Research Summary
Research topic: Investigating the pathology behind periprosthetic knee infections by identifying the characteristics of bacterial biofilms that make them resistant to antibiotics, and developing possible treatments to improve the lives of patients
Research results: Understanding of the mechanisms that make bacteria biofilms resistant to antibiotic treatment
Patient care application of results: New options for targeting antibiotic-resistant bacteria to eradicate periprosthetic joint infections
Simplified patient care application: Better treatment options for patients who develop infections after a joint replacement
It Takes a Biofilm
OREF grant recipient characterizes antibiotic tolerance in periprosthetic infections
Jay D. Lenn
More than 600,000 total knee replacement surgeries are performed each year. Although fewer than 2 percent of the procedures result in serious complications, the most common problem—infection in the knee joint—is the most difficult to treat.1-3 The standard intervention for such periprosthetic joint infections, irrigation and debridement of the joint, has a failure rate around 50 percent.4,5
A growing body of evidence points to particular features of bacterial biofilm—a collection of bacteria that can grow on the joint surface—as the culprit in making infections difficult to treat. Bacterial biofilms are persistent despite robust antibiotic treatments. A better characterization of biofilms may explain their persistence and suggest how to crack this barrier to successful outcomes. Kenneth L. Urish, MD, PhD, associate professor of orthopaedic surgery at the University of Pittsburgh, has made this research his mission.
"Our work is essentially trying to understand from a molecular perspective why biofilm is so antibiotic tolerant,” Dr. Urish said. “Why is it so hard to get rid of? How can we look at the basic science of biofilm to come up with new ideas around how we change treatments?"
Dr. Urish's research is supported in part by a 2018 Orthopaedic Research and Education Foundation (OREF) Total Joint Replacement Research Grant in Honor of Jorge O. Galante, MD. This $300,000, three-year grant supports the work of clinician-scientists establishing their research careers. Additional support includes a 2018 OREF Mentor Clinician-Scientist Grant, a one-year $20,000 award that promotes career development and supplements the salary of an investigator who has secured mentored-research funding from the National Institutes of Health.
Bacterial neighbors—not just strength in numbers
Biofilm is not simply an accumulation of bacteria. It's more an integrated bacterial community that self-produces its own matrix and adheres to a surface, for example, the surface of an artificial knee joint. Importantly, bacteria in biofilm behave differently than planktonic bacteria—the independent organisms just floating about in their environment. These differences matter in antibiotic treatment.
Antibiotics work by attacking some feature of metabolically active bacteria, cells that are dividing and growing. Bacteria in biofilm are able to switch to an inactive state, and this dormancy may be the primary reason biofilm is tolerant of antibiotics.
Dr. Urish explained, "A good example is penicillin, which disrupts cell wall synthesis. Penicillin shows up. If bacteria are actively making cell walls, the penicillin is going to disrupt the process. The bacteria will try to keep growing, but if they can't make cell walls, they are going to die. If the bacteria are dormant—if they’re not producing cells walls—there’s nothing for the penicillin to disrupt, and it doesn't matter how much penicillin we throw at them, they won’t die."
The mechanism of switching biofilm bacteria to a dormant state is believed to be regulated by a toxin-antitoxin system in which a toxin disrupts an essential cellular process and an antitoxin prevents toxin activation. When the biofilm encounters a stressor, such as penicillin, a toxin is generated that deactivates metabolic activity. Dr. Urish stated, "When the metabolism for a bacterial cell shuts down, it doesn't matter how much stress there is. The cell just hangs out, and then at some point—we're not sure why—an antitoxin reverses the dormant state. But by then, the penicillin is gone. The bacteria have survived."
The nature of biofilm tolerance
Dr. Urish's 2018 OREF grant is supporting his work to characterize the antibiotic tolerance of biofilm and the role of the toxin-antitoxin system. One goal is to compare differences between strains of persistent Staphylococcus aureus from periprosthetic knee infections and persistent S. aureus from nasal tissues. Dr. Urish expects to observe a persistence in nasal S. aureus due to genetic variation—tolerance to antibiotics due to genetic selection (for example, increased antibiotic tolerance attributed to the overuse or misuse of antibiotics). The tolerance of periprosthetic S. aureus, on the other hand, is expected to be a phenotypic difference, a change in gene expression—affecting how the cell acts—and a subsequent change in cell function.
A second line of investigation will characterize the expression of genes that are likely regulators of toxin-antitoxin activity in S. aureus. In vitro experiments will quantify changes in toxin-antitoxin gene expression in planktonic and biofilm cells before and after exposure to a number of antibiotics. An in vivo study will quantify changes in mice with a 3D-printed titanium tibia implant. After the knee joints are inoculated with S. aureus, the researchers will compare the toxin-antitoxin gene expression between antibiotic-treated and untreated mice.
Similarly, the researchers will conduct experiments to characterize the phenotypic changes in biofilm using strains of bacteria in which toxin-antitoxin genes have been knocked out. In vitro experiments will measure factors—such as planktonic growth, biofilm production and biofilm mass—in wildtype and toxin-antitoxin knockout S. aureus. They will then compare changes in these factors when the wildtype and knockout bacteria are exposed to doses of different antibiotics.
The knockout strain that's least tolerant of antibiotic exposure will then be studied in the mouse model. The investigators will compare the effect of antibiotic treatment on the persistence of biofilm in mice with wildtype bacteria and mice with the knockout bacteria.
A final aim of the research will test a recently developed compound, an antimicrobial peptide designed to work against persistent bacteria because it's not dependent on active metabolism. The peptide, which has been studied in other animal models, is designed to bind selectively to bacteria and create pores in bacterial membranes. Dr. Urish's team will study an optimum dosing schedule of this compound in their mouse model. Because research in a larger animal model is critical for clinical trials, they will gather additional treatment data in a similar rabbit model of periprosthetic joint infection.
OREF funding—Turbocharging research
Dr. Urish first received OREF funding in 2016 with a New Investigator Grant. Noting the importance of this grant, he stated, "There's not a lot of support for new investigators to get going. OREF is the linchpin. OREF grants support people getting started, so that they can hit those early milestones in career development and become established researchers."
He said that OREF grants are also critical in building a research team, bringing in partners with needed expertise, and expanding ideas. "OREF is like the supplemental turbocharge. OREF grants allow us to take a bigger risk or make a bigger investment in an idea we really think is going to pay off."
Jay D. Lenn is a contributing writer for OREF. He can be reached at communications@oref.org.
References
1. “Total Knee Replacement.” OrthoInfo, American Academy of Orthopaedic Surgeons, June 2019, https://orthoinfo.aaos.org/en/treatment/total-knee-replacement.
2. “Joint Replacement Infection.” OrthoInfo, American Academy of Orthopaedic Surgeons, June 2019, https://orthoinfo.aaos.org/en/diseases--conditions/joint-replacement-infection/.
3. Bozic KJ, Kurtz SM, Lau E, et al. The epidemiology of revision total knee arthroplasty in the United States. Clin Orthop Relat Res 2010;468:45-51.
4. Urish KL, Bullock AG, Kreger AM, et al. A Multicenter Study of Irrigation and Debridement in Total Knee Arthroplasty Periprosthetic Joint Infection: Treatment Failure Is High. J Arthroplasty 2018;33:1154-9.
5. Romano C, Logoluso N, Drago L, Peccati A, Romano D. Role for irrigation and debridement in periprosthetic infections. J Knee Surg 2014