To examine the role of the accessory gene regulator (agr) in staphylococcal osteomyelitis, we compared a Staphylococcus aureus osteomyelitis isolate (UAMS-1) with a derivative of the same strain (UAMS-4) carrying an inactivated agr locus. Virulence was assessed with a rabbit model of acute, exogenous osteomyelitis. Bacteria were delivered by microinjection into the midradial region of the forelimb. After 4 weeks, UAMS-1 was identified in the bone of 12 of 13 rabbits infected with >2 ؋ 10 6 CFU and 5 of 6 infected with <2 ؋ 10 5 CFU. In contrast, UAMS-4 was found in 6 of 13 infected with the higher dose and 1 of 6 infected with the lower dose. Additionally, on the basis of a five-point scale assessing radiographic evidence of disease, rabbits infected with UAMS-1 had average scores of 2.64 ؎ 0.30 (high dose) and 1.43 ؎ 0.39 (low dose) while rabbits infected with UAMS-4 had average scores of 0.95 ؎ 0.23 (high dose) and 0.63 ؎ 0.20 (low dose). Uninfected controls had an average score of 0.53 ؎ 0.08. The results obtained with UAMS-1 were significantly different from those obtained with UAMS-4 at both doses (P < 0.047). The results obtained with UAMS-4 were not significantly different from those obtained with the controls at either dose of UAMS-4 (P > 0.150). On the basis of a similar five-point scale assessing histopathological evidence of disease, rabbits infected with UAMS-1 had average scores of 2.31 ؎ 0.22 (high dose) and 1.96 ؎ 0.36 (low dose) while rabbits infected with UAMS-4 had average scores of 1.58 ؎ 0.29 (high dose) and 0.83 ؎ 0.32 (low dose). Controls had an average score of 0.33 ؎ 0.05. The results obtained with UAMS-1 were significantly different from those obtained with UAMS-4 at both doses (P < 0.040). However, the results obtained with UAMS-4 were significantly different from the controls only at the high dose of UAMS-4 (P ‫؍‬ 0.025). We conclude that mutation of agr reduces the incidence and severity of disease but does not eliminate the ability to colonize bone and cause histopathological evidence of osteomyelitis.

Biofilm-associated implant-related bone and joint infections are clinically important due to the extensive morbidity, cost of care and socioeconomic burden that they cause. Research in the field of biofilms has expanded in the past two decades, however, there is still an immense knowledge gap related to many clinical challenges of these biofilm-associated infections. This subject was assigned to the Biofilm Workgroup during the second International Consensus Meeting on Musculoskeletal Infection held in Philadelphia USA (ICM 2018) (https://icmphilly.com). The main objective of the Biofilm Workgroup was to prepare a consensus document based on a review of the literature, prepared responses, discussion, and vote on thirteen biofilm related questions. The Workgroup commenced discussing and refining responses prepared before the meeting on day one using Delphi methodology, followed by a tally of responses using an anonymized voting system on the second day of ICM 2018. The Working group derived consensus on information about biofilms deemed relevant to clinical practice, pertaining to: (1) surface modifications to prevent/inhibit biofilm formation; (2) therapies to prevent and treat biofilm infections; (3) polymicrobial biofilms; (4) diagnostics to detect active and dormant biofilm in patients; (5) methods to establish minimal biofilm eradication concentration for biofilm bacteria; and (6) novel anti-infectives that are effective against biofilm bacteria. It was also noted that biomedical research funding agencies and the pharmaceutical industry should recognize these areas as priorities. ß

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To determine whether the ability of Staphylococcus aureus to bind collagen involves an adhesin other than the collagen adhesin encoded by cna, we examined the collagen binding capacity (CBC) of 32 strains of S. aureus. With only two exceptions, a high CBC corresponded with the presence ofcna. Both exceptions involved cna-positive strains with a low CBC. The first was a single strain (ACH5) that encoded but did not express cna. The second were the mucoid strains Smith diffuse and M, both of which encoded and expressedcna but bound only minimal amounts of collagen. Analysis of capsule mutants suggests that the reduced CBC observed in the mucoid strains was due to masking of the collagen adhesin on the cell surface and that this masking effect is restricted to heavily encapsulated strains. Differences in the CBC of the remainingcna-positive strains were correlated to variations in the level of cna transcription and were independent of the number of B domain repeats in the cna gene. In allcna-positive strains other than ACH5, cnatranscription was temporally regulated, with cna mRNA levels being highest in cells taken from exponentially growing cultures and falling to almost undetectable levels as cultures entered the post-exponential growth phase. The CBC was also highest with cells taken from exponentially growing cultures. Mutation of agrresulted in a slight increase in cna transcription and a corresponding increase in CBC during the exponential growth phase but did not affect the temporal pattern of cna transcription. Mutation of sar resulted in a more dramatic increase in CBC and a delay in the post-exponential-phase repression of cnatranscription. Mutation of both sar and agr had an additive effect on both CBC and cna transcription. We conclude that (i) cna encodes the primary collagen-binding adhesin in S. aureus, (ii) sar is the primary regulatory element controlling expression of cna, and (iii) the regulatory effects of sar and agr oncna transcription are independent of the interaction between sar and agr.