All the chemical reagents used in the synthesis reactions were analytical-grade and available from commercial resources without further purification. All the reactions were monitored by analytical thin-layer chromatography (TLC) using silica gel TLC plates (GF254). Silica gel (200–300 mesh) was used for chromatography. The ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy were recorded on a JEOLECA400 spectrometer, with TMS as an internal standard at ambient temperature. All coupling constants were reported in Hertz. Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). High-resolution mass spectra (HRMS) were obtained by electrospray ionization (ESI) using an Agilent TOF G6230A mass spectrometer. The synthesis of compound Str7410 was shown in Scheme 1. Intermediates 1, 2, and 3 were prepared by the following routes.

To a solution of N-tert-butoxycarbonyl-1,2-ethylenediamine (4.49 g, 28.0 mmol) in CH₃CN (60 ml) was added K₂CO₃ (1.55 g, 11.2 mmol), then the mixture was heated to 65°C. At this temperature, 1-bromoheptane (1 g, 5.6 mmol) in CH₃CN (10 ml) was added dropwise to the mixture. The mixture was stirred at 80°C for 6 h and cooled to room temperature. The solvent was concentrated in vacuo. The residue was dissolved in CHCl₃, washed with brine, dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by flash silica gel chromatography (1% triethylamine, petroleum ether: ethyl acetate = 1: 3) to afford 1 as colorless oil (0.91 g, 63%). ¹H NMR [400 MHz, dimethylsulfoxide (DMSO)-d₆] δ 6.69 (t, J = 6.8 Hz, 1H), 2.98 (q, J = 6.2 Hz, 2H), 2.53–2.48 (m, 2H), 2.46 (t, J = 7.0 Hz, 2H), 1.37 (s, 11H), 1.25 (s, 8H), and 0.86 (t, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 156.14, 77.94, 49.52, 31.83, 30.10, 29.21, 28.75, 27.29, 22.59, and 14.46. HRMS (ESI) of C₁₄H₃₀N₂O₂ [M + H]⁺ calcd., 259.2381; found 259.2380.

To a cold (0°C) solution of trifluoroacetic acid (2.5 ml) and CH₂Cl₂ (7.5 ml) was added tert-butyl (2-(heptylamino)ethyl)carbamate (0.5 g, 1.9 mmol), then the mixture was warmed to room temperature and stirred for 2 h. The solution was concentrated in vacuo and used directly in the next step without further purification.

To a solution of N¹-heptylethane-1,2-diamine (0.3 g, 1.9 mmol) in CH₃CN (40 ml) was added K₂CO₃ (1.05 g, 7.6 mmol) and ethyl 2-bromoacetate (0.98 g, 5.9 mmol). Then, the mixture was stirred at 45°C for 3 h and cooled to room temperature. The solvent was concentrated in vacuo. The residue was dissolved in CHCl₃, washed with brine, dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by flash silica gel chromatography (1% triethylamine, CH₂Cl₂: CH₃OH = 20: 1) to afford 3 as colorless oil (0.44 g, 56%). ¹H NMR (400 MHz, DMSO-d₆) δ 4.09 (t, J = 7.1 Hz, 6H), 3.57 (s, 4H), 3.37 (s, 2H), 2.93 (d, J = 5.0 Hz, 2H), 2.89 (d, J = 4.9 Hz, 2H), 2.86–2.81 (m, 2H), 1.50 (m, 2H), 1.27 (s, 8H), 1.21 (t, J = 7.1 Hz, 9H), and 0.87 (d, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 171.47, 169.40, 60.46, 56.23, 55.06, 54.58, 52.49, 50.32, 31.64, 28.90, 26.73, 25.54, 22.53, 14.60, and 14.43. HRMS (ESI) of C₂₁H₄₀N₂O₆ [M + H]⁺ calcd., 417.2959; found 417.2960.

