Introduction

Salmonella typhimurium is an important foodborne pathogen of humans and animals, attracting considerable concern for public health and the food industry (Friedman 2015). S. typhimurium invades intestinal epithelial cells, establishes biofilms, and induces gastroenteritis and colitis. The invasion is a crucial step in the pathogenesis of infections. This requires the expression of invasion genes, encoded by Salmonella pathogenicity island 1 (SPI-1), which mediates the initial penetration of intestinal epithelial cells. SPI-1 encodes a specialized type III secretion system (TTSS-1), and this system consists of structural components (e.g., needle complex) that make up the apparatus used to inject effector proteins into the cytoplasm of host cells. The effector proteins are translocated by the TTSS1 into the intestinal epithelial cells, inducing actin cytoskeletal rearrangements, generating profuse membrane ruffling at the site of interaction, and driving bacterial entry (Lawhon et al. 2002). The biofilm aggregations of S. typhimurium are involved in persistent colonization of a new host and are known to facilitate bacterial persistence by increasing antimicrobial resistance and interfering with the host immune response (Koopman et al. 2015). Flagella are involved in the motility and biofilm formation of S. typhimurium and favor the interaction with the intestinal epithelium (Elhadad et al. 2015). Therefore, SPI-1 and flagella play a major role in the virulence of S. typhimurium and may be targets for treating Salmonella.

Recently, many natural products that inhibit bacterial virulence, such as berberine, carvacrol and thymol, have been used to prevent and treat Salmonella infections (Barreca et al. 2014; Bian et al. 2016; Si et al. 2006; Wu et al. 2005). Phloretin (Fig. 1), a natural component of apple and pear, exhibits anti-inflammatory, anti-oxidant (de Oliveira 2016), and anti-virulence effects (Barreca et al. 2014). In previous studies, phloretin inhibited alpha-hemolysin expression in methicillin-resistant Staphylococcus aureus USA300 and Escherichia coli O157:H7 biofilm formation (Lee et al. 2011; Zhou et al. 2015). In this article, the effect of phloretin on the virulence of S. typhimurium was investigated.

Fig. 1
figure 1

The chemical structure of phloretin

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Materials and methods

Reagents

Phloretin was purchased from Chengdu Must Bio-technology Co., Ltd. (Chengdu, China). Luria-Bertani (LB) medium was obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and RPMI-1640 were obtained from Gibco (Invitrogen S.r.l., Milan, Italy). TRIzol reagent and PrimeScript™ RT Reagent Kit with gDNA Eraser were purchased from TaKaRa (Da Lian, Liaoning, China).

Bacterial strain and growth conditions

S. typhimurium CVCC541 was purchased from the Chinese Veterinary Culture Collection Center. Diluted (100-fold in LB) overnight cultures of S. typhimurium were incubated in 5-ml centrifuge tubes with 80, 40, and 20 μg/ml of phloretin at 37 °C. The OD600 was monitored for 18 h, at 60-min intervals, with a Synergy™ HT Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT).

Crystal violet biofilm assay

A static biofilm formation assay was performed in 96-well polystyrene plates. Briefly, overnight cultures were diluted to an OD600 of 0.05 in LB medium containing 0, 20, 40, or 80 μg/ml of phloretin and were incubated for 48 h without shaking at 37 °C. After washing three times with PBS, 0.1 % crystal violet was added to each well. After 20 min at room temperature, the microplate was emptied and washed three times with PBS. Finally, ethanol was added to resolve the stained biofilm cells, and total biofilm cells were measured at 570 nm.

Motility assay

S. typhimurium was inoculated with a sterile toothpick in the center of the LB agar (0.3 %) assay plates supplemented with 0, 20, 40, or 80 μg/ml of phloretin and then incubated at 37 °C. After incubation for 12 h, motility halos were measured.

