Medical Professional Publications

Researchers Find Key Factors Driving Development of Protective Structures in Biofilms

(From the February 2018 issue of Research Now)

Findings are a step toward developing therapies for hard-to-treat ear infections

Extracellular bacterial DNA (eDNA) and type IV pilus expression appear to regulate the biochemical reactions that form biofilms and the structure that protects nontypeable Haemophilus influnenzae (NTHI) from antibiotics and antibodies, teams from two laboratories at Nationwide Children's Hospital have found.

By combining computer analysis with imaging of lab-grown biofilms, the researchers built a mathematical model that revealed the roles of the eDNA and pili in creating fractal structures that comprise the biofilm.

Fractals are repeated shapes of varying scales. The structure may promote NTHI survival by increasing the surface area of the biofilm, which in turn exposes the bacteria to more nutrients, the teams say. The structure also appears to facilitate quorum signaling, which bacteria use to communicate and function together.

Biofilms formed by NTHI in the middle ear are the primary factor making otitis media infections chronic, recurrent and resistant to treatment. The research is published in MBio.

"When the three-dimensional structure forms, gene expression of the bacteria changes and as a result it's hard to eliminate it using antibiotics," says Jayajit Das, PhD, a principal investigator in the Battelle Center for Mathematical Medicine in The Research Institute at Nationwide Children’s and lead author of the study. "We want to know the underlying mechanisms and characteristics of the structure in order to find targets and strategies for therapies."

“The model adds data to our contention that extracellular DNA and type IV pilus are essential to biofilm production and important to the way the bacterium causes disease,” says Lauren Bakaletz, PhD, a microbiologist and director of the Center for Microbial Pathogenesis in The Research Institute.

Dr. Das and members of his lab first discussed the idea of this research with Dr. Bakaletz and members of her lab during a meeting between their research centers about three years ago.

“These are two disparate disciplines, but an ideal collaboration,” Dr. Bakaletz says. “Biophysicists and mathematicians working with vaccine developers to make development of a vaccine easier.”

The teams built and validated the model going back and forth between the labs, using images of biofilms grown from healthy bacteria and biofilms made with mutant bacteria to develop the math that quantifies the process.

The investigators predicted and confirmed that bacteria unable to release extracellular DNA and that have a poorly functioning type IV pilus generated a biofilm with substantially fewer fractal patterns and a smoother surface.

“The biofilm is a very complicated system created by multiple bacterial cells,” Dr. Das says. “They disperse throughout the fractal surfaces they create, which appears to help them survive in the hostile environment of the middle ear.”

Drs. Das and Bakaletz are now seeking a grant to add more information to the mathematical model by testing it against biofilms formed in the middle ear of animal models. The ultimate goal is to improve the model so they can use it in a predictive way, to guide them in vaccine development.

Among the answers they seek is whether the structure that exposes bacteria to scarce nutrients in the ear may also protect them against an antibiotic, Dr. Das says. Nutrients and antimicrobial proteins come in different sizes, and the gateways in the film that allow the transport of nutrients may be too small to transport the antimicrobials.

Citation: Das J, Mokrzan E, Lakhani V, Rosas L, Jurcisek JA, Ray WC, Bakaletz, LO. Extracellular DNA and Type VI Expression Regulate the Structure and Kinetics of Biofilm Formation by Nontypeable Haemophilus influnenzae. MBio 2017 Dec 19;8(6). Pii; e01466-17.


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