Antibacterial and anti-virulence effects of furazolidone on Trueperella pyogenes and Pseudomonas aeruginosa

Background Trueperella pyogenes and Pseudomonas aeruginosa are two important bacterial pathogens closely relating to the occurrence and development of forest musk deer respiratory purulent disease. Although T. pyogenes is the causative agent of the disease, the subsequently invaded P. aeruginosa will predominate the infection by producing a substantial amount of quorum-sensing (QS)-controlled virulence factors, and co-infection of them usually creates serious difficulties for veterinary treatment. In order to find a potential compound that targets both T. pyogenes and P. aeruginosa, the antibacterial and anti-virulence capacities of 55 compounds, which have similar core structure to the signal molecules of P. aeruginosa QS system, were tested in this study by performing a series of in vitro screening experiments. Results We identified that furazolidone could significantly reduce the cell densities of T. pyogenes in mono-culture or in the co-culture with P. aeruginosa. Although the growth of P. aeruginosa could also be moderately inhibited by furazolidone, the results of phenotypic identification and transcriptomic analysis further revealed that sub-inhibitory furazolidone had remarkable inhibitory effect on the biofilm production, motility, and QS system of P. aeruginosa. Moreover, furazolidone could efficiently protect Caenorhabditis elegans models from P. aeruginosa infection under both fast-killing and slow-killing conditions. Conclusions This study reports the antibacterial and anti-virulence abilities of furazolidone on T. pyogenes and P. aeruginosa, and provides a promising strategy and molecular basis for the development of novel anti-infectious drugs to dealing with forest musk deer purulent disease, or other diseases caused by T. pyogenes and P. aeruginosa co-infection. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-022-03216-5.


