30-minute Caltech test identifies antibiotic resistant bacteria

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BacteriaA new test developed at the California Institute of Technology identifies antibiotic-resistant bacteria in as little as 30 minutes, allowing medical professionals to better choose which antibiotics to treat an infection with.

When doctors treat patients with bacterial infections, they often skip over first-line antibiotics like methicillin or amoxicillin – drugs that bacteria are more likely to be resistant to – and go straight for stronger second-line antibiotics, like ciprofloxacin. This practice increases the chance that the treatment will be effective, but it is not ideal. That’s because the increased use of second-line antibiotics makes it more likely that bacteria also will become resistant to these stronger drugs.

“Right now, we’re overprescribing, so we’re seeing resistance much sooner than we have to for a lot of the antibiotics that we would otherwise want to preserve for more serious situations,” says Nathan Schoepp, a Caltech graduate student and co-author of the study.

The problem is that there has not been a quick and easy way for a doctor to know if their patient’s infection is resistant to particular antibiotics. To find out, the doctor would have to send a sample to a testing lab, and wait two to three days for an answer.

“Therapies are driven by guidelines developed by organisations like the World Health Organisation or the US Centres for Disease Control and Prevention without knowing what the patient actually has, because the tests are so slow,” says Rustem Ismagilov, Caltech’s Ethel Wilson Bowles and Robert Bowles professor of chemistry and chemical engineering and director of the Jacobs Institute for Molecular Engineering for Medicine. “We can change the world with a rapid test like this. We can change the way antibiotics are prescribed.”

Ismagilov, Schoepp, Caltech graduate student Travis Schlappi, who is also a co-author, and their fellow researchers aimed to develop a test that could be completed during a single visit to the doctor’s office. They focused on one of the most common types of infections in humans, urinary tract infections (UTIs), which 50% of women contract during their lifetimes. UTIs result in 8m doctor visits and 1m ER visits each year in the US alone.

The researchers’ new test works like this: A sample of urine (which may contain bacteria) collected from a patient with a UTI is divided into two parts. One part is exposed to an antibiotic for 15 minutes, while the other part incubates without antibiotics. The bacteria from each sample then are broken open (lysed) to release their cellular contents, which are run through a process that combines a detection chemistry technique called digital real-time loop-mediated isothermal amplification, or dLAMP, with a device called a SlipChip (SlipChips are a previous invention of Ismagilov and his Caltech colleagues). This combination replicates specific DNA markers so they can be imaged and individually counted as discrete fluorescent spots appearing on the chip.

The test operates on the principle that typical bacteria will replicate their DNA (in preparation for cellular division) less well in an antibiotic solution, resulting in the presence of fewer DNA markers. However, if the bacteria are resistant to the antibiotic, their DNA replication will not be hampered and the test will reveal similar numbers of DNA markers in both the treated and untreated solutions.

When used on 54 samples of urine from patients with UTIs caused by the bacteria Escherischia coli, the test results had a 95% match with those obtained using the standard two-day test, which is considered the gold standard for accuracy.

Ismagilov and Schoepp plan to begin running the test on other types of infectious bacteria to see how well it performs. They also hope to tweak the testing procedures to work with blood samples. Blood infections are more difficult to test because the bacteria are present in much lower numbers than they are in urine, but such a test could help reduce mortality from blood-borne infections, which can turn fatal if not treated quickly.

Rapid antimicrobial susceptibility testing (AST) is urgently needed for informing treatment decisions and preventing the spread of antimicrobial resistance resulting from the misuse and overuse of antibiotics. To date, no phenotypic AST exists that can be performed within a single patient visit (30 min) directly from clinical samples. We show that AST results can be obtained by using digital nucleic acid quantification to measure the phenotypic response of Escherichia coli present within clinical urine samples exposed to an antibiotic for 15 min. We performed this rapid AST using our ultrafast (~7 min) digital real-time loop-mediated isothermal amplification (dLAMP) assay [area under the curve (AUC), 0.96] and compared the results to a commercial (~2 hours) digital polymerase chain reaction assay (AUC, 0.98). The rapid dLAMP assay can be used with SlipChip microfluidic devices to determine the phenotypic antibiotic susceptibility of E. coli directly from clinical urine samples in less than 30 min. With further development for additional pathogens, antibiotics, and sample types, rapid digital AST (dAST) could enable rapid clinical decision-making, improve management of infectious diseases, and facilitate antimicrobial stewardship.

Nathan G Schoepp, Travis S Schlappi, Matthew S Curtis, Slava S Butkovich, Shelley Miller, Romney M Humphries, Rustem F Ismagilov

California Institute of Technology material
Science Translational Medicine abstract

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