Bacteria virulence genes assay by qRT-PCR

S. typhimurium (108 CFU/ml) was cultured in LB medium with or without phloretin (20, 40, or 80 μg/ml) at 37 °C for 3 h. After incubation for 3 h at 37 °C in 5 % CO2, the bacterial media were centrifuged at 12,000×g for 10 min, and bacterial cells were then collected for RNA extraction. Total RNA from S. typhimurium was isolated using TRIzol reagent according to standard procedures. For each RT-PCR reaction, 2 μg of total RNA was used to synthesize cDNA using a PrimeScript™ RT reagent kit with gDNA Eraser. The quantification of relative mRNA concentrations was detected by qRT-PCR using a 7500 Fast Real-Time PCR System (Applied Biosystems) with the SYBR green Plus reagent kit (Roche) and 30 pmol primers for target sequences (Table 1). The relative change in transcripts due to phloretin with respect to the control was calculated using the comparative CT method. The change in gene expression was calculated using the formula: phloretin/control = \( - 2^{{\Delta \Delta C_{\text{T}} }} \), where ΔΔC T = ΔCT for phloretin −ΔCT for control, and ΔCT is the difference between the CT value of the target gene and the normalization gene (16S rRNA). The mean ± standard deviation (SD) of three biological replicates is presented.

Table 1 Sequences of primers used for RT-PCR
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Bacterial adherence and invasion assay

S. typhimurium was grown to log phase in the presence of 0, 20, 40, or 80 μg/ml phloretin for 3 h. Bacteria were centrifuged and resuspended in plain DMEM medium to a density of 108 CFU/ml. IEC-6 cells were grown to confluency in 24-well tissue culture plates, washed three times with plain medium, and incubated with 500 ml of the bacterial solution for 3 h at 37 °C. The monolayers were washed three times with PBS to remove nonadherent bacteria and lysed in 500 μl 1 % Triton X-100 at room temperature for 5 min to release the bacteria. The suspensions were serially diluted, and bacterial cell counts were obtained by plating serial dilutions of bacteria on LB agar plates. In the invasion assay, the IEC-6 monolayers in the wells were washed once with 1 ml warm plain medium after a 3-h incubation with bacteria and then incubated for 2 h with 500 μl of 100 μg/ml gentamicin in warm plain DMEM to kill extracellular bacteria. Cells were washed three times with PBS and finally lysed in 500 μl 1 % Triton X-100. The intracellular bacterial cell counts were obtained by plating serial dilutions of bacteria on LB agar plates as described above.

LDH assay

The susceptibility of IEC-6 cells to the cytotoxic effects of phloretin was evaluated using the LDH assay. IEC-6 cells were grown to confluency in 24-well tissue culture plates and subjected to 0–80 μg/ml of phloretin in plain DMEM (without serum, l-glutamine, or antibiotics) for 3 h. After incubation for 3 h, the supernatants were collected by centrifugation at 12,000×g for 10 min. The LDH activity was determined according to the manufacturer’s protocol (Jiancheng Technology Co., Nanjing, China).

The cytotoxic effect of phloretin-treated S. typhimurium on IEC-6 cells was also evaluated using the LDH assay. S. typhimurium was grown to log phase in the presence of 0, 20, 40, or 80 μg/ml of phloretin for 3 h. Bacteria were centrifuged and resuspended in plain DMEM medium to a density of 108 CFU/ml. IEC-6 cells were grown to confluency in 24-well plates, washed three times with plain medium, and incubated with 500 μl of the bacterial solution for 3 h at 37 °C. The LDH activity was determined as described above.

In vivo protection studies

ICR mice were divided into five groups, and each group consisted of five mice. One day prior to infection, food and water were withdrawn for 4 h, and mice were orally gavaged with 100 μl streptomycin (15 mg) in water 24 h prior to infection; 24-h post-streptomycin treatment, food, and water were withdrawn for 4 h and mice infected with 108 cfu S. typhimurium in 400 μl of PBS containing 0, 20, 40, or 80 μg/ml of phloretin by gavage. Food and water were returned 2 h after infection and mice monitored for 48 h. Mice were killed by CO2 asphyxiation, and ceca were harvested aseptically, weighed, and homogenized in cold, sterile PBS for bacterial enumeration. Tissue Salmonella loads were quantified by plating serial dilutions of tissue homogenate on MacConkey agar plates supplemented with streptomycin (50 μg/ml).

Statistical analyses

Data are shown as the mean ± SD. Fisher’s least significant difference (LSD) test was used to evaluate significant differences between groups. P values of <0.05 were considered statistically significant.

