NEW YORK (GenomeWeb) – Researchers in Sweden have created an antibiotic susceptibility test that combines very short culture times with padlock probes detecting bacterial ribosomal RNA, and demonstrated detection of antibiotic-resistant bacteria in urine samples from urinary tract infection patients in less than four hours.
The investigators published their research in the Journal of Clinical Microbiology in November, and are developing the method in tandem with a more extensive pathogen profiling diagnostic platform from Swedish biotech start-up Q-linea.
The classical method of antibiotic susceptibility testing looks at cultured bacterial colonies. It typically takes one to two days to get enough cells to see a colony, explained Dan Andersson, a professor of microbiology at Uppsala University and one of the authors of the JCM study.
The new method is also essentially a phenotypic test to see if the bacteria grow with or without antibiotics, Andersson said.
However, because the subsequent step is a molecular test, "we only have to follow growth for perhaps a couple of hours," he said.
In addition, Andersson noted that the method is "completely general," and could be used on any pathogen or antibiotic combination. "You really don't have to know anything about resistance mechanisms," he said.
Other commercially available phenotypic tests only measure growth, while molecular tests are specific for particular known resistance genes that may or may not actually confer antibiotic resistance in a given circumstance. Combining phenotype and identification assays, however, also has the potential advantage of distinguishing whether there are many different types of bacteria in a single sample and pinpointing which are resistant to antibiotics and which are not.
Andersson said he'd been thinking for a long time about how to do faster antibiotic susceptibility testing. Meanwhile, Mats Nilsson, the other senior author on the study, had been developing a sensitive detection system using padlock probes, a well-established molecular technology first described by Nilsson in a Science paper twenty years ago.
In the new assay, padlock probes are designed to detect a species-specific 16S rRNA region, and reacted probes are amplified by rolling circle amplification, or RCA, with rolling circle products detected as blobs of fluorescence under the microscope.
In the proof-of-principle study, Andersson and his colleague evaluated 88 urine samples from patients with diagnosed urinary tract infections for the presence of Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis. They measured growth in the presence of two antibiotics, ciprofloxacin and trimethoprim, and also assayed samples spiked with bacteria. All samples were cultured for two hours, lysed in boiling hydroxide, and subjected to padlock probe-mediated RCA.
Although E. coli causes about 80 percent of UTIs, Andersson noted, "it would be very easy to multiplex" the assay further to detect many bacteria since the padlock probes have such high specificity. This could be important, as a recent study in Infection Control and Hospital Epidemiology showed both a rising number of UTIs requiring hospitalization in the US as well as shifting patterns in the antibiotic resistant bacteria causing them.
The padlock-probe assay could also be applied to other types of samples and infections, such as respiratory or wound infections, Andersson said. These are particularly vulnerable to P. aeruginosa, which behaves synergistically in coinfections, and some studies suggest differential expression of metabolic genes, rather than genotype, may differentiate acute infections from chronic ones.
Andersson said he envisions building the UTI test into a kit or panel, detecting the most common pathogens first, with additional panels for more rare isolates. This may then be commercialized via an ongoing collaboration between researchers at Uppsala University and Q-linea.
"It's basically going to be automated to the point that all the technician has to do is take the sample and put it in a tube," Andersson said.
Molecular vs. phenotypic testing
Molecular testing requires knowledge of the genes that confer resistance in a particular organism, and designing probes specific for that sequence. Cepheid's GeneXpert MTB/RIF assay, for example, tells clinicians whether a patient is infected with a strain of TB that has a gene conferring resistance to rifampicin. Other groups are now attempting to characterize additional resistance genes in TB and develop array-based molecular diagnostics for multi-drug resistant infections.
For blood stream infections, or sepsis, diagnostic speed is especially critical. Culture-based tests can take one to two days. Methods such as Abbott's Iridica can find the cause of sepsis in about six hours after positive blood culture. However, "If you can find out within two or three hours what these bacteria are susceptible to, that would be an enormous advantage," Anderson said.
In an email to GenomeWeb, Q-linea CEO Jonus Jarvius said that the company's first product will indeed be focused on diagnosing sepsis, with a UTI product coming later. The sepsis test will also use a phenotypic assay of antibiotic susceptibility coupled with padlock probes for identification of bacteria, Jarvius confirmed. He added that he expects the product to launch in 2017.
A recent review in the European Journal of Microbiology and Immunology describes different molecular diagnostic tests for sepsis that use either positive blood cultures or whole blood. Roche's SeptiFast, for example, tests for 25 pathogens using PCR of whole blood, and has been subject to over 60 clinical evaluations, according to the review. Other blood pathogen tests in development or currently on the market include T2 Biosystems' T2Candida panel, Nanosphere's Gram-Positive Blood Culture Test, and Cepheid's Xpert MRSA/SA BC and BioFire's FilmArray Blood Culture Identification Panel. Analytik Jena acquired a PCR- and array-based sepsis assay called Vyoo last year, and Anagnostics and Mobidiag are also developing sepsis arrays.
For Q-linea's test, the absolute limit on the speed of combined phenotypic and molecular method will hinge on how fast the bacteria in question divide, Andersson explained. "You have to have two to three cell divisions," he said, in order to get a readout using padlock probes. Fast-growing bacteria take about 20 minutes to divide, "so in principle you could get down to 40 minutes," he said. TB bacteria has a generation time of 24 hours, however, so would require two days of growth. This would still be much faster than the two-week culture time currently required for thorough resistance testing of TB, he said.
And if a particular resistance phenotype could be caused by ten different genetic mechanisms, it would require ten molecular tests, Andersson said, adding "that's why I think, in the long run, the phenotypic tests are going to win over the molecular tests … because they give the most relevant information for the physician … whether the strain is susceptible or not." Andersson's lab is now working on adapting its combined assay to develop a TB susceptibility test, with preliminary results showing they can get a profile in three to four days, he said.
Along with the sepsis diagnostic, Q-linea is also using padlock probes to develop biosecurity assays, and recently tested its biothreat detection system, Aquila, in the subway system in Prague.