Diethyl 2,2'-((2-((2-ethoxy-2-oxoethyl)(heptyl)amino)ethyl)azanediyl) diacetate (0.2 g, 0.5 mmol) was added to concentrated hydrochloric acid (15 ml). The mixture was heated to 100°C and stirred for 12 h. Then, the residue was cooled to room temperature and concentrated in vacuo. The residue was added to concentrated hydrochloric acid (15 ml), and the mixture was stirred at 100°C for 12 h and concentrated in vacuo. The residue was recrystallized with ethanol (5 ml) and methyl tert-butyl ether (MTBE, 45 ml), filtered, and washed with MTBE to afford 4 as white solid (86.2 mg, 54%). ¹H NMR (400 MHz, DMSO-d₆) δ 4.23 (s, 2H), 3.63 (s, 4H), 3.37 (t, J = 5.6 Hz, 2H), 3.26–3.19 (m, 2H), 3.16 (t, J = 5.9 Hz, 2H), 1.71–1.59 (m, 2H), 1.26 (s, 8H), and 0.89–0.83 (m, 3H). ¹³C NMR (101 MHz, DMSO) δ 172.64, 168.12, 55.11, 54.97, 53.39, 51.70, 49.28, 31.51, 28.67, 26.34, 23.68, 22.49, and 14.44. HRMS (ESI) of C₁₅H₂₈N₂O₆ [M + H]⁺ calcd., 333.2020; found 333.2019.

Vibrio harveyi BB170 (ATCC BAA-1117), S. aureus ATCC 25923, P. aeruginosa PAO1, and E. coli OP50 were used in this study. AB medium contained 0.3 M NaCl, 0.05 M MgSO₄, and 2% acid-hydrolyzed casein (without vitamins), adjusted to pH 7.5 with 1 M KOH. After autoclaving at 121°C, 10 ml of 1 M potassium phosphate buffer (K₂HPO₄/KH₂PO₄, pH 7.0), 10 ml of 0.1 M sterile arginine solution (L-Arg), and 20 ml of 50% sterile glycerol were added to the medium (per 1 L). LB medium contained 1% tryptone, 1% NaCl, and 0.5% yeast extract. TSB medium contained 1.5% tryptone, 0.5% soy peptone, and 0.5% NaCl (pH = 7.2 ± 0.2). PB medium contained 2% peptone, 1% K₂SO₄, and 0.3% MgCl₂. PTSB medium contained 5% peptone, 1.7% tryptone, 0.5% NaCl, 0.25% K₂HPO₄, 0.25% glucose, and 0.3% soy peptone. PGS-agar plates contained 1% peptone, 0.15 M sorbitol, 1% NaCl, 1% glucose, and 1.7% agar. Swimming solid medium contained 0.8% nutrient broth, 0.5% glucose, and 0.3% agar. Swarming solid medium contained 0.8% nutrient broth, 0.5% glucose, and 0.5% agar.

Test compounds were dissolved in DMSO to a stock concentration of 65 mM. Antibiotics [tobramycin base, meropenem trihydrate (MEPM), ceftazidime, amikacin, colistin sulfate, and ciprofloxacin] were purchased from Sigma and stored at 6.4 mg/ml at −20°C.

The concentration of compounds that resulted in 50% maximum V. harveyi BB170 bioluminescence (the IC₅₀ value) was tested as described previously (Collins et al., 2015). Vibrio harveyi BB170 was grown in AB medium (14 h, 30°C) to OD_600 nm = 1.5. The cells were diluted into fresh AB medium (1:2,500) and added (100 μl/well) to serially diluted test compounds in AB medium (100 μl/well); the final DMSO concentration was 0.5%. The plate was incubated at 30°C for 8 h, and then the bioluminescence (OD_460 nm) and cell density (OD_600 nm) were measured. The values of OD_460 nm and OD_600 nm were normalized and IC₅₀ values were calculated using GraphPad Prism 5 software.

Pseudomonas aeruginosa PAO1 and S. aureus ATCC 25923 were inoculated into LB medium, respectively, and cultured at 37°C at 150 rpm until OD_600 nm = 0.5. Then, the cultures were diluted with fresh LB medium to OD_600 nm = 0.05. Next, the bacteria were cultured in the presence of compound Str7410 (80, 40, 20, 10, and 1 μM), and OD_600 nm was measured at intervals of 2 h for up to 24 h using a spectrophotometer (UV-1800, Bio-Rad Smart Spec Plus, United States). All experiments were performed three times, independently (Wang et al., 2016b).

