Inhibition of Cronobacter Sakazakii Biolm Formation and Expression of Virulence Factors by Coenzyme Q 0

Objectives(cid:0)Here, we investigated the inhibitory effects of coenzyme Q 0 (CoQ 0 ) on biolm formation and the expression of virulence genes by Cronobacter sakazakii. Results(cid:0)We found that the minimum inhibitory concentration of CoQ 0 against C. sakazakii strains ATCC29544 and ATCC29004 was 100 μg/mL, while growth curve assays showed that sub-inhibitory concentrations (SICs) of CoQ 0 for both strains were 6.4, 3.2, 1.6 and 0.8 μg/mL. Assays exploring the inhibition of specic biolm formation showed that SICs of CoQ 0 inhibited biolm formation by C. sakazakii in a dose-dependent manner, which was conrmed by scanning electron microscopy and confocal laser scanning microscopy analyses. CoQ 0 inhibited the swimming and swarming motility of C. sakazakii and reduced its ability to adhere to and invade HT-29 cells. In addition, CoQ 0 impeded the ability of C. sakazakii to survive and replicate within RAW 264.7 cells. Finally, real time polymerase chain reaction analysis conrmed that nine C. sakazakii genes associated with biolm formation and virulence were down-regulated in response to CoQ 0 treatment. Conclusion(cid:0)Overall, our ndings suggest that CoQ 0 is a promising antibiolm agent and provide new insights for the prevention and control of infections caused by C. sakazakii.

stresses [14]. As such, compared with planktonic cells, bio lm-based C. sakazakii infections are signi cantly more di cult to resolve [15]. In an industrial setting, bio lm formation by C. sakazakii increases the risk of contamination of foodstuffs, which has signi cant health and nancial repercussions [16].
Virulence factors are gene-mediated molecules produced by microorganisms that enhance their ability to invade a host, cause disease, or evade host defenses [17]. Functions such as motility are also considered virulence factors. C. sakazakii produces a variety of virulence factors, including proteins involved in motility, host cell adhesion and invasion [18], and replication and survival within macrophages [19].
Motility is essential for the virulence of C. sakazakii, allowing it to pass through the intestine and colonize more favorable host environments such as mucous membranes, the gastric and intestinal epithelia, and endothelial tissues [20][21][22]. Outer membrane proteins OmpA and OmpX were shown to play an important role in C. sakazakii adhesion to and invasion of human intestinal epithelial Caco-2 and HT-29 cells [23]. Further study con rmed that OmpA contributes signi cantly to the pathogenicity of C. sakazakii as an essential factor in the invasion of various epithelial and endothelial cells of human and animal origin [24], while OmpX plays a crucial role in the basolateral invasion of host cells [25]. The ability of C. sakazakii to survive and replicate inside immune cells such as macrophages is critical for establishing infection and is the rst step in the development of severe illnesses such as sepsis and meningitis [26].
Over the past decade, the use of plant-derived compounds as alternative antimicrobials has gained signi cant attention as a result of increasing concerns over the safety of synthetic antimicrobial agents and the emergence of antibiotic-resistant bacteria [27]. Coenzyme Q 0 (CoQ 0 ; 2,3-dimethoxy-5-methyl-1,4benzoquinone; C 9 H 10 O 4 ) is a benzoquinone compound extracted from the fungus Antrodia camphorata [28]. It has demonstrated anti-tumor, anti-in ammatory, and anti-angiogenic properties [29], as well as antibacterial activity against Staphylococcus aureus and Listeria monocytogenes [30,31]. In addition, CoQ 0 successfully inhibited bio lm formation by Salmonella enterica serovar Typhimurium [32].
Although multiple reports show that CoQ 0 has inhibitory effects against a variety of microorganisms, few studies have examined the effects of CoQ 0 on C. sakazakii bio lm formation or the expression of virulence factors. In the current study, we rst determined the minimum inhibitory concentration (MIC) and sub-inhibitory concentration (SIC) of CoQ 0 against several C. sakazakii strains. Crystal violet staining, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM) were then used to study the effects of SICs of CoQ 0 on C. sakazakii bio lm formation. We then assessed the effects of CoQ 0 on various C. sakazakii virulence traits, including motility, adhesion to and invasion of HT-29 cells, and survival and replication within macrophages. Finally, the expression of genes associated with bio lm formation and other virulence factors following CoQ 0 treatment was examined using real time polymerase chain reaction (RT-PCR) analyses.

