Strains and reagents
E.coli K12BW25113 was obtained from Guangdong Microbial Culture Collection Centre. Ampicillin and chloramphenicol were purchased from Sangon Biotech (Shanghai, China). Cefpirome, cefazolin, kanamycin, amikacin, tetracycline, erythromycin, ofloxacin and ciprofloxacin were purchased from Macklin (Shanghai, China). SYBR Green was purchased from Biosharp (Anhui, China).
Determination of minimum inhibitory concentration
Single colonies were chosen and incubated in Lysogeny Broth (LB) medium until saturation, transferred to fresh LB medium at 1:100 (v/v), incubated until the OD600 was in the range of 0.5, and the bacterial solution was diluted 100 times with saline. In a 96-well plate, 160 μL of LB medium was added to the first column and 90 μL of medium was added to subsequent columns. Antibiotic solution (20 μL) was added to the first column and mixed by pipetting, then 90 μL of solution was aspirated from the first column, added to the second column, and mixed by pipetting, and so on. Finally, 90 μL was aspirated from the last column and 10 μL of the bacterial solution was added to each well and incubated at 37 °C for 12 h. After incubation, the medium in the wells presented a clarified minimum concentration of the antibiotic as the MIC value of the strain for that antibiotic.
Subculture of artificially resistant bacteria
After E. coli was cultured to saturation in LB medium, the strains were transferred to fresh LB medium containing 1/2 MIC antibiotics at 1:100 (v/v) for generations. This process yielded the first generation after culture at a constant temperature (37 °C) at 220 rpm for 12 h. The MIC of each strain generation was measured until 4-, 16-, and 64-fold resistant bacteria were obtained. Thereafter, the first generation was used as the control group and generation 4-, 16-, and 64-fold resistant bacteria were used as experimental groups in this study.
Oxford cup method inhibition circle test
A single bacterial colony was removed from the plate, placed into 5 mL LB liquid medium, and incubated overnight at 220 rpm (37 °C) until OD600 was 1.0. The cultured bacterial solution (100 μL) was diluted 10 times with sterilized saline, then 80 μL of the bacterial solution was aspirated, added to 420 μL of sterilized saline and mixed. The plate was then shaken horizontally to ensure even distribution of the bacterial solution in each corner of the plate. Oxford cups (inner diameter 6 mm, outer diameter 8 mm, height 10 mm) were carefully placed on solid medium with forceps, 50 μL of antibiotics (ampicillin concentration gradient from 0, 2.5, 5, 10, 20, 40, 80, 160, 320, 640, 1280 and 2560 μg) were added to the Oxford cups, and the plates were incubated at 37 °C for 12 h . After incubation, the plates were removed, the diameter of the inhibition circle was measured using Vernier callipers, the data were recorded, and the plates were photographed.
Growth curve
To generate a curve showing the dilution and OD600, the suspensions were diluted 1/1.5/2/3/4 times, and the dilution times and specific OD600 values were recorded. The dilution times and OD600 values were used as horizontal and vertical coordinates to create the relationship curve between dilution and OD600, respectively, where (where y is the OD600 after dilution, x is the dilution, A and b are constants, A is the OD600 before dilution of the bacterial solution, and b is related to the strain).
To plot growth curves, single colonies were chosen from the plates and inoculated into 5 mL LB liquid medium at 220 rpm (37 °C) overnight. Bacteria were incubated until OD600=1.0 and transferred to 50 mL LB liquid medium at 1:100 (v/v). The optical density values of the culture solution were measured (OD600) using a spectrophotometer from 0 h to 16 h (measured every 2 h). From the sixth hour, 1 mL of fresh LB medium was aspirated into an EP tube, 500 μL of bacterial solution was added to it, the optical density values were measured after mixing, and the data were recorded. The data from the sixth hour and later were calculated by substituting to obtain a value at a dilution of 1. The actual OD600 value of the original bacterial solution was taken as the growth curve, with the OD600 of the first four hours as the vertical coordinate and time as the horizontal coordinate.
Test of colony morphology
A single colony was removed from the plates and placed into 5 mL LB medium for 12 h for activation. Thereafter, the activated strains were transferred to new medium at 1:100 (v/v) and incubated to OD600 values of 0.5. The bacterial broth was centrifuged at 8, 000 rpm for 5 min, the supernatant was discarded, and the wash was resuspended by adding an equal volume of saline and repeated twice. The wash was repeated twice with an equal volume of physiological saline and then diluted 40, 000 times with physiological saline. The diluted bacterial solution was taken (10 μL), spotted on solid medium, and incubated at 37 °C. Next, the colony morphology was recorded using photography at the 8th, 10th, 12th, and 14th hours.
Determination of intracellular ampicillin
To prepare cell-free extracts, single colonies of 64-fold MIC strains were removed from the plates overnight in advance and inoculated into 50 mL of LB medium for overnight activation. The activated bacterial solution (30 mL) was centrifuged at 8, 000 rpm for 5 min at room temperature, the supernatant was discarded, and the precipitate was washed twice with saline resuspension solution. After the last wash, the precipitate was resuspended in M9 medium, the bacterial solution was adjusted to OD600=0.5, 50 mL of the bacterial solution was transferred to a 100 mL conical flask, the final concentration of 320 μg/mL ampicillin was added, and bacteria were incubated at 37 °C, 220 rpm in a shaker for 4 h. After 4 h of incubation, the supernatant was discarded by centrifugation at 8, 000 rpm for 5 min at room temperature. Phosphate-buffered saline (PBS; 1 mL) was added to resuspend the bacterial solution, and the solution change was repeated twice. Finally, the cell suspension was sonicated in ice to clarify and centrifuged at 4 °C, 12, 000 rpm for 5 min, and the supernatant was collected to obtain a cell-free extract. According to the instructions of the BCA protein concentration determination kit (Shanghai Beyotime Biotechnology Co., Ltd.), liquid A and liquid B were mixed at 50:1 (v/v), 20 μL of the sample was added to the 96-well plate, and then 200 μL of the mixture was added. The protein concentration was calculated according to the standard curve by measuring the absorbance at 562 nm with an enzyme standard meter, and the sample was diluted with PBS until the protein concentration was standardised. The intracellular concentration of ampicillin in the strains was determined using a competitive ELISA, according to the instructions of the Ampicillin Rapid Test Kit (Green Spring Biotechnology Co., Ltd. Shenzhen, China).
Cell membrane permeability assay
A single colony was chosen from the plates and activated in 5 mL LB medium for 12 h. Then, the bacterial solution was centrifuged at 8, 000 rpm for 5 min, the supernatant was discarded, and the wash was repeated twice by adding an equal volume of PBS. The bacterial suspension was resuspended in PBS and adjusted to an OD600 of 0.2. SYBR Green (10, 000 ×) was diluted 100 times with DMSO and 1 μL was added to an opaque 96-well plate. The bacterial solution (99 μL) was added to the wells and mixed. The 96-well plate was incubated at 37 °C for 10 minutes, and the absorbance at 485/515 nm was measured using an enzyme marker.