Jeffrey S. Buguliskis Ph.D. Technical Editor Genetic Engineering & Biotechnology News
A Unique Approach toward Antibiotic Susceptibility Testing Could Have Significant Benefits for Patients
In 1928, a Scottish pharmacologist, with some untidy workshop etiquette, had returned to his London laboratory and noticed some mold growing on the bacterial plates he had left in the sink before he embarked on a two-week holiday. Thankfully for much of humankind, Alexander Fleming was an observant scientist, if not the neatest, and noticed that the mold growth inhibited the spread of the Staphylococcus strain that he was studying—penicillin was subsequently born and the world was vaulted into a new pharmaceutical drug age.
Although we have enjoyed decades of being able to easily treat communicable diseases that were besieging the population at the beginning of the 20th century, we now face a rapidly growing threat that could possibly leave us without a single drug to combat many of the most common microbial diseases. Antibiotic resistance is on the rise, and it’s creating strains of superbugs that are easily circumventing some of the last-line drugs that clinicians employ.
Obviously, addressing the overuse of antibiotics in both the clinical setting and within the food supply is essential, but additional measures also need to be taken to rapidly identify those infected with resistant microbial strains. With newly emerging resistance strains, such as methicillin-resistant Staphylococcus aureus (MRSA) and extensively drug-resistant tuberculosis (XDR-TB), which can disseminate rapidly and cause life-threating illness often measured in hours, the time to diagnosis is imperative. To address these issues, many companies over the past several years have been in the process of developing rapid diagnostic tools for determining the antibiotic susceptibility of various bacteria.
Antibiotic Susceptibility Testing
Traditionally, antibiotic susceptibility testing (AST) has been performed using two methods that have been taught in microbiology laboratories since the 1950s. The first technique, often called the disc-diffusion or Kirby-Bauer method, is performed using small discs that are impregnated with different antibiotics that are placed onto agar plates where bacteria are growing. A clear ring, or zone of inhibition, becomes visible if the antibiotic is effective at preventing the growth of the bacterial strain.
The second method, called agar or broth dilution, is often used to determine the minimum inhibitory concentration (MIC) for the antibiotic being tested. This technique utilizes serial dilution measures to find the lowest concentration at which a particular antibiotic will prevent the growth of the bacterial strain being tested. Both techniques are extremely reliable, accurate, and represent the gold standard for AST testing. However, they have two main drawbacks: their lack of speed and ability to be scaled up for high-throughput assays.
There are a variety of new approaches for expediting AST testing, from multiplexed PCR-based assays designed to amplify various resistance biomarkers, to mass spectrometry techniques like MALDI-TOF for rapid identification of unknown organisms. Another unique approach toward AST uses real-time imaging techniques and analysis software to distinguish live resistant cells from the milieu of microbes typically contained within clinical microbiology samples.
Time-Lapse Imaging Microscopy
“We were looking to understand how to use time-lapse imaging microscopy to better appreciate how bacteria behave in the presence of antibiotics for resistance and susceptibility profiling,” says Joen Johansen, head of marketing at Accelerate Diagnostics. “We ended up developing a fully automated platform for doing quantitative identification and antibiotic susceptibility testing right from clinical samples such as positive blood cultures, respiratory, and other critical microbiological samples where you need a fast identification and susceptibility result.”
The foundation of the technology Johansen says relies first on the accurate identification of the organisms in a sample through the use of fluorescent in situ hybridization (FISH) and subsequently using the molecular identification to guide which antibiotic to test in a susceptibility study—providing specific results that are relevant for the identified microbes. The second stage of Accelerate Diagnostics’ technology employs time-lapse imaging of the bacteria as they are growing in the presence of antibiotics and quantifying the various parameters such as rate of growth, cell shape, and fluorescent intensity from various dyes for cell viability in order to generate an algorithm for determining MIC.
So far, the Accelerate ID/AST System has proven its worth, as the company recently announced positive findings from a multicenter pilot study to evaluate the external performance of the ID/AST System and Blood Culture Assay Kit. Consequently, based on the results of the recent study, plans to initiate a clinical trial for submission to the FDA were announced.
“We believe the results of the study are promising and support the potential of the system and kit in a clinical setting,” said Beth Lingenfelter, head of clinical and scientific affairs at Accelerate Diagnostics. “Across 273 samples, the overall sensitivity and specificity for identification were 96.6% and 99.4% respectively; while essential agreement (EA) and categorical agreement (CA) for AST averaged 94.3% and 91.3% across all drugs.”
Lawrence Mehren, president and CEO of Accelerate Diagnostics added, “We are encouraged by the results of the pilot study. While small, the pilot, combined with the extensive external preclinical work we have done, provides additional confidence in our investigational system, our first investigational test kit, and our trial intended to support FDA clearance.”
If the clinical trial proves successful, the landscape surrounding antibiotic resistance could change dramatically as clinicians will not only be able to rapidly identify seriously ill patients with resistant microbes, it may be possible to recognize sooner when the efficacy of specific antibiotics are beginning to wane. This would allow the medical community to cycle antibiotic use and prevent the selection of resistant populations of bacteria, which at the very least could slow the exponential rise of drug-resistance phenotypes.