Colistin sulfate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Difco^TM Mueller Hinton Broth (MHB) was ordered from BD (Franklin Lakes, NJ, USA). All other chemicals and reagents used were of analytical grade.

The colistin-resistant E. coli strain SHP45 was preserved in National Risk Assessment Laboratory for Antimicrobial Resistance of Microorganisms in Animals, South China Agricultural University, Guangzhou, China. E. coli ATCC 25,922 obtained from the Chinese National Center for Surveillance of Antimicrobial Resistance (Beijing, China) was used as a reference strain.

L-thanatin (GSKKPVPIIYCNRRTGKCQRM) and Stomoxyn [21] (RGFRKHFNKLVKKVKHTISETAHVAKDTAVIAGSGAAVVAATG) was synthesized by GL Biochem (Shanghai) Ltd. (Shanghai, China) using classic Fmoc methodology. The synthetic peptide was purified to over 90% chromatographic homogeneity by reverse-phase high-performance liquid chromatography (RP-HPLC). The purified peptide was then analyzed using a MALDI-TOF mass spectrometer. The molecular masses of the detected L-thanatin and stomoxyn were 2435.95 and 4474.19, which were in good agreement with the calculated masses (2435.97 Da and 4479.22).

Fresh E. coli colonies were inoculated into 5 ml sterile MHB and grow at 37˚C for 16 h. The bacteria cells were collected by centrifugation and resuspended with saline, the cell density was adjusted to approximately 2.5 × 10⁹ CFU/ml. 50 µl of the suspension was then mixed with 50 ml of autoclaved MHA maintained at 50˚C. 10 ml of the resulting mixture was poured into each 10-cm-diameter sterile petri dishes. The dishes were left ajar in a super-clean bench for 1 h until the agar solidified. Wells of 2.7 mm diameter were made with a sterile puncher. 5 ul of colistin, stomoxyn and thanatin solution at the same concentration (1 mg/ml in water) were added to separate well. Sterile deionized water was used as the negative control, stomoxyn was used as a positive control. The plates were incubated at 37˚C for 16 h and the clear zones of inhibition were observed and the diameters of the zones were measured. Data were reported as mean and standard deviation (SD).

The minimum inhibitory concentration (MIC) and the bactericidal concentration (MBC) of colistin and L-thanatin were determined by microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guideline M31-A3 (National Committee for Clinical Laboratory Standards (NCCLS): 2004, Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals, informational supplement M31-S1. NCCLS, Wayne, PA). Briefly, Mueller-Hinton (MH) broth (100 µl) containing E. coli (5 × 10⁵ CFU/ml) was added to 100 µl of the culture medium containing colistin (0.25 to 128 µg/ml in serial 2-fold dilutions) thanatin (0.39 to 100 µg/ml in serial 2-fold dilutions). The plates were incubated at 37˚C for 20 h in an incubator. The MBC was reported as the lowest concentration producing a 99.9% reduction in bacterial viable count in the sub-cultured well contents, relative to the initial inoculum. Concisely, immediately after inoculation, 0.1 ml of the positive control well were subcultured onto Mueller-Hinton agar (MHA) plates and incubated at 37˚C for 16 h. Likewise, immediately after MIC testing, 0.1 ml of bacterial cultures that showed negative bacteria growth were spread evenly on MHA plates and were grown under the same condition as the positive control. The colonies on each plate were enumerated.

To monitor the bacterial growth responding to the treatments of antibiotic substances, equal volume of 2 × L-thanatin solutions were added to separate wells bacteria cultures (OD600 = 0.04) to achieve different final concentrations (0.25, 0.5, 1.0, 1.5 μg/ml) with the addition of MHB as a control. The optical densities at 600 nm for the colistin-resistant strain E. coli SHP45 and the reference strain E. coli ATCC 25,922 were recorded using a Biotek Synergy 2 microplate reader at 37˚C at 2-h intervals for 24 h. The growth curves were drawn by plotting optical densities on the Y-axis and the growth times on the X-axis. Duplicate experiments were done and for the construction of growth curves.

Chequerboard assay was employed to study the interaction between L-thanatin and colistin against E. coli according to the protocol published by others [21] . Serial twofold dilutions of the antimicrobials were combined in each well of a 96-well microtitre plate so that each row (and column) contained a fixed concentration of one agent (50 µl) and increasing concentrations of the other agent (50 µl). Mueller-Hinton (MH) broth (100 µl) containing E. coli (5 × 10⁵ CFU/ml) was then added to each well. The MICs for each combination were determined as described above.

Fractional inhibitory concentration (FIC) was calculated based on the MIC data. The FIC of an individual antimicrobial compound is defined as the ratio of the concentration of the antimicrobial in the inhibitory concentration with a second compound to the concentration of the antimicrobial by itself [16] . The formula is as follows: FIC_A = MIC of A with B/MIC of A. The FIC index for each combination was then calculated as the sum of FIC_A and FIC_B. The interaction was defined as synergistic if the FIC index was ≤0.5, additive if the FIC index was >0.5 and ≤1.0, indifferent if the FIC index was >1.0 and ≤2.0, and antagonistic if the FIC index was >2.0 [22] .

Clear zones of inhibition were observed for Stomoxyn, colistin and thanatin spotted wells except the negative control well (Figure 1). The zone diameter generated by thanatin was comparable to that produced by colistin. But upon close inspection, the transparency of inhibition zone produced by thanatin was lower than that generated by colistin. In addition, both colistin and thanatin produced larger inhibition zones against ATCC25922 than SHP45 (Table 1).

As shown in Table 2, both E. coli strains ATCC 25922 and SHP45 had identical MIC and MBC values towards colistin. However, the colistin MIC/MBC values between the two strains were significantly different. The MIC/MBC value of SHP45 to colistin (4 µg/ml) was 3-fold higher than that of ATCC 25922 (1 µg/ml). L-thanatin exerted potent antibacterial effects against both colistin-susceptible E. coli ATCC25922 and colistin-rsistant E. coli SHP45. The MIC values for these two strains were the same (3.12 µg/ml). Notably, the MBC value for ATCC 25922 was same as its MIC value; but the MBC value for SHP 45 was 6.25 ug/ml, which was 1-fold higher than its MIC.

*: Data were obtained from three repetitive experiments.

On evaluating the effect of different concentrations of thanatin on the growth of E. coli, it was found that 1 µg/ml L-thanatin significantly inhibited the growth of both ATCC25922 and SHP45 strains. Total growth inhibition of both strains was achieved at dosages of 1.5 ug/ml (Figure 2).

*: all values were obtained from three repetitive experiments.

To test the interaction of L-thanatin and colistin against E. coli, the checkerboard assay was conducted to test the effect of different combinations of the above two antimicrobials on the growth of the colistin-susceptible ATCC25922 reference strain and the colistin-resistant SHP45 strain. As a result, different modes of interaction (additive and indifferent) were observed. However, no synergism or antagonism was obtained with all combinations. The combinations that had additive effect were summarized in Table 3. The best calculated FICI for ATCC 25922 was 0.625 obtained with 1.56 µg/ml of thanatin and 0.125 µg/ml of colistin. The best calculated FICI for SHP45 was same as ATCC25922, but was obtained with 1.56 µg/ml of thanatin and 0.25 µg/ml of colistin or with 2 µg/ml of colistin and 0.39 µg/ml of thanatin.