A General Method for Rapid Determination of Antibiotic Susceptibility and Species in Bacterial Infections

Abstract

To ensure correct antibiotic treatment and reduce the unnecessary use of antibiotics, there is an urgent need for new rapid methods for species identification and determination of antibiotic susceptibility in infectious pathogenic bacteria. We have developed a general method for the rapid identification of the bacterial species causing an infection and the determination of their antibiotic susceptibility profiles. An initial short cultivation step in the absence and presence of different antibiotics was combined with sensitive species-specific padlock probe detection of the bacterial target DNA to allow a determination of growth (i.e., resistance) and no growth (i.e., susceptibility). A proof-of-concept was established for urinary tract infections in which we applied the method to determine the antibiotic susceptibility profiles of Escherichia coli for two drugs with 100% accuracy in 3.5 h. The short assay time from sample to readout enables fast appropriate treatment with effective drugs and minimizes the need to prescribe broad-spectrum antibiotics due to unknown resistance profiles of the treated infection. Overprescription and extensive use of antibiotics have selected for resistant bacteria at an alarmingly rapid rate, and we are now facing one of the greatest medical challenges of our time (1). Today, both the diagnosis of bacterial infections and determination of antibiotic susceptibility profiles (ASP) are slow and tedious processes. As a consequence, a patient might be given an antibiotic that has no effect on infections caused by resistant bacteria. Thus, there is a considerable need for new techniques enabling quick and specific diagnosis along with characterization of an ASP in order to guide correct treatment, reduce the use of broad-spectrum antibiotics, and slow the development of resistance.In the last few decades, we have seen an amazing development of novel molecular methods to detect bacterial pathogens and their resistance genes and resistance mutations (2-5). These new hybridization/PCR-based methods are generally faster and more sensitive than are the classical phenotypic methods, but they also suffer from serious drawbacks that have often reduced their general use. An intrinsic limitation of all genotypic methods that identify resistance mutations or genes is that they detect only the potential for resistance (i.e., presence of a resistance gene/mutation), whereas phenotypic methods detect the realization of susceptibility (i.e., no growth in the presence of antibiotic). For a clinician, the realization of susceptibility measure is far more relevant as a basis for a therapeutic decision.Padlock probes are oligonucleotides with target-specific ends, which upon perfect target recognition can be enzymatically joined (6). Reacted probes can be amplified by rolling circle amplification (RCA). RCA is a linear amplification technique for the replication of DNA circles, such as reacted padlock probes, and the product is a single-stranded DNA concatemer containing around 1,000 copies of a 100-mer t...

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