Results

Effect of phloretin on the growth of S. typhimurium

The growth rates of S. typhimurium in the presence of 20, 40, and 80 μg/ml of phloretin were determined by measuring the OD600 up to 18 h. As shown in Fig. 2, growth of S. typhimurium was not inhibited by 20, 40, and 80 μg/ml of phloretin (P > 0.05).

Fig. 2
figure 2

Growth curves of S. typhimurium in LB in the presence of different concentrations of phloretin. S. typhimurium was grown for 18 h in the absence or presence of phloretin (20, 40, and 80 μg/ml), and then optical density (OD600 nm) measurements were obtained automatically every 60 min. # P < 0.05, ## P < 0.01

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Effect of phloretin on bacterial biofilm formation and motility of S. typhimurium

The biofilm formation of S. typhimurium in 96-well plates and motility in soft agar plates were measured in the presence of phloretin. As shown in Figs. 3 and 4, the S. typhimurium biofilm formation in 96-well plates was significantly inhibited by 40 and 80 μg/ml of phloretin (Fig. 3, P < 0.05), whereas the swimming motility halos of S. typhimurium in soft agar plates were significantly reduced in the presence of 80 μg/ml of phloretin (Fig. 4, P < 0.05).

Fig. 3
figure 3

Bacterial biofilm formation of S. typhimurium in the presence of different concentrations of phloretin. S. typhimurium was grown in 96-well plates without shaking for 48 h in the absence or presence of phloretin (20, 40, and 80 μg/ml), and bacterial biofilm formation was measurement using the crystal violet method. # P < 0.05, ## P < 0.01

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Fig. 4
figure 4

Swimming motility of S. typhimurium in soft agar plates supplemented with different concentrations of phloretin. S. typhimurium was inoculated with sterile toothpicks in the center of LB agar (0.3 %) plates supplemented with 0, 20, 40, and 80 μg/ml of phloretin, and then motility halos were measured after incubation for 12 h at 37 °C. # P < 0.05, ## P < 0.01

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Effect of phloretin on the expression of flagella-related genes

The effect of phloretin on the expression of flagella-related genes was determined using qRT-PCR. When S. typhimurium was grown in the presence of 80 μg/ml of phloretin, the transcriptional levels of fliA, fliC, and flhD were significantly decreased (Fig. 5, about 40 % of control, P < 0.05). In addition, the transcriptional levels of fliA, fliC, and flhD in 40 μg/ml of phloretin-treated S. typhimurium were about 70 % of control.

Fig. 5
figure 5

Flagella-related gene expression of S. typhimurium in LB medium with different concentrations of phloretin. S. typhimurium was grown in LB medium with 0, 20, 40, and 80 μg/ml of phloretin for 3 h, and then the bacteria cells were collected by centrifuging. The mRNA expression levels of fliA, fliC, and flhD in phloretin-treated S. typhimurium were determined by qRT-PCR. # P < 0.05, ## P < 0.01

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Effect of phloretin on the expression of SPI-1-related virulence genes

The effect of phloretin on the expression of SPI-1-related genes was determined using qRT-PCR. After exposure to 20, 40, and 80 μg/ml of phloretin for 3 h, the gene expression levels of sipA, sopB, and sopE2 were significantly repressed (Fig. 6a, P < 0.05). In addition, the gene expression levels of SPI-1 transcriptional regulators hilC, hilD, hilA, rtsA, and invF were also significantly repressed by 20, 40, and 80 μg/ml of phloretin (Fig. 6b, P < 0.05).

