Twelve isolates of XDRAB were derived from the bacterial specimens of patients from the critical care units, internal medicine, and emergency departments at the First Affiliated Hospital of Chengdu Medical College from 2018 to 2020 (Chengdu, China). Standard laboratory methods and ATB New (BioMérieux, France) were used to identify these isolates, which were also confirmed by PCR of the genes 16S rRNA and blaOXA-51 (Chang et al., 2015); Chromobacterium violaceum 026 (CV026) was purchased from Biobw (Beijing, China); A. baumannii ATCC 17978 and Escherichia coli DH5a were preserved in our laboratory.

Berberine, ferulic acid, baicalin, baicalein, ursolic acid, naringenin, hesperidin, gallic acid, malic acid, resveratrol, myricetin, vanillin, phloretin, capsaicin, osthole, andrographolide, parthenolide, magnolol, and ellagic acid were purchased from Meilun Biological (Dalian, China). Meanwhile, ampicillin, piperacillin, ampicillin sulbactam (2:1), cefoperazone sulbactam (2:1), efotaxime, ceftazidime, tetracycline, doxycycline, amikacin, gentamicin, ciprofloxacin, levofloxacin, imipenem, meropenem, tigecycline and polymyxin B were purchased from Meilun Biological (Dalian, China); Linoleic acid, kaempferol, 4-terpineol, coumarin, linalool, naringin, matrine, and cinnamaldehyde were purchased from Macklin Biochemical (Shanghai, China); Eugenol, citral, vanillin, cinnamic acid, hordenine, and nootkatone were purchased from Yuanye Biochemical (Shanghai, China); Furanone C30 and C6-HSL were purchased from Sigma-Aldrich (Taufkirchen, Germany); RNA isolation kit, cDNA synthesis kit, and fluorescent dye SYBR Color qPCR Master Mix were purchased from Vazyme Biological (Nanjing, China). Cation-adjusted Muller-Hinton broth (CAMHB) (Comprised of beef extract, caseins hydrolyzates, starch and calcium chloride), Luria Bertani (LB) broth medium (Comprised of tryptone, yeast extract, sodium chloride and glucose), LB agar medium (Comprised of tryptone, yeast extract, sodium chloride and agar) and tryptone soy broth (TSB) medium (Comprised of tryptone, soya peptone, sodium chloride, glucose and dipotassium hydrogen phosphate) were purchased from Haibo Biotechnology (Qingdao, China). Dimethyl sulfoxide (DMSO) and phosphate buffer saline (PBS) were purchased from Kelong Chemical (Chengdu, China).

The minimal inhibitory concentration (MIC) of thirty-four TCMMs for CV026 and XDRAB were determined by using the broth microdilution method. Briefly, CV026 and XDRAB were inoculated in LB agar medium at 30°C and 37°C for 16–20 h, respectively. And then resuspended in saline (0.9% sodium chloride) to produce a 0.5 McFarland turbidity standard, followed by a 20-fold dilution. TCMMs were initially dissolved in dimethyl sulfoxide (DMSO) and diluted with PBS into a series of solutions at different concentrations. CAMHB medium, bacterial suspension and TCMMs at different concentrations were added into 96-well cell culture plates in groups. The CV026 and XDRAB were incubated at 30°C and 37°C for 16–20 h, respectively. Less than OD600 of 0.1 were regarded as no bacterial growth, and colored drugs were observed macroscopically and data were recorded (Peng et al., 2020; Humphries et al., 2021).

CV026 is used as a biosensor strain to detect anti-QS activity. A culture-activated CV026 strain (5 mL) was added to 50 mL of LB nutrient agar medium containing kanamycin and C6-HSL (final concentration of 20 μg/mL), mixed and poured well, then punched and subinhibitory TCMM concentrations were added into the wells. After 24 h of incubation at 30°C, plates were observed for the inhibition of violacein. At the same time, the culture-activated CV026 bacterial solution was added to a 6-well cell culture plate, and then LB broth medium containing kanamycin, C6-HSL, and TCMM solutions with subinhibitory concentrations were added. 0.9% normal saline was used as the blank control, and furanone C30 was used as the positive control. After 24 h of incubation at 30°C, 1 mL of the bacterial solution from each well was centrifuged (13,000 g, 5 min) and DMSO was added to the cell pellets to dissolve violacein pigment. Supernatants (containing dissolved violacein) were transferred to a 96-well cell culture plate, and the absorbances were measured at 585 nm (Lou et al., 2019).

Taking the 96-well cell culture plate, 170 μL TSB medium was added into each well, and then diluted bacterial solution and TCMMs solution with the different concentrations (1 MIC, 1/2 MIC, 1/4 MIC, and 1/8 MIC) were added. The value of OD₅₇₀ was measured after incubation at 37°C every 6 h. The growth curves of XDRAB within 24 h were plotted, respectively. Concentrations that did not affect bacterial growth were finally selected as subsequent working concentrations (Aranda et al., 2011).

Twelve activated XDRAB strains were diluted 1:1,000 and added into confocal dishes, then 1 mL of working concentration TCMMs were added to each well and incubated at 37°C for 4 h. After rinsing twice with PBS and sonicating for 10 min, PBS was added again to detach the bacteria adhering to the culture dish. 100 μL of detached bacteria were diluted 10-fold and spread on LB plates and cultured at 37°C for 24 h before counting. The relative value method (lg10 (N×10 × 1,000)) was used to assess the effect of TCMMs on XDRAB adhesion (Fan et al., 2018).