Background
Forest musk deer (Moschus berezovskii) is an important economic animal endemic in China, and has been categorized as first-class key species of wildlife protected by Chinese legislation in 2002. The musk secreted by male forest musk deer is a precious Chinese medicine and important raw material of high-grade spice. As a kind of solitary small ruminant, forest musk deer are timorous and fiddle-footed and easily hurt themselves while leaping. These casual injuries frequently lead to the occurrence of purulent disease, which normally manifests as suppurative lesions on the epidermis, uterus, and internal organs [1,2]. Previous studies have revealed that purulent disease is one of the main reasons hindering the increase of forest musk deer population and causes more than 50% of the total deaths [3,4]. In a farm with ≈ 400 forest musk deer in Sichuan Province (China), 8-10 individuals are raised in each fold about 200 m 2 . According to the statistical report of forest musk deer therapy, we find that among the 467 treatment records (including 100 death cases) in about 2 years, 165 of them (35.3%) were related to purulent disease, and 41 death cases were diagnosed as severe internal suppuration, especially in the lung. Compared to the body surface abscesses which can be easily observed and removed artificially, the internal suppurative lesions are usually fatal and difficult to be detected in time [1,2,5].
Our prior work has shown that Trueperella pyogenes and Pseudomonas aeruginosa are two main bacterial pathogens in the respiratory suppurative lesion of forest musk deer. T. pyogenes is considered as the primary pathogen, while P. aeruginosa become the dominant species in the lateral stage [1,4,6]. The detection rate of T. pyogenes in the samples from surface abscesses and internal suppurative lesions of forest musk deer was 100% (28/28). Other bacterial species, such as P. aeruginosa (21.4%), Escherichia coli (7.2%), Bacillus cereus (14.3%), and etc., could only be isolated from the internal suppurative lesions [2]. T. pyogenes is a Gram-positive, pleomorphic, non-spore forming, inactive, non-enveloped and facultative anaerobic bacterium belonging to the family Actinomycetaceae, and a resident bacterium in the skin and mucous membranes of animal respiratory tract, digestive tract, and genitourinary tract [7][8][9][10]. The biosynthesis of the key virulence factor pyolysin is depended on fermentation metabolism of T. pyogenes. This process can reduce the oxygen content in the infection site and thus benefits the growth of other anaerobic or facultative anaerobic bacterial species [10][11][12].
P. aeruginosa is a ubiquitous opportunistic Gramnegative bacterium that can infect a variety of host tissues and cause acute and chronic infections [13,14]. It is well-recognized that quorum-sensing (QS) system plays an important role in the processes of bacterial invasion and cell-cell communications. The QS system of many Gram-negative bacterial species is activated by specific signal molecules (acyl-homoserine lactones, AHLs), and coordinates the production of diverse virulence factors [5,[15][16][17]. In P. aeruginosa, the QS system is composed of three hierarchically arranged regulatory networks. The las-and rhl-QS systems have complete signal molecule synthesis proteins (LasI/RhlI) and regulatory proteins (LasR/RhlR). The activation of rhl is largely depended on the las system. The pqs system only has the regulatory protein pqsR, and the activation of which requires Pseudomonas quinolone signal from other pathways co-regulated by lasR and rhlR [16].
In the past years, aminoglycosides and cephalosporins were the antibiotics routinely used to treat the infection diseases of forest musk deer. However, the increased frequency of treatment failure caused by bacterial antibiotic resistance has brought huge challenge and economic losses to forest musk deer breeding industry [1,2]. It is considered that inhibiting the virulence of multi-resistant bacteria by targeting the QS system, rather than killing them, is a promising strategy for the development of novel anti-infectious drugs, namely QS inhibitors or anti-virulence drugs [18]. P. aeruginosa has robust innate resistance to a wide range of antibiotics, and is a model species in the screening of anti-infectious compounds because of its well-characterized QS system [13,15,17]. We have previously shown that the natural AHL signals of P. aeruginosa QS system could inhibit the growth and virulence of multi-resistant T. pyogenes [19]. P. aeruginosa has strong competitive advantage over T. pyogenes when any of the three QS regulators of P. aeruginosa was knocked out [20]. Therefore, we hypothesize that there might be a kind of compound that has the potential to simultaneously control T. pyogenes and P. aeruginosa, either by inhibiting the growth or virulence of them. In this study, we tested the antibacterial and anti-QS activities of 55 small molecule compounds with similar core structure (furan, benzofuran, or flavonoids) to the AHL signals of P. aeruginosa (Additional file 1 Supplementary  Fig. S1), or to the QS inhibitors identified previously [18]. Finally, we identified that furazolidone had strong inhibitory activity on T. pyogenes and moderately inhibited the growth of P. aeruginosa. Moreover, sub-inhibitory furazolidone showed a strong inhibitory activity on the QS system of P. aeruginosa.

Screening of compounds inhibit the growth of T. pyogenes
For the 55 compounds with similar core structure to the AHL signals of P. aeruginosa QS system, we first tested the inhibitory effects of them on the growth of T. pyogenes TP13 in brain heart infusion medium containing 5% fetal bovine serum (BHI-FBS). The result showed that compared with the control, 9 compounds were found to have significant growth inhibition effect on T. pyogenes TP13 and 8 have significant growth enhancement effect (Additional file 1 Supplementary Table S1). Nitrofurantoin, nitrofurazone, ronidazole, and furazolidone, which could significantly inhibit T. pyogenes TP13 in a doseand time-dependent manner ( Fig. 1), were selected to treat co-cultured T. pyogenes and P. aeruginosa. Equal amount of T. pyogenes TP13 was cultured at 37 °C for different time durations in brain heart infusion medium containing 5% fetal bovine serum (BHI-FBS) and different concentrations of (A, E) nitrofurantoin, (B, F) ronidazole, (C, G) nitrofurazone, or (D, H) furazolidone. Cell densities were determined by measuring the optical density (left Y-axis) at 600 nm (OD 600 ) and colony forming units (CFUs) enumeration on BHI-FBS agar plates (right Y-axis). Data shown are the mean ± standard deviation (SD) of three independent experiments, compare to the untreated control. One-way ANOVA, * p < 0.05, *** p < 0.001, **** p < 0.0001. Small letters a and b above the symbols indicate p < 0.001 and p < 0.0001, respectively

