Bacterial strains and chemicals
A total of 8 non-duplicated clinical COL-R P. aeruginosa were recovered from the First Affiliated Hospital of Wenzhou Medical University in China. Among them, except for TL1671 derived from trauma seepage surface, the other 7 COL-R P. aeruginosa strains were isolated from the sputum surface. These strains were all identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; bioMérieux, Lyon, France). P. aeruginosa ATCC27853 was used as the control. Colistin and AA were purchased from Wenzhou Kangtai Biological Technology Co., Ltd (Zhejiang, China) and Sigma-Aldrich (Saint Louis, USA), respectively.
Antimicrobial susceptibility test
The minimum inhibitory concentration (MIC) of colistin and AA were determined by using the cationic adjusted Mueller–Hinton broth (CAMHB) microdilution method . Respectively, colistin or AA was diluted twice and subjected to serial two-fold dilutions from 128 to 0.0625 μg/mL for colistin and 5–0.005% (v/v) for AA prepared in CAMHB 96-well microtiter plates. A final bacterial suspension of 1.5 × 106 CFU/mL was added to each well, and the plate was incubated with colistin or AA at 37℃ for 18 h. The interpretation of the antimicrobial susceptibility test was based on the breakpoint point of antibiotics provided by 2020 CLSI (intermediate ≤ 2 μg/mL; resistant ≥ 4 μg/mL), with each MIC test verified as a duplicate .
Bacterial growth monitoring
To assess the effect of AA on COL-R P. aeruginosa growth, the bacterial cultures were grown in the absence or presence of AA at 37℃ for 24 h, followed by measurement of absorbance at 600 nm every 2 h. The bacterial growth curve for untreated cultures (control condition) and treated cultures was determined by plotting the values against time .
Biofilm-formation inhibition assay
P. aeruginosa was cultured overnight on Columbia blood plates. The bacterial suspension was prepared and adjusted to 0.5 McFarland and diluted 1:100 in fresh Luria–Bertani (LB) broth. The suspension was then spread on 96-well plates and cultured overnight in the absence or presence of AA at 37℃ for 24 h. The culture supernatant was discarded after incubation. The plates were washed thrice with water to remove any remaining planktonic cells. Biofilms formed on the plates were stained for 15 min with 1% crystal violet, the excess dye was removed by washing thrice, and the bound crystal violet was solubilized in 95% ethanol. The absorbances were measured at 600 nm .
Biofilm eradication assays
The removal of preformed biofilms by using AA was performed as described earlier . Briefly, the bacterial suspension prepared in LB broth was added to a 96-well microtiter plate and incubated for 24 h for biofilm formation. Then, the planktonic cells were removed after incubation. The established biofilm cells were treated with or without AA within a fresh LB medium. Then, the microtiter plate was incubated at 37 °C for 24 h, and the biofilm cells were quantified after staining with 0.1% crystal violet according to the procedure described previously . The experiments were conducted in triplicate. p < 0.05 was considered to indicate statistical significance.
P. aeruginosa has a single polar flagellum and type-IV pili that enables them to swim in low-agar (< 0.4%) medium and propagate at the surface interfaces. In addition to swimming and twitching, P. aeruginosa can propagate on semisolid surfaces (i.e., 0.4–1.0% agar) in a coordinated manner through swarming motility . The effect of AA on COL-R P. aeruginosa swimming, swarming, and twitching motilities were determined as described previously . To monitor the swimming activity, 2 μL of the bacterial suspension was cultured overnight and spotted onto plates containing 0.3% (w/v) Bacto agar, 0.2% casamino acids (w/v), and 30 mM glucose in the presence or absence of subinhibitory concentrations (1/2 × MIC, 1/4 × MIC, 1/8 × MIC, 1/16 × MIC) of AA or hydrochloric acid (HA) corresponding to the pH of each concentration of AA, followed by incubation for 24 h at 37 °C. To monitor the swarming activities, 2 μL of the bacterial suspension cultured overnight was spotted onto the centers of treated and untreated swarming plates composed of 0.4% (w/v) Bacto agar and LB supplemented with 0.5% (w/v) casamino acids and 0.5% (w/v) glucose, followed by incubation at 37 °C for 24 h. For the twitching assay, P. aeruginosa was inoculated onto the bottom of a Petri dish containing subinhibitory concentrations (1/2 × , 1/4 × , 1/8 × , 1/16 × MIC) of AA or HA corresponding to the pH of each concentration of AA by stabbing a toothpick through a 2-mm thin layer of LB medium supplemented with 0.2% casamino acids, 30 mM glucose, and 1.5% Bacto agar. After incubation for 24 h at 37 °C, the agar was gently removed, and the Petri dish was air-dried. Then, 1% crystal violet solution was used to stain the plate agar interface for 10 min. Finally, the Petri dish was rinsed, and the crystal violet-stained twitching pattern was evaluated. The migration distance around the incubation site was also measured. The migration distance was found to be directly proportional to the motility ability.