Quantitative analysis of biofilms used crystal violet assay (Armbruster et al., 2016). First, we used fresh LB medium to dilute P. aeruginosa PAO1 and S. aureus ATCC 25923 cultures in the logarithmic growth phase to OD_600 nm = 0.05, and then mixed them in equal proportions in the same volume and added them to a 96-well plate (100 μl/well, three parallel wells for each group). The final test compound concentrations were 80, 40, 20, 10, and 1 μM. The 96-well plate was incubated at 37°C for 24 h, then the bacterial culture was removed, and the plate was washed three times with phosphate-buffered saline (PBS) and dried. Next, 0.1% (w/v) crystal violet solution was added for 10 min. After washing three times with PBS, the crystal violet-stained biofilms were air-dried. To quantify the biofilm biomass, the crystal violet was removed by adding 150 μl of 33% glacial acetic acid, and OD_595 nm of the solubilized dye was determined using a Biotek multifunction microplate reader. The biofilm inhibition rate was defined as (OD_{595 blank} − OD_{595 sample})/OD_{595 blank} × 100%. Each crystal violet assay was run in triplicate, with a minimum of three replicates per assay.

The determination of minimal inhibitory concentrations (MICs) was performed as described previously (Peeters et al., 2009; Guo et al., 2016). Pseudomonas aeruginosa PAO1 and S. aureus ATCC 25923 were, respectively, cultured in LB medium to OD_600 nm = 0.8. Pseudomonas aeruginosa PAO1 alone or in co-culture with S. aureus ATCC 25923 (mixed in equal proportions) were diluted 1,000-fold with fresh LB medium and added to a 96-well plate. According to the broth dilution method, Antibiotics concentrations ranged from 64 to 0.125 μg/ml. Finally, the plates were incubated at 37°C for 18 h, and OD_590 nm was determined using a Biotek multifunction microplate reader.

Determination of minimum biofilm inhibitory concentrations (MBICs) of antibiotics (Wang et al., 2016a). Based on the above method, 100 μl of diluted bacteria was added to each well of a 96-well plate and cultured at 37°C for 48 h. After removing the bacterial culture, the biofilms were washed twice using PBS. Antibiotics and fresh LB medium were added to the 96-well plate, which was incubated at 37°C for 24 h. The lowest drug concentration without turbidity well is MBIC value of antibiotics.

For the minimum biofilm eradication concentrations (MBECs) of compound for biofilm cells (Tsukatani et al., 2016, 2020), the 96-well plate containing 100 μl diluted bacterial solution of each well was incubated at 37°C for 24 h. After removing the bacterial culture, the biofilms were washed three times using PBS, then 180 μl reaction solution of 2-fold serial dilutions of compound (range 1,024–1 μg/ml) in fresh LB medium added to 96-well plate. After 20 h challenge at 37°C, the reaction solution were removed and washed three times with sterile PBS, then 150 μl fresh LB medium added to each well of 96-well plate and incubated for a further 24 h at 37°C. After removing the LB medium, the biofilms were washed three times using PBS and dried, and then 150 μl 0.1% (w/v) crystal violet solution was added for 10 min. After washing three times with PBS, adding 150 μl of 33% glacial acetic acid to 96-well plate, and the OD_460 nm was measured using a Biotek multifunction microplate reader. The MBEC represents the lowest concentration of constituents for which the biofilm eradication activity was >99%. Biofilm eradication activity (%) was determined using the following formula:

Treatment of biofilms and quantification of cells was performed as follows. Dual-species biofilms were grown in 96-well plate. The liquid medium was decanted every 2 days leaving the attached cells at the bottom of the plate, and fresh LB medium was added (Armijo et al., 2020). After 6 days, the liquid culture was removed and the biofilms were slowly washed twice with PBS. In the test compound + antibiotic group, the test compound (1 μl, 10 μM final concentration), antibiotic (1 μl of MEPM, 0.5 μg/ml final concentration), and LB medium (98 μl) were added to each well. In the test compound or antibiotic alone groups, 99 μl of LB medium and 1 μl of test compound or MEPM were added, respectively. In the antibiotic + sonication group, bacteria in the biofilm were detached by sonication (40 kHz, 300 W, and 5 min) before adding 99 μl of LB medium and 1 μl of MEPM. In the control group, 100 μl of LB medium was added. After incubation for 3.5 h, biofilm cells were detached by sonication (40 kHz, 300 W, and 5 min) and the number of colony-forming units was determined by plating the resulting suspension (Brackman et al., 2011; Guo et al., 2016).

