Albumin in Tears Modulates Bacterial Susceptibility to Topical Antibiotics in Ophthalmology

Abstract

Bacterial keratitis is a serious and vision-threatening condition in veterinary and human patients, one that often requires culture and susceptibility testing to adjust therapy and improve clinical outcomes. The present study challenges the antimicrobial susceptibility testing (AST) paradigm in ophthalmology, enabling more accurate in vitro-to-in vivo translation by incorporating factors normally present during host-pathogen interactions in clinical patients. Thirty bacteria (10 Staphylococcus pseudintermedius, 10 Streptococcus canis, 10 Pseudomonas aeruginosa) were isolated from canine patients with infectious keratitis. For each isolate, commercial plates (Sensititre™ JOEYE2) were used to assess the minimal inhibitory concentration (MIC) of 17 different antibiotics in the absence (0% albumin, control) or presence of canine albumin (0.01–2%). For Staphylococcus pseudintermedius, the experiment was repeated with actual tear fluid collected from canine eyes with ocular surface inflammation. Kruskal-Wallis, Wilcoxon signed rank test and Spearman's correlation tests were used for statistical analysis. Clinical outcomes were unfavorable in selected canine patients with bacterial keratitis (e.g., globe perforation, graft dehiscence) despite standard AST (i.e., 0% albumin in test medium) confirming that most corneal infections (93%) were susceptible to ≥1 topical antibiotics used at the initial visit. Albumin levels ≥0.05% increased MICs in a dose-dependent, bacteria-specific, and antibiotic-specific manner. No significant differences (P = 1.000) were noted in MICs of any antibiotic whether albumin or tear fluid was added to the Mueller-Hinton broth. Percent protein binding inherent to each antibiotic was associated with clinical interpretations (Spearman's rho = −0.53, P = 0.034) but not changes in MICs. Albumin in tears impacted the efficacy of selected ophthalmic antibiotics as only the unbound portion of an antibiotic is microbiologically active. The present findings could improve decision making of clinicians managing bacterial keratitis, reduce development of antimicrobial resistance, influence current guidelines set by the Clinical and Laboratory Standards Institute, and serve as a reference for bacteriological evaluations across medical fields and across species.

Kinetics and minimal inhibitory concentrations of ceftiofur in tear film following extended-release parenteral administration (Excede®) in dogs

“…Blood-tear barrier breakdown: Dogs with ulcerative keratitis (or other inflammatory conditions) often develop concurrent conjunctivitis as bystander inflammation, a condition that promotes leakage of compounds from the blood-to-tear compartment ( 15 , 30 ). This physiological process was shown in several studies for large proteins such as albumin ( 11 , 14 , 15 , 31 ) but could also be true for smaller compounds such as xenobiotics. In the present study, tear film concentrations of ceftiofur were higher in the canine eye with conjunctivitis compared to the contralateral healthy eye at all time points investigated (0.5–24 h), with a difference in tear concentrations up to ~29-fold.…”

“…Antibiotic-protein binding in tear film: Diseased eyes have elevated levels of albumin in the tear film, leading to protein-antibiotic binding that could modulate drug efficacy on the ocular surface. In one study, albumin levels > 0.05% increased MICs of canine ocular pathogens in a dose-dependent, bacteria-specific, and antibiotic-specific manner ( 14 ). Here, 1% canine albumin was added to bacterial wells to optimize in vitro susceptibility testing with in vivo conditions.…”

“…Bacterial isolates ( n = 30) cultured from canine patients were revived from the −80°C freezer by thawing at room temperature and grown on tryptic soy with 5% sheep blood agar plates. Bacterial broths containing 4 × 10 7 colony forming units per milliliter (CFU/mL) were prepared using Mueller-Hinton broth (MHB; catalog #T3462, Thermo Scientific Inc.) for Staphylococcus pseudintermedius and Pseudomonas aeruginosa , or Mueller-Hinton broth with lysed horse blood (catalog # CP114-10, Thermo Scientific Inc.) for Streptococcus canis , as previously described ( 14 ). Albumin 4% solution was prepared by mixing canine albumin powder (Animal Blood Resources International, Stockbridge, MI) with deionized water.…”

“…Using one blank plate (Corning 96-well Clear Polystyrene Microplates, Corning Inc.) for each bacterial species, 15 μL of albumin solution was pipetted into 80 wells (10 columns x 8 rows), 15 μL of a given bacterial isolate was pipetted in the 8 wells of a given column ( e.g ., column 1 for isolate #1, column 2 for isolate #2, …., column 10 for isolate #10), and 30 μL of a given ceftiofur concentration was pipetted in the 10 wells of a given row ( eg , row 1 for 40 ng/mL, row 2 for 80 ng/mL, …, row 8 for 5,120 ng/mL). Accounting for the dilutions resulting from mixing the 3 different solutions in the wells, bacterial load was set to the standard for susceptibility testing (10 7 CFU/mL), albumin level was set to 1% (average albumin levels in canine tear film), ( 14 , 15 ) and ceftiofur concentrations ranged from 20 ng/mL to 2,560 ng/mL.…”

“…The second objective was to determine the MICs of ceftiofur against common ocular pathogens in dogs, allowing for a preliminary pharmacokinetic-pharmacodynamic (PK-PD) analysis of subcutaneous ceftiofur as a potential therapeutic option for canine patients with bacterial keratitis. Notably, results were made as clinically relevant as possible by (i) assessing tear film concentrations in the presence of compromised blood-tear barrier, ( 10 ) and (ii) assessing MICs in the presence of albumin to account for protein-binding in tears ( 14 ).…”