by Michael Schmidt
Every now and then you read a paper and think to yourself, this might transform patient care. Such was the case when I read a paper suggested to me by one of our faithful TWiM (This Week in Microbiology) listeners, Dr. Volkan Ozenci of the Karolinska University Hospital in Sweden. The paper, Antibiotic susceptibility testing in less than 30 minutes using direct single-cell imaging, appeared in a recent issue of PNAS. The authors take us on a remarkable journey, whereby in the not too distant future it may be possible to assess within 30 minutes the antibiotic susceptibility of microbes responsible for the over 100 million urinary tract infections (UTI) across our globe each year. Their healthcare provider will be able to prescribe a suitable drug almost as fast as the patient can get dressed.
Resistance of microbes to broad-spectrum antibiotics represents a clear and present danger to human health. In order to mitigate this danger a strategy of "the right drug for the right bug for the right time" represents one of the best approaches to limit the emergence of antibiotic resistance. Yet, increased antibiotic resistance is a fact of modern medicine. It has grown at an alarming rate since the wide spread introduction of these miracle drugs in the 1950s through indiscriminate prescribing, incorrect use and, an absence of new antibiotics in the pipeline.
Today, the time required to assess antimicrobial susceptibility often requires greater than 48 hours from the time of initial specimen collection. There are two principal methods to measure susceptibility, Kirby-Bauer (disk diffusion) or broth dilution. Both methods require a pure culture to determine susceptibility to the antibiotic. Each method is dependent on the principle that when susceptible microbes are exposed to antibiotics, their growth will slow or stop. If the microbe is resistant to the compound they will grow, generally at the same rate or a bit slower, should resistance to the antibiotic impose a fitness penalty on growth of the microbe. The method described in the paper here is no different and relies on the same principle of assessing susceptibility but greatly compresses the time required to reach a decision. How the authors accomplished time compression is given away in their title. Rather than measuring growth susceptibility of a population, they measure the growth of single cells exposed to the antibiotic. Critical to their strategy is monitoring the growth of up to 4,000 single cells. Then, susceptibility profiles could be assessed by statistically comparing the differences between growth rates of individual microbes when they were exposed or not exposed to the antibiotic.
As you can imagine monitoring the growth rate of 4,000 individual bacteria by routine techniques is not trivial and would likely not result in time compression, at least not in my lab. However, the innovation they applied was accomplished by the use of a microfluidic chip of a unique design, where the chip has two rows to trap bacteria, each row having 2,000 cell traps. Each trap measures 1.25 x 1.25 x 50 µm, which is of sufficient size to capture one bacterial cell. Each cell trap was engineered with a constriction or bottleneck to prevent the bacteria from passing to the back channel, while allowing medium to flow around the cells (Figure 1). Their device is similar in design to the Mother Machine pioneered by Jun's lab at the University of California, San Diego. In order to assess susceptibility, they only needed to monitor how quickly individual bacteria in the traps divided, thus progressed down the constricted channel. They monitored all of this through phase-contrast microscopy, where measuring elongation or division of individual cells in their respective traps, was captured through automated computer control enabling an algorithm to calculate the speed with which each cell was dividing. In one row they provided nutrient medium without the antibiotic and the other they provided the same medium with the drug (Figure 2). Then, based on how fast the individual cells divided, it was possible to quickly assess sensitivity or resistance with a sufficient degree of confidence. The entire assessment lasts only 30 minutes, with an image being captured automatically each 30 or 60 seconds. They named their system 'Fast detection of Response to Antibiotic Treatment', or fASTest.
You can witness for yourself the elegant simplicity of their system in action by viewing a series of time-lapse movies where you will marvel how they evaluated the susceptibility of E. coli (MG1655) to ciprofloxacin in the Growth Condition Mother Machine (2GMM) chip. In this remarkable 11-second movie, 120 phase contrast images were captured using their automated system at 30-second intervals. Even a cursory comparison of the top frame of cells against the bottom will convince you that ciprofloxacin (1µg/ml) does indeed significantly slow the growth rate of the E. coli. If you were wondering will this work beyond E. coli, they provide two other movies using an 100x objective, offering you a magnified view of how easily it is able to visualize growth of individual cells using another Gram negative rod, Klebsiella pneumoniae (Movie S2) or a Gram positive coccus, Staphylococcus saprophyticus (Movie S3).
The key to time compression for the fASTest assay is in how quickly individual cell traps of the modified mother cell chip can be loaded. Even when the concentration of bacteria in patient samples was low, ~104 cfu/ml, it was possible to collect 100 bacteria within 5 minutes. While this concentration of bacteria is considered to be in the lower range considered significant for clinically relevant UTIs, it was still possible to assess sensitivity of the microbe to an antibiotic. Even at concentrations as low as 10 cfu/µl, pre-cultivation of the patient urine samples was not required, enabling assessment of susceptibility in as few as 100 cells.
Upon taking the system to clinic, they learned that when blindly screening 50 clinical isolates of uropathogenic E. coli (UPEC), fASTest was as effective as the gold standard disk diffusion test for ciprofloxacin recommended by European Committee on Antimicrobial Susceptibility Testing and practiced by their clinical microbiology laboratory. All of the 24 resistant strains and 25 of the susceptible were correctly assessed by fASTest in less than 30 minutes.
While fASTest worked for urine containing as few as 100 cells, further testing is required to demonstrate its ability to be used for sepsis, mastitis, or meningitis. Yet, this system offers hope in our battle against antimicrobial resistance. Hopefully, in the not too distance future, healthcare providers will be able to offer patients the right drug, for the right bug, before they leave the exam room for the pharmacy.