P. aeruginosa produces a secondary metabolite, pyocyanin, which can change the level of intracellular redox and induce oxidative damage to the host, making it one of the main causes of death in patients infected with P. aeruginosa . The effect of AA on pyocyanin production was measured as described previously . P. aeruginosa cultures were grown for 16–20 h in the presence or absence of subinhibitory concentrations (1/2 × , 1/4 × , 1/8 × , 1/16 × MIC) of AA. The pyocyanin concentration was estimated by vortexing 7.5 mL of filtered supernatant with 4.5 mL of chloroform until the color changed to greenish blue. The samples were then centrifuged (10,000 × g for 10 min), and 3 mL of the resultant blue-colored liquid was transferred into a fresh tube containing 1.5 mL of 0.2 M HCl and agitated until the blue color changed to pink. The absorbance of the pink layer was measured at 520 nm after it was transferred into a cuvette . The absorbance obtained was proportional to the pyocyanin content.
Elastase is a protease secreted by P. aeruginosa that interacts with the host during pathogen infection and plays a key role in invasiveness . The effect of AA on elastase production by P. aeruginosa was measured as described elsewhere . Briefly, P. aeruginosa cultures were grown for 16–20 h with subinhibitory concentrations (1/2 × , 1/4 × , 1/8 × , 1/16 × MIC) of AA or no AA (control). Filtered supernatants were mixed in the ratio 2:1 with phosphate buffer (0.1 M, pH 6.3) and 2 mg/mL elastin Congo red (Sigma). For 24 h, the mixture was incubated at 37℃ under 200 rpm shaking. A spectrophotometer zeroed on an elastin Congo red sample cultured with medium alone was used to determine the absorbance of the supernatant at 495 nm after centrifugation . The absorbance was proportional to the elastase content.
Cell viability assay
RAW 264.7 macrophages (5 × 104 macrophages in 100 μL of complete DMEM) were seeded in a 96-well tissue culture plate and incubated at 37℃ under a 5% CO2 atmosphere. After 12 h of incubation, 10 μL of AA prepared in a series of concentrations (0.312%, 0.156%, 0.078%, 0.039%, 0.0195%, and 0.010% were added to the cell culture medium. P. aeruginosa supernatant treated with or without AA was added to the cell culture media. As a control, LB media was added to the cell culture media and incubated for 12 h. After 12 h, the cells cultured with the tested compounds were washed with phosphate-buffered saline (PBS) and incubated for 4 h in 100 μL of 10% CCK8 solution. The absorbance of the converted dye in living cells was measured at the wavelength of 450 nm .
The reducing potential was determined with Resazurin (PrestoBlue; ThermoFisher Scientific, Waltham, Massachusetts, USA), wherein resazurin was reduced by the cell metabolic activity. The mid-log-phase cells were transplanted into a black polystyrene 96-well plate containing varied concentrations (1 × , 1/2 × , 1/4 × MIC) of AA. AA (prepared in a series of concentrations) was added, and the plate was incubated with shaking for 30 min. Then, 20 μL Resazurin was added to the plate 5 min before the indicated time point . The plate was then incubated with shaking in the dark at room temperature for 5 min. Finally, fluorescence readings were taken (excitation wavelength[ex], 570 nm/emission wavelength[em], 650 nm) using the BioTek Synergy H1 plate reader .
