Properties of an Antimicrobial Molecule Produced by an Escherichia coli Champion

Abstract

Over recent decades, the number and frequency of severe pathogen infections have been increasing. Pathogen mitigation strategies in human medicine or in livestock operations are vital to combat emerging arsenals of bacterial virulence and defense mechanisms. Since the emergence of antimicrobial resistance, the competitive nature of bacteria has been considered for the potential treatment or mitigation of pathogens. Previously, we identified a strong E. coli competitor with probiotic properties producing a diffusible antimicrobial molecule(s) that inhibited the growth of Shiga toxin-producing E. coli (STEC). Our current objective was to isolate and examine the properties of this antimicrobial molecule(s). Molecules were isolated by filter sterilization after 12 h incubation, and bacterial inhibition was compared to relevant controls. Isolated antimicrobial molecule(s) and controls were subjected to temperature, pH, or protease digestion treatments. Changes in inhibition properties were evaluated by comparing the incremental cell growth in the presence of treated and untreated antimicrobial molecule(s). No treatment affected the antimicrobial molecule(s) properties of STEC inhibition, suggesting that at least one molecule produced is an efficacious microcin. The molecule persistence to physiochemical and enzymatic treatments could open a wide window to technical industry-scale applications.

Keywords: E. coli; Shiga toxin; antimicrobial molecule; enzyme resistance; heat resistance; inhibition; microcins; pH resistance.

Figure 1

Schematic of the Molecule Isolation Protocol and Inhibition Assay. (A): Isolation and Treatment. E. coli O103F was grown overnight on a MacConkey Agar plate, and a single colony was grown in EC for 12 h. Cells were pelleted at 10,000× g for 10 min. The cell pellet was discarded, and the supernatant was filter-sterilized using a 0.22 µm filter. The sterilized supernatant was then treated to examine, heat, pH, or protease digestion. Note: SPENT was also prepared using the same protocol. (B): Inhibition Assay. O157A was grown overnight for 12 h in EC. The cells were then diluted to an OD_600nm of 0.1 and grown for 3 h. This culture was then used to inoculate the AMMO and the controls. OD_600nm measurements were taken at 0, 2, 4, 6, and 8 h. Note: (1) O103F was also prepared using the same protocol when utilized in the experiment. (2) AMMO is the cell-free supernatant collected after 12 h E. coli O103F growth. SPENT is the cell-free supernatant collected after 12 h E. coli O157A growth.

Figure 2

The molecule isolation protocol confirmation results for E. coli O157A grown in AMMO and SPENT in comparison to E. coli O103F grown in SPENT and AMMO. (A): The difference between the cell numbers for either O157A or O103F in the SPENT and in AMMO. (B): The OD_600nm data for O157A in AMMO, O157A in SPENT, O103F in SPENT, and O103F in AMMO and * control in fresh EC as a numerical value below the bars. Symbols: α and β denote a significant difference between growth in AMMO and SPENT for O157A and O103F, respectively (p < 0.05). A comparison of O103F growth in AMMO to O157A growth in SPENT revealed they are not significantly different. Note: (1) AMMO is the cell-free supernatant collected after 12 h E. coli O103F growth. SPENT is the cell-free supernatant collected after 12 h E. coli O157A growth. (2) Bars are the calculated standard deviation for AMMO and SPENT in each experiment (O157A and O103F) at each time point.

Figure 3

The effect of pH treated AMMO on growth inhibition of E. coli O157A at two pH’s 3 and 11. (A): The difference between the cell numbers for O157A in SPENT and in AMMO at pH 3, pH 11, and untreated supernatant. (B): The OD_600nm data for O157A grown in AMMO, in SPENT (pH 3, pH 11, and untreated) and * control in fresh EC as a numerical value below the bars. Symbols: α, β and ɣ denote a significant difference between O157A grown in AMMO and in the SPENT for pH 3, untreated, and pH 11 supernatants, respectively (p < 0.05). Note: (1) AMMO is the cell-free supernatant collected after 12 h E. coli O103F growth. SPENT is the cell-free supernatant collected after 12 h E. coli O157A growth. (2) Bars are the calculated standard deviation for the treated or untreated AMMO and SPENT at each time point.

Figure 4

The effect of autoclaving AMMO on growth inhibition of E. coli O157A. (A): The difference between the cell numbers for O157A in SPENT and in AMMO with treated and untreated supernatant. (B): The OD_600nm data for O157A grown in AMMO, in SPENT (treated and untreated), and * control in fresh EC as a numerical value below the bars. Symbols: α and β, denote a significant difference between O157A grown in AMMO and in SPENT for treated and untreated supernatants, respectively (p < 0.05). Note: (1) AMMO is the cell-free supernatant collected after 12 h E. coli O103F growth. SPENT is the cell-free supernatant collected after 12 h E. coli O157A growth. (2) Bars are the calculated standard deviation for the treated or untreated AMMO and SPENT at each time point.

Figure 5

The effect of trypsin protease digestion of AMMO on growth inhibition of E. coli O157A over time. (A): The difference between the cell numbers for O157A in SPENT and in AMMO treated and untreated supernatant. (B): The OD_600nm data for O157A grown in AMMO, in SPENT (treated and untreated), and * control in fresh EC as a numerical value below the bars. Symbols: α and β, denote a significant difference between O157A in AMMO and in SPENT for treated and untreated supernatants, respectively (p < 0.05). Note: (1) AMMO is the cell-free supernatant collected after 12 h E. coli O103F growth. SPENT is the cell-free supernatant collected after 12 h E. coli O157A growth. (2) Bars are the calculated standard deviation for the treated or untreated AMMO and SPENT at each time point.

Figure 6

The effect of chymotrypsin protease digestion of AMMO on growth inhibition of E. coli O157A over time. (A): The difference between the cell numbers for O157A in SPENT and in AMMO with treated and untreated supernatants. (B): The OD_600nm data for O157A grown in AMMO, in SPENT (treated and untreated), and * control in fresh EC as a numerical value below the bars. Symbols: α, β and ɣ, denote a significant difference between: O157A in AMMO and in SPENT for 1×, 10×, and untreated supernatants, respectively (p < 0.05). Time-point = 2 difference in cell number data not shown for chymotrypsin treated AMMO and SPENT (1× and 10×) due to parallel OD_600nm data, and the difference is ~zero. Note: (1) AMMO is the cell-free supernatant collected after 12 h E. coli O103F growth. SPENT is the cell-free supernatant collected after 12 h E. coli O157A growth. (2) Bars are the calculated standard deviation for the treated or untreated AMMO and SPENT at each time point.

Figure 7

Comparison of average inhibition activity against E. coli O157 of treated AMMO across all treatments versus untreated AMMO (control) at time point 4, 6, and 8 h. Note: (1) Bars are the calculated standard deviation for the treated or untreated AMMO, respectively, at each time point. (2) AMMO is the cell-free supernatant collected after 12 h E. coli O103F growth.