Pseudomonas aeruginosa PAO1 and S. aureus ATCC 25923 were cultured to OD_600 nm = 2.0 at 37°C in TSB medium, respectively. Then, after mixing in equal proportions, 20 μl of culture were spread on PGS-agar plates containing antibiotic or test compound. The plates were placed in an incubator at 37°C and cultured for 18 h to allow the formation of a bacterial lawn. Then, 50 synchronized nematodes (L4 stage of wild-type C. elegans N2) were selected and placed on a plate. The plates were incubated at 20°C and the number of living and dead nematodes was counted every 24 h using a stereomicroscope. A nematode was defined as dead when it no longer responded to touch. Any nematodes that died as a result of getting stuck to the wall of the plate were excluded from the analysis. Survival curves were drawn for analysis (Rajkumari et al., 2018).

Pyocyanin was quantified as follows: P. aeruginosa PAO1 and S. aureus ATCC 25923 were, respectively, cultured in LB medium at 37°C, 150 rpm, to the logarithmic growth phase. After mixing in equal proportions, the culture was diluted tenfold with fresh PB medium. Bacterial cultures (5 ml) with and without the test compound (40, 20, and 10 μM) were incubated at 37°C and 150 rpm for 16 h. To extract pyocyanin, the culture was centrifuged at 1,665 × g for 10 min, and the supernatant was collected and extracted with 3 ml of chloroform. Then, 1 ml of 0.2 M HCl was mixed with the chloroform layer, and the upper (aqueous) phase was collected by centrifugation at 1,665 × g for 10 min. The absorbance was measured at 520 nm (Essar et al., 1990). The inhibition rate of pyocyanin production was calculated as (OD_{520 blank} − OD_{520 sample})/OD_{520 blank} × 100%.

Elastase was determined using the Elastin-Congo red assay with modifications, and the final absorbance was read at 495 nm (Pearson et al., 1997). Pseudomonas aeruginosa PAO1 and S. aureus ATCC 25923 were, respectively, cultured in PTSB medium at 37°C and 150 rpm to the logarithmic growth phase. After mixing in equal proportions, co-cultures were divided into the experimental group and the control group, and then incubated at 37°C and 150 rpm for 18 h. Then, the cultures were centrifuged at 9,590 × g for 5 min and the supernatant was collected. The bacteria were filtered through a 0.22-μm pore-size filter. Then, 200 μl of the filtrate was added to 1 ml of Elastin-Congo red reaction solution [20 mg/ml Congo red, 0.1 M Tris–HCl (pH 7.2), and 1 mM CaCl₂], and the mixture was shaken for 18 h at 37°C. Stop-reaction solution (ethylenediaminetetraacetic acid, 0.1 ml, and 0.12 M) was added and the mixture was incubated on ice for 5 min, and then centrifuged at 9,590 × g for 10 min. The supernatant was collected and OD_495nm was determined.

Pseudomonas aeruginosa PAO1 was shaken in LB medium at 37°C until OD_600 nm = 1.0. Bacterial culture (1 μl) was inoculated into the center of cooled swimming solid medium and further cultured at 30°C for 16 h. Staphylococcus aureus ATCC 25923 was shaken at 37°C in LB medium to OD_600 nm = 0.5. The culture was centrifuged at 9,590 × g for 5 min, and the supernatant was filtered (0.22-μm pore-size). The filtered supernatant was added to thawed swarming solid medium at a ratio of 1:10, mixed, poured into a plate, and left to stand for 8 h to dry. Finally, colonies were picked from the P. aeruginosa PAO1 swimming solid medium, added to the center of the swarming solid plate, and cultured at 30°C for 16 h (Tremblay and Deziel, 2008; Pallett et al., 2019).

Pseudomonas aeruginosa PAO1 and S. aureus ATCC 25923 cultured overnight for 16 h were diluted with fresh LB medium to OD_600 nm = 0.05. After mixing in equal proportions, they were divided into a control group and an experimental group (with test compound added) and grown at 37°C and 150 rpm for 10 h. Pseudomonas aeruginosa PAO1 was also grown in monoculture. After centrifugation (9,590 × g, 5 min) to retain the bacterial cells, a one-step cDNA kit was used [5× All-In-One RT MasterMix (With Excellent Lysis Kit, abm, Canada) for cDNA Extraction]; the DNA was stored at −20°C.

