as direct transmission to humans remains very difficult to demonstrate. However, if, say, water were to be identified as a major Klebsiella reservoir with frequent transmission to and from the clinic, this might in turn impact on waste water treatment procedures. Similarly, if cockroaches are found to be associated with Klebsiella this should lead to increased pest control both in hospitals and in the community.

To address this, we will take samples in order to identify Klebsiella from multiple clinical, community, agricultural, veterinary and environmental settings. Then we will use a novel approach based on whole genome sequencing to provide a much more efficient means to understand ecological adaptation, the distribution of resistance and virulence genes in the bacteria, and to identify key environmental settings from which these Klebsiella bacterial clones emerge. This approach will involve sequencing multiple colonies of bacteria simultaneously in order to detect a wider sample of genes present within a given sample. We will build a comprehensive, data-driven, model of adaptation, diversification, gene flow and transmission dynamics between all relevant ecological and clinical compartments. This will then pave the way for the development of targeted disease management strategies.

Technical Summary

Klebsiella pneumoniae (Kp) is a leading cause of multidrug resistant hospital-acquired infections globally, and is responsible for an increasing public health burden in the community. In order to control the spread of Kp through targeted surveillance and intervention policies it is necessary to identify the sources of emergent community and health-care associated infection from the interlinked and varied niches encompassing "the environment". To address this, we will sample from multiple clinical, community, agricultural, veterinary and environmental settings in and around a single town, Pavia, in Northern Italy, and supplement these data with matched samples from France and elsewhere. We will use whole genome sequencing of community (mixed-colony) samples to assay accessory gene abundance and distribution. This contrasts with the more common approach based on phylogenetic analysis of single colonies, which would be of limited utility over broad environmental scales due to the complexity of transmission chains, environmental dormancy, and high rates of recombination.

This gene-centric approach provides a much more efficient means to understand ecological adaptation, the distribution of resistance and virulence genes, and to identify key environmental reservoirs from which clinical clones emerge. A key deliverable of this project will be the establishment of a pan-genome database ('pangenomium') that will integrate with both existing Kp genome community resources established by project partners (BIGSdb-kp, and wgsa.net). We will also develop mathematical models to further explore gene flow between environmental compartments, and will use the Oxford Nanopore Minion TM platform to characterise AMR plasmids, and understand their distribution and transfer, using hybrid assemblies.

Planned Impact

Klebsiella pneumoniae (Kp) is a highly diverse pathogen that is ubiquitous in the environment. This high level of variation will seriously hinder attempts to infer transmission dynamics between different environmental niches (including animal hosts) using approaches based on SNP variation within the core genome. This is due to factors such as the complex fitness landscape of the wider environment, the high level of genomic diversity (even within single hosts), high rates of co-colonisation, environmental dormancy, recombination, complex transmission chains and the simultaneous transmission of multiple variants. These issues would lead to inevitable gaps in sampling and statistical uncertainties stemming from the chance sequencing of one colony over another for any given sample.

This project will address these difficulties by providing a novel framework for understanding transmission on the basis of the distribution of accessory genes, rather than core genome variation, and in doing so will be of direct benefit to medical microbiologists and bacterial genomicists working on Kp, as well as the broader microbial genomics community. Our approach will be to construct a database of all known genes in this species, then used mixed colony sequencing to assay the distribution of these genes across multiple ecological niches within and around a single town (Pavia, northern Italy). This will enable us to identify niche specific genes, which will then be used as markers to determine the principal routes by which Kp moves through the environment and into healthcare settings. The identification of specific environmental settings enriched with particular AMR or virulence genes will inform on community risk assessment and policy guidelines. The project will involve the development of novel statistical and bioinformatics approaches and mathematical models, and all pipelines and tools will be readily accessible to the broader community via MRC-CLIMB. The resultant database and analysis tools will provide the means for users to infer the likely environmental source of query genomes by automated mining for the presence of our niche-specific marker genes. This will provide critical knowledge for future source attribution studies of Kp infections and to prioritize interventions to limit the spread of MDR-Kp from its natural reservoirs to human carriage and infections. Our team includes the developers of both Kp-BIGsdb and wgsa.net genomic platforms, and working to common standards between these two systems will be of further broad utility to the community at large. We will also develop and utilise our hybrid assembly methods to combine long read data generated by the Oxford Nanopore Minion with short read Illumina data which can be used for the complete assembly of plasmids (Bayliss et al, Gigascience 2017). In this way we will provide critical data concerning plasmid composition and mobility; understanding the genetic bases of (in)compatibilities between resistance genes, mobile elements and host chromosomes will play a key role in mitigating their spread. In addition to shedding light on plasmid transfer and evolution in Kp, our project will also provide important methodological advances for studying accessory elements on population scales.