Furazolidone inhibits the growth of T. pyogenes and P. aeruginosa
T. pyogenes TP13 and P. aeruginosa PAO1 were wellmixed into different ratios (1:1, 1:9, and 9:1) and co-cultured on BHI-FBS agar supplemented with 200 μM of nitrofurantoin, ronidazole, furazolidone, or nitrofurazone. In agreement with our prior finding that P. aeruginosa had an innate growth advantage in the competition with T. pyogenes [19], P. aeruginosa would always be the dominant species under co-culture condition with T. pyogenes, irrespective of their initial ratios (Fig. 2). The addition of nitrofurantoin, ronidazole, furazolidone, or nitrofurazone significantly reduced the number of T. pyogenes TP13 cells in all the polymicrobial colonies compared to the control. Differently, compared to the co-culture of T. pyogenes TP13 and P. aeruginosa PAO1 started from the ratio of 9:1 on blank BHI-FBS plates, there were more P. aeruginosa PAO1 cells in the polymicrobial colony on the plates supplemented with nitrofurantoin or nitrofurazone. Notably, among the 4 tested compounds, only furazolidone could simultaneously inhibit T. pyogenes TP13 and P. aeruginosa PAO1 compared to the control, and the growth inhibition of furazolidone on T. pyogenes TP13 was stronger than that on P. aeruginosa PAO1 (Fig. 2).

Furazolidone inhibits the QS-related phenotypes of P. aeruginosa
We found that furazolidone also showed a dose-and time-dependent growth inhibition on P. aeruginosa PAO1. However, the cell densities of P. aeruginosa PAO1 decreased slowly and remained relatively stable when the concentration of furazolidone reached 50 μM (Additional file 1 Supplementary Fig. S2 and S3). Considering the similar core structure of furazolidone and AHL signals, we then tested the possibility that furazolidone might negatively regulate the QS system of P. aeruginosa PAO1. Rapid population proliferation of P. aeruginosa using adenosine or skim milk as the sole carbon source requires the QS induced intracellular hydrolase or extracellular proteases, respectively [21]. Therefore, the growth status of P. aeruginosa under these conditions can be used to preliminarily evaluate the performance of QS system. As shown in Table 1, furazolidone significantly inhibited the growth of P. aeruginosa PAO1 on M9-adenosine plates, and had a dose-dependent inhibition effect on the production of extracellular proteases on M9-skim milk plates. Moreover, the production of biofilm and pyocyanin, and the swimming and twitching motilities of P. aeruginosa PAO1 could also be significantly inhibited by furazolidone (Fig. 3).

Furazolidone inhibits the QS-regulation of P. aeruginosa
To further investigate the effect of furazolidone on the QS-regulation of P. aeruginosa, RNA-sequencing was then used to profile the global transcription of furazolidone-treated P. aeruginosa PAO1. The results showed that compared to the control, 465 up-regulated genes T. pyogenes TP13 and P. aeruginosa PAO1 were mixed and co-cultured on BHI + 5% FBS plates containing 200 μM of nitrofurantoin, ronidazole, nitrofurazone, or furazolidone from the initial ratios of (A) 1:1, (B) 1:9, and (C) 9:1, and cultured overnight at 37 °C. Data shown are the mean ± SD of three independent experiments, compare to the untreated control. One-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 Table 1 Inhibitory effect of furazolidone on the growth of P. aeruginosa PAO1 on M9-adenosine and M9-skim milk plates a "+", Normal growth. "-", Inhibited growth b Data shown are the diameters of proteolytic ring (includes the diameters of colony), compare to the untreated control. Mean ± standard deviation (n = 9). One-way ANOVA, **** p < 0.0001 Prediction of KEGG pathway revealed that the functions of flagellar assembly, bacterial chemotaxis, propanoate metabolism, ribosome, and degradation of valine, leucine and isoleucine were significantly enriched among the upregulated genes (p < 0.05), while QS system was the sole significantly enriched KEGG term among the down-regulated genes (Fig. 4B). We then explored the inhibitor effect of furazolidone on P. aeruginosa QS system in more detail by applying all the significantly changed genes to the list of QSinduced genes previously released by Schuster et al. [22]. The result that among the 315 QS-induced genes, 63 of them including the typical genes relating to the common QS-activated phenotypes, were screened from the 107 down-regulated genes (Fig. 4C). The result of quantitative PCR further confirmed that compared to the control, the expression levels of three key regulatory genes (lasR, rhlR, and pqsR) and their downstream functional genes (lasB, rhlA, pqsA, pqsD, pqsE, hcnA, and phzA) were all down-regulated by 1.5-8.2 fold in furazolidone-treated P. aeruginosa (Fig. 4D). Moreover, we also checked the effect of furazolidone on the expression levels of the main virulence factors of T. pyogenes TP13 and found that, the supplementation of furazolidone significantly decreased the expression of plo, ploS, ploR, cbpA, fimA, nanH, and nanP by 1.7-10.7 fold compared to that of untreated group (Additional file 1 Supplementary Fig. S4).