Quantitative reverse transcriptase PCR (qRT-PCR) was performed with a Roche LightCycler® 480 instrument and EvaGreen 2× qPCR MasterMix. The 20-μl reaction system contained 0.6 μl each of forward and reverse primers, 1 μl template cDNA, 10 μl EvaGreen 2× qPCR MasterMix, and 7.8 μl nuclease-free H₂O. The primer sets used for these analyses are listed in Table 1. A no-template control was included during each qPCR experiment. The thermal cycling program started with 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. After qPCR amplification, the comparative threshold method (ΔΔCt analysis) was applied to evaluate relative changes in gene expression. The experiment used GraphPad Prism 8 software for mapping (Conway et al., 2012). ΔΔCt = ΔΔCt, _sample – ΔΔCt, _reference.

All assays were performed with three replicates and the values obtained are expressed as the mean ± SD. Differences in data were compared with the untreated control at each time-point and considered significant when p < 0.05 (^*p < 0.05, ^**p < 0.01, and ^***p < 0.001) by one-way ANOVA with t-tests using GraphPad Prism 8 software.

Synthesis of compound Str7410 was accomplished in four steps from the commercially-available 1-bromoheptane (Scheme 1). Single substitution of N-tert-butoxycarbonyl-1,2-ethylenediamine with 1-bromoheptane generated intermediate 1. Removal of the Boc amino-protecting group followed by reaction with ethyl 2-bromoacetate afforded intermediate 3. Acidic hydrolysis twice afforded the desired compound Str7410.

We used V. harveyi BB170 (a luxN mutant) as a reporter strain to evaluate the AI-2 QS inhibitory activity of test compounds. The IC₅₀ values of compounds were tested in terms of V. harveyi BB170 bioluminescence. Bromo-furanone C56 (Zang et al., 2009) was used as the positive control. As shown in Table 2, Str7410 had the best inhibitory effect on AI-2 QS of V. harveyi BB170 (IC₅₀ = 0.3724 ± 0.1091 μM). Based on this, compound Str7410 was selected for further research.

The main difference between QSIs and antibiotics is that QSIs inhibit the production of virulence factors without killing pathogenic bacteria, so that the bacteria do not develop drug-resistance mutations (Allen et al., 2014). Therefore, we analyzed the effect of the putative QSI compound Str7410 on the growth of P. aeruginosa PAO1 and S. aureus ATCC 25923. As Figure 1 shows, compound Str7410 affected the growth of neither bacterium.

Bacterial resistance to antibiotics is related to the formation of biofilms (Kalia, 2013). We used the crystal violet assay to evaluate the effect of compound Str7410 on formation of biofilms by P. aeruginosa PAO1 and S. aureus ATCC 25923 mixed in equal proportion. As shown in Figure 2, significantly more biofilm was formed by the mixture of species than by either species alone. When compound Str7410 was added to the co-culture, the formation of biofilm was significantly reduced [range 9.75% (p < 0.05)–48.40% (p < 0.001)]. The inhibitory effect of compound Str7410 was concentration-dependent. When the concentration of Str7410 was 40 μM, the inhibition rate of biofilm formation was 40.58% (p < 0.01).

First, the MIC and MBIC values of antibiotics for single and mixed bacteria were determined when P. aeruginosa PAO1 was cultured alone or in co-culture with S. aureus ATCC 25923. As shown in Table 3, compared with planktonic cells, biofilm cells showed significantly increased resistance to antibiotics. The MBIC values of MEPM against biofilm cells increased 32 and 16-fold, respectively, compared with the MIC values for planktonic cells, when P. aeruginosa PAO1 was cultured alone and co-cultured with S. aureus ATCC 25923. Moreover, planktonic cells or biofilm cells from co-cultures of P. aeruginosa PAO1 and S. aureus ATCC 25923 showed significantly increased resistances to some antibiotics (ceftazidime, colistin sulfate, ciprofloxacin, and MEPM) compared with that of P. aeruginosa PAO1 monoculture. These data indicate that cultures of multiple bacterial species and the formation of biofilms can increase resistance to antibiotics. We also evaluated the MBEC of the compound Str7410 against the formed biofilm. As shown in Supplementary Table S1, the compound Str7410 had no eradication effect on the formed biofilm.

