A novel molecule developed by chemically linking (conjugate) an amino acid (glycine) to a polymer has been found to possess high antibacterial activity against multidrug-resistant Acinetobacter baumannii while showing no toxicity whatsoever to human cells. The antibacterial activity was also seen against clinical isolates of A. baumannii that are multidrug-resistant.
The team led by Jayanta Haldar from the Antimicrobial Research Laboratory at the Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bengaluru, found that the molecule has several other salient properties. The most important being its ability to kill even the dormant bacteria.
At 16 microgram per ml concentration, the molecule took about two hours to completely kill the actively dividing bacteria that are sensitive to drugs and two-four hours to kill drug-resistant strains of A. baumannii . However, at the same concentration, the molecule needed less than two minutes to kill the dormant, drug-sensitive bacteria, and about five minutes to kill the dormant, drug-resistant bacteria. “At this point in time we don’t know the reason behind the rapid killing of the dormant bacteria,” says Prof. Haldar.
Disrupting biofilm
Since A. baumannii bacteria display great propensity to form biofilm, the researchers tested the ability of the molecule to disrupt biofilm. They found that the molecule could disrupt the biofilm formed by both drug-sensitive and drug-resistant A. baumannii. “At 64 microgram per ml concentration, the efficacy of the molecule to disrupt the biofilm was comparable with the last-resort antibiotic colistin,” says Prof. Haldar.
“One dose of the molecule was able to achieve 65% biofilm disruption when the concentration used was 64 microgram per ml. If we use the molecule repeatedly then it can completely disrupt the biofilm,” says Swagatam Barman from JNCASR, the first author of a paper published in the journal ACS Applied Materials & Interfaces.
The toxicity of the molecule was tested using human embryonic kidney cell line. Not a single cell was killed when 8-16 microgram per ml concentration was used. Even when the concentration was increased to 500 microgram per ml, about 80% cells were still not killed. “The molecule did not show any toxicity at the concentration needed to kill both drug-sensitive and drug-resistant bacteria,” Barman says.
The researchers also tested if the molecule killed red blood cells. Even at 1,000 microgram per ml concentration, which is several times the concentration needed to kill the bacteria, only about 1-3% of red blood cells were destroyed.
Killing mechanism
A. baumannii bacteria are already resistant to most antibiotics. So to evaluate how quickly the bacteria would develop resistance against the synthesised molecule, the researchers exposed the bacteria to the molecule for 14 days. “The bacteria did not develop any resistance against the molecule at the end of 14 days, while the bacteria exhibited 250-fold resistance against meropenem drug,” says Prof. Haldar. “The bacteria developed a high level of resistance against the last resort antibiotic colistin too.”
The molecule is able to destroy the integrity of the membranes thus killing even the multidrug-resistant A. baumannii, Prof. Haldar says. Gram-negative bacteria have an outer and an inner membrane thus making it difficult for drugs to effectively kill them unlike in the case of Gram-positive bacteria that have only one membrane.
The molecule was found to destroy the integrity of the outer and inner membrane and permeate through the membranes even at a low concentration of 5 microgram per ml. The permeability increased when higher concentration of 20 microgram per ml was used.
“We will soon be studying the efficacy of the molecule in animal models,” Prof. Haldar says. “Based on the in vitro studies, we feel this molecule has immense potential for being developed as a future therapeutic agent.”