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Chemicals and reagents CoQ 0 (HPLC ≥99%, CAS 605-94-7) was obtained from J&K Scienti c Co., Ltd (Beijing, China) and dissolved in dimethyl sulfoxide (DMSO) for use in all experiments. The nal concentration of DMSO in all sample solutions was 0.1% (v/v), which has no apparent effect on the growth of C. sakazakii. All other chemicals were of analytical grade and were unaltered.
Bacterial strains and culture conditions C. sakazakii strains ATCC 29004 and ATCC 29544 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Strain ATCC 29004, which is a relatively strong bio lm producer, was used in the bio lm assay. Prior to each assay, bacteria were inoculated onto tryptic soy agar (TSA) medium and incubated at 37°C for 12 h. To obtain fresh overnight cultures, a single colony was inoculated into 30 mL of tryptic soy broth (TSB) medium and incubated with shaking at 130 rpm for 12 h at 37°C. Following incubation, cultures were centrifuged (4°C, 8000 × g, 5 min), washed three times with sterile phosphate-buffered saline (PBS), and diluted in TSB medium to an optical density at 600 nm (OD 600 ) of 0.5 (approximately 4 × 10 8 colony-forming units (CFU)/mL).

MICs and SICs determinations
The MICs and SICs of CoQ 0 against C. sakazakii ATCC 29004 and ATCC 29544 were determined as described previously [33], with some modi cations. Brie y, overnight bacterial culture was diluted 400× in TSB medium (approximately 1 × 10 6 CFU/mL) before 125 μL of the diluted culture were added to individual wells of a 96-well plate. Equal volumes of CoQ 0 solution were gently added to each well to achieve nal CoQ 0 concentrations of 0 (control), 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, and 51.2 μg/mL. TSB medium containing 0.1% DMSO was used as the negative control. Plates were incubated at 37°C for 24 h, and cell growth was monitored at 600 nm at 1-h intervals using a microplate reader (Model 680; Bio-Rad, Hercules, CA, USA). The MIC of CoQ 0 was de ned as the lowest concentration at which there was no visible growth of C. sakazakii. SICs of CoQ 0 were de ned as concentrations at which no signi cant inhibition of C. sakazakii growth was observed.

Inhibition of speci c bio lm formation (SBF) assay
The inhibition of SBF assay was carried out using a crystal violet staining method as described previously [34], with minor modi cations. Brie y, an overnight culture of C. sakazakii ATCC 29004 was diluted in TSB medium to an OD 600 of 1.0. CoQ 0 was added to culture aliquots to obtain nal concentrations of 0 (control), 1.6, 3.2, and 6.4 μg/mL. Aliquots (200 μL) of the mixtures were then pipetted into individual wells of a 96-well plate. Uninoculated TSB containing 0.1% DMSO was used as the negative control. Plates were incubated statically at 37°C or 25°C for 24, 72, or 120 h and the absorbance of the mixtures was monitored using a microplate reader (Model 680; Bio-Rad) at 630 nm.
Following incubation, bacterial cultures were aspirated and the plates were washed with 300 μL of distilled water before being air-dried for 30 min. The wells were stained with 250 μL of 1% (w/v) crystal violet (Tianjin Kermel Chemical Regent Co., Ltd, Tianjin, China) for 20 min and any excess stain was removed using sterile distilled water. After drying for 30 min, 250 μL of 33% (v/v) glacial acetic acid was added to each well and the plates were shaken at ambient temperature for 20 min. The bio lm biomass in each well was quanti ed by measuring the OD at 570 nm. The SBF was determined from the ratio of the OD 570 and OD 630 values.

SEM observation
Con rm the effects of CoQ 0 on bio lm formation, the C. sakazakii ATCC 29004 bio lms were further analyzed by SEM as described previously [31], with minor modi cations. Brie y, C. sakazakii ATCC 29004 cultures (OD 600 = 1.0) mixed with CoQ 0 solution (6.4, 3.2, 1.6, or 0 μg/mL) were added to individual wells of a 24-well plate containing sterile glass slides of the same diameter as the bottoms of the wells. Culture without CoQ 0 was used as a control. Following incubation at 25°C for 48 h, culture supernatants were removed, and the samples were xed with 2 mL of 2.5% (v/v) glutaraldehyde overnight at 4°C. The glass slides were then removed and washed with sterile PBS, followed by treatment with 1% (v/v) osmic acid at 4°C for 5 h. Subsequently, the glass slides were dehydrated using a graded ethanol series (30%, 50%, 60%, 70%, 80%, 90%, and 100%). After being dried and coated with gold, the slides were examined under a eldemission scanning electron microscope (S-4800; Hitachi, Tokyo, Japan) at 4000× and 1500× magni cation.