Next, we evaluated the effects of compound Str7410 on the susceptibility of mixed-species biofilms to antibiotics. We tested the inhibitory effects of MEPM and compound Str7410 alone, and MEPM combined with compound Str7410. MEPM in combination with sonication was used as a reference treatment. As Figure 3 shows, compound Str7410 when applied alone had no inhibitory effect on biofilms of mixed P. aeruginosa PAO1 and S. aureus ATCC 25923 cells. Treatment with MEPM alone and MPEM combined with sonication significantly decreased the number of biofilm cells (p < 0.05 and p < 0.01, respectively). The combination of MEPM with Str7410 also had a significant inhibitory effect on biofilm cells (p < 0.01). Notably, compound Str7410 increased the inhibitory effect of MEPM toward biofilm cells (p < 0.05), and the level of inhibition by Str7410 + MEPM was greater than that by MEPM + sonication.

We used wild-type C. elegans N2 as a model to evaluate the effect of compound Str7410 on survival rates following co-infection with P. aeruginosa PAO1 and S. aureus ATCC 25923 in vivo.

After 240 h of incubation, the survival rate of nematodes decreased by 2.55% for C. elegans co-infected with P. aeruginosa PAO1 and S. aureus ATCC 25923 compared with that of nematodes infected with P. aeruginosa PAO1 alone (Figure 4). This indicates that the pathogenicity caused by multispecies infection was greater than that by a single bacterial species.

Compared with the positive control group co-infected by P. aeruginosa PAO1 and S. aureus ATCC 25923, the survival rate of the infected group treated with compound Str7410 (40 μM) increased by 12.67% (p < 0.05), and the survival rate of the infected group treated with MEPM (0.5 μg/ml) increased by 14.67% (p < 0.001). Furthermore, the survival rate of the group treated with the combination of Str7410 and MEPM increased by 12.67% (p < 0.05) and 10.67% (p < 0.05) compared with the Str7410-alone and MEPM-alone treatments, respectively (Figure 4). Although compound Str7410 inhibited the growth of neither P. aeruginosa PAO1 nor S. aureus ATCC 25923 (e.g., Figure 1), it decreased the pathogenicity of these bacteria by inhibiting interspecific QS. In addition, the combination of compound Str7410 with MEPM increased the therapeutic effect of MEPM.

The effect of Str7410 on virulence factors of P. aeruginosa PAO1 in multispecies infection was investigated. We quantitatively analyzed production of the virulence factors pyocyanin and elastase by P. aeruginosa PAO1 when this strain was cultured alone or co-cultured with S. aureus ATCC 25923. As Figure 5 shows, the production of pyocyanin and elastase was significantly increased (p < 0.001 and p < 0.05, respectively) when P. aeruginosa PAO1 was co-cultured with S. aureus ATCC 25923 compared with P. aeruginosa PAO1 monoculture. However, treatment with Str7410 significantly reduced the production of pyocyanin and elastase in co-cultures compared with the untreated controls; this effect of Str7410 was concentration dependent. When the concentration of Str7410 was 40 μM, the inhibition of production of pyocyanin and elastase was 22.07% (p < 0.01) and 24.85% (p < 0.05), respectively, compared with no treatment with Str7410.

Bacterial motility is important for pathogenicity and antibiotic resistance. Swarming motility, an important virulence factor of P. aeruginosa, is mainly regulated by QS-related genes, such as lasB and pvdQ (Tremblay and Deziel, 2008). Furthermore, S. aureus culture supernatant can promote the swarming motility of P. aeruginosa PAO1 under normoxia (Pallett et al., 2019). Similarly, in this study, culture supernatant of S. aureus ATCC 25923 promoted the swarming motility of P. aeruginosa PAO1 (Figures 6A,B). However, when compound Str7410 was added, the swarming motility was significantly inhibited (Figures 6B,C).

From the data above, we found that S. aureus ATCC 25923 can increase the production of virulence factors and the swarming motility of P. aeruginosa PAO1 on co-culture of strains PAO1 and ATCC 25923. Compound Str7410 significantly inhibited this. These findings indicate that in multispecies infections, bacteria can enhance pathogenicity through their interactions, potentially via AI-2 QS systems. Therefore, we quantified the relative expression of QS-related genes of P. aeruginosa PAO1 in co-culture of P. aeruginosa PAO1 with S. aureus ATCC 25923. As shown in Figure 7, the expression of PAO1 QS-related genes (lasI, lasR, rhlI, rhlR, pqsH, and pqsR) and virulence factor-related genes (elastase, lasB; pyocyanin, and phzH) was significantly upregulated in the co-culture group compared with that in P. aeruginosa PAO1 monoculture. When Str7410 (40 μM) was added, the expression of all the strain PAO1 QS genes was significantly downregulated in co-culture compared with that in the absence of Str7410.