Journal: PLoS Pathogens

Article Title: A Product of Heme Catabolism Modulates Bacterial Function and Survival

doi: 10.1371/journal.ppat.1003507

Figure Lengend Snippet: (A) An equal number of CFUs of E. faecalis or E. coli (O157:H7 86-24) were mixed with increasing amounts of bilirubin (0, 100, 200, 300, 400, and 500 µM), biliverdin (500 µM), or α-tocopherol (500 µM), before spotting onto LB agar. Plates were grown at 37°C overnight. (B) Densitometry of bacterial lawns was measured using ImageJ software. (C) E. faecalis strains OG1RF (oral origin), X33 (fecal origin), UWH 1936 (blood origin), and NJ-3 (peritoneal fluid origin) were exposed to increasing concentrations of bilirubin (100, 200, or 300 µM) prior to spotting onto an LB-agar plate. Bacterial growth was captured by imaging the plates, and images were modified by adjusting brightness and contrast to best display colony formation (darker areas of image). (D) Growth of E. faecalis with bilirubin (yellow squares) or without bilirubin (white diamonds) was quantified by CFU plating. Bilirubin was titrated into E. faecalis cultures initially with bilirubin (red squares) and initially without bilirubin (orange squares) after 4 hours (marked by arrows). Error bars represent ± one standard deviation, n = 3, and (*) denotes a significant (P≤0.05) difference between treated samples and solvent-treated samples.

Article Snippet: Bacterial strains used in this study included Escherichia coli serotype O157:H7 (EHEC) strain EDL933 (ATCC# 700927) and 86-24 , , E. coli serotype O104:H4 2011 German outbreak (EAEC) (generously provided by Dr. Alison Obrien, Uniformed Health Services), Enterococcus faecalis strain OG1RF (ATCC# 47077) , E. faecalis strain X33 (ATCC# 27274), E. faecalis strain UWH 1936 (ATCC# 49533), E. faecalis strain NJ-3 (ATCC# 51299), Staphylococcus aureus strain MW2 (ATCC# BAA-1707), and Bacillus cereus strain NRS 248 (ATCC# 10987).

Techniques: Software, Imaging, Modification, Standard Deviation, Solvent

Journal: PLoS Pathogens

Article Title: A Product of Heme Catabolism Modulates Bacterial Function and Survival

doi: 10.1371/journal.ppat.1003507

Figure Lengend Snippet: Fold reduction in viability of bacteria exposed to bilirubin.

Article Snippet: Bacterial strains used in this study included Escherichia coli serotype O157:H7 (EHEC) strain EDL933 (ATCC# 700927) and 86-24 , , E. coli serotype O104:H4 2011 German outbreak (EAEC) (generously provided by Dr. Alison Obrien, Uniformed Health Services), Enterococcus faecalis strain OG1RF (ATCC# 47077) , E. faecalis strain X33 (ATCC# 27274), E. faecalis strain UWH 1936 (ATCC# 49533), E. faecalis strain NJ-3 (ATCC# 51299), Staphylococcus aureus strain MW2 (ATCC# BAA-1707), and Bacillus cereus strain NRS 248 (ATCC# 10987).

Techniques: Bacteria

Journal: PLoS Pathogens

Article Title: A Product of Heme Catabolism Modulates Bacterial Function and Survival

doi: 10.1371/journal.ppat.1003507

Figure Lengend Snippet: (A) Bacteria including EHEC (86-24), E. faecalis , S. aureus , and B. cereus were incubated with heme, biliverdin, bilirubin, and bilirubin ditaurate (0, 1, 5, 10, 20, 50, 75, 100, 250 and 500 µM for heme and bilirubin (B,C), or 500 µM for biliverdin and bilirubin ditaurate (A)) and membrane permeability monitored by propidium iodide fluorescence. (D) E. faecalis OG1RF was cultured to mid-log phase and exposed to bilirubin, alpha-tocopherol, and CCCP (each 100 µM). DiSC 3 (5) (1 µM final concentration), a fluorescent compound which increases in intensity when associated with polarized membranes, was supplemented into cultures before quantifying the fluorescence intensity at excitation 622 nm and emission 670 nm. Error bars represent ± one standard deviation, n = 3, and (*) denotes a significant (P≤0.05) difference while (**) denotes a non-significant difference (P>0.05) between treated samples and solvent-treated samples.

Article Snippet: Bacterial strains used in this study included Escherichia coli serotype O157:H7 (EHEC) strain EDL933 (ATCC# 700927) and 86-24 , , E. coli serotype O104:H4 2011 German outbreak (EAEC) (generously provided by Dr. Alison Obrien, Uniformed Health Services), Enterococcus faecalis strain OG1RF (ATCC# 47077) , E. faecalis strain X33 (ATCC# 27274), E. faecalis strain UWH 1936 (ATCC# 49533), E. faecalis strain NJ-3 (ATCC# 51299), Staphylococcus aureus strain MW2 (ATCC# BAA-1707), and Bacillus cereus strain NRS 248 (ATCC# 10987).

Techniques: Bacteria, Incubation, Membrane, Permeability, Fluorescence, Cell Culture, Concentration Assay, Standard Deviation, Solvent

Journal: PLoS Pathogens

Article Title: A Product of Heme Catabolism Modulates Bacterial Function and Survival

doi: 10.1371/journal.ppat.1003507

Figure Lengend Snippet: (A) E. coli (86-24), E. faecalis , B. cereus , and S. aureus were supplemented with resazurin and incubated with heme (50 µM), biliverdin (500 µM), bilirubin (50 µM), and bilirubin ditaurate (500 µM) for either 30 minutes or 2 hours. Unreduced resazurin was monitored by absorbance at 600 nm. (B) E. faecalis cultures were supplemented with resazurin and solvent (NaOH, Sol.), bilirubin (100 µM, BR), biliverdin (100 µM, BV), or TTF (1 mM, a known inhibitor of succinate dehydrogenase) while incubated in 1× PBS with 0.5% sucrose for 30 minutes at 37°C. (C) Similar to panel B, E. faecalis cultures were supplemented with resazurin and either superoxide dismutase (SOD, 1000 U/mL) or heat-inactivated superoxide dismutase (SODi, 1000 U/mL). (D) E. faecalis supplemented with resazurin and diluted into 1× PBS with 0.5% sucrose (+Suc.) or without sucrose (−Suc.) and incubated for 30 minutes at 37°C. (E) E. faecalis supplemented with resazurin and increasing amounts of bilirubin (1, 5, 10, 20, 30, 50, and 100 µM) in similar conditions as panels B and C. Error bars represent ± one standard deviation, n = 3, and the (*) denotes a significant (P≤0.05) difference while (**) denotes a non-significant difference (P>0.05) between treated samples and solvent-treated samples.

Article Snippet: Bacterial strains used in this study included Escherichia coli serotype O157:H7 (EHEC) strain EDL933 (ATCC# 700927) and 86-24 , , E. coli serotype O104:H4 2011 German outbreak (EAEC) (generously provided by Dr. Alison Obrien, Uniformed Health Services), Enterococcus faecalis strain OG1RF (ATCC# 47077) , E. faecalis strain X33 (ATCC# 27274), E. faecalis strain UWH 1936 (ATCC# 49533), E. faecalis strain NJ-3 (ATCC# 51299), Staphylococcus aureus strain MW2 (ATCC# BAA-1707), and Bacillus cereus strain NRS 248 (ATCC# 10987).

Techniques: Incubation, Solvent, Standard Deviation