IIT Delhi researchers have used microgels to encapsulate bacteria of interest and carbon dots. Carbon dots indicate bacterial growth through a change in emission ratio in response to a change in the pH. The platform requires less time and lower concentration of bacteria for culturing, and can be used for carrying out drug susceptibility testing within a short time.
A two-member team from the Indian Institute of Technology (IIT) Delhi has used a novel method to culture bacteria and determine its growth at much lower concentration in relatively less time — four-six hours. E. coli and S. aureus bacteria were studied. Currently available clinical methods require more than 10 hours to culture and observe growth of pathogenic bacteria and a higher bacterial concentration for laboratory confirmation.
While the new method will not be useful in identifying the species of bacteria isolated from a patient sample, it will help in early detection of the presence of bacteria and carrying out drug susceptibility testing within a short time period.
The team led by Dr. Neetu Singh from the Centre for Biomedical Engineering at IIT Delhi prepared alginate microgels that encapsulate E. coli bacteria and carbon dots. The microgel was prepared using sodium alginate solution to which the bacteria and carbon dots were added. Micron-sized droplets of the solution were produced using static electricity and dropped into a solution of calcium chloride for crosslinking.
The carbon dots used are pH sensitive. They emit light of two different wavelengths (450 nm and 550 nm) but the intensity of only one wavelength (550 nm) changes in response to a change in the pH. Measuring the ratio of the intensity of emission of light at two different wavelengths helps in detecting any change in the pH.
Change in pH
The microgels were found to support bacterial growth and colony formation, and the pH changes in response to bacterial growth. Generally, the pH becomes acidic when bacteria grow and multiply. But in some cases, the pH could become alkaline (basic) too. “The change in the pH, either acidic or basic, in response to bacterial growth can be detected by the change in the emission ratio of the two wavelengths,” says Dr. Singh.
Unlike conventional methods that require 105 CFU (colony forming units) and typically use 107 CFU for detecting bacterial growth and take about 10 hours, the microgels needed only 104 CFU. “A change in the pH was seen in about four-six hours when 104 CFU were used and about eight hours when 103 CFU were used,” says Anil Chandra from the Centre for Biomedical Engineering at IIT Delhi and first author of a paper published in the journal Chemical Communications.
The researchers found smaller the droplet size the faster was the change in the pH. “The hypothesis is that a small number of bacteria encapsulated in smaller microgel can efficiently change the pH of a small volume in less time compared to a bulk culture,” says Chandra.
Testing antibiotic sensitivity
The team used the platform to test for antibiotic sensitivity by treating E. coli with ampicillin drug of different concentrations (0 to 400 microgram per millilitre). The amount of ampicillin required to achieve IC50 value (concentration of compound needed to reduce the growth of bacteria by 50% of the maximum possible growth rate) was very close to the value reported in the literature. “The microgel is porous and drugs could easily diffuse through the microgel,” says Dr. Singh.
While the drug-sensitive E. coli showed less growth and produced only 5% change in fluorescence emission when ampicillin was added, the drug-resistant bacteria exhibited as high 35% change in emission. “This suggests that the bacteria were growing even in the presence of ampicillin and hence were drug-resistant,” says Chandra.
“Our platform will help in simultaneously studying resistance to different drugs, combination of drugs and resistance to different concentrations of drugs,” says Dr. Singh. “But it cannot be used when the drug has a buffering effect or when it is acidic, as the drug itself will change the pH of the microgel.” The team will be collaborating to develop a portable device with multiple functionalities and be testing clinical samples soon.