CLSM observation
To examine the effects of CoQ 0 on the viability of bio lm-associated C. sakazakii ATCC 29004 cells, bio lms were next strained using a LIVE/DEAD BacLight Bacterial Viability Kit (Thermo Fisher Scienti c, Waltham, MA, USA), consisting of SYTO 9 and propidium iodide (PI) dyes. Bio lms were cultured on stainless steel coupons in a 24-well plate in the presence or absence of CoQ 0 , as described in section 2.5.
Following removal of the supernatant, the plate was washed twice with sterile water. Stainless-steel coupons were then stained with STYO 9 and PI in the dark for 8 min as per the manufacturer's instructions. After being washed with sterile water, the stained bio lms on the stainless-steel coupons were examined using a confocal laser scanning microscope (A1; Nikon, Tokyo, Japan).

Motility assay
Swimming and swarming motility assays were conducted as described previously [35], with minor modi cations. Bacterial swimming motility assays were conducted using 20 mL of LB broth containing 0.3% (w/v) agar, while swarming motility assays were conducted in 20 mL of LB broth supplemented with 0.5% (w/v) agar and 0.5 (w/v) glucose. CoQ 0 was added to the warm media (45°C) to achieve nal concentrations of 0 (control), 1.6, 3.2, and 6.4 μg/mL, and the resulting mixtures poured into Petri dishes. The resulting agar plates were dried at ambient temperature for 1 h. Aliquots (5 μL) of bacterial cultures (OD 600 = 0.5) were then inoculated onto the center of each plate and incubated upright at 37℃ for 7 h. Images of the resulting bacterial halos and swarm areas were obtained using a Gel Imaging System (Bio-Rad).

Adhesion and invasion assay
The effects of CoQ 0 on bacterial adhesion and invasion were examined as described previously [36], with some modi cations. For both assays, HT-29 cells in DMEM/F-12 were inoculated into 24-well plates (10 5 cells/well) and incubated overnight before being washed twice with sterile PBS. Aliquots of C. sakazakii ATCC 29544 culture (OD 600 = 0.5) were mixed with CoQ 0 solution (6.4, 3.2, 1.6, 0.8, or 0 μg/mL nal concentration) and cultured at 37°C for 6 h. Bacterial cultures were then washed with sterile PBS and the cell pellets resuspended in TSB medium to an OD 600 of 0.5 (approximately 4 × 10 8 CFU/mL). Cultures were diluted 40× in DMEM/F-12 and inoculated onto the HT-29 cell monolayers at a multiplicity of infection (MOI) of 10. Following centrifugation (600 × g, 5 min), the plates were incubated in a humidi ed, 5% CO 2 incubator at 37°C for 2 h.
For adhesion assays, the incubated plates were rinsed three times with sterile PBS and the cells lysed by the addition of 1 mL of 0.1% (v/v) Triton X-100 (Amresco, Solon, OH, USA) followed by incubation at 4°C for 20 min. The lysed cells were then serially diluted in sterile PBS and plated on TSA for colony counting. For invasion assays, the incubated plates were washed once with sterile PBS and 1 mL of DMEM/F-12 containing gentamicin (100 μg/mL; Amresco) was added to each well. The plates were then incubated for a further 45 min to kill the extracellular bacteria. Following incubation, the cells were washed three times before being lysed by the addition of 1 mL of 0.1% (v/v) Triton X-100 followed by incubation at 4°C for 20 min. The lysed bacteria were then serially diluted and plated on TSA for colony counting. Results were expressed as the percentage of colonies on the treatment plates relative to those on the control plates.

Intracellular survival and replication assay
The effects of CoQ 0 on the intracellular survival and replication of C. sakazakii ATCC 29544 in RAW 264.7 cells were examined as described previously [34], with some modi cations. RAW 264.7 cells cultured in DMEM were seeded into 24-well plates (10 5 cells/well) and incubated at 37°C in a humidi ed atmosphere with 5% CO 2 for 16 h. Aliquots of bacterial suspension (OD 600 = 0.5) were mixed with CoQ 0 at a nal concentration of 0 (control), 0.8, 1.6, 3.2, or 6.4 μg/mL and incubated at 37°C for 6 h. Following incubation, the suspensions were washed once with sterile PBS and the resulting cell pellets resuspended in TSB to an OD 600 of 0.5 (approximately 4 × 10 8 CFU/mL). The cell suspensions were then diluted in DMEM to a density of 1 × 10 6 CFU/mL and inoculated onto the RAW 264.7 cell monolayers at a MOI = 10.
Following incubation at 37°C in the presence of 5% CO 2 for 2 h, the plates were rinsed once with sterile PBS, and 1 mL of DMEM containing gentamicin (100 μg/mL) was added to each well. The plates were then incubated for a further 45 min.
For the intracellular survival assays, the incubated plates were rinsed with sterile PBS and the cells lysed by the addition of 1 mL of 0.1% (v/v) Triton X-100 followed by incubation at 4°C for 20 min. The lysed cells were serially diluted before being plated on TSA plates. The results are expressed as the number of viable C. sakazakii cells (CFU/mL) before and after CoQ 0 treatment. Infected cells that were not treated with CoQ 0 were used as the control. For the intracellular replication assay, the incubated plates were again rinsed with sterile PBS and 1 mL of DMEM containing gentamicin (10 μg/mL) was added to each well. Following incubation at 37°C in the presence of 5% CO 2 for either 24 h or 48 h, the cells were lysed, serially diluted, and plated on TSA plates, as described in the intracellular survival assay.

