Date of Award

2013

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Dr Deirdre Fitzgerald-Hughes

Second Supervisor

Professor Hilary Humphreys

Third Supervisor

Dr Marc Devocelle

Funder/Sponsor

Health Research Board in Ireland

Keywords

Bacterial Infections, prevention and control, microbiology

Abstract

Herein we studied the epidemiology of two species of ESBL-producing Enterobacteriaceae causing clinical infections and investigated the potential of a novel host defence peptide conjugate as a therapeutic agent against these infections. We first characterised 100 ESBL-producing E. coli (ESBL-EC) collected over two years that caused infections in patients of Beaumont Hospital and in community patients attending general practitioners or residing in long-term care facilities (LTCFs) in North Dublin. Using molecular typing techniques, we found that this cohort of isolates was both clonal and sporadic in nature and that the pandemic ST131 clone was dominant in community and hospital patients and comprised 54% of isolates overall. We found UK epidemic strain A, which belongs to this clone and is the most frequently identified strain in the United Kingdom, was responsible for 34% of infections locally. LTCFs were identified as a reservoir of ST131 and we provided evidence for the spread of this clone via the hospitalisation of LTCF residents.

Detailed molecular characterisation of the ESBL-EC collection was then carried out to identify the specific ESBL genes present and the mechanisms involved in their mobilisation, expression and dissemination in North Dublin. As previously reported nationwide, CTX-M genes of groups 1 or 9 were the most common. Almost half were associated with the ISEcpl insertion sequence, which facilitated high-level expression of the blact x -m gene by the ISEcpl promoter. Presence of the insertion sequence IS26 within ISEcpl was characteristic of UK strain A isolates and was associated with reduced expression of blact x - m - Class 1 integrons, genetic platforms for the expression of mobile resistance genes, were present in two thirds of ESBL-EC, contained genes for resistance to trimethoprim, aminoglycosides and sulphonamides and had a reservoir in local livestock and companion animals. Four isolates contained complex class one integrons, bearing CTX-M-9 or CTX-M-2 alleles downstream of the ISCR1 insertion sequence, a feature of these elements. ESBL plasmids were transferred in 33% of isolates and the most prevalent incompatibility types were varied multireplicon IncF plasmids and Incll plasmids. These plasmids were similar to those previously identified in E. coli of human and animal origin throughout Western Europe in recent years. However, some new combinations of plasmid types and CTX-M genes were also noted. The spread of CTX-M-1 was associated with Incll and IncN plasmids commonly associated with food animal strains. Our data supports significant roles for both horizontal spread of CTX-M via conjugative IncF, Incll and IncN plasmids and vertical spread of CTX-M via clonal expansion of ST131 in North Dublin.

Non-typhoidal Salmonella enterica (NTS) resistant to third generation cephalosporins are an emerging problem because they are frequently MDR and therefore therapeutic options to treat infections with these bacteria are limited. The mechanisms of cefotaxime resistance in 121 cefotaxime-resistant NTS collected over two years throughout England and Wales were examined. We found that the recent increase in cefotaxime resistance was mediated predominantly by group 1 CTX-M enzymes and AmpC enzymes of the CMY variety. Commonly identified ESBL genes matched those detected amongst food-producing animals and human clinical isolates in Europe. A high rate of multidrug-resistance was identified and almost half of NTS were co-resistant to ciprofloxacin, a favoured therapeutic option. Our data support travelassociated spread of resistant strains to the UK from locally endemic areas and local acquisition of strains producing food-animal associated ESBL and AmpC genes.

Finally, we evaluated a novel hybrid therapeutic agent, Bac8cP, which is composed of a synthetically-optimised host defence peptide that is conjugated to the cephalosporin antibiotic cephalothin. This molecule was designed as a prodrug to achieve targeted activation at the site of bacterial infection, where interaction with p-lactamase or penicillin-binding proteins releases the cephalothin promolety, thus activating the host defence peptide. The conjugate had good in vitro activity against ESBL-producing Enterobacteriaceae and also against MDR Gram-positives. However, further investigation of its mechanism of action against isogenic derivatives of E. coli BL21, which differed in their expression of CTX-M-15, showed that the hybrid molecule also had residual activity in its conjugated form. Nonetheless, this information is crucial to the optimisation of future host defence peptide conjugates of this nature, which may be developed as broad-spectrum antimicrobials for parenteral administration against MDR bacteria.

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.

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Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2013.

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