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Objectives: This study describes the microbiological spectrum of chronic osteomyelitis and so guides the choice of empirical antibiotics for this condition., Methods: We performed a prospective review of a 166 prospective patient series of chronic osteomyelitis from Oxford, UK in which a standardised surgical sampling protocol was used., Results: Staphylococcus aureus was most commonly isolated (32%) amongst a wide range of organisms including gram negative bacilli, anaerobes and coagulase negative staphylococci. Low grade pathogens were not confined to patients with a history of metalwork, a high proportion of cases were polymicrobial (29%) and culture negative cases were common (28%). No clear predictors of causative organism could be established. Many isolates were found to be resistant to commonly used empirical anti-microbial regimens., Conclusions: The wide range of causative organisms and degree of resistance to commonly used anti-microbials supports the importance of extensive intra-operative sampling and provides important information to guide clinicians' choice of empirical antibiotics., (Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.)