08 November 2016

Escherichia coli (E. coli) are diverse bacteria, with pathogenic and non-pathogenic strains. The rod-shaped bacterium is a major cause of diarrheal diseases, peritonitis, colitis, urinary tract infections and bacteraemia, costing billions of dollars to treat annually and claiming approximately 2 million lives 1. A BBC News article recently highlighted E. coli as the major cause of antibiotic-resistant infections, overtaking the more publicised MRSA and clostridium 2.

Pathogenic strains of E. coli can be categorized based on bacterial surface characteristics, pathogenicity mechanisms, clinical symptoms or virulence factors. The most notorious of these is E. coli O157: H7, an enterohemorrhagic strain which causes 73,000 illnesses, 2,200 hospitalizations, and 60 deaths annually, attributing to $405 million dollars of healthcare costs in the United States alone 3.

With the health risks of intestinal-borne bacteria, various methods have been developed for their diagnosis. As mentioned in our previous blogs (MRSA , Pneumoniae), culture-based methods for assay of E. coli are time-consuming and are not amenable to real-time bacterial detection (Figure 1).

timeline-1

Figure 1. Timeline for reporting cases of E.coli O157 infection 4.

Immunoassays and PCR-based assays require specialized instrumentation and can be laborious. Other methods employ an enrichment step of bacterial culturing to specifically promote E. coli growth, usually taking 2 to 3 days to yield results. E. coli detection in the clinic therefore remains suboptimal and there’s an urgent need for faster, more sensitive detection methods.

Aptamers targeting E. coli

Various studies have demonstrated the selection of DNA aptamers against E. coli isolates including intestinal K88 and urinary tract NSM59, and a fecal E. coli isolate 5. Similarly, aptamers against other variants, such as the laboratory strain DH5α or O157:H7, have also been reported as having high affinity and specificity, with potential uses in clinical diagnosis and therapeutics 4. Marton et al. have successfully isolated four single stranded DNA aptamers that bind strongly to E. coli cells (ATCC generic strain 25922), with Kd values in the nanomolar range. More importantly, tests revealed one aptamer to be highly specific to meningitis/sepsis associated E. coli (MNEC) clinical isolates; the first aptamer described with potential for diagnosing MNEC infections 5.

Wu et al. were able to construct a biosensor based on label-free aptamers and gold nanoparticles (AuNPs) for detecting E. coli O157:H7. As illustrated in Figure 2, target bacteria-binding aptamers are adsorbed on the surface of unmodified AuNPs. Upon binding to the bacterium the aptamers are desorbed from the surface of the AuNPs. The AuNPs consequently clump together and with a salt treatment change colour from red to purple.

This approach enables detection of as little as 105 CFU ml-1 of bacteria within 20 minutes and is absolutely specific to the target 5. The detection limit is significantly lower than or comparable to, the variety of antibody-based biosensors currently available for E. coli. The most important aspect of this aptasensor is direct detection of the whole bacterium without specialized instrumentation. A further advantage of the AuNP biosensor is easy modification for detection of other species by incorporating different aptamers 6.

Figure 2 principle of target bacteria detection using aptamer and AuNP (6)

Figure 2 principle of target bacteria detection using aptamer and AuNP 6.

Poor diagnosis of bacterial infection has obvious implications in patient welfare but also in combatting antibiotic resistant bacteria. Rapid identification of E. coli enables equally rapid development of a treatment strategy, including the type of antibiotic to be used and appropriate dosing. The result would be improved patient outcomes, reduced healthcare cost and tighter control over nosocomial infection causing pathogens.

To learn more about the Aptamer Group’s Diagnostic arm, please click the following link Aptadx.