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Solving the Infection Puzzle

Catherine G. Ambrose, PhD OREF Prospective Clinical Research Grant
Research Summary:

Research Topic: Used molecular techniques to investigate which species of bacteria are responsible for osteomyelitis.

Research Results: Identified species of bacteria present in osteomyelitis and the patterns they follow, leading to a more accurate understanding of the infection

Patient care application of results: Improved treatment for osteomyelitis patients that reduces the risk of unnecessary antimicrobial therapy and development of antibiotic-resistant bacteria

Solving the infection puzzle

OREF grant recipient researches bacterial causes of osteomyelitis

Amy Kile

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

“The diagnosis and management of osteomyelitis has long been a challenge for orthopaedic surgeons. The absolute identification of bacterial strains in any infection will help to design improved treatment regimes, reduce the risk to patients of unnecessary antimicrobial therapy, and possibly reduce the risk of antibiotic-resistance in bacteria,” said Catherine G. Ambrose, PhD, associate professor and director, Biomechanics Lab at The University of Texas Health Science Center (UTHSC) at Houston.

Diagnosis difficulties

Identifying the root cause of infection can be difficult. Laboratory test results are only positive for infection when living bacteria are present in a tissue sample. If the treating physician prescribes broad spectrum antibiotics to immediately treat an obvious infection, and takes a tissue sample days later, bacterial populations may already have been significantly reduced. Even when a tissue sample is taken immediately, if the conditions are not ideal for bacterial survival and reproduction—an anaerobic species stored in a container that isn’t airtight, for example—they won’t live long enough to result in a positive test.

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

“There are many patients being given broad spectrum antibiotics, which may be increasing the amount of bacteria that are resistant to treatment,” said Dr. Ambrose.
“There are a lot of patients who have chronic infections that are not responding to antibiotic treatment. I think if we had a clear picture of what kind of bacteria were in these wounds, it would be a lot easier to eradicate the infections.”

With support from an OREF Prospective Clinical Award, Dr. Ambrose began working with co-principal investigators and UTHSC colleagues Terry A. Clyburn, MD, assistant professor and director of research, Department of Orthopaedic Surgery, and Heidi Kaplan, PhD, associate professor, Department of Microbiology and Molecular Genetics, to research types of bacteria that cause osteomyelitis.

Bacterial Identity

To learn what bacteria are present in osteomyelitis, surgeons who are treating patients for implant infection, septic arthritis, or deep infection extract tissue samples to be tested as part of the research study in addition to traditional culture analysis. Dr. Kaplan, who is a microbiologist, isolates bacterial DNA, separates DNA 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 extracts 16S DNA from the tissue samples to identify the strains of bacteria contained in the sample.  The 16S gene is common to all bacteria but not found in mammalian cells. DNA sequencing tells researchers if more than one species is present. If there is more than one, the research team separates the DNA into bands, which are then divided and sequenced until each species has been identified.

To identify what they’ve found, Dr. Ambrose and her research team compare each species’ DNA sequence using the Basic Local Alignment Search Tool (BLAST), to search a database that holds every 16S gene that has been sequenced.

Infection Trends

In addition to identifying the species of bacteria present, Dr. Ambrose and her research nurse, Thea Troetscher, RN, and research assistants, Shidrokh Ardestani and Karen Gomez, are also looking for trends in infection by comparing the DNA tests with patient records. They review culture results; serum tests; antibiotics used in treatment; other tests run, including clinical tests to diagnose infection; how many times a patient had to return to the operating room to have his or her wound cleaned; and whether the infection responded to treatment or if the patient needed an amputation or died as a result of the infection. Knowing these trends and identifying species that cause the infection would enable orthopaedists to target specific bacteria, possibly reducing the number of patients for whom broad spectrum antibiotics are ineffective.

So far, DNA analysis shows 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 kind of patient, which would lead to a more accurate understanding of what is happening in these infections.”

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

“When we’re done with this study, and can say that the culture was a false negative and there are actually bacteria present, hopefully these patients won’t need to come back so many times,” said Dr. Ambrose.

Putting together results of three studies

“The OREF Prospective Clinical Award,” Dr. Ambrose said, “has been instrumental in providing resources to cover the number of people involved in this study.  Until we received this award, we could only test a small number of subjects.”

Dr. Ambrose and her research team plan to apply for National Institutes of Health (NIH) funding using data obtained from the OREF study, along with what they’ve gathered from related research on biofilms associated with implant-related infections, and drug delivery systems to treat infections.
“I think all three of these projects can be put together in a nice package for an NIH grant. The OREF Prospective Clinical Award has clearly been an important part of our investigations of the big puzzle that is orthopaedic infections.”

Dr. Ambrose hopes orthopaedic surgeons and others involved in the specialty recognize the important role OREF plays in funding research.

“OREF supports residents. It supports orthopaedists who make time for research in their careers at a time when it’s all about bringing in clinical dollars. Research doesn’t have any benefit if it doesn’t ever get out of the lab and to patients, but the only way to do translational research is to involve orthopaedic surgeons, and the only way to do that is to support them. OREF has been a consistent funding source for many people. I know many surgeons who started their research careers with OREF funding and moved on to very productive research.”

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