Table 1.
Bacterial strains and plasmids used in this study.
Fig 1.
Hierarchical cluster analysis of the bactericidal effects of CA and its derivatives on E. coli cells in exponential growth phase.
Cell survival percentage after treatment was calculated based on the colony counts on the LB agar plates. Resulting tree figure was obtained using Cluster 3.0 software and Java Treeview. The cluster color bar was shown as [log (cell survival percentage ×100)-2]. If the given amount of drug failed to completely inhibit the bacterial growth, the corresponding values in the bar would be positive in red color; If the given amount of drug showed the bactericidal effects, the corresponding values in the bar would be negative in green color.
Fig 2.
Determination of MIC (A) and MBC (B) of Br-CA against E. coli.
MIC was defined as the lowest concentration of Br-CA to completely inhibit cell growth at 37°C in 96 wells plates for 24 hour and OD600 was monitored with the microplate reader to indicate the cell growth. MBC was defined as the lowest concentration of Br-CA to completely eradicate all the cells incubated at 37°C in 96 wells plates for 24 hour and surviving cells were evaluated using drop plating method after serial dilutions. ID stood for the initial cell density. Each data point or bar was indicated as the means of three replicates ± standard deviation.
Fig 3.
Killing curves of MG1655 cells in the stationary phase by Br-CA, Cipro and Amp.
Cell survival percentage after treatment was calculated based on the colony counts on the LB agar plates. Each data point or bar was indicated as the means of three replicates ± standard deviation.
Fig 4.
The persisters induced by toxin overexpression were eradicated by Br-CA.
(A) The persisters were induced by RelA overexpression and the effects of IPTG on the persister formation in MG1655/pCA24N; (B) The persisters were induced by TisB overexpression and the effects of IPTG on the persister formation in MG1655/pZS*24; (C) The effects of IPTG on the persister formation in MG1655. Cell survival percentage after treatment was calculated based on the colony counts on the LB agar plates. The arrows indicated that persister cells were eradicated completely and different colors showed different treatments. Each data point or bar was indicated as the means of three replicates ± standard deviation. Asterisks indicate a significant difference from their controls (P <0.05).
Fig 5.
Persister cells induced by chemicals were challenged by Br-CA.
Cells were pretreated with Rifa, Tetra or CCCP, respectively except the control group and then exposed to Cipro, Amp and Br-CA for 4 hour. Cell survival percentage after treatment was calculated based on the colony counts on the LB agar plates. The arrows indicated that persister cells were eradicated completely and different colors showed different treatments. Results are means from three independent experiments ± standard deviation. Each bar was indicated as the means of three replicates ± standard deviation. Asterisks indicate a significant difference from their controls (P <0.05).
Fig 6.
Thiourea and DTPA have no effect on the eradication of persisters by Br-CA.
The initial bacterial suspension (OD600 = 0.05 or OD600 = 1.0) was pretreated with or without Thio or DTPA iron chelator and then exposed to different concentrations Br-CA. Cell survival percentage after treatment was calculated based on the colony counts on the LB agar plates. The arrows indicated that cells were killed completely by Br-CA and different colors showed different treatments. Each bar was indicated as the means of three replicates ± standard deviation. Survival post treatment with Br-CA alone at the given concentration were set as the control in the corresponding group. Asterisks indicate a significant difference from their controls (P <0.05).