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Title: Investigation, using an in-vitro alginate biofilm model, into locally delivered antibiotic combinations to treat staphylococcal prosthetic infection
Author: Dall, Graham Fraser
ISNI:       0000 0004 7429 8874
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2014
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Introduction: Joint replacement is a common and effective procedure but unfortunately, a small proportion of patients develop Prosthetic joint Infection (PJI). The bacteria responsible for these infections exist within a surface-associated community known as a biofilm. When this biofilm phenotype is expressed, it allows the organisms to resist phagocytic host defenses, tolerate the stresses induced by antimicrobials and colonize peri-prosthetic niches. PJI is invariably refractory to standard therapies and clinicians are required to use a combination of systemic and local antimicrobials, repeated debridement and prosthesis exchange to treat the patient resulting in significant morbidity. Standard antibiotic sensitivity tests offer little insight into bacterial susceptibilities in the biofilm state. Furthermore, they neither test at the higher levels, nor in the combinations of antibiotics that are commonly locally delivered during surgery. The aim of this thesis was to develop and validate a biofilm model, and to use it to test staphylococcal biofilms with clinically achievable concentrations of antibiotics in combination. Methods: Sodium alginate gel was chelated with a calcium chloride solution to form bullet shaped beads with a surface area of 161mm2 These beads were then removed and their surface inoculated with either a methicillin sensitive Staphylococcus aureus (ATCC 29213) or a clinical strain of coagulase negative staphylococcus. After being incubated aerobically for 20 hours in a 48 -well micro -titre plate, growth controls were sampled and enumerated. The remaining beads were washed to remove non-adherent bacteria and placed into fresh broth containing antibiotics. After 3 hours of antibiotic challenge, they were removed, washed and the biofilm detached by dissolving the bead in a citric acid and Na2CO3 solution. Viable organisms were enumerated after micro-dilution and Miles Misra plating onto agar. Results: Cryo-scanning electron microscopy demonstrated the model allowed a biofilm to develop on the surface of alginate beads. Overall the technique performed with satisfactory resemblance of the control data and acceptable responsiveness after disinfection. The repeatability of disinfection was found to be most variable around the level used to define bacterial eradication. The model was used to compare the minimum inhibitory (MIC) and biofilm eradication concentrations (MBEC) of seven commonly used antibiotics. Poor correlation was found between the susceptibility of the standard planktonic cultures to antibiotics and those that were effective against organisms in biofilm. Gentamicin and daptomycin were found to be the only mono-therapies that were effective against the biofilm at clinically achievable levels. Combining antibiotics that were ineffective as single agents did not confer additional benefit. Interestingly despite gentamicin being effective when tested alone, combining it with clindamycin, rifampacin or linezolid reduced the bactericidal effect markedly in both strains. This phenomenon was investigated further by varying the concentration of antibiotics within the combinations. Combining the bactericidal antibiotics tested with gentamicin had an additive or synergistic effect. More importantly, a strong antagonistic effect was observed, with between 8 and 32 times more gentamicin being required, when it was combined with antibiotics considered bacteriostatic. Conclusion: Standard microbiology laboratory testing is inadequate to guide clinical treatment of PJI. Testing of biofilm susceptibility to combinations of antimicrobials at high concentrations should be included in the laboratory testing of PJI. Further research should be directed towards understanding the mechanisms in which bacteriostatic antibiotics induce the organisms to become more tolerant to other antibiotics. If the antagonistic effect is confirmed in -vivo then it is logical to study a two stage antimicrobial strategy, avoiding potential antagonism by only introducing bacteriostatic agents when the bactericidal drugs dip below their minimum biofilm eradication concentration.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available