Determination of Tobramycin and Vancomycin Exposure Required to Eradicate Biofilms on Muscle and Bone Tissue In Vitro.

Vajra Badha,Rex Moore,John Heffernan,Paulo Castaneda,Alex Mclaren,Derek Overstreet

doi:10.7150/jbji.29711

Abstract

Background: Bacterial biofilms cause chronic orthopaedic infections. Surgical debridement to remove biofilm can be ineffective without adjuvant local antimicrobials because undetected biofilm fragments may remain in the wound and reestablish the infection if untreated. However, the concentrations and duration of antimicrobial exposure necessary to eradicate bacteria from clinical biofilms remain largely undefined. In this study, we determined the minimum biofilm eradication concentration (MBEC) of tobramycin and vancomycin for bacterial biofilms grown on bone and muscle in vitro.Methods: Biofilms of pathogens found in musculoskeletal infections (S. aureus, S. epidermidis, E. faecalis, P. aeruginosa, and E. coli) were established for 72 hr on rabbit muscle and bone specimens in vitro and characterized by SEM imaging and CFU counts. Biofilm-covered tissue specimens were exposed to serial log2 dilutions (4000-31.25 µg/mL) of tobramycin, vancomycin, or a 1:1 combination of both drugs for 6, 24, or 72 hr. Tissues were subcultured following antimicrobial exposure to determine bacterial survival. The breakpoint concentration with no surviving bacteria was defined as the MBEC for each pathogen-antimicrobial-exposure time combination.Results: All tested pathogens formed biofilm on tissue. Tobramycin/vancomycin (1:1) was the most effective antimicrobial regimen with MBEC on muscle (10/10 pathogens) or bone (7/10 pathogens) generally in the range of 100-750 µg/mL with 24 or 72 hr exposure. MBEC decreased with exposure time for 53.3% of biofilms between 6 and 24 hr, 53.3% of biofilms between 24 and 72 hr, and for 76.7% of biofilms between 6 and 72 hr. MBECs on bone were significantly higher than corresponding MBECs on muscle tissue (p < 0.05). In most cases, tissue MBECs were lower compared to previously published MBECs for the same pathogens on polystyrene tissue-culture plates.Conclusions: The majority of MBECs for orthopaedic infections on bone and muscle are on the order of 100-750 µg/mL of vancomycin+tobramycin when sustained for at least 24 hr, which may be clinically achievable using high-dose antimicrobial-loaded bone cement (ALBC).

Highlights

The majority of surgical site infections including bone, joint, and implant-related infections are caused by biofilms, which are communities of bacteria in a polysaccharide matrix that is self-produced following bacterial adhesion to implant or compromised tissue surfaces.[1]
We developed a protocol for determining minimum biofilm eradication concentration (MBEC) by establishing biofilms on aseptically harvested rabbit muscle and bone specimens in vitro, and treating those biofilms with antimicrobials for 6-72 hr to simulate the duration of exposure provided by local delivery
Synergy between tobramycin and vancomycin only occurred for S. epidermidis 29886 (FIC index 0.18) and there was no antagonism between tobramycin and vancomycin for any of the organisms. (Figure 1B); CFUs for biofilms on bone and muscle specimens were not significantly different (p = 0.2742; two-tailed paired t-test)

Summary

Introduction

The majority of surgical site infections including bone, joint, and implant-related infections are caused by biofilms, which are communities of bacteria in a polysaccharide matrix that is self-produced following bacterial adhesion to implant or compromised tissue surfaces.[1] Biofilms are characteristically tolerant to antimicrobials and present a major challenge to successful clinical management.[2,3] For planktonic infections, the inhibition or reduction of bacteria by systemic antimicrobials combined with a normal host http://www.jbji.net immune response can effectively cure the infection.[4]. While accepted treatment of established orthopaedic infections includes surgical debridement of infected tissue, these procedures may leave biofilm along the debridement margins or fragments in the surgical wound.[4,8] Retention of biofilm bacteria in the postsurgical wound increases the risk that infection will recur.[4] Local delivery of antimicrobials is commonly performed with the goal of eradicating bacteria, including any remaining biofilm, after debridement. We determined the minimum biofilm eradication concentration (MBEC) of tobramycin and vancomycin for bacterial biofilms grown on bone and muscle in vitro

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