Rapid in vitro antimicrobial screening assay against Campylobacter

M. Navarro, R. Stanley, Y. Sultanbawa (QAAFI, The University of Queensland) - A. Cusack (Department of Agriculture, Fisheries & Forestry)

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Research was undertaken to adapt a multi-well antimicrobial assay as a fast and reliable method to screen large numbers of plant extract treatments against multiple Campylobacter strains.

Campylobacter cultures, isolated from chicken faecal droppings were used to trial and validate the assay. The strains were recovered by growing in 7% sheep blood agar at 42°C for 24 hours in 5%CO2. Cultures were then placed in nutrient broth No 2 (Oxoid CM0067) enriched with Campylobacter growth supplement (Oxoid SR0232E). An innoculum of 104 cfu/ml was obtained by dilution in the broth and measured by Optical Density (OD). Samples of 10 essential oils, 6 phytochemicals, 2 plant by-product extracts and 5 organic acids were evaluated for activity against all the Campylobacter strains. Essential oils were diluted with agar and the rest of compounds in sterile water.

The screening assay was carried out using flat bottom 96-well sterile microtiter plates. Each plate had rows of wells containing the treatments and their replicates with the first and last row containing sterile culture media and bacteria culture tested as negative and positive controls respectively. OD of the wells was determined at 595 nm prior to incubation for 48 hours. The growth/inhibition value was calculated by the following formula: % Inhibition = (1-(OD T48-OD T0)/(OD C48-OD C0)) x 100, were T is the treatment and C the positive control. The minimum inhibitory concentration (MIC) was considered the concentration of the compound tested that first gave 100% inhibition of the growth of the bacteria.

The MIC of Campylobacter by treatments was relatively independent of the strain. Oregano, thyme and their purified constituents carvacrol and thymol showed the strongest inhibition with the MIC > 0.0004% w/w. Cinnamon, cinnamaldehyde and anise myrtle had MIC values between 0.001% and 0.004%. The rest of the essential oils and phytochemicals tested (lime, sage, lemon, garlic, tea tree oil, eugenol, citral, and vanillin) all had MIC>0.01% w/w. The MIC of the sorghum extract was > 5% while olive leaf extract showed no antimicrobial activity. Of the organic acids citric, lactic and benzoic gave MIC values between 0.03% and 0.06%, while ascorbic and caprylic acids were higher than 0.25%. The results obtained with this new screening method were in agreement with existing literature validating the assay.
Future research will survey more strains with combinations of extracts and acids to find MIC compositions that can be achieved in vivo in the chicken intestinal tract and caecum.