We performed biofilm formation assays using crystal violet. Briefly, using the 96-well cell culture plate, 170 μL of TSB medium and 10 μL of PBS (control group) or TCMM solution at the working concentration were added into each well, followed by the inoculation of 20 μL of bacterial suspension (OD₆₀₀ of 0.12). Escherichia coli DH5a was used as a negative control and six replicate wells were set for each strain. The plates were incubated at 37°C for 24 h, then the wells were voided and washed three times with PBS. After complete dryness, 0.1% crystal violet was added for staining for 20 min, then PBS was washed three times, dried in air for 30 min, dissolved in 95% ethanol for 15 min, and finally, the absorbance at 570 nm was measured (Farshadzadeh et al., 2018).

The effect of TCMMs on XDRAB QS-regulated genes and virulence factor-related genes abaR, abaI, csuE, bap, and pgaA was investigated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the gene primer sequences are presented in Table 1. The 16S rRNA gene was used for the internal standard of mRNA quantification, and the expression levels of virulence factor genes were detected using the fluorescent dye SYBR Color qPCR Master Mix. According to the kit instructions, RNA was extracted and reverse transcribed into cDNA, followed by a 20 μL system used for the reaction. The cycling conditions of qRT-PCR were as follows: pre-denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 5 s. The expression level of each gene was normalized and the relative expression was calculated as 2^−ΔΔCT. Fold changes in gene expression from TCMM-treated cells were compared to untreated cells that were propagated under the same conditions (Luo et al., 2015; Navidifar et al., 2022).

The mean and standard deviation of the mean were calculated using SPSS 26.0. GraphPad Prism 8.0 was used for mapping. The statistical analysis of the data was performed using the Student’s t-test to compare the treated group with the control group. Data was considered significant when p < 0.05.

The MIC of thirty-four TCMMs against strain CV026 ranged from 16 to 1,024 μg/mL (Table 2). Cinnamic aldehyde, carvacrol, and resveratrol showed obvious antibacterial activity, and the MICs were 32 μg/mL, 32 μg/mL and 16 μg/mL, respectively. The MIC of furanone C30 was 64 μg/mL. Twelve isolates of A. baumannii were susceptible to polymyxin B or tigecycline, but resistant to other antimicrobial agents, including aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, and tetracyclines (Table 3). Such resistance profiles classified these isolates as XDRAB.

Thirty-four TCMMs showed different effects on the QS activity of CV026 (Table 4). Twenty-one TCMMs, including coumarin, 4-terpineol, and hordenine, formed turbid inhibition circles on purple plates and showed different degrees of QS inhibition effects, which belonged to QS activity inhibitors. In contrast, the remaining thirteen TCMMs and blank controls did not form turbid or only transparent diaphragms, indicating no QS inhibitory activity. Thus, twenty-one of thirty-four TCMMs showed different degrees of QS inhibitory activity.

Nine of twenty-one TCMMs could inhibit violacein production to varying degrees at sub-inhibitory concentrations and decrease in a concentration-dependent manner (Figure 1). Caffeic acid, vanillin, cinnamic acid, matrine, hordenine, kaempferol, coumarin, 4-terpineol, and myricetin showed relatively obvious QS inhibitory activity, with significant differences at the concentrations of both 1/4 MIC and 1/8 MIC (p < 0.05), and the inhibitory effects were better than furanone C30. In addition, caffeic acid, vanillin, kaempferol, myricetin, and coumarin also showed inhibitory effects at a concentration of 1/16 MIC (p < 0.05). Thus, nine TCMMs, including caffeic acid, vanillin, and cinnamic acid, have inhibitory effects on the QS system without affecting the normal growth of the strains, which can be regarded as potential QSIs.

The MICs of nine TCMMs against twelve XDRAB strains ranged from 256 to 2048 μg/mL. We found that myricetin, cinnamic acid, caffeic acid, vanillin, hordenine, kaempferol, matrine, coumarin, 4-terpineol, and furanone C30 did not affect the growth of bacterial cells at the 1/8 MIC concentration of 32, 256, 256, 256, 128, 64, 256, 32, 64, and 4 μg/mL, respectively. The growth curve of XDRAB by TCMMs is shown in Figure 2.

Nine TCMMs could inhibit the adhesion of twelve XDRAB strains to confocal dishes at a concentration of 1/8 MIC and significantly reduce the number of colonies adhering to the culture dishes (p < 0.01). After 1/8 MIC of kaempferol and 4-terpineol, the relative numbers of bacteria adhering to the culture dishes were 3.52 ± 0.09 and 3.70 ± 0.17, respectively, compared with 6.99 ± 0.02 in the blank control group (Figure 3).

Nine TCMMs could inhibit the biofilm formation of XDRAB to different extents. At a concentration of 1/8 MIC, 4-terpineol, coumarin, hordenine, caffeic acid, matrine, kaempferol, and vanillin could significantly inhibit the biofilm formation of XDRAB (p < 0.05). Myricetin, cinnamic acid, and furanone C30 had relatively weak inhibitory effects (p > 0.05). Overall, TCMMs showed stronger anti-biofilm effects compared with furanone C30 (Table 5).

The expression levels of QS-regulated genes abaI, abaR and virulence factor-related genes csuE, bap, and pgaA in biofilm strongly positive strains were analyzed by qRT-PCR at a concentration of 1/8 MIC (Table 6). Compared with the control group, the expression levels of QS genes abaI and abaR, pilus regulatory gene csuE, and biofilm-related genes bap and pgaA were significantly decreased by the eight TCMMs at the concentration of 1/8 MIC, except coumarin, which showed no effect on the biofilm-related gene bap. Therefore, nine TCMMs downregulated the QS-related genes abaI, abaR and the virulence factor-associated genes csuE, bap, and pgaA, except coumarin, which upregulated the gene bap.