## 3 Results  
### Competitive Exclusion in Transwell Culture Plates  
Early preliminary experiments indicated that the supernatant from the strain NH culture was effective at inhibiting the growth of _Campylobacter jejuni_ and _Listeria monocytogenes_ by up to 9 log (CFU/mL), while _Salmonella_ enterica serovars Typhimurium, Enteritidis, Kentucky, Heidelberg, and _Escherichia coli_ STEC serotype O157:H7 exhibited minimal inhibition by strain NH (ca. 1 log CFU/mL). A method was needed to determine whether the inhibition of _C. jejuni_ and _L. monocytogenes_ was actively caused by an antagonistic substance secreted from NH cells or merely a depletion of nutrients. Transwells are six-well culture plates that contain 0.4-micrometer pore size filters at the bottom of the inserts that fit inside each well. These filters were employed to indicate whether the inhibitory activity was due to a substance that was secreted from NH cells and whether it could cross a 0.4-micrometer membrane. Both _L. monocytogenes_ and _C. jejuni_ growth were inhibited when strain NH was inoculated in the same well, regardless of whether or not NH was inoculated into the same side of the membrane as the pathogens or was inoculated on the side of the membrane separated from the pathogens by the membrane. In control wells, strain NH cells were inoculated and grown alone in the inner chamber and were unable to cross the 0.4 \(\upmu\)m membrane to the outer chamber (Figure 1C). These results indicated that strain NH secretes an inhibitory substance that can cross a 0.4-micrometer membrane and significantly reduce populations of _C. jejuni_ and _L. monocytogenes_. A bactericidal effect of this substance on _L. monocytogenes_ cells was further evident from the very marked lysis of _L. monocytogenes_ cells co-cultured either on the same side of the membrane as strain NH or separated from NH by the membrane (Figure 2). Figure 1: Competitive exclusion in transwells of _Listeria monocytogenes_ (**A**) and _Campylobacter jejuni_ (**B**) by strain NH. When strain NH is grown in the transwell, it cannot cross the membrane of the transwell (**C**). “<LOD” = none detected, below limit of detection (10 CFU/mL). Data points and brackets represent the mean and standard deviation, respectively, of three independent experiments. Asterisks represent significantly different means at timepoints indicated compared to control cultures (pathogen alone or strain NH alone) (\(p<0.05\)). Characterization of the Size of the Antagonistic Substance and Correlation of Bactericidal Activity with Protein Concentration  
Preliminary experiments indicated that the bactericidal substance was protease-sensitive, consistent with it being a protein. The inhibitory substance was therefore further characterized by collecting the cell-free culture supernatant by vacuum filtration through 0.2-micrometer vacuum filters, followed by ammonium sulfate precipitation. Preliminary experiments indicated that salting out by the addition of crystalline, proteomics-grade ammonium sulfate to a 60% final concentration precipitated essentially all of the bactericidal activity. This precipitate was dialyzed three times, which was followed by ultrafiltration. The flow-through and retentate fractions from differently sized ultrafiltration devices were then tested for bactericidal activity by measuring the inhibitory endpoint in microtiter plate dilutions of the fractions (Figure 3A) and by comparing the specific activity of the fractions (Figure 3B), calculated as the endpoint dilution factor divided by the mg of protein present. These experiments indicated that the bactericidal substance was present in the retentate from the 10 kDa cutoff ultrafiltration device but was not present in the filtrate from this same device (Figure 3A), suggesting that the substance exceeds 10 kDa in size. Further, the specific activity of the bactericidal substance was found to be directly proportional to the protein concentration in each fraction, which is consistent with the hypothesis that the substance is a protein (Figure 3B).