Development of multiplex real-time PCR assays for the detection and identification of ESKAPE pathogens and their closely related species complexes: Toward reducing the occurrence of healthcare-associated infection using enhanced surveillance of clinical and environmental samples
Mc Andrew, Grainne
Mc Andrew, Grainne
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Publication Date
2023-08-18
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Thesis
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Abstract
Microorganisms have the ability cause a wide range of infections in humans - from superficial skin infections to invasive pneumonia and sepsis. Advancements in medical technology, as well as an expanding global population, mean that increasing numbers of people receive life-saving treatment and interventions each year. However, the increased use of invasive medical procedures and antimicrobial treatments can result in an increased risk of patient infection. Infections which occur during or after contact with healthcare facilities are known as healthcare-associated infections (HAI). HAI are a significant cause of morbidity and mortality worldwide. A small number of bacterial species cause the majority of HAI: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter. These species have been grouped together under the acronym ESKAPE due to their ability to “eskape” conventional treatments. They frequently cause severe, multidrug-resistant infections in patients who are already seriously ill, immunocompromised, or undergoing invasive treatment such as mechanical ventilation. Relative to other pathogenic bacteria, the ESKAPE pathogens are particularly adept at survival in healthcare settings. In addition to displaying high levels of antimicrobial resistance, the ESKAPEs have been shown to persist on inanimate surfaces in the hospital environment. Currently, routine diagnostics for ESKAPE HAI and for hospital environmental surveillance are culture-based. These methods lack speed, sensitivity, and accuracy. This study describes the development of a series of real-time multiplex PCR assays for specific identification of each ESKAPE, as well as methodology for environmental sample collection. This enables the rapid and sensitive identification of pathogens from clinical and environmental samples from healthcare settings. In addition to identifying the primary causes of HAI - the ESKAPE pathogens - this study includes methods to detect the often-misdiagnosed species complexes which are closely related to the ESKAPEs. Namely, the species contained in the A. baumannii group (A. baumannii, Acinetobacter pittii, Acinetobacter nosocomialis, Acinetobacter seifertii and Acinetobacter lactucae) and the K. pneumoniae species complex (K. pneumoniae, Klebsiella quasipneumoniae, Klebsiella variicola, Klebsiella quasivariicola, and Klebsiella africana). A. baumannii and K. pneumoniae are considered critically important human pathogens. However, they exist within closely related, and poorly understood species complexes. Currently, there are no rapid real time PCR diagnostics to accurately identify these species. Existing data pertaining to the clinical relevance of these species is limited to a small number of studies which have identified isolates to the species level using sequence-based techniques. These data have shown that A. baumannii group and K. pneumoniae complex members have inherently different antimicrobial resistance profiles which affects disease prognosis and patient outcomes. As such, the aim of this study was to create novel real-time PCR diagnostics which would provide rapid and accurate information on the occurrence of ESKAPE pathogens with improved species identification capabilities.
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NUI Galway