Furazolidone protects C. elegans from P. aeruginosa-T. pyogenes co-infection
We then tested the in vivo protection activity of furazolidone against P. aeruginosa infection by using C. elegans as a model. In the fast-killing assay which mimics the acute infection condition, all the C. elegans were killed by mono-cultured P. aeruginosa PAO1 or co-cultured P. aeruginosa PAO1 and T. pyogenes TP13 (1:1) in 80 h in the untreated group (Fig. 5A, B), while furazolidone treatment significantly increased the survival rates of C. elegans under both infection conditions (p < 0.0001 and p = 0.0003, respectively). In the slow-killing assay which mimics the chronic infection condition, all the C. elegans were killed by mono-cultured P. aeruginosa PAO1 in 8 days on blank plates (Fig. 5C), and by co-cultured P. aeruginosa PAO1 and T. pyogenes TP13 (1:1) in 10 days (Fig. 5D). Expectedly, the presence of furazolidone significantly protected C. elegans from the challenges of pure P. aeruginosa PAO1 (p = 0.0102) and co-cultured P. aeruginosa PAO1 and T. pyogenes TP13 (p = 0.0157), albeit Fig. 3 Inhibitory effect of furazolidone on the A biofilm formation, B pyocyanin production, C swimming motility and D twitching motility of P. aeruginosa PAO1. Data shown are the mean ± SD of three independent experiments, compare to the untreated control. One-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 the protection effect was not comparable to that in fastkilling assay.

