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Chronic Infections and Biofilms

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Indiscriminate antibiotic use has caused a dramatic rise in antibiotic-resistant bacteria. To determine which antibiotic is most appropriate, many veterinarians will send samples for culture and sensitivity testing. The classic antibiotic sensitivity testing assay is based on the disk diffusion method, whereby antibiotic-soaked disks are placed on plates inoculated with the sample bacteria. The size of the zone of inhibition (an area of no growth surrounding the disk, which results from bacterial sensitivity to the antibiotic) is measured. Additional assays using microtiter plates or automated antibiotic-sensitivity test cards are also commonly used.

Although rapid and reproducible, these tests only determine the effectiveness of antibiotics on bacteria present in the planktonic or free-floating state. It is now known that in most cases of chronic infection, the bacteria are not in the planktonic state but are primarily present as a biofilm, a complex community of bacteria encased in an extracellular matrix composed of proteins, DNA and polysaccharides. This matrix acts as a physical barrier, protecting the bacteria inside from a variety of environmental insults.

Because of this protective matrix, as well as the associated intercellular signaling that occurs between bacteria in the community, biofilm bacteria are significantly more resistant to antibiotics (in some cases more than 1,000-fold) than planktonic bacteria. Although antibiotic treatment may initially decrease symptoms, once it is discontinued the remaining bacteria will renew the biofilm and symptoms may recur.

The recently developed becSCREEN assay is designed to determine the antibiotic resistance profile of both planktonic and biofilm bacteria. It features a microtiter plate containing 96 pegs projecting from the lid. These pegs allow for bacterial adherence and biofilm formation so the antibiotic sensitivity analysis may be performed.

Following biofilm formation, the pegs are incubated in growth media containing several concentrations of a variety of antibiotics. The pegs are then washed and further incubated in a recovery plate. The sensitivity or resistance of the bacteria to each antibiotic is assessed by observing the growth in the microtiter plate. Once completed, the assay gives antibiotic sensitivity results for sample bacteria in both planktonic and biofilm states, allowing for easy comparison.

This assay has been used to successfully treat a variety of chronic infections including recurrent pyoderma and pyogenic otitis externa in dogs, and hemorrhagic cystitis and sinusitis in cats. In each case, traditional antibiotic testing was performed and the initial therapeutic course of action failed to resolve the condition. Following becSCREEN testing, an alternate therapeutic course of action was determined and, in all cases, the condition was resolved within two months.

chronic_diagram
Regeneration of biofilms following antibiotic treatment. Taken from:
Drenkard, E. “Antimicrobial Resistance of Pseudomonas Aeruginosa Biofi lm.” Microbes and Infection, 2003, 5: 1213-9.


Kim Perkins joined the Spectrum Group in early 2012 and is currently the Laboratory Director where he oversees the development, optimization and manufacture of veterinary diagnostic assays. He obtained his PhD from Arizona State University while studying the molecular mechanisms of viral pathogenesis, focusing on the role viral dsRNA-binding proteins play in disease. He has presented at several conferences and was an invited speaker at the International Angiogenesis Conference in Greece.