Fig. 6
figure 6

SPI-1-related virulence gene expression of S. typhimurium in LB medium without or with different concentrations of phloretin. S. typhimurium was grown in LB medium with 0, 20, 40, and 80 μg/ml of phloretin for 3 h, and the bacteria cells were collected by centrifuging. The SPI-1-related virulence gene expression of phloretin-treated S. typhimurium was determined by qRT-PCR. # P < 0.05, ## P < 0.01

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Effect of phloretin on S. typhimurium-infected IEC-6 cells

The adhesion and invasion characteristics of phloretin-treated S. typhimurium in IEC-6 cells were measured (Fig. 7). Bacterial adhesion and invasion assays revealed that 20, 40, and 80 μg/ml of phloretin significantly inhibited the adhesion and invasion of S. typhimurium in IEC-6 cells (P < 0.05). The virulence potential of phloretin-treated S. typhimurium was also determined by examining LDH release from cells. As shown in Fig. 8, the LDH release by IEC-6 cells was not affected in the presence of 20, 40, and 80 μg/ml of phloretin (Fig. 8a, P > 0.05), whereas 20, 40, and 80 μg/ml of phloretin significantly reduced the LDH release by IEC-6 cells induced by S. typhimurium (Fig. 8b, P < 0.05).

Fig. 7
figure 7

Adhesion and invasion of IEC-6 cells by phloretin-treated S. typhimurium. S. typhimurium was grown in LB medium with 0, 20, 40, and 80 μg/ml of phloretin for 3 h, and then the bacteria cells were collected by centrifuging. The confluent IEC-6 cells were infected with phloretin-treated S. typhimurium (108 CFU/ml) for 3 h, and then the bacterial counts for adhesion (a) and invasion (b) were detected using the spread plate method. # P < 0.05, ## P < 0.01

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Fig. 8
figure 8

Cytotoxicity of phloretin and phloretin-treated S. typhimurium to IEC-6 cells. a IEC-6 cells were grown to confluency in 24-well plates and subjected to 0, 20, 40, and 80 μg/ml of phloretin in plain DMEM for 3 h. After incubation for 3 h, the culture supernatants were collected for the determination of LDH. # P < 0.05, ## P < 0.01. b S. typhimurium was grown in LB medium with 0, 20, 40, and 80 μg/ml of phloretin for 3 h, and then the bacterial cells were collected by centrifuging. The confluent IEC-6 cells were infected with phloretin-treated S. typhimurium (108 CFU/ml) at 37 °C for 3 h, and the culture supernatants were collected for the determination of LDH. * P < 0.05, ** P < 0.01 vs. IEC-6 control group. # P < 0.05, ## P < 0.01 vs. S. typhimurium group

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Effect of phloretin on S. typhimurium infection in vivo

Quantitation of Salmonella recovered from cecum homogenates 48 h post-infection of mice was determined using the spread plate method. As shown in Fig. 9, the numbers of Salmonella in the cecum were dramatically affected. The numbers of S. typhimurium were lower in cecum tissues from mice infected with S. typhimurium in PBS containing phloretin compared with the control group (Fig. 9, P < 0.05).

Fig. 9
figure 9

Bacteria burdens in the cecum of the mice infected with phloretin-treated S. typhimurium. ICR mice were infected with 108 CFU S. typhimurium in 400 μl of PBS containing 0, 20, 40, or 80 μg/ml of phloretin by gavage 24-h post-streptomycin treatment. Ceca from these mice were harvested 48 h post-infection, and bacterial counts were detected through the spread plate method (n = 5)

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Disscusion

Anti-virulence strategies are now gaining interest as an alternative strategy to developing new types of anti-infective agents. Because phloretin demonstrated inhibition of the virulence of S. aureus and E. coli, we wondered whether phloretin has inhibitory effects on the virulence of S. typhimurium. In this study, we have demonstrated that subinhibitory concentrations of phloretin attenuated the virulence of S. typhimurium and protected against S. typhimurium.

Biofilm formation and cell motility are important contributors to the pathogenicity of S. typhimurium. Biofilms of S. typhimurium are known to facilitate bacterial persistence by increasing antimicrobial resistance and interfering with the host immune response (Koopman et al. 2015). Motility is strongly associated with the host cell invasion by S. typhimurium. To further study the effect of phloretin on S. typhimurium, biofilm formation in 96-well plates and motility in soft agar plates were measured. These results showed that the S. typhimurium biofilm formation in 96-well plates was inhibited by 40 and 80 μg/ml of phloretin, whereas the swimming motility halos were significantly reduced in the presence of 80 μg/ml of phloretin. Interestingly, these results suggested that at concentrations where growth of Salmonella was not inhibited, phloretin significantly inhibited bacterial biofilm formation and cell motility. Flagellin is required for host cell invasion and is possibly associated with motility (Singer et al. 2014a) and biofilm formation (Ahmad et al. 2013; Elhadad et al. 2015). The synthesis and function of the flagellum require the expression of a set of genes that encodes the structural proteins of the flagella (Chilcott and Hughes 2000; Fabrega and Vila 2013). We observed that phloretin repressed the gene expression of fliA (2.8-fold), fliC (2.7-fold), and flhD (2.1-fold) (Fig. 5), which may be one possible reason for the inhibition of biofilm formation and cell motility.

