Researchers at The University of Nottingham and the Massachusetts Institute of Technology have built a high throughput screening platform to identify polymers that are resistant to bacterial colonization.1 The technique has yielded a new class of compounds that could prevent biofilm formation on the surface of medical devices.

Biofilms are slow-growing, hardy, drug-resistant bacterial conglomerates that can form on the surface of catheters and other surgical implants. The films can lead to local inflammation and serve as reservoirs of bacteria that cause local or systemic infection.

Venous catheters are especially prone to biofilms, and about 1% of patients implanted with i.v. catheters develop systemic infections with high risk of mortality.

"The current of standard care is to give preoperative i.v. antibiotics just prior to surgery and any implantation," said David Grainger, professor of bioengineering and professor and chair of pharmaceutics and pharmaceutical chemistry at The University of Utah.

If an infection arises, Grainger said patients "receive a six-week course of antibiotics and then typically require removal and replacement of the implant." He added that these patients run high risks of reinfection upon reimplantation because of changes in the local tissue environment at the implant site that promote biofilm growth.

Patients with urinary catheters also have a high rate of infection, although such infections typically are not life threatening and can be treated with oral antibiotics.

Regardless, Morgan Alexander, professor of biomedical surfaces and head of the Division of Biophysics and Surface Analysis at the University of Nottingham and lead author of the manuscript, would like to forestall the need for antibiotics. The goal, he said, is "to prevent the attachment of bacteria, which is distinct from most strategies that attempt to kill bacteria with antibiotics."

Alexander reasoned that if catheters were coated with a bacteria-resistant polymer, the bugs would be unable to form a hardy biofilm and would instead be destroyed by the host's immune system.

"If you don't form a biofilm in the first place, it may be easier to deal with bacteria near the surface of the implant without resorting to antibiotics," he said.

Biofilm club

The challenge was to find a suitable polymer. Prior efforts by industry and by Alexander's collaborators at MIT focused on bactericidal metal coatings and protein-blocking zwitterionic materials, respectively. As an alternative, Alexander focused on hydrophobic materials that prevent microbial attachment by repelling water from coated surfaces.

The group created a library of 576 acrylate-based polymers and spotted them onto a chemically activated microscope slide to create a microarray of covalently attached polymers.

The team then soaked these microarrays in cultures of three different bacterial pathogens-Pseudomonas aeruginosa, Staphylococcus aureus and pathogenic Escherichia coli-that were labeled with GFP.

When visualized under a fluorescent microscope, spots with lower than average fluorescence indicated the polymer coating on those spots made it difficult for bacteria to take root. The compounds coating the least luminous spots were isolated, modified with further polymerization and then rescreened for even greater prevention of bacterial growth.

With a set of lead polymers from this recursive microarray-based screen in hand, the team assessed the molecules in an in vitro assay of urinary catheter biofilm formation. Catheters coated with the most promising polymers had 30-fold fewer bacterial colonies growing on them than a silver-coated catheter, which is the standard of care.

The team saw similar results in a mouse model of urinary tract infection. Mice implanted with a polymer-coated catheter and challenged with a high dose of S. aureus had lower bacterial titers at the catheter implantation site, surrounding tissue and distal organs than animals implanted with an uncoated control.

Results were reported in Nature Biotechnology. The findings are patented and are available for licensing.

Ex catheter declaration

Christopher Loose, cofounder and CTO of Semprus BioSciences Corp., said that Alexander's materials add to a growing menu of potentially useful device coatings.

"This is a new class of materials that couldn't have been predicted without this screening technique," said Loose.

Semprus is developing zwitterionic surface modification materials for implantable devices. The company was cofounded by Robert Langer, professor of chemical and biomedical engineering at MIT, who is a coauthor of Alexander's report.

In July, Semprus was acquired by medical device maker Telefelx Inc.

Loose said Semprus' zwitterionic surface modifications work by a different principle than the hydrophobic materials described in the paper. Instead of excluding water from the surface, Semprus' coatings attract water but block protein attachment to the device surface.

The company's coated peripherally inserted central catheter (PICC) has received European CE Mark approval and is under review in the U.S.

Alexander said his lead compounds-a family of polymers with methacrylate or cyclic and aromatic acrylate groups-could be suitable for clinical development, but the polymers will need further testing in the context of real-world implantable devices.

For example, he noted that visualizing systemic bacterial infection in the mouse catheterization assay required "injecting a large dose of bacteria," more than would be found in a real infection, so further dose-ranging studies will be needed.

Grainger wanted to know whether Alexander's coating could prevent a modest number of bacteria from taking hold and proliferating over the course of several weeks because the coating reduced but did not completely eliminate bacterial growth.

"They use an immense amount of bacteria in their in vivo assay," said Grainger. "While they reduced the bacterial burden after four days, there's still an enormous burden remaining. They might get this initial knockdown, but it's possible that by day 10 or day 20, the infection comes back."

Another question is whether the hydrophobic coatings would be suitable for use on something besides urinary catheters. Grainger said venous catheters are prone to causing bacterial infections and to accumulation of thrombi. The ideal coating would be resistant to both bacteria and blood clots.

Alexander said his team is now testing whether the coatings are resistant to encrustation by urinary minerals, another common cause of catheter blockage and tissue inflammation.

Another concern is whether the most promising coatings can be applied consistently over the surface of the device.

"This study focuses on creating new types of coatings, but the next stage is to see how this behaves when you put it onto top-performing devices" such as market-leading venous and urinary catheters, said Loose. "In real devices, you have multiple types of materials and process steps that make it difficult to come up with surface modifications."

Alexander's team is performing manufacturing and toxicology studies to determine how best to apply the new materials.

Grainger and Loose both said the screening platform could be further exploited to identify other biofilm-resistant compound classes and to probe the mechanism of bacterial attachment and pathogenesis at device surfaces.

"This study will pique further interest in studies about how these surfaces resist biofilm formation," said Grainger. "Adhesion doesn't necessarily lead to infection, and the mechanism of this virulent transition is a black box. This technique could allow us to correlate material biophysical properties of a material with its propensity for infection."

Osherovich, L. SciBX 5(33); doi:10.1038/scibx.2012.860
Published online Aug. 23, 2012

REFERENCES

1.   Hook, A.L. et al. Nat. Biotechnol.; published online Aug. 12, 2012; doi:10.1038/nbt.2316
Contact: Morgan R. Alexander, The University of Nottingham, Nottingham, U.K.
e-mail: morgan.alexander@nottingham.ac.uk

COMPANIES AND INSTITUTIONS MENTIONED

      Massachusetts Institute of Technology, Cambridge, Mass.

      Semprus BioSciences Corp., Cambridge, Mass.

      Teleflex Inc. (NYSE:TFX), Limerick, Pa

      The University of Nottingham, Nottingham, U.K.

      The University of Utah, Salt Lake City, Utah