Date of Award

2011

Document Type

Doctoral Thesis

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Dr. Lesley Cotter

Second Advisor

Dr. Brigid Lucey

Third Advisor

Dr. Aidan Coffey

Abstract

Clinical microbiology has been revolutionised in recent years by the introduction of molecular biology. Traditionally, the gold standard for diagnosis in the clinical microbiology laboratory has been culture, which in some cases, is gradually being replaced by molecular detection methods, particularly where the pathogen is difficult to grow or to identify, or when the significance of an organism is dependent on the detection of specific markers of pathogenicity, such as toxins. Many fastidious bacteria which are difficult to culture and present public health implications, i.e. Mycobacterium tuberculosis. Chlamydia trachomatis and Neisseria gonorrhoea, can now be detected using commercial molecular assays which have the advantage of avoiding the delays in diagnosis of days or weeks. Molecular techniques should not take the place of conventional methods but rather address laboratory testing problems that cannot be approached or would be more labour intensive, expensive, and/or time consuming to address by these conventional techniques.

Molecular advances in microbiology have also played a key role in gathering important epidemiological data through extensive characterisation of pathogens using various typing and subtyping systems. The most widely used molecular typing methods are the DNA-based methods, such as plasmid profiling, restriction endonuclease analysis of plasmid and genomic DNA, Southern hybridization analysis using specific DNA probes, and chromosomal DNA profiling using either pulsed-field gel electrophoresis or polymerase chain reaction-based methods.

In terms of epidemiologic surveillance, molecular typing methods may be used to monitor geographic spread and prevalence shifts of epidemic and endemic clones. The possible applications of molecular techniques that could be utilised in laboratory testing and in epidemiologic surveillance are numerous and collectively have the potential to change the course of clinical medicine dramatically in the future.

This study focused on three selected Gram-positive pathogens, namely Methicillin resistant Staphylococcus aureus (MRSA), Lancefield’s Group B Streptococcus (GBS) and Clostridium difficile.

The archetypal hospital "superbug", MRS A, frequently attracts media interest and is a serious pathogen in both the nosocomial and community setting. Its impact on public health over the past five decades, since it was first recognised in 1961, has been immense and its emergence in the community further heightened its significance. The impact of MRSA is considerable; in Ireland approximately 40-50% of isolates of 5. aureus recovered from bloodstream infections have recently been methicillin resistant, and this is significantly higher than in some European countries such as the Netherlands and the Scandinavian countries. MRSA strains show a high degree of clonality, and molecular epidemiological testing enables these clones to be determined and differentiated. The dynamic nature of MRSA necessitates constant research and surveillance to be carried out on this pathogen. With this in mind, two individual studies were designed to investigate two key properties of MRSA; the first study examined the prevalence of CA-MRSA in the south of Ireland followed by extensive characterisation of these strains using molecular techniques. The second study investigated alternative treatment options for S. aureus and MRSA by investigating a panel of chemical derivative compounds for antistaphylococcal activity.

GBS is not generally regarded as a “superbug”; however, it has emerged as a major pathogen causing a variety of bacterial infections among neonates, pregnant women, non-pregnant adults, and the elderly. In neonatal infections, GBS is the leading cause of bacterial sepsis, pneumonia and meningitis, showing a mortality rate of 4-6% in Europe and the United States. Universal screening of antenatal mothers for GBS has been an area of contention worldwide, resulting in diverse policies for screening. Also, molecular investigation of GBS has been limited and as a result there are many unanswered questions about this bacterium. This lack of knowledge certified the need for an epidemiological study to be carried out to determine the prevalence and serotype distribution of GBS in the south of Ireland.

C. difficile has become a fully-fledged member of the group of microorganisms portrayed to the general public as “superbugs” and this organism has been responsible for outbreaks of hospital-acquired infections that show high morbidity and mortality rates. Although C. difficile infection (GDI) is historically considered to be a healthcare-associated infection; recent reports have highlighted its increasing prevalence in the community. The economic burden of this nosocomial pathogen for hospitals has proven to be costly; studies have reported the median cost per case between $9179 and $11,456. C. difficile has become a conundrum for healthcare professionals in the past decade not only in relation to cost but also due to the inadequacy of current detection methods (EIA toxin testing), and the need for alternative antimicrobial treatment options, particularly as there has been a recent increased incidence in first line antibiotic (metronidazole) treatment failure. The widespread use of EIA toxin testing for GDI detection has largely been due to issues of cost, ease of use and turnaround time. As a result, two individual studies were carried out; the first study investigated a new two step algorithm for the detection of GDI while the second study examined the benefit of including a pre enrichment step; using a C. difficile selective broth, prior to EIA toxin testing to improve GDI detection.

The use of molecular epidemiological testing of C. difficile has the potential to assist our understanding of the complexities of GDI. This is seen particularly since the emergence of hypervirulent type strains such as ribotype 027 and 078. These strains have caused severe outbreaks of GDI, associated also with a high relapse rate and increased mortality. Molecular epidemiology allows the prevalence and spread of these strains to be monitored and also allows the detection of any emerging hypervirulent strains. This in turn allows the implementation of appropriate infection control measures and optimal patient care to prevent or control C. difficile outbreaks. C. difficile is an evolving pathogen and will continue to pose a serious public health concern for years to come. As a result, it is imperative that molecular epidemiological surveillance studies are continued and expanded worldwide. In Ireland, the absence of a mandatory reporting system and national reference laboratory for C. difficile means that there is limited national data available on this pathogen. As a result, an epidemiological study was designed to achieve enhanced surveillance and molecular characterisation of C. difficile in the south of Ireland.

The overall purpose of this research was to employ microbiological and molecular techniques for the detection, characterisation and epidemiological analysis of the three pathogens outlined above to facilitate enhanced surveillance and establish important epidemiological data for these organisms in the south of Ireland.

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info:eu-repo/semantics/openAccess

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