Outer membrane permeability assay
Outer membrane permeability was assessed by the 1-N-phenylnaphthylamine (NPN) uptake assay . The fluorescence of this probe increases when incorporated into the hydrophobic core of a permeabilized outer membrane. A graded series of AA concentrations was prepared (1 × , 1/2 × , 1/4 × , 1/8 × , 1/16 × MIC). HA corresponding to the pH of each concentration of AA and colistin (2 μg/mL) served as the positive control was added to mid-log-phase cells. The cultures were then sampled at 2 h. The cells were pelleted, washed twice, and resuspended in PBS. Finally, the NPN solution (final concentration, 30 μM) was added to the cells and incubated at 37 °C for 30 min. The fluorescence was immediately measured using the BioTek Synergy H1 plate reader (λexc/λem: 340/410 nm).
Inner membrane permeability assays
To measure the disruption of the inner membrane barrier function, the membrane integrity dye propidium iodide (PI) was used. PI is a nucleic acid dye that cannot pass through normal membranes owing to their barrier action, but it can dye the nucleus red in necrotic cells with altered membrane permeability . A graded series of AA concentrations (1 × , 1/2 × , 1/4 × , 1/8 × , 1/16 × MIC) were prepared. HA corresponding to the pH of each concentration of AA and colistin (2 μg/mL) served as the positive control was added to mid-log-phase cells. The cultures were sampled at 2 h. The cells were pelleted, washed twice, and resuspended in PBS. Finally, PI (50 μg/mL, Life Technologies) was added and the cells were incubated at 37 °C for 30 min and then monitored (ex, 535/em, 617 nm) using the BioTek Synergy H1 plate reader . Fluorescent images were obtained using a fluorescence microscope (Nikon Eclipse 80i, Nikon, Tokyo, Japan) using red filters to visualize the PI-stained cells. This microscope was equipped with the Nikon DS-Ri2 high-definition color digital camera and the Nikon software NIS-elements F imaging software for subsequent analyses .
Membrane potential assays
We monitored the effects of AA exposure by fluorescence assay using the carbocyanine dye, 3,3'-diethylthiacarbocyanine iodide [DisC2(3)] (Invitrogen), which is a good indicator of membrane potential. A graded series of AA concentrations was prepared (1 × , 1/2 × , 1/4 × , 1/8 × , 1/16 × MIC). HA corresponding to the pH of each concentration of AA and colistin (2 μg/mL) served as the positive control was added to mid-log-phase cells. The cultures were sampled at 2 h. The membrane potential was measured using the potentiometric fluorescent probe [DisC2(3)]. Mid-log-phase cells were diluted to an OD600 of 0.4. The culture was incubated at 37℃ for 15 min after [DisC2(3)] was added to a final concentration of 2 mM. The plates were monitored (ex, 488 nm; em, 620 nm) on the BioTek Synergy H1 plate reader .
Quantitative reverse transcription PCR (qRT-PCR)
The effects of AA on the expression levels of P. aeruginosa QS circuit genes (i.e., lasR, rhlR, rhlI, rhlA, and pqsA) and T3SS circuit genes (i.e., exoT, exoS, exsA, and exoY) and the flagellin gene (fliC) were evaluated by qRT-PCR, as described previously [35,36,37,38]. P. aeruginosa TL1671 and TL2314 was incubated in fresh LB broth at 37℃ under 180 rpm until reaching the logarithmic growth phase (OD600 0.5–0.6). The cultures were then treated with 1/2 × MIC of AA or no AA (control) for 4 h. Total RNA was extracted from planktonic bacteria using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s instructions. Purified RNA was reverse transcribed into cDNA using the Cdna Synthesis Kit (TaKaRa, Tokyo, Japan) in accordance with the manufacturer’s instructions. The gene expression levels were measured with qRT-PCR using the TB Green Premix Ex Taq II (Tli RNase H Plus) (2 ×) (Takara) with specific primers listed in Table S2. We use the rpsL gene as the reference gene. In the analysis of qRT-PCR results, according to the reference [36, 39], normalized expression of each gene was calibrated against corresponding mRNA expression by control group (no AA treated group). Control group as an internal standard was assigned a value of 1. 2−ΔΔCt was used to calculate the multiples of gene expression in the 1/2MIC group relative to the control group.
The statistical significance of the differences between the control and experimental groups was evaluated by the Student’s t-test. For all analyses: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns P > 0.05.