Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

Abstract

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition.

Cultivable microbiota associated with Aurelia aurita and Mnemiopsis leidyi

“…Negative as well as positive bacterial interactions based on competition/cooperation can be monitored with growth inhibition assays as described by Moran et al . (Moran et al, 2016). The assay is used to detect growth inhibition and its degree or growth promotion of the neighboring bacteria.…”

“…First, all 231 isolated bacterial strains were grown on MB agar plates at 30 °C overnight (Moran, Crank, Ghabban, & Horsburgh, 2016). Out of those 231 strains, 203 formed compact bacterial lawns on the agar plates under the selected growth conditions and were subsequently used for the growth inhibition assay as follows.…”

Corynebacterium accolens Has Antimicrobial Activity against Staphylococcus aureus and Methicillin-Resistant S. aureus Pathogens Isolated from the Sinonasal Niche of Chronic Rhinosinusitis Patients

“…The antagonistic activity of all C. accolens clinical strains and a culture collection strain, C. accolens ATCC 49726 was evaluated against S. aureus clinical isolates and S. aureus ATCC 25923 (from ATCC, Manassas, VA, USA) using deferred growth inhibition assays as described previously [ 34 ] with modifications. Briefly, a 48 h C. accolens culture (20 µL, approximately 10 8 cells) in TSB (test inhibitor strains) was pipetted onto the centre of a TSA plate supplemented with 0.8% Tween 80 and incubated for 48 h at 37 °C with 5% CO 2 .…”

Evidence of Biocontrol Activity of Bioinoculants Against a Human Pathogen, Listeria monocytogenes

“…It confirmed that the impact of ABP on the populations of L. monocytogenes varied according to the conditions of its environment. The reason for visible inhibition at 1/10th and 1/100th strength of TSA may be attributed to competitive exclusion, i.e., increased competition for the available nutrients in the medium (Moran et al, 2016). In a study conducted by Buchanan and Bagi (1999), it was observed that P. fluorescens was able to inhibit L. monocytogenes, and this inhibition was dependent on temperature, pH and sodium chloride levels in brain-heart infusion broth.…”