LIMR Clinical Gastroenterology: Investigation of Fidaxomicin Resistance Mechanisms in Clostridium difficile

Clostridium difficile is a leading pathogen in healthcare-associated diarrheal infections. C. difficile has a plastic genome with multiple mobile genetic elements and recognized capacity to acquire genes involved in resistance and virulence. Antibiotic treatment is a major risk factor for C. difficile infection (CDI), with resistance to multiple antibiotics becoming frequent in newly emergent strains. CDI antibiotic treatment is currently limited to vancomycin and fidaxomicin, with the later showing high CDI resolution rates and reduced risk of recurrent infection1. Fidaxomicin is the first new antimicrobial agent licensed for use in CDI treatment for 30 years. This antibiotic can persist in the faeces for ~3 weeks following the cessation of dosing, and does not necessarily lead to eradication of C. difficile spores in patients. Furthermore, a recent novel extended dosage of fidaxomicin has been trialled, which involves a 25 day administration period (as opposed to the current standard 10 day regimen). Thus, there is a potential for C. difficile exposure to the antibiotic for ~9 weeks, increasing the risk of resistance emergence. We need to understand why resistance may develop in clinical C. difficile strains and what the potential implications are for clinical treatment of the disease.

<p paraeid="{b4e1bbac-b927-40d0-9cf8-ba49febf3fb3}{44}" paraid="355252497">Clinical strains with increased minimum inhibitory concentration (MIC) consistent with in vitro resistance or reduced susceptibility to fidaxomicin are emerging. In a pan-European surveillance study1, we identified one isolate showing and MIC of >4 mg/L to fidaxomicin, with one further isolate showing an MIC of 0.5 mg/L. Laboratory selection assays have implicated the RNA polymerase gene rpoB, and a transcriptional regulator gene marR, in fidaxomicin resistance in vitro2. However, it is unknown whether the same mechanisms occur in vivo, and their clinical implications in the treatment of CDI remain unclear. This project will investigate fidaxomicin resistance in clinical C. difficile isolates, assessing the stability of fidaxomicin resistance using MIC assays, and will use whole-genome sequencing to identify potential gene mutations associated with the high MIC phenotype. The student will also create mutants in fidaxomicin susceptible C. difficile strains for the genes of interest rpoB, marR and others possibly identified by the whole-genome sequencing analysis, and study the resulting phenotype. Mutations in multidrug efflux transporters can confer increased tolerance to macrocyclic antibiotics in other microorganisms. By over-expressing these systems in fidaxomicin susceptible strains, we will be able to infer other mechanisms or stress responses that may occur in C. difficile down the line. The fitness cost of these mutations regarding toxin production, sporulation and competitive growth will also be investigated.  </p> <p paraeid="{1d77f8e1-c9f8-4f35-a904-fa2187f08c2b}{43}" paraid="1373313647">In order to understand the clinical implications of fidaxomicin resistance, the student will use a clinically reflective chemostat model of the human colon, developed by the HCAI group3. This model accurately represents the human gut microbiota and has been used in multiple studies resulting in >30 publications, helping define the UK prescribing regulations for CDI. Fidaxomicin resistant C. difficile strains will be inoculated in this model to investigate their potential to cause CDI in presence of drug concentrations equivalent to those found in the human colon. </p> <p paraeid="{1d77f8e1-c9f8-4f35-a904-fa2187f08c2b}{43}" paraid="1373313647">Techniques associated with project</p> <p paraeid="{1d77f8e1-c9f8-4f35-a904-fa2187f08c2b}{43}" paraid="1373313647">Bacterial culture and microbial identification, MIC determination, whole-genome sequencing analysis, genetic modification of class II microorganisms, assembly and use of a chemostat model. </p> <p paraeid="{1d77f8e1-c9f8-4f35-a904-fa2187f08c2b}{43}" paraid="1373313647">References</p> <p paraeid="{1d77f8e1-c9f8-4f35-a904-fa2187f08c2b}{161}" paraid="451320423">1. Freeman J, Vernon J, Pilling S, Clark E, Nicholson S, Morris K, Fabrega AP, Wilcox MH. 2018. Pan-European Longitudinal Surveillance of Antibiotic Resistance among Prevalent Clostridium difficile Ribotypes Study Group. The ClosER study: results from a three-year pan-European longitudinal surveillance of antibiotic resistance among prevalent Clostridium difficile ribotypes, 2011-2014. Clin Microbiol Infect 24:724–31.  </p> <p paraeid="{1d77f8e1-c9f8-4f35-a904-fa2187f08c2b}{209}" paraid="1234391083">2. Leeds JA, Sachdeva M, Mullin S, Barnes SW, Ruzin A. 2013.?In vitro selection, via serial passage, of?Clostridium difficile?mutants with reduced susceptibility to fidaxomicin or vancomycin.?J Antimicrob Chemother?69:41–44. </p> <p paraeid="{f922a7c7-c502-40a4-96e4-d9a5db6534b0}{2}" paraid="319939329">3. Chilton CH, Crowther GS, Freeman J, Todhunter SL, Nicholson S, Longshaw CM, Wilcox MH. 2014. Successful treatment of simulated Clostridium difficile infection in a human gut model by fidaxomicin first line and after vancomycin or metronidazole failure. J Antimicrob Chemother. 69:451–62.  </p> <p paraeid="{f922a7c7-c502-40a4-96e4-d9a5db6534b0}{2}" paraid="319939329">This project is part of the <a href="https://medicinehealth.leeds.ac.uk/leeds-institute-research-st-james/doc/international-phd-academy-medical-research">International PhD Academy: Medical Research</a></p> <p paraeid="{f922a7c7-c502-40a4-96e4-d9a5db6534b0}{2}" paraid="319939329"><strong>In line with the bespoke nature of our International PhD Academy a modified PhD project can be proposed dependent on students interests and background.</strong></p>

<p>Please note these are not standalone projects and applicants must apply to the PhD academy directly.</p> <p>Applications can be made at any time. You should complete an <a href="https://medicinehealth.leeds.ac.uk/faculty-graduate-school/doc/apply-2">online application form</a> and attach the following documentation to support your application. </p> <ul> <li>a full academic CV</li> <li>degree certificate and transcripts of marks (or marks so far if still studying)</li> <li>Evidence that you meet the programme’s minimum English language requirements (if applicable, see requirement below)</li> <li>Evidence of funding to support your studies</li> </ul> <p>To help us identify that you are applying for this project please ensure you provide the following information on your application form;</p> <ul> <li>Select PhD in Medicine, Health & Human Disease as your planned programme of study</li> <li>Give the full project title and name the supervisors listed in this advert</li> </ul>

A degree in biological sciences, dentistry, medicine, midwifery, nursing, psychology or a good honours degree in a subject relevant to the research topic. A Masters degree in a relevant subject may also be required in some areas of the Faculty. For entry requirements for all other research degrees we offer, please contact us.

Applicants whose first language is not English must provide evidence that their English language is sufficient to meet the specific demands of their study. The minimum requirements for this programme in IELTS and TOEFL tests are: • British Council IELTS - score of 7.0 overall, with no element less than 6.5 • TOEFL iBT - overall score of 100 with the listening and reading element no less than 22, writing element no less than 23 and the speaking element no less than 24.

<p>Informal enquires about regarding the bespoke taught first year of the PhD programme and research projects can be made by contacting LIMRPhD@leeds.ac.uk.</p> <p>Enquiries regarding the application process should be directed to the Faculty of Medicine and Health Graduate School e: <a href="mailto:fmhpgradmissions@leeds.ac.uk">fmhpgradmissions@leeds.ac.uk</a></p>