Isolation of RNA and RT-PCR assays
To assess the effects of CoQ 0 on the expression of virulence genes ( as per the manufacturer's instructions. The quality and concentration of the extracted RNA were determined using a nucleic acid and protein spectrophotometer (Nano-200; Aosheng Instrument Co., Ltd, Hangzhou, China). RNA was then reverse-transcribed into cDNA using a Takara PrimeScript RT Reagent Kit (Takara, Dalian, China) as per the manufacturer's instructions. RT-PCR assays were carried out in 25-μL reaction volumes with SYBR Green reagents (Takara) using an IQ 5 Multicolor Real-Time PCR Detection System (Bio-Rad). The reaction parameters included an initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s, with a dissociation step of 95°C for 15 s and 60°C for 30 s. Speci c primers corresponding to each of the target genes (Table 1) were based on those from previous reports [24,37]. The 16S rRNA gene was used as an internal control for normalization of gene expression. The 2 −∆∆Ct method was used to compare the expression of genes from different samples [38]. The means and standard deviations were calculated from triplicate experiments. Table 1 Primers used in this study.

Primers
Gene ampli ed Primer sequences (5′-3′) All experiments were carried out independently three times. The data were presented as means ± standard deviations (SD) and analyzed using SPSS 23.0 software (IBM, New York, NY, USA). A Student's ttest was used to analyze differences between means. Differences were considered statistically signi cant at P<0.05 and extremely signi cant at P<0.01.

MICs
CoQ 0 showed strong antibacterial activity against C. sakazakii ATCC 29004 and ATCC 29544, with an observed MIC of 100 μg/mL for both strains (data not shown).

SICs and growth curve analyses
The effects of CoQ 0 on the growth of C. sakazakii strains ATCC 29004 and ATCC 29544 are shown in Fig.   1. At a CoQ 0 concentration of 51.2 μg/mL, the lag phase of both C. sakazakii cultures was longer than that of the control culture grown in the absence of CoQ 0 . However, the growth curves of C. sakazakii ATCC 29004 and ATCC 29544 treated with 6.4, 3.2, 1.6, or 0.8 μg/mL CoQ 0 were not signi cantly different from that of the untreated control (P>0.05). Therefore, these concentrations were used as SICs in subsequent bio lm formation, virulence, and RT-PCR assays.

Effects of CoQ0 on C. sakazakii bio lm formation
The effects of CoQ 0 on the ability of C. sakazakii ATCC 29004 to form bio lms following incubation at 25°C or 37°C for 24, 72, or 120 h are shown in Fig. 2. The total bio lm biomass was signi cantly (P<0.05) decreased compared with the control following treatment with CoQ 0 at 6.4 μg/mL and incubation at 25°C ( Fig. 2A). At 37°C, bio lm biomass was signi cantly (P<0.05) decreased compared with the control at a CoQ 0 concentration of 1.6 μg/mL, with further decreases in biomass observed with increasing CoQ 0 concentration (Fig. 2B).
SEM observation SEM images of CoQ 0 -treated C. sakazakii bio lms at 4000× and 1500× magni cation are shown in Fig. 3.
In the absence of CoQ 0 , the bio lms exhibited a typical three-dimensional morphology with thick aggregates. In comparison, the bio lms became monolayers and cell clusters became looser with increasing CoQ 0 concentrations, indicative of signi cant disruption.

CLSM observation
As shown in Fig. 4A, the untreated bio lm appeared almost completely green, indicative of live cells, with few bacteria exhibiting PI uorescence. However, increasing levels of red uorescence (PI) were observed with increasing concentrations of CoQ 0 , suggesting a breakdown in membrane integrity. The highest level of red uorescence intensity was observed at a CoQ 0 concentration of 6.4 μg/mL (Fig. 4d).

Motility
As shown in Fig. 5A, CoQ 0 treatment resulted in a decrