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
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Date
2023-08-18Embargo Date
2025-08-17
Author
Mc Andrew, Grainne
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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.