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Home / Equine Research Bank / Equine Vet J / Use of in vitro assays to identify antibiotics that are cyto...
Equine veterinary journal2020; 53(3); 579-589; doi: 10.1111/evj.13314

Use of in vitro assays to identify antibiotics that are cytotoxic to normal equine chondrocytes and synovial cells.

Abstract: Intra-articular (IA) antibiotic usage is prevalent in equine practice. However, recent emergence of antimicrobial resistance prompts re-evaluation of antibiotic selection, particularly when used prophylactically. Furthermore, many commonly used antibiotics exert direct cytotoxicity to equine cells, and appropriate IA doses have not been defined. Objective: To screen antibiotics in vitro as an initial assessment of cytotoxicity against normal equine joint cells in monolayer culture and explant tissues. Methods: In vitro experimental study. Methods: Chondrocytes and synovial cells were harvested from three horses and plated on 24-well plates (100 000 cells/wells in triplicate) for 48 hours prior to addition of antibiotics. Joint cells were exposed to antibiotics (n = 15) at various doses (25-0.39 mg/mL in complete DMEM media) for 24 hours and viability was assessed by trypan blue dye exclusion. The half maximal inhibitory concentration (IC50) was determined for each antibiotic. Cartilage explants were obtained from 3 horses, minced and exposed to antibiotics (n = 5) for 72 hours. Live/dead staining was performed, and fluorescence was visualised using Olympus IX83 spinning disk confocal microscope. Percentage of live vs dead cells was quantified. Results: Antibiotics from different antimicrobial classes expressed dose-dependent but variable cytotoxicity to equine joint cells in vitro. Aminoglycosides and doxycycline had the lowest IC50 (most toxic). Ampicillin sulbactam, imipenem, tobramycin, ceftiofur sodium and amoxicillin had IC50 > 25 mg/mL for at least one cell line, representing potentially less cytotoxic alternatives. Conclusions: Further studies are necessary to extrapolate these in vitro data results to the in vivo joint environment. Conclusions: Targeted IA antibiotic therapy would involve selection of the safest antibiotics (highest IC50) with efficacy based on bacterial culture/sensitivity. Antimicrobial selection and evidence-based dosing may minimise damage to native articular cartilage and synovial cells and development of antimicrobial resistance when IA antibiotics are used in equine practice.
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Publication Date: 2020-07-03 PubMed ID: 32544273PubMed Central: PMC7738387DOI: 10.1111/evj.13314Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article evaluates the potential cytotoxicity of various antibiotics on equine joint cells to determine safer alternatives for intra-articular (IA) antibiotic therapy in horses. The study emphasizes the need to minimize damage to cartilage and synovial cells and combat increasing rates of antimicrobial resistance.

Methodology

  • The research involved in vitro tests with chondrocytes and synovial cells collected from three horses. These cells were grown in 24-well plates with a density of 100,000 cells per well in triplicate and cultured for 48 hours before the addition of different antibiotics.
  • A total of 15 various antibiotics at different doses (ranging from 25 – 0.39 mg/mL in complete DMEM media) were tested on joint cells in a 24-hour duration. Cell viability post-exposure was evaluated using the trypan blue dye exclusion method.
  • The level of cytotoxicity was identified by determining the concentration of each antibiotic that caused half-maximal inhibition or IC50.
  • Furthermore, cartilage explants were obtained from three horses, crushed, and exposed to five different antibiotics over 72 hours. A live/dead stain was used, and fluorescence was examined using the Olympus IX83 spinning disk confocal microscope. The number of live and dead cells was then quantified.

Findings

  • The research disclosed that antibiotics from varied antimicrobial classes showed variable, dose-dependent cytotoxicity to equine joint cells in vitro.
  • Among all, aminoglycosides and doxycycline exhibited the lowest IC50, implying these were the most toxic to the cells.
  • On the other hand, ampicillin sulbactam, imipenem, tobramycin, ceftiofur sodium, and amoxicillin showed an IC50 greater than 25 mg/mL for at least one cell line, indicating that these were less cytotoxic and thus could be safer alternatives.

Conclusions

  • The study suggests the importance of further research to relate these in vitro findings to in vivo joint environment conditions.
  • It concludes that a targeted IA antibiotic therapy should consider the safest antibiotics (those with highest IC50 levels), combined with the efficacy determined through bacterial culture and sensitivity tests.
  • By selecting suitable antibiotics and adopting evidence-based dosing, this can limit the potential damage to the cartilage and synovial cells, as well as minimizing the likelihood of antimicrobial resistance when intra-articular antibiotics are used in equine practice.

Cite This Article

APA
Pezzanite L, Chow L, Piquini G, Griffenhagen G, Ramirez D, Dow S, Goodrich L. (2020). Use of in vitro assays to identify antibiotics that are cytotoxic to normal equine chondrocytes and synovial cells. Equine Vet J, 53(3), 579-589. https://doi.org/10.1111/evj.13314

Publication

Equine veterinary journal
ISSN: 2042-3306
NlmUniqueID: 0173320
Country: United States
Language: English
Volume: 53
Issue: 3
Pages: 579-589

Researcher Affiliations

Pezzanite, Lynn
  • Translational Medicine Institute, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.
Chow, Lyndah
  • Translational Medicine Institute, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.
Piquini, Gabriella
  • Translational Medicine Institute, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.
Griffenhagen, Gregg
  • Translational Medicine Institute, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.
Ramirez, Dominique
  • Translational Medicine Institute, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.
Dow, Steven
  • Translational Medicine Institute, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.
Goodrich, Laurie
  • Translational Medicine Institute, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.

MeSH Terms

  • Animals
  • Anti-Bacterial Agents / toxicity
  • Cartilage, Articular
  • Chondrocytes
  • Horses

Grant Funding

  • TL1 TR002533 / NCATS NIH HHS
  • USDA National Institute of Food and Agriculture
  • Colorado State University Young Investigator Award in Companion Animal Studies
  • TL1TR002533 / CCTSI NIH/NCATS CTSA
  • NI18AHDR1019785G010 / Animal Health and Disease
  • Colorado State University CVMBS Veterinary Summer Scholars Program
  • Shipley Foundation
  • Verdad Foundation

Conflict of Interest Statement

Authors’ declaration of interests. The authors have no competing interests.

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Citations

This article has been cited 1 times.
  1. Pezzanite L, Chow L, Hendrickson D, Gustafson DL, Russell Moore A, Stoneback J, Griffenhagen GM, Piquini G, Phillips J, Lunghofer P, Dow S, Goodrich LR. Evaluation of Intra-Articular Amikacin Administration in an Equine Non-inflammatory Joint Model to Identify Effective Bactericidal Concentrations While Minimizing Cytotoxicity.. Front Vet Sci 2021;8:676774.
    doi: 10.3389/fvets.2021.676774pubmed: 34095281google scholar: lookup
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