Date of Award

2016

Document Type

Doctoral Thesis

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Abstract

The work presented in this thesis focuses on clinically significant staphylococci (with an emphasis on coagulase-negative staphylococci) in terms of their isolation, identification, characterisation, and biocontrol.

CoNS are increasingly being recognised as true nosocomial pathogens yet remain under-characterised, particularly from an Irish clinical perspective. We examined a large cohort of clinically-derived CoNS, isolated from blood (n=l35), identifying them to species level. Extensive antibiotic resistance against nine clinically-relevant antibiotics was recorded. Additionally, widespread biofilm forming capabilities associated with increased virulence was detected both phenotypically and genotypically. We identified multiple species of CoNS in circulation in an Irish healthcare setting, and determined valuable insight into their virulence potential. Consequently, a need for their rapid identification to species level upon isolation from blood should be supported. Furthermore, MALDl-TOF MS was compared against 16S rRNA typing, and proposed as a cost- and time-efficient alternative to conventional phenotypic clinical-based tests for the identification of Staphylococcus epidermidis (the most frequently isolated and virulent of the CoNS group).

From an Irish perspective, we reported the earliest incidence of meticillin resistant S. epidermidis clinical isolates to demonstrate both mutational and transmissible mechanisms of linezolid resistance. This characterisation has provided significant epidemiological insight into this rare yet emerging resistance to an important anti-staphylococcal antibiotic. Furthermore, a novel HRM-PCR assay was designed to detect S. epidermidis isolates resistant to linezolid. We delivered a faster more reliable means of detecting the G2576T point mutation (most frequently encountered mechanism of linezolid resistance). This work can be used to more rapidly detect and perhaps prevent further dissemination of linezolid resistance in clinical settings.

We carried out a study, the first of its kind in Ireland, which reported the superior ability of culture from sonicate fluid combined with MALDI-TOF MS to detect periprosthetic joint infection (PJl), compared with conventional methods in

current clinical practise. Notably, two hospitals are looking into changing laboratory protocol based on our findings. We reported that 16S rRNA screening of sonicate fluid from resected hip and knee prostheses allowed the identification of “true” aseptic failure as opposed to clinically-undetected PJI, which if implemented for routine in diagnostic laboratories could circumvent the unnecessary administration of broad spectrum antibiotics. We found the application of 16S-cloning and sequencing from sonicate to further enhance the detection rate of bacteria from the resected prosthetic joints. Additionally, the microbial diversity associated with failed hip and knee prostheses was revealed, another first from an Irish perspective.

Finally, a bacteriophage (phage) demonstrating excellent staphylolytic activity was isolated and purified. The results here are significant as phage B.l proved highly lytic against S. aureus PJI isolates and meticillin-resistant S. aureus (MRSA) strains from the National MRSA reference bank (2008), all of which expressed resistance to multiple antibiotics. While preliminary, we have exhibited the potential for phage as an adjunct or alternative to antibiotics for the treatment of infections such as PJIs, quelling the spread of multidrug resistance.

Access Level

info:eu-repo/semantics/openAccess

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