Abstract
Pseudomonas aeruginosa is an opportunistic pathogen that causes significant morbidity and mortality in immunocompromised patients, particular cystic fibrosis sufferers, burns victims, diabetics and neonates. It thrives in moist places where it forms biofilms that are exceedingly difficult to eradicate on hospital surfaces, in water supplies and implanted biomaterials. Using a live cell SELEX approach we selected DNA aptamers to P. aeruginosa grown as biofilms in microfluidic cells. From a pool of aptamer candidates showing tight binding a stem-loop structure was identified as being important for binding. Enhanced binding and increased specificity was achieved by truncating structures and generating chimeric aptamers from the pool of top candidates. The top candidates have low nanomolar binding constants and high discrimination for P. aeruginosa over other Gram-negative bacteria. The aptamers bind both planktonic grown and biofilm grown cells. They do not have intrinsic bacteriostatic or bactericidal activity, but are ideal candidates for modification for use as aptamer-drug conjugates and in biosensors.
Highlights
Pseudomonas aeruginosa is a clinically important, opportunistic, gram-negative bacterium that is responsible for a wide variety of severe hospital-acquired infections
In this study we describe the selection and characterisation of DNA aptamers selected to live biofilm derived P. aeruginosa cells that bind with a high affinity and specificity to P. aeruginosa but not to a selection of other gram-negative bacteria
The bacterial strain used for SELEX selection was P. aeruginosa 692 (PA692) (ATCC 14502) obtained from New Zealand Culture Collection (Porirua, NZ)
Summary
Pseudomonas aeruginosa is a clinically important, opportunistic, gram-negative bacterium that is responsible for a wide variety of severe hospital-acquired infections. It largely effects immune-compromised patients, those with cystic fibrosis, as well as causing infections in burn wounds, and forming biofilms on implanted devices such as urinary catheters and heart stents [1]. Resistance is further accentuated by the formation of biofilms composed of a matrix of excreted exopolysaccharides, proteins and DNA that helps protect the bacteria inside from antibiotic action [3]. Bacteria within the biofilm exhibit adaptive changes in gene expression and a shift to a metabolically less active state making the infections harder to clear, which in part accounts for the recalcitrance of many P. aeruginosa infections to antibiotic therapy. Aptamers are selected using a SELEX (Systematic Evolution of Ligands by Exponential Enrichment) methodology [4], that can be modified to select aptamers
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