Bacterial strains and antimicrobial susceptibility profiling
Four multidrug-resistant P. aeruginosa strains (TL2941, TL3147, TL3445 and TL3581) clinically isolated from different diabetic foot ulcers from the First Affiliated Hospital of Wenzhou Medical University in China, and P. aeruginosa strain PAO1 were used in this study. Identification and antimicrobial susceptibility testing were conducted on clinical isolates using VITEK MS system and VITEK2 system, respectively. All strains were kept in our laboratory and propagated in LB medium (1% tryptone, 1.0% NaCl, 0.5% yeast extract) at 37 °C.
Determination of minimum inhibitory concentration
The minimum inhibitory concentration (MIC) values of glucose (D-(+)-Glucose, Sigma, St. Louis, USA) were determined as previously published methods, with some modifications . Briefly, bacterial suspension (108 CFU/ mL) of P. aeruginosa PAO1 and multidrug-resistant P. aeruginosa strain were added to LB broth (1%, v/v) with glucose at concentration gradients (0, 50, 100, 150, 200, 250, 300 and 350 mg/mL) in 96-well microtiter plates, then incubated at 37 °C for 18 h. The MIC was the lowest concentration of glucose with visible inhibition of cell growth. The MICs of glucose against all tested P. aeruginosa are recorded in Table 1. All further experiments in this study were conducted at sub-minimum inhibitory concentrations (sub-MICs).
Growth curves asssay
Effects of glucose on the growth of all tested P. aeruginosa strains were determined according to the method described previously, with some modifications . Briefly, the overnight cultures were diluted 1:100 into fresh LB broth for incubation and OD600 values of subcultures were adjusted to 0.1, then added to the 96-well microtiter plates containing LB with different final concentrations of glucose (0, 50, 100, 150 and 200 mg/mL). LB medium and LB with glucose were used as negative control. The 96-well microtiter plates were incubated at 37 °C. The absorbance of each sample at OD600 nm was measured every 1 h.
Biofilm formation inhibition
Crystal violet method was used to test the biofilm forming capacity of all P. aeruginosa. Briefly, all selected strains were grown overnight in LB broth. The overnight cultures were then diluted 1:100 in fresh LB broth supplemented with different glucose concentrations (0, 50, 100, 150 and 200 mg/mL). A total of 100μL of each dilution were added to a 96-well microtiter plate and incubated at 37 ℃ for 24 h. Wells containing media alone were used as blank. Planktonic cells were removed and the wells were washed twice with sterile water, then the wells were stained with 150 μL 0.1% crystal violet for 10 min and rinsed twice with sterile water. Stained biofilms were solubilized with 95% ethanol and quantified by measuring the OD595 using a microplate reader.
Swimming motility assay
The swimming assay was conducted as prior published methods, with some modifications . Briefly, the filtered different concentrations of glucose were added to molten swimming agar (pH 7.2), which consisted of 0.1% tryptone, 0.05% yeast extract, 0.5% NaCl, 0.3% Bacteriological agar. The molten agar was then dispensed onto Petri dishes after gentle mixing. Once the agar with glucose (0, 50, 100, 150, 200 and 300 mg/mL) was solidified, 2µL of P. aeruginosa culture was inoculated in the center of the agar and then incubated at 37 ℃ for 24 h. We used swimming agar and swimming agar with 300 mg/mL glucose as the control.
Pyocyanin production was examined according to a protocol described previously with modification . 1 ml of the bacterial cultures under different concentrations of glucose conditions (0, 50, 100, 150 and 200 mg/mL) were subject to centrifugation at 13,000 rpm for 5 min. The supernatant was collected and extracted with 600 μl chloroform following vortex for 10s twice. After centrifugation at 13,000 rpm for 5 min, the chloroform phase was transferred into a sterile tube, and subsequently mixed with 0.5 ml 0.2 M HCl followed by gentle shaking to transfer the pyocyanin to the aqueous phase. Pyocyanin was determined by measuring the absorbance of the aqueous phase at 510 nm.
Elastase activity assay
The elastolytic activity of the cell-free culture supernatant of P. aeruginosa was determined following the method described previously by using Elastin-Congo red (ECR; Sigma, St. Louis, USA) as the substrate . Then, 100 μL supernatants of P. aeruginosa incubated in different concentrations of glucose (0, 50, 100, 150 and 200 mg/mL) were added to 900 μl of ECR buffer (100mM Tris, 1mM CaCl2, pH 7.5) containing 10 mg of ECR and incubated at 37 °C for 3 h. The reaction was terminated by adding 1ml of 0.7 M sodium phosphate buffer (pH 6.0) and the tubes were placed in cold water bath. The insoluble ECR was removed by centrifugation at 10,000 rpm for 10 min and then the absorbance was measured at OD495 nm.
Infection model of Galleria mellonella larvae
mellonella killing assays were carried out on the P. aeruginosa as described previously, with some modifications . Nine larvae weighing between 200 mg-250 mg were randomly selected for each strain. The overnight cultures were diluted 1:100 in fresh LB broth supplemented with different concentrations of glucose (0, 100, 150 and 200 mg/mL) and incubated at 37 ℃ for 8h. Then the bacteria were rinsed three times using phosphate-buffered saline (PBS) and resuspended in PBS. A 10 μL of bacterial suspension (107 CFU/mL) was injected into the last left proleg using a 25 μL Hamilton precision syringe. Larvae injected with 10 μl PBS were used as control. The insects were incubated at 37 ℃ in the dark and observed after 24 h, 48 h and 72 h. Larvae were considered dead when they repeatedly failed to respond to physical stimuli. The primary outcome for the insect model was rapidity and extent of mortality of G. mellonella assessed with Kaplan-Meier analysis and log-rank test.
Quantitative reverse transcription PCR (qRT-PCR)
The effects of hypertonic glucose on the expression levels of P. aeruginosa major QS circuit genes (lasI, lasR, rhlI, and rhlR) were evaluated using quantitative reverse transcription PCR (qRT-PCR). For RNA extraction, P. aeruginosa isolates were grown in fresh LB medium with or without 200 mg/mL glucose at 37 ℃ for 17 h. Total RNA was extracted using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The extracted RNA samples were stored at −80 ℃. Purified RNA was reverse transcribed into cDNA for qRT-PCR analysis using a cDNA synthesis kit (TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions. Gene expression levels were measured with qRT-PCR using a 7500 RT-PGE system (TOYOBO, Osaka, Japan) and SYBR Green qRT-PCR Kit (TOYOBO) with the specific primers listed in Table 2. The rpsL gene was used as an internal control to normalize the data. Gene expression levels were calculated using 2−△△Ct method.
All experiments were conducted independently with at least two replicates on different days, and results were expressed as mean ± standard deviation. The total area under the curve was calculated for analysis on growth. T-test was used to evaluate the significance of differences between two groups. One-way analysis of variance (ANOVA) was performed to analyze the significance among more groups. Statistical significance was determined at P < 0.05. Statistical analyses were performed using SPSS version 17.0 statistical software.