Discussion
Purulent disease is a common disease greatly limits the population increase of forest musk deer and the development of musk-related industrials. The rapid emergence and spread of bacterial antibiotic resistance bring a huge challenge for the treatment of infectious diseases [23,24]. Our prior work identifies that T. pyogenes is the causative agent of forest musk deer purulent disease, while P. aeruginosa dominates the development of respiratory suppurative lesions and leads the forest musk deer to an untimely death [2]. In the present study, we further report that furazolidone was capable of simultaneously inhibiting the growth and virulence of T. pyogenes and P. aeruginosa, and thus might be a promising candidate for the treatment of infections caused by the two species.
T. pyogenes is a Gram-positive bacterium with small genome size (≈ 2.3 Mbp) and commonly inhabits in the mucosal tissues of ruminant animals [6,7,10]. By contrast, the Gram-negative bacterium P. aeruginosa has a relatively large genome size (≈ 6.3 Mbp) and complex transcriptional network. These characters provide P. aeruginosa an innate advantage in colonizing different environments and resisting the clearance of antibiotics [14,16,25]. The present study confirmed the competitive advantage of P. aeruginosa over T. pyogenes by showing that, P. aeruginosa was always the dominant species in the co-culture with T. pyogenes, irrespective of their initial ratios (Fig. 2). This also provides an explanation for the replacement of dominant bacteria from T. pyogenes to P. aeruginosa during the development of suppurative lesions in forest musk deer lungs.
Reducing bacterial virulence by antagonizing the QS system, rather than killing bacterial cells or halting the growth of them, has been suggested to be an . D Expression of typical QS-regulated genes of furazolidone-treated PAO1 determined by qPCR. Data shown are the mean ± SD of three independent experiments, compare to the untreated control. One-way ANOVA, ** p < 0.01, *** p < 0.001, **** p < 0.0001 evolutionarily robust anti-infectious strategy [18,26]. Previous studies have identified that the AHL signals of P. aeruginosa QS system could inhibit the growth and virulence of T. pyogenes in vitro and in vivo [4,6,19,27]. In the present study, we first tested the antibacterial activities of 55 compounds on mono-cultured T. pyogenes and co-cultured T. pyogenes and P. aeruginosa. Furazolidone, which could profoundly inhibit the growth of T. pyogenes TP13 and slightly inhibit P. aeruginosa PAO1, was finally screened. Moreover, compared to the control groups, 200 μM of furazolidone was found to be capable of inhibiting the growth of T. pyogenes and P. aeruginosa in the mixed colonies started from any ratios, albeit P. aeruginosa was always the dominant species (Figs. 1, 2, and Additional file 1 Supplementary Fig. S3).
Furazolidone is a nitrofuran antibiotic that can be used to treat gastrointestinal diseases such as dysentery, enteritis, and gastric ulcer caused by bacteria and protozoa [28]. In this study, we further discovered that sub-inhibitory furazolidone could significantly reduce the production of QS-regulated virulence factors, biofilm formation, and cell motilities of P. aeruginosa PAO1 (Table 1 and Fig. 3). It has been reported that the commonly used clinical antibiotics azithromycin, ceftazidime, and ciprofloxacin could function as QS inhibitors of P. aeruginosa to improve the clinical outcome of patients [29][30][31]. Some natural products such as the extract of Dalbergia Trichocarpa bark, baicalin (an active natural compound extracted from the traditional Chinese medicinal Scutellaria baicalensis), and sodium ascorbate could also inhibit the QS-related virulence of P. aeruginosa [32][33][34]. Our transcriptional analysis further revealed that (Fig. 4), furazolidone could simultaneously inhibit the expression levels of all the three core regulatory genes and their downstream functional genes of P. aeruginosa PAO1. Additionally, the expression levels of the known virulence factor-encoding genes in T. pyogenes were also inhibited by furazolidone (Additional file 1 Supplementary Fig. S4). Therefore, the results indicated that furazolidone might be a good candidate to inhibit the virulence of T. pyogenes and P. aeruginosa.
On the other hand, it is noticed that furazolidone significantly up-regulated the expression of some genes relating to the flagellar assembly and chemotaxis of P. aeruginosa PAO1 as determined by RNA-sequencing (Fig. 4B). This is contradictory to the reduced swimming and twitching phenotypes of P. aeruginosa PAO1 by the presence of furazolidone (Fig. 4C, D). We reasoned that this might be due to the reduced the number of P. aeruginosa cells in the colony by sub-inhibitory furazolidone (Additional file 1 Supplementary Fig. S2), which could promote the expression of a partial of cell motility-related genes (14 out of 46) but limit the expansion of the whole population. Nevertheless, the result of C. elegans killing experiments demonstrated that furazolidone could efficiently protect C. elegans from P. aeruginosa infection and T. pyogenes + P. aeruginosa co-infection, especially in the fast-killing assay (Fig. 5). We failed to measure the protection of furazolidone on C. elegans against T. pyogenes challenge in this study due to the low virulence of T. pyogenes compared to the common bacterial pathogens. The C. elegans models grew well even when the inoculum dose of T. pyogenes was up to 1.0×10 9 CFUs (Data not shown).

Conclusion
Collectively, this study finds that furazolidone can simultaneously inhibit the growth and virulence of the two important bacterial pathogens of forest musk deer, T. pyogenes and P. aeruginosa. It is concluded that furazolidone can be considered as a promising candidate with antibacterial and anti-virulence capacities for the treatment of purulent disease of forest musk deer. The functional identification of furazolidone also provides a structural basis for development of novel anti-infectious drugs in the future.