In sum, this project will define, at what we believe is an unprecedented scale, the distribution of Kp lineages and genes in various environments. The project will also contribute to strengthen the bioinformatics infrastructures (wgsa.net, BIGSdb-Kp) used by the global microbiology and surveillance communities to communicate on the epidemiology and population biology of Kp. These advances will prove critical in the ongoing battle to mitigate the global spread of this serious pathogen.
 
Description GCRF
Amount £1,351,083 (GBP)
Funding ID EP/P028403/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 05/2017 
End 04/2020
 
Description One Health Drivers of Antibacterial Resistance in Thailand
Amount £2,919,177 (GBP)
Funding ID MR/S004769/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 05/2018 
End 04/2021
 
Title "Pangenomium" bioinformatics tool for deducing gene and strain abundance profiles from mixed colony short read sequencing data 
Description A pipeline has been developed to analyse "plate sweep" data, whereby multiple colonies (in this case belonging to the Klebsiella genus) are harvested and sequenced en masse. The pipeline works by mapping these short reads to a pan-reference database containing all known genes in Klebsiella genomes (>100,000 genes). This then provides abundance profiles of each gene which can then be used to detect genes that are enriched in specific niches (using a machine-learning approach). The pipeline is also being used to define species and sub-species clusters using a combination of two recently published tools, MSweep and PopPunk. 
Type Of Material Improvements to research infrastructure 
Year Produced 2019 
Provided To Others? No  
Impact None yet 
 
Title Improvement to MSweep bioinformatics tool 
Description Improvement of method to determine bacterial community profiles from plate sweep data based on the published method : DOI: 10.1099/mgen.0.000075 
Type Of Material Improvements to research infrastructure 
Year Produced 2019 
Provided To Others? No  
Impact More accurate and efficient categorisation of short sequencing reads to species and sub-species level. Designed for use on large datasets (eg the 1000 plate sweep datasets generated in SpARK). 
 
Title Genome sequence database 
Description ~3,500 genomes of Klebsiella and Raoultella species sequenced on Illumina (short read) platform from multiple clinical and non-clinical sources in and around Pavia. ~1,000 metagenomics "plate sweeps" of Klebsiella and Raoultella species sequenced. ~100 genomes completed on long-read (Nanopore) platform. 400 in progress being sequenced on Pac. Bio. platform 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? No  
Impact Phylogenetic and AMR analyses ongoing 
 
Title PIGGY 
Description This bioinformatics pipeline provides a means to cluster and align intergenic region (IGR) sequences from large genome datasets. This allows the detection of IGR "switching", a process whereby a single gene is associated with different intergenic regions (hence promoters and other regulatory elements) - this has been shown to be associated with changes in gene expression. This work has been accepted for publication in Gigascience. 
Type Of Material Data analysis technique 
Year Produced 2018 
Provided To Others? Yes  
Impact The work has received a great deal of interest form the scientific community. 
URL http://gigadb.org/dataset/100410
 
Title PIRATE: pipeline for exploring the pan-genome 
Description A new bioinformatics pipeline for exploring bacterial pan-genomes using different thresholds of sequence identity to define gene homology groups. 
Type Of Material Data analysis technique 
Provided To Others? No  
Impact Manuscript in preparation 
 
