The spread of antibiotic resistant Enterobactericeae in hospital sink drain traps

Lead Research Organisation: University of Surrey
Department Name: Microbial & Cellular Sciences


This studentship focuses on the spread of antibiotic resistant Enterobactericeae in hospital sink drain traps. Following initial genetic and phenotypic characterisation, real-life sink drain trap microbial communities will be exposed to various cleaning regimes and the survival and transmission of resistance elements and populations determined over time.


10 25 50
Description Microbiology Society conference grant
Amount £240 (GBP)
Organisation Microbiology Society 
Sector Learned Society
Country United Kingdom
Start 03/2020 
End 04/2020
Title Use of MinION for DNA barcoding and sequencing of bacterial isolates in house 
Description One of the objectives to our project is to assess the genetic basis of CPE isolated from the sink environment. To provide comprehensive reads from CPE isolates, a combination of both long and short read sequencing has been performed. Short reads were generated from NOVEGENE wereby DNA was sequneiced usinf the Novaseq 6000 (illumina). Long read sequences were performed in house using the MinION (Oxford Nanopore). DNA was extracted from isolates using a commersial kit and were subject to DNA repair, ligation and barcoding of individual samples, clean-up of prepared DNA before sequencing of library prepped DNA in efforts to use long read sequencing reads to fill in the gaps of DNA reads from illumina sequences during the assesbly step of raw sequence data. 
Type Of Material Biological samples 
Year Produced 2021 
Provided To Others? Yes  
Impact Hybrid sequencing permits genome assembly from both supplementing short, accurate second-generation sequencing data Illumina ) with long less accurate third generation sequencing data (i.e. from PacBio RS) to resolve complex repeated DNA segments. Sequentianlly, generating comprhensive genome assemblies for publication and submission into NCBI database. 
Title 16S rRNA data processing of enviromental isolates 
Description DNA was extracted from hospital sink environmental samples between 2018 and 2019, these DNA extracts underwent 16S rRNA sequencing. This was to target the aim of the project which was to investigate the composition, diversity and stability of bacterial communities associated with hospital sink waste traps. Following sequencing which was outsourced, raw datasets were provided and I have devised an operation pipeline in processing raw sequence data for quality control, error correction and taxonomic profiling of cleaned datasets to a reference database (SILVA database was selected for the purposes of our research). This pipeline used Linux operating system and the Qiime2 bioinformatics pipeline. I have also learn 'R' which is a language and environment for statistical computing and graphics. This is for the standardisation of cleaned OTUs (using iNEXT and Vegan packages in R) and created visualization plots of cleaned OTU reads including bar plots of abundance data, NMDS and ordination plots, stressplots to test ordination dimensions, boxplots to show diversity data. 
Type Of Material Data analysis technique 
Year Produced 2018 
Provided To Others? No  
Impact The impact of the resulting datasets will be by examining the sink-trap microbiome at a metagenetic level, we can characterise this environment generally thus commenting on commonly abundant organisms which may be implicated in HAI outbreaks. This information may contribute to our understanding of the spread and transfer of organisms from the sink community into the nosocomial environment. This dataset is intended to be published. 
Title WGS and plasmid sequencing workflow for assembly and gene annotation of genomes 
Description Selected CPE isolates underwent WGS using short read sequencing (Illumina) and long read sequencing (Nanopore).The aim of the project was to assess the CPE populations and their genetic content from both chromosomal DNA and extracellular DNA in the form of plasmids. I have developed a pipeline for the assembly and annotation of WGS from exclusively illumine reads and will be working on a pipeline that assembles and annotates whole genomes from both long read sequencing input and short read sequencing input. This pipeline uses as linux operating system and the following bioinformatics platforms: SPAdes, Prokka, Abricate and Ugene. 
Type Of Material Data analysis technique 
Year Produced 2019 
Provided To Others? No  
Impact WGS will be submitted onto NCBI database, publication will also follow characterising CPE populations amongst the sink community. 
Title Working with big datasets 
Description Bioinformatics research is characterized by voluminous datasets and complex data analytics methods. The university has a research IT department that specialises in handling of big datasets, thus we have collaborated with this department. Thus, I have learnt to use big data tools perform computation in batch mode. This optimises not only memory usage but increases the efficiency of my work since I am able to access multiple virtual computers and perform multiple commands simultaneously. 
Type Of Material Data handling & control 
Year Produced 2021 
Provided To Others? Yes  
Impact We have collaborated with the research IT department at the University of Surrey and have set up a pipeline and infrastructure to perform high throughout bioinformatics and computational work should any other member within the Ritchie research group wish to use. 
Description Sample collection and microbial profiling of hospital hand-washing sink waste traps (January-February 2018 and June-August 2019) 
Organisation Public Health England
Country United Kingdom 
Sector Public 
PI Contribution The contributions I have made thus far to the project include learning basic microbiological manipulation techniques for the isolation of specific resistant communities Carbapenemase-Producing Enterobacteriaceae (CPE) from a complex environmental reservoir that is the sink environment. This includes safe sample collection, selective culturing of bacteria and enumeration of isolated organisms of interest. The method for isolation has been optimised following weekly sampling collection performed by the research team (based at Porton Down) and I. Following the isolation of CPE, I have performed phenotypic assays for the basic characterisation of CPE positive isolated including the generation of resistance profiles, growth rates through growth curves and biofilm formation potential through biofilm assay under standard microbial growth conditions and selective pressures including changes in calcium carbonate concentration to simulate soft and hard water conditions. I am devising code to operate the QIIME2 pipeline so 16S can be cleaned and processed independently by myself generate complex statistical graphs displaying relative diversity within the sink environment. This is following the extraction of DNA from environmental samples (from waste water and biofilm samples) for downstream analysis performed by myself. I have also performed genotypic characterisation of CPE positive isolates for basic profiling including carbapenamase and ESBL typing. Expertise from the research group include changing the direction of the project, to hypothesise potential resistance transfer mechanisms which may promote the spread and transfer of mobile genetic elements harbouring resistance genes such as KPC. Furthermore, through discussion with my principle supervisor (Dr Jennifer Ritchie) whom specialises in antimicrobial resistance, it was agreed to focus on the KPC allele. Also, through Dr Jennifer Ritchie's expertise in AMR and plasmids, it was made clear to plasmid characterise CPE isolates for which KPC, an example of carbapenemase are typically encoded on. Through characterisation of plasmids, it is possible to establish common plasmids in the sink environment which could suggest resistance trasnfer mechanisms such as conjugation. Since conjugation could be a potential mechanism of resistance transfer, it was suggested to perform conjugation on wildtype CPE positive sink isolates in vitro to determine conjugation potential. This is ongoing. I have also been trained by Dr Emma Laing whom specialises in computational bioinformatics and am in training for bioinformatics analysis. Her input influenced decisions including where to send samples for sequencing (Novagene) and the appropriate parameters needed for optimal 16S amplicon sequencing (such as which hyper variable region to target (V3-V4) and the number of tags to use) as well as making the sequencing pipeline understandable for the entire research group.
Collaborator Contribution The collaboration was fundemental to ensuring the correct permissions were made before the collection of samples from the hospital environment, also the replacement of missing traps were immediately tended to by the TRACE groups connection with external plumbers. PHE provided expertise, training and specialised laboratory equipment for the enumeration, isolation and identification of CPE organisms such as CARB agar for selection and MALDI-TOF for quick identification of isolated colonies. It was only through the generation of the model sink system by the TRACE group that we could continue to simulate a real life environment for the samples. The model sink system includes a set of 12 sinks in the lab that are subjected to daily automatic flushing regiments and temperature controlled tap operation to best replicate the hospital setting, the water hardness can also be changed which was a parameter changed .
Impact Observation that reduced water hardness may be implicated in enhanced biofilm formation in the sink microbiota including resistant communities (CPE) Revisited traps (2018 vs 2019) identify re-colonisation of CPE organisms Awareness of the process of environmental sampling to identify high risk areas in a clinical setting (Hospital microbiological laboratory) and in a reference laboratory (PHE) Trained in MALDI TOF (can use independently)
Start Year 2017
Description Attendance and participation of NERC Metagenomics 3-Day Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact I attended a 3-day bioinformatics course is presented by the Centre for Genomic Research and funded by the Natural Environment Research Council (NERC). This was based at the university of Liverpool. It primarily focused on practical computer sessions designed to provide delegates with hands-on experience of the informatics involved in analysing metagenomic and metagenetic datasets.16S rRNA gene profiling (metagenetics) and shotgun sequence based metagenomics are now well established and can be utilised to characterise complex populations. This was a completely new area of research for myself but an important skill to learn since I have and will continue to process such data sets. It is a now a skill expected of those who explore environmental micriobiome to independently process 16S amplicon data and I wanted to indenpendently process 16S data to add as a skill for later research and work opportunities The course provided help in conceptual and practical issues involved in analysing such datasets, generated from the Illumina NGS platforms, and comprises the following sessions:

• Sequence data QC and preparation for downstream analyses
• Metagenomic shotgun sequence analysis.
• Taxonomic profiling (MetaPhlAn)
• Metagenome assembly (MEGAHIT) and annotation (Prokka)
• Functional annotation (Cognizer)
• Data visualisation
• Analysis of 16S rRNA data with QIIME
• Laboratory tour of sequencing laboratory
• Metagenome functional prediction using metagenetic data
Year(s) Of Engagement Activity 2019
Description Presentation of research locally to fellow collaguaes at the University of Surrey 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact The university hosts a departmental seminar which gives researchers including PhD students a platform to presenting their research projects. The concept behind these talks are to promote current research within the university environment and receive constructive feedback from people of multiple disciplines such as immunology, virology, bacteriology and biochemistry. It was also an opportunity to showcase my entire progress thus far in the PhD in front of my entire supervisory team who commended my hard work, input and effort into the research. The main outcome of the presentation was increased awareness of my current project and promoting the awareness for exploring environmental microbiomes, highlighting the sink as a source for antimicrobial resistant organisms which harbour resistance genes. I was questioned how I would explore the potential mechanisms for the spread and transfer of resistance to which I responded, "through plasmid characterisation and assessment of plasmid diversity." Should identical plasmids be carried amongst different microbial communities in the sink environment, this could suggest horizontal gene transfer. It was also to showcase my effort in learning to process (and continue to process) raw 16s amplicon sequence data and highlight the importance in maintaining a good support network in the university environment for bioinformatics work since this field is increasing rapidly in the microbial research field.
Year(s) Of Engagement Activity 2019,2020
Description Public Health England Phd student event 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Public Health England (PHE) host a PhD student day where student are able to present posters or deliver a talk about their research. I was successful in submitting an abstract for a poster presentation. The event takes place at the PHE site in Collindale, Oxfordshire and hosts more than 50 students who too are affiliated with PHE. Research areas are diverse and range from the use of e-cigarettes to the relationship between unemployment and drinking motivations. Students from multiple disciplines and from across the UK were able to network, this was the main outcome I achieved from this event . This allowed feedback from multiple disciplines which broadened my perspective of which direction I wish to take future research, whether that be focused on practical approaches such as transmission routes from the sink to the patient or mechanistic approaches such as investigation into the dissemination of resistance genes.
The event was also a place to meet colleagues from PHE and learn more about research groups that investigate water microbiology, I have built contacts within PHE from this event.
Year(s) Of Engagement Activity 2019