Bacterial strains and media
T. pyogenes TP13 isolated from the lung pus of forest musk deer [6] and wild-type (WT) P. aeruginosa strain PAO1 were preserved in the lab and used elsewhere [17]. All the strains were routinely cultured in brain heart infusion with 5% fetal bovine serum (BHI-FBS) or in lysogeny broth (LB) from a single colony.

Culture conditions
A total of 55 compounds (Additional file 1 Supplementary Table S1) with similar core structure to the Acylhomoserine lactones (AHL) signals of P. aeruginosa QS system were selected and purchased from MedChemExpress (Shanghai, China). Overnight cultured T. pyogenes TP13 was diluted to optical density of 1.0 at wavelength of 600 nm (OD 600 = 1.0) by sterile saline solution. Equal amount of T. pyogenes (10 μL) was inoculated in 200 μL of BHI-FBS medium containing different concentrations (0, 50, 100, and 200 μM) of compounds and cultured overnight at 37 °C. The cell densities of the culture were determined by measuring the OD 600 and counting the colony forming units (CFUs) on BHI-FBS agar plates after appropriate dilution. Subsequently, T. pyogenes TP13 and P. aeruginosa PAO1 were mixed (1:9, 1:1, and 9:1) and co-cultured overnight on BHI-FBS agar containing 200 μM of the compounds with significant growth inhibition activities on T. pyogenes. The composition of T. pyogenes and P. aeruginosa in the co-culture were determined by counting the CFU of them on BHI-FBS agar plates after appropriate dilution, because the phenotypes of T. pyogenes and P. aeruginosa are significantly different and can be easily discriminated. Finally, the compounds that could inhibit the growth of T. pyogenes and P. aeruginosa were added (0, 50, 100, and 200 μM) to 200 μL of LB medium containing 10 μL of P. aeruginosa and cultured overnight at 37 °C. The cell densities were determined at OD 600 , and the colony forming units (CFUs) were counted on LB agar plates after appropriate dilution. All the experiments above were independently repeated for three times.

Quorum-sensing inhibition assay
M9-adenisine (0.1%, wt/v) and M9-skimmed milk (0.5%, wt/v) agar medium were used to evaluate the inhibitory activity of compounds on P. aeruginosa QS regulation [21]. Overnight cultured P. aeruginosa PAO1 was adjusted to OD 600 = 1.0 and inoculated (5 μL) on M9-adenisine agar and M9-milk agar containing different concentrations (0, 50, 100, and 200 μM) of compounds. The growth status of P. aeruginosa on M9-adenisine plates and the diameter of proteolytic circle formed on M9-milk plates were determined after 24 h. The experiments were independently repeated for nine times.

Biofilm production assay
Equal amount (20 μl, OD 600 = 1.0) of P. aeruginosa PAO1 was inoculated in glass tubes containing 2 mL of LB broth supplemented with different concentrations (0, 50, 100, and 200 μM) of compounds, and overnight cultured at 37 °C with shaking (220 rpm). The cell density was measured at OD 600 . After the culture solution and unadhered biofilm were gently removed, the adhered biofilm on the tube wall was stained with crystal violet (0.1%) for 30 min and washed with PBS buffer for three times. Subsequently, the stained biofilm was dissolved by 95% of ethanol solution and quantified at OD 595 . The experiments were independently repeated for three times.

Pyocyanin production assay
Equal amount (20 μl, OD 600 = 1.0) of P. aeruginosa PAO1 was inoculated in glass tubes containing 2 mL of LB broth supplemented with different concentrations (0, 50, 100, and 200 μM) of compounds, and overnight cultured at 37 °C with shaking (220 rpm). After the cell density was equalized with fresh LB broth, 200 μL of bacterial solution was taken out to extract the pyocyanin by chloroform and 0.2 N HCl and measured at OD 520 as described by Essar [35]. The experiments were independently repeated for three times.