The effect of phloretin on the expression of SPI-1-related genes was determined using qRT-PCR. As shown in Fig. 4, after exposure to 20–80 μg/ml of phloretin for 3 h, the gene expression levels of sipA, sopB, and sopE2 in S. typhimurium were significantly repressed (Fig. 6a). Consistent with the SPI-1 gene levels, SPI-1 gene transcriptional regulators HilC, HilD, HilA, rtsA, and invF were also significantly repressed (Fig. 6b). The invasion of intestinal epithelial cells by Salmonella requires the TTSS-1 to encode by SPI-1, and the TTSS-1 secretes and translocates bacterial virulence factors directly into the cytosol of epithelial cells, where they promote actin rearrangement and engulfment of the bacterium (Hung et al. 2013). The secretion and effector proteins encoded by the SPI-1 include SipA, SopE, and SopE2 as well as others. SipA, SopB, and SopE2 trigger cytoskeletal rearrangements that internalize the pathogen by binding host actin or activating small GTPases, including Rac1, Cdc42, and RhoG (Lilic et al. 2003; Zhou et al. 2001; Zhou and Galan 2001). After bacterial invasion, SptP, a GTPase-activating protein, recovers the activated cell membrane (Fu and Galan 1999). Here, the gene levels of SipA, SopB, and SopE2 were reduced after exposure to phloretin (Fig. 6a). The SPI-1 gene expression also requires many transcriptional regulators. Most SPI-1 genes are arranged in operons that are coordinately regulated by the SPI-1-encoded protein HilA. HilA regulates the TTSS apparatus encoded by the prg/org and inv/spa genes by directly binding to the HilA box (Lostroh et al. 2000; Lostroh and Lee 2001). By binding upstream of invF, HilA directly activates the expression of invF. invF regulates the transcription of several effector genes that encode proteins secreted by the TTSS-1 apparatus (Akbar et al. 2003). In turn, HilA and invF are governed by three other transcriptional regulators, HilC, HilD, and rtsA (Ellermeier et al. 2005; Saini et al. 2010; Singer et al. 2014b). The results indicate lower gene expression of HilC/HilD/HilA/rtsA/invF in the presence of phloretin (Fig. 6b), suggesting that phloretin exerts its effect on SPI-1 possibly by regulating expression of HilC/HilD/HilA/rtsA/invF.

Infection of intestinal epithelial cells is dependent on the SPI-1-encoded type III secretion system and flagellar motility. Inhibition of Salmonella motility, biofilm formation, and the mRNA expression of SPI-1 and flagella genes by phloretin suggests that phloretin can attenuate the virulence of S. typhimurium. To test this hypothesis, the effects of phloretin on IEC-6 cells infected with S. typhimurium and in vivo S. typhimurium infection were measured. Bacterial adhesion and invasion assays revealed that 20, 40, and 80 μg/ml of phloretin inhibit the adhesion and invasion of S. typhimurium in IEC-6 cells (Fig. 7). The virulence potential of S. typhimurium also can be determined by examining LDH release from cells. In this study, at the doses that did not inhibit cell viability, phloretin reduced the LDH release by IEC-6 cells induced by S. typhimurium (Fig. 8). Moreover, phloretin-supplemented mice decreased Salmonella colonization in the cecum (Fig. 9). These results reveal that phloretin powerfully protects mice against S. typhimurium infection.

In conclusion, subinhibitory concentrations of phloretin attenuated S. typhimurium virulence and protected mice against S. typhimurium infection. These results suggest that phloretin could potentially be used as a food additive to prevent gastrointestinal disease caused by S. typhimurium, providing new insights into the function of fruit.