Most people have encountered the black, grey, or pink stains of bacterial biofilms built up on the bathroom tiles or kitchen sink. Even with vigorous scrubbing and strong cleaning chemicals, this grime can be difficult to remove and often returns with vengeance. A new study, published in Chemical Engineering Journal, reports a novel, two-step method to effectively dismantle bacterial biofilms and prevent regrowth.
"Biofilms are everywhere, from bathrooms to food factories," said Hyunjoon Kong (M-CELS leader/EIRH/RBTE), a professor of chemical and biomolecular engineering. "Biofilms are also responsible for cross contamination between patients at hospitals, and they can be found on medical instruments like tooth or bone implants."
Bacterial infections are often thought to be caused by individual, free floating cells; in reality, bacteria form complex communities, supported by an external 3D matrix that provides structural scaffolding and defense mechanisms to protect the colony.
Compared to isolated bacterial cells, biofilms demonstrate increased resistance to antimicrobials, including antibiotics and chemical disinfectants like bleach. Often, only cells on the outer layers of the biofilm are killed by these antimicrobials, while bacteria embedded deep in the 3D matrix are protected and survive.
"In order to effectively kill bacteria in the biofilms, we have to figure out how to weaken the interaction between the cells and matrix," Kong said.
Led by first authors of the publication Yu-Heng Deng and Joohun Lee, former and current graduate students in Kong's research group respectively, the team began to investigate this by treating Pseudomonas aeruginosa biofilms with chemical disinfectants that can kill bacterial cells. The biofilms were either treated with no disinfectant, hydrogen peroxide (H2O2), or a mixture of H2O2 and peroxyacetic acid (PAA). They then counted the number of colony-forming bacteria present immediately after treatment and 24 hours later.
Their results demonstrated that most of the P. aeruginosa cells were killed by the chemical treatments, but by the next day both had regrown biofilms.
Kong said, "We thought that maybe these chemicals kill the cells but don't effectively damage the matrix. The cells that managed to stay alive during the treatment may use the matrix to help regrow the biofilm."
Using an advanced software to process immunostained microscope images, Kong's group constructed 3D renderings of the biofilms to evaluate the interactions between the bacterial cells and their surrounding matrix. Specifically, they compared the spatial distribution of the extracellular polymeric substances (EPS)—structural components of the matrix—and determined the area and mass of EPS associated with residual bacteria. Their analyses showed that while the chemicals removed more than 50 % of the entire biofilm, including both cells and EPS, a large portion of the matrix was left behind, as suspected.
"On top of this, we also found that the cells are associating this matrix more strongly. These chemicals tend to condense the matrix, which provides a better environment for the cells to survive," Kong said. "So how can we develop a method that prevents biofilm regrowth?"
The researchers took advantage of self-locomotive antibacterial microbubblers, or SLAM, previously developed by the Kong group to disrupt and displace the EPS of biofilm. When activated with H2O2, the SLAM particles generate oxygen bubbles which expand and rupture in the biofilm, leading to mechanical disruption. They hypothesized that a combined approach using SLAM particles to weaken the cell to matrix interactions followed by a mixture of H2O2 and PAA could effectively prevent regrowth.
"The SLAM particles remove more than 95 % of the matrix and cells. However, the biofilm regrows because 5 % are left, with some remaining matrix. So, the mixture of H2O2 and PAA can be applied as a final step to remove residual cells," Kong said.
24 hours after sequential treatment, there was no observable P. aeruginosa regrowth, and biofilm resurgence was further prevented for over two months. Moving forward, Kong looks to translate this new technology for real world applications. Currently, his group is working on adapting the method for disinfecting dental implants. They are also developing low-cost strategies to manufacture SLAM particles on a larger scale.
But overall, Kong believes there is still more work to be done.