Motility assay
For the swimming motility assay, 5 μl (OD 600 = 1.0) of P. aeruginosa PAO1 was inoculated on the surface of LB plates containing 0.5% of agar supplemented with different concentrations (0, 50, 100, and 200 μM) of compounds and cultured at 37 °C for 24 h. For the twitching motility assay, 2 μl (OD 600 = 1.0) of P. aeruginosa PAO1 was stabbed into the bottom of LB plates containing 1.0% of agar supplemented with different concentrations (0, 50, 100, and 200 μM) of compounds and cultured at 37 °C for 24 h. The motilities of P. aeruginosa PAO1 were determined by measure the diameters of colony on the surface (swimming motility) or the thin film region on the bottom (twitching motility). All the experiments were independently repeated for six times.

Transcriptomic analysis
Bacterial cells of furazolidone-treated (200 μM) and -untreated P. aeruginosa PAO1 were harvested for total RNA isolation using TRIzol reagents (Invitrogen), respectively. RNAs samples were conducted for library construction and RNA-sequencing (RNA-seq) by Novogene Bioinformatics Technology Company using prokaryotic strand-specific Illumina-based RNA-Seq technology (HiseqTM2500 platform). The obtained clean reads were mapped to the reference genome of PAO1 (NCBI accession number: AE004091) by the software Tophat2 [36]. SOAP2 program [37] and Cufflinks [38] were used to calculate the expected fragments per kilobase of transcript per million fragments (FPKM) sequenced, and the differentially expressed transcripts were presented and analyzed by EdgeR [39]. Differentially expressed gene with false discovery rate p < 0.05 was thought to be significantly different. The significantly differently expressed genes were mapped to the list of QS-activated genes reported by Schuster et al. [22] using Venn Diagrams (http:// bioin forma tics. psb. ugent. be/ webto ols/ Venn/).

Quantitative PCR
Total RNAs of furazolidone-treated and -untreated P. aeruginosa PAO1 were isolated by using TRIzol reagents, and the cDNA was synthesized by reverse transcription using a high-capacity cDNA Reverse Transcriptase kit Specific with gDNA removal (Takara). Quantitative PCR was performed by using an iTaq ™ universal SYBR ® Green Supermix (Bio-Rad) and the CFX Connect Real-Time PCR Detection System to validate the expression of typical QS-activated genes including lasR, rhlR, pqsR, lasB, rhlA, pqsA, pqsD, pqsE, hcnA, and phzA (Additional file 1  Supplementary Table S2). Gene expression was calculated by the 2 −ΔΔCT method using 16S rRNA as reference.

Caenorhabditis elegans assay
For the fast-killing assay, 20 μl (OD 600 = 1.0) of P. aeruginosa PAO1 and mixture of T. pyogenes TP13 and P. aeruginosa PAO1 (1:1) were spread on peptone-glucose-sorbitol (PGS) agar media with and without small molecule drug respectively, and cultured overnight at 37 °C. The naturally cooled plates were seeded with 10 newly cultured adult C. elegans (L4 stage) and further incubated at 25 °C for 96 h. For the slow-killing assay, to prevent C. elegans from laying eggs, 40 μL of 5-fluoro-2′-deoxyuridine solution (40 μg/mL) was evenly coated on the surface of nematode growth medium (NGM). Subsequently, 20 μl (OD 600 = 1.0) of P. aeruginosa PAO1 and mixture of T. pyogenes TP13 and P. aeruginosa PAO1 (1:1) were spread on NGM plates with and without small molecule drug and cultured overnight at 37 °C. The naturally cooled plates were seeded with 10 newly cultured adult C. elegans and further incubated at 25 °C for 10 days. The survival status of C. elegans in each experiment were observed and recorded. Growth of C. elegans on PGS agar plates or NGM plates feed with uracil auxotrophy Escherichia coli OP50 were set as controls.

Statistical analyses
Data analysis and statistical tests were performed by using Graphpad Prism version 9.0 (San Diego, CA, USA). Mean values of standard deviation were compared by using two-tailed unpaired t-test or One-way ANOVA. The survival curves of C. elegans were compared by using Log-rank (Mantel-Cox) test.