Description Antimicrobial treatment, resistome and microbiome 
Organisation University of Glasgow
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in diversity analysis
Collaborator Contribution Expertise in epidemiology of mastitis and AMR
Impact Funding application to BBSRC responsive mode
Start Year 2015
 
Description Aquaculture and antimicrobial resistance 
Organisation University of Plymouth
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in diversity analysis and statistics of diversity
Collaborator Contribution Expertise in aquaculture and microbiome analysis
Impact Funding application to the BBSRC NEWTON fund for the UK-China AMR initiative.
Start Year 2016
 
Description BMC 
Organisation Bugando Medical Centre
Country Tanzania, United Republic of 
Sector Hospitals 
PI Contribution We initiated collaboration with Bugando Medical Centre in Mwanza, Tanzania. We visited BMC and met with key administrative contacts. We have since set up an MoU with BMC and involved BMC as a partner on an MRC grant application.
Collaborator Contribution Professor Stephen Mshana from Bugando Medical Centre participated in several workshops on AMR, hosted a visit by delegates from the University of Glasgow, facilitated the signing of an MoU and contributed to the writing of a grant proposal that is under consideration by MRC. His team will also contribute Klebsiella isolates to the MRC funded SpARK project, funded as JPI-AMR project and led by Prof. Ed Feil at the University of Bath.
Impact This collaboration is multi-disciplinary and involves one or more representatives of the following disciplines: anthropology, epidemiology, genomics, health economics, human geography, mathematics, medical science, microbiology, pharmacology, sociology, veterinary medicine
Start Year 2017
 
Description JPIAMR 7th Joint Programme Initiative on AMR - SOLIDNESS 
Organisation University of Groningen
Country Netherlands 
Sector Academic/University 
PI Contribution This network has just started (kick off meeting in March)
Collaborator Contribution This network has just started (kick off meeting in March)
Impact This network has just started. Kick off meeting in March.
Start Year 2019
 
Description KlebNet: a One Health network bridging science and surveillance on antimicrobial resistant Klebsiella 
Organisation Pasteur Institute, Paris
Country France 
Sector Charity/Non Profit 
PI Contribution Attended kick-off meeting in April 2019. I have been invited to present at the next meeting in April 2020 in Paris
Collaborator Contribution 31 leading experts in Kp in distinct sectors located in 12 different countries (Europe, Africa, North America, South America, Asia). Consortium headed by Slyvain Brisse who is a SpARK partner
Impact Kick off meeting last year resulting in new collaborations. I have been invited to present outputs from the the SpARK project at this years meeting.
Start Year 2019
 
Description Landscape ecology of antimicrobial resistance 
Organisation University of Glasgow
Department School of Mathematics and Statistics
Country United Kingdom 
Sector Academic/University 
PI Contribution We have established a new collaboration with the statistics department to develop novel statistical tools to analyse emerging regional and national scale antimicrobial resistance datasets. We bring expertise in the analysis of diversity and epidemiology of antimicrobial resistance spread.
Collaborator Contribution Advanced statistical tools
Impact Fully funded studentship to commence October 2016 plus a Pump-priming NERC application
Start Year 2015
 
Description Nor-Kleb-Net and Kleb-Gap 
Organisation University of Tromso
Country Norway 
Sector Academic/University 
PI Contribution I am on the advisory board of Nor-Kleb-Gap. I have provided advise and data for the Nor-Kleb and Kel-Gap networks and will be attending a workshop in June.
Collaborator Contribution NA
Impact This is a multidisciplinary partnership covering all aspects of Klebsiella population biology, with a focus on Norway.
Start Year 2019
 
Description STARCS consortium 
Organisation Utrecht University
Country Netherlands 
Sector Academic/University 
PI Contribution I am attending and presenting at the final meeting of the STARCS consortium on the outputs of the SpARK project
Collaborator Contribution NA
Impact I will present at the final meeting in June 2020
Start Year 2019
 
