Land use influences the composition and antimicrobial effects of propolis

Propolis Collection and Extraction Propolis was collected from five different apiaries of a commercial beekeeping operation in Fayette and Clayton counties in Iowa. Samples were collected from 5–10 colonies at each location, yielding a total of 45 samples. Propolis was collected from the front rim of the top lid of each hive. This location on the hive was chosen because it is the area of most active recent propolis placement by honey bees, due to frequent beekeeper disturbance. Each propolis sample was collected into sterile plastic tubes and stored at −80 °C until further use. Propolis samples were then pulverized with a mortar and pestle, and diluted in 100% ethanol (0.2 g propolis in 1.8 g ethanol). Samples were then mixed at 500 rpm at 30 °C for 48 h in the dark. Samples were filtered using a 0.22 μm syringe filter (Fisher Scientific, Hampton, NH, USA). Filtered samples were then diluted according to specific needs for chemical or antimicrobial analysis. MIC50 The antimicrobial effects of the propolis samples were measured by performing a MIC50 assay, according to Wiegand et al. [34] with modifications. Briefly, propolis samples were serially diluted with phosphate buffered saline (PBS) to yield a diluted range of 0.1 mg propolis/mL to 17 mg propolis/mL, a range previously observed to be inhibitory to bacterial growth [7,35,36]. Ethanol serially diluted in PBS was used as the negative control. Fifty μL of each propolis sample or negative control dilution was then plated on a 96 well plate in triplicates. Paenibacillus larvae (ATCC 9545) and Lysinibacillus sphaericus (ATCC 4525) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and Serratia marcescens and Klebsiella pneumoniae were obtained from Carolina Biological Supply Company (Burlington, NC, USA). Bacterial suspensions were prepared by transferring two colonies of bacteria from agar media into tryptic soy broth (S. marcescens and K. pneumoniae) or brain heart infusion broth (P. larvae and L. sphaericus). Viability of bacteria was determined on a hemocytometer using erythrosine B. A 1 × 105 cells/ml bacterial solution was then prepared using the appropriate liquid broth medium. Next, 100 μL of each bacteria suspension was added per well in a 96 well plate. The plates were then incubated at 30 °C (S. marcescens and L. sphaericus) and 37 °C (K. pneumoniae and P. larvae) for 24 h. Following incubation, 0.75 μL from each well was diluted in 50 μL of PBS and transferred to a well in a 12 well plate with the appropriate agar media for each bacterium. These plates were then incubated at 30 °C (S. marcescens and L. sphaericus) and 37 °C (K. pneumoniae and P. larvae) for 24 h. Colonies of bacteria were then counted and the MIC50 of each sample for each bacterium was determined as the lowest propolis concentration to inhibit 50% of the bacterial growth. Statistical Analyses Effectiveness of Propolis against Pathogens In this analysis, we constructed a series of linear models between apiary and MIC50 scores, using apiary as the explanatory variable and the MIC50 score for each hive’s propolis against each pathogen as the response variable (resulting in one linear model for each pathogen). In the event of significant models, we used a Tukey HSD test to identify which apiaries were significantly different from one another.