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Solving the infection puzzle

By Amy Kile

OREF grant recipient researches bacterial causes of osteomyelitis

At least 12,000 cases of osteomyelitis are treated annually. Although the number may not seem significant, considering the hundreds of thousands of orthopaedic surgeries performed each year, the risk of further complications—including amputation and death—is high, and osteomyelitis can be a serious problem.

Dr. Ambrose (far right) in the laboratory with members of her research team (from left): research assistant Karen Gomez; microbiologist Heidi Kaplan, PhD; and research nurse Thea Troetscher, RN. Not pictured: Terry A. Clyburn, MD, and research assistant Shidrokh Ardestani.

“The diagnosis and management of osteomyelitis has long been a challenge for orthopaedic surgeons,” notes Catherine G. Ambrose, PhD, associate professor and director of the Biomechanics Lab at the University of Texas Health Science Center (UTHSC). “If we can identify the bacterial strains in an infection, we will be able to design improved treatment regimens, reduce the risk to patients of unnecessary antimicrobial therapy, and possibly reduce the development of antibiotic resistance in bacteria.”

Diagnosis difficulties
Identifying the root cause of an infection can be difficult. Laboratory test results are only positive for infection when living bacteria are present in the tissue sample. If the treating physician prescribes broad-spectrum antibiotics to treat an obvious infection immediately and takes a tissue sample days later, bacterial populations may already have been significantly reduced.

Even tissue samples taken immediately may result in false negatives, if the conditions are not ideal for bacterial survival and reproduction. For example, an anaerobic species stored in a container that isn’t airtight won’t live long enough to result in a positive test.

But diagnosing and prescribing treatment for infection based on cues other than laboratory results could lead to long-term negative consequences.

“Many patients are being given broad-spectrum antibiotics, which may be increasing the numbers of bacteria that are resistant to treatment,” said Dr. Ambrose. “Many patients with chronic infections are not responding to antibiotic treatment. If we had a clearer picture of the type of bacteria in these wounds, it would be a lot easier to eradicate the infections.”

With support from a Prospective Clinical Award from the Orthopaedic Research and Education Foundation (OREF), Dr. Ambrose began working with coprincipal investigators and UTHSC colleagues Terry A. Clyburn, MD, and Heidi Kaplan, PhD, to research types of bacteria that cause osteomyelitis.

Bacterial identity
As part of a research study to learn what bacteria are present in osteomyelitis, tissue samples are taken from patients being treated for implant infections, septic arthritis, or deep infections. Dr. Kaplan, who is a microbiologist, isolates the bacterial DNA, separates it into different species, and uses DNA sequences to identify the bacteria causing the infection. If bacterial DNA is detected, the researchers know that bacteria are present in the tissue, even if they aren’t alive.

The research team uses a specific gene—16S DNA—to identify the bacterial strains in the tissue sample. The 16S gene is common to all bacteria but is not found in mammalian cells. DNA sequencing is used to determine whether more than one bacterial species is present. If so, the research team separates the DNA into bands, which are then divided and sequenced.

To identify what they’ve found, Dr. Ambrose and her research team compare the various DNA sequences against a database of every 16S gene that has been sequenced.

Infection trends
In addition to identifying the species of bacteria present, Dr. Ambrose and her team are looking for trends in infection by comparing the bacterial DNA tests with patient records. They review culture results, serum tests, the antibiotics used in treatment, and other tests, including clinical tests, to diagnose infection. They also determine how many times a patient had to return to the operating room to have the wound cleaned and what the eventual outcome was (a positive response to treatment, an amputation, or death). Knowing these trends and being able to identify the species that cause the infection would enable orthopaedists to target specific bacteria, possibly reducing the number of patients who don’t respond to broad-spectrum antibiotics.

So far, DNA analysis has shown that orthopaedic infections tend to be polymicrobial. With enough samples, Dr. Ambrose and her research team believe they will start seeing patterns.

“We might notice that two types of bacteria are always together in a specific type of infection,” she explains. “That would lead to a more accurate understanding of what is happening in these infections.”

According to Dr. Ambrose, bacteria build a community in a specific progression. One strain begins the infection process, then another appears, followed by a third strain, and the infection quickly progresses from mild to severe. “If infections could be treated when only one strain is present,” she says, “they would be easier to stop.

“If the treating physician knows exactly which bacteria are in a patient’s wound, he or she will be able to treat that patient more effectively and efficiently.”

Several patients in this study have needed their wounds cleaned multiple times.

“If this study provides us with a way to identify false negatives in tissue cultures, hopefully these patients won’t need to come back so many times,” says Dr. Ambrose.

Combining three studies
“The OREF Prospective Clinical Award,” Dr. Ambrose says, “has been instrumental in providing resources to enable us to expand the number of people involved in this study. Until we received this award, we could only test a small number of patients.”

Dr. Ambrose and her research team plan to apply for funding from the National Institutes of Health (NIH), using data obtained from the OREF-funded study, along with what they’ve gathered from related research on biofilms associated with implant-related infections and drug delivery systems to treat infections.

“These three projects can be packaged nicely for an NIH grant,” says Dr. Ambrose. “The OREF Prospective Clinical Award has clearly been an important part of our investigations of the big puzzle that is orthopaedic infections.

“Research doesn’t have any benefit if it doesn’t ever get out of the lab and to patients,” she continues, “but the only way to do translational research is to involve orthopaedic surgeons, and they need support. OREF has been a consistent funding source for many orthopaedic surgeons who have moved on to very productive research.”

Amy Kile is the OREF publications manager. She can be reached at kile@oref.org

AAOS Now
August 2010 Issue
http://www.aaos.org/news/aaosnow/aug10/research1.asp