Description Sharing/collaboration on Klebsiella sequences 
Organisation Washington State University
Country United States 
Sector Academic/University 
PI Contribution A BBSRC funded project in Tanzania has provided Klebsiella isolates for sequencing which will provide input into SPARK plus and a currently funded MRC funded project in Tanzania looking at the drivers of antibtioic resistance.
Collaborator Contribution A BBSRC funded project in Tanzania has provided Klebsiella isolates for sequencing which will provide input into SPARK plus and a currently funded MRC funded project in Tanzania looking at the drivers of antibtioic resistance.
Impact Analysis of sequence data in progress.
Start Year 2019
 
Description WSU 
Organisation Washington State University
Country United States 
Sector Academic/University 
PI Contribution I led the write up of the grant proposal HAZEL, submitted under the ZELS call, with contributions from my research team incl. Dr Margaret Davis at WSU. Prof Douglas Call, also at WSU, led a NSF-BBSRC project on AMR in Northern Tanzania and Klebsiella isolates or sequences from that project will be included in data analysis under SpARK.
Collaborator Contribution Dr. Margaret Davis contributed to the writing of the grant proposal HAZEL; that was submitted under the ZELS call. Dr Davis contributes intellectual input into project activities and attends annual in-person meetings and monthly skype calls. Conference Presentations: G. Prinsen, J. Benschop, N. French, E. Swai, R. Zadoks, J. Sharp, L. Waldman. Food Safety Awareness and Practices in Urban and Rural Butcheries and Eateries in Northern Tanzania. 34th Tanzania Veterinary Association Conference, 6-8 December 2016, Arusha, Tanzania. Audience: veterinarians, policy makers, PhD students, researchers. G. Prinsen, J. Benschop, N. French, E. Swai, R. Zadoks, J. Sharp, L. Waldman. Food Safety Awareness and Practices in Urban and Rural Butcheries and Eateries in Northern Tanzania. 9th Biennial Conference New Zealand International Development Studies Network, 5-7 December 2016, Wellington, New Zealand. Audience: researchers, development experts. R. Zadoks, K. Thomas, G. Barker, J. Benschop, J. Buza, S. Cleaveland, M. Davis, N. French, E. Karimuribo, R. Kazwala, B. Mmbaga, N. Mtui, G. Prinsen, J. Sharp, E. Swai, L. Waldman, J. Crump. Of Meat and Men, Models and Microbes - Understanding Salmonella risks in Tanzania's Meat Supply Chain. . 34th Tanzania Veterinary Association Conference, 6-8 December 2016, Arusha, Tanzania. Audience: veterinarians, policy makers, PhD students, researchers. Since then, the collaboration has been extended to include Professor Douglas Call, who has shared a collection of ~600 AMR resistant E. coli isolates with us to inform writing of a follow-on grant proposal. He is also contributing material to the Klebsiella-focussed SpARK project, funded by MRC through a JPI-AMR grant.
Impact (1) Successful application for funding of the HAZEL project. This is a multi-disciplinary collaboration involving medics, veterinarians, quantitative scientists, social scientists, and molecular epidemiologists. (2) Submission of a grant application to MRC. This is a multi-disciplinary collaboration involving representatives of the following disciplines: anthropology, bioinformatics, health economics, epidemiology, human geography, mathematics, medicine, microbiology, pharmacology, sociology, veterinary medicine
Start Year 2012
 
Description Outreach event for local schools 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact In my capacity as Head of Outreach for the Milner Centre for Evolution, I organised a major outreach activity in September 2018. 150 local school children (year 4-6) were invited from local schools to take part in a range of activities, including the use of a lego DNA "sequencer", and understanding AMR and bacteria.
Year(s) Of Engagement Activity 2018
URL https://www.youtube.com/watch?v=TkwIiB72itY&feature=youtu.be
 
Description Public Lecture (Norwich) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact I gave an invited lecture at the Quadram Institute in Norwich on 12 Feb 2020 as part of the "Darwin Day" symposium. The lecture was on bacterial adaptation and included some discussion of the research output form SpARK and other ongoing projects.
Year(s) Of Engagement Activity 2020
 
Description open symposium 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Open symposium on project progress at the University of Pavia
Year(s) Of Engagement Activity 2017