P. aeruginosa strain PAO1, and four multidrug-resistant P. aeruginosa strains (TL2941, TL3147, TL3445 and TL3581) isolated from different diabetic foot ulcers from patients at the First Affiliated Hospital of Wenzhou Medical University in China were used in this study. Identification and antimicrobial susceptibility testing were conducted on clinical isolates using the VITEK MS and VITEK2 systems, respectively. All strains were cultured in LB medium (1% tryptone, 1.0% NaCl, 0.5% yeast extract) at 37 °C.
Minimum inhibitory concentration assay
The minimum inhibitory concentration (MIC) values of glucose (D-(+)-glucose; Sigma, St. Louis, USA) were determined using previously published methods, with some modifications . Briefly, bacterial suspensions (108 CFU/mL) of P. aeruginosa PAO1 and multidrug-resistant P. aeruginosa strains were added to LB broth (1%, v/v) that contained glucose at concentrations of 0, 50, 100, 150, 200, 250, 300 and 350 mg/mL) in 96-well microtiter plates, and then incubated at 37 °C for 18 h. The MIC was the lowest glucose concentration that caused complete inhibition of cell growth. All further experiments in this study used sub-minimum inhibitory concentrations (sub-MICs) of glucose.
Growth curve assay
The effects of glucose on the growth rate of P. aeruginosa strains were determined according to a method described previously, with some modifications . Briefly, overnight cultures were diluted 1:100 into fresh LB broth and incubated at 37 °C for 4 hours before adjusting the OD600 values to 0.1. A 100 μL aliquot of the subcultures were added to 96-well microtiter plates containing 100 μL LB with different concentrations of glucose (0, 100, 200, 300 and 400 mg/mL). LB medium and LB with glucose were used as negative controls. The microtiter plates were incubated at 37 °C, with shaking at 180 rpm. The absorbance of each sample at OD600 was measured every 1 h.
Biofilm formation assay
The crystal violet method was used to measure the biofilm-forming capacity of P. aeruginosa strains. Strains were initially grown in LB broth for 17 h, which were then diluted 1:100 in fresh LB broth supplemented with glucose (0, 50, 100, 150 and 200 mg/mL). A 100 μL aliquot of each sample was then transferred to a 96-well microtiter plate and incubated at 37 ℃ for 24 h. Wells containing media alone were used as references. Planktonic cells were removed, and the wells were washed twice with sterile water. Wells were then 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 described elsewhere, with some modifications . Briefly, glucose solutions at different concentrations (0, 50, 100, 150, 200 and 300 mg/mL) were added to molten swimming agar, which consisted of 0.1% tryptone, 0.05% yeast extract, 0.5% NaCl and 0.3% bacteriological agar (pH 7.2). A 2 µL aliquot of P. aeruginosa culture (≈107 CFU) was inoculated in the center of the agar and then incubated at 37 ℃ for 24 h.
Pyocyanin production was examined as described previously with modifications . Bacterial cultures containing different concentrations of glucose (0, 50, 100, 150 and 200 mg/mL) were grown for 17 h, and 1 mL samples were then centrifuged at 13,000 rpm for 5 min. The supernatant was collected and extracted with 600 μL chloroform, and then mixed by vortex (2 × 10 s). After centrifugation at 13,000 rpm for 5 min, the chloroform phase was transferred to a sterile tube and mixed with 0.5 mL of 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 OD510.
Elastase activity assay
The elastolytic activity of the cell-free culture supernatant of P. aeruginosa was determined using Elastin-Congo red (ECR; Sigma) as the substrate . 100 μL supernatants of P. aeruginosa incubated in different concentrations of glucose (0, 50, 100, 150 and 200 mg/mL) for 17 h were added to 900 μL of ECR buffer (100 mM Tris, 1 mM CaCl2, pH 7.5) containing 10 mg of ECR and incubated at 37 °C for 3 h. The reaction was terminated by adding 1 mL of 0.7 M sodium phosphate buffer (pH 6.0) and the tubes were placed in a cold water-bath. The insoluble ECR was removed by centrifugation at 10,000 rpm for 10 min, and the absorbance was measured at OD495.
Galleria mellonella larvae infection model
G. mellonella killing assays were performed as described previously, with some modifications . Eight larvae weighing between 200–250 mg were randomly selected for each P. aeruginosa strain. Overnight bacterial cultures were diluted 1:100 in fresh LB broth and incubated at 37 ℃ for 4 h. Cell suspensions were washed three times with phosphate-buffered saline (PBS) and resuspended in PBS to 103 CFU/mL. A 10 μL of bacterial suspension (103 CFU/mL) was injected into the last left proleg using a 25 μL Hamilton precision syringe and a 10μL of glucose at 200 mg/mL (or a 10μL of PBS as a control) was injected into the last right-side proleg at 2 h post infection. Larvae injected with 10 μl PBS were used as controls. G. mellonella were incubated at 37 ℃ in the dark and observed after 24 h, 48 h, 72 h and 96 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, as assessed with Kaplan-Meier analysis and a log-rank test.
Quantitative reverse transcription 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). RNA was extracted from P. aeruginosa isolates that were grown in fresh LB medium with or without 200 mg/mL glucose at 37 ℃ for 17 h. Total RNA was extracted from planktonic bacteria using an 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 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 a SYBR Green qRT-PCR Kit (TOYOBO) with specific primers listed in Additional Table S1. The rpsL gene was used as an internal control to normalize the data. Gene expression levels were calculated using the 2−△△Ct method.
All experiments were conducted independently with at least three replicates on different days, and results were expressed as mean ± standard deviation. The total area under the curve was calculated for the growth rate analysis. The results of the pyocyanin and elastase assays are presented as production percentage of the untreated (0 mg/ml) samples. A 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 than two groups. Statistical significance was determined at P < 0.05. Statistical analyses were performed using SPSS version 17.0 statistical software.