Figure 1.
Ciclopirox and ciprofloxacin MICs for E. coli clinical isolates.
The left y-axis is the average MIC in µg/ml for ciclopirox (light gray bar) and ciprofloxacin (dark gray bar). The clinical resistance breakpoint for ciprofloxacin is 4 µg/ml and an asterisk indicates isolates with MICs below this breakpoint. MIC measurements were repeated three times, and the average value and standard deviation is shown. The right y-axis denotes the number of antibiotic classes (aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, monobactams, nitrofurans, penicillins, combination penicillins, and sulfamethozazole-trimethoprim) each isolate is resistant to, as indicated by the solid line.
Figure 2.
Time-kill and growth curves for E. coli with increasing ciclopirox concentrations.
ATCC® 25922™ E. coli were grown with the indicated ciclopirox concentrations. (A) Cell densities were measured for three independent cultures by counting the number of colony forming units per 1 ml of culture for 12 hours. (B) The corresponding OD600 of growing cultures were measured over 12 hours. Error bars are the standard deviation from the mean.
Figure 3.
Effects of metals on exposure to ciclopirox for E. coli.
Growth curves measured for three independent cultures of ATCC® 25922™ E. coli grown in (A) LB alone or (B) LB supplemented with 5 µM FeCl3, (C) 50 µM FeCl3, (D) 500 µM ZnCl2, (E) 500 µM MgCl2, (F) or 500 µM NiCl2. Error bars are the standard deviation from the mean.
Figure 4.
Effects of hydrogen peroxide exposure on bacterial response to ciclopirox.
ATCC® 25922™ E. coli were grown to mid-logarithmic phase (OD600 = 0.4) and then subjected to either water or 5 µM H2O2 for 20 minutes. Colony forming units (CFUs) were measured per 1 ml of E. coli grown with either 0, 2.5, 5.0, or 7.5 µg/ml ciclopirox or 1.0 µg/ml chloramphenicol. Each triangle represents a result from an independent culture and the gray bar shows the average. Statistical significance was measured using Student’s t-test.
Figure 5.
Effect of GalE on ciclopirox inhibition of bacterial growth.
Growth curves for three independent cultures of TransforMax™ EC100™ Electrocompetent E. coli (A) without overexpression plasmid, (B) with empty plasmid pCA24N, or (C) with pCA24N-galE grown with the indicated ciclopirox concentrations. Error bars are the standard deviation from the mean. (D) Schematic representation of GalE epimerization of UDP-galactose to UDP-glucose coupled to the activity of UDP-glucose dehydrogenase (UGD). (E) Using the assay schematized in D, the average rate of NADH formation with or without ciclopirox was measured three independent times per ciclopirox concentration. Error bars are the standard deviation from the mean.
Figure 6.
Effect of ciclopirox on strains involved in the galactose and lipopolysaccharide biosynthesis pathways.
(A) Schematic representations of the galactose salvage pathway, (B) LPS biosynthesis pathway proteins for E. coli K-12 strain. Note the O-antigen is not present in K-12 strains, but is depicted for reference for where the O-antigen can attach. Abbreviations include fructose (Fru), glucose (Glc), galactose (Gal), heptose (Hep), N-acetylglucosamine (GlcN), 3-deoxy-d-manno-oct-2-ulopyranosonic acid (Kdo), and phosphate (P). (C) Ciclopirox MICs for Keio parent, BW25113, and single gene Keio deletion strains were measured in three independent cultures. These genes encode enzymes involved in the galactose metabolism and LPS biosynthesis pathways. Student’s t-test was used to assess significance.
Figure 7.
Effect of ciclopirox on E. coli LPS structure.
LPS was purified from E. coli clinical isolate ATCC®25922™ or K-12 strain MG1655 that had been either incubated with 9 µg/ml ciclopirox as indicated. LPS was subjected to 12.5% Tris-glycine-SDS-PAGE. Lanes 1 and 2 are LPS from two independent LPS purifications. This result was repeated six times with the same results.
Table 1.
Ciclopirox and other Antibiotic MICs in K. pneumoniae, A. baumannii, and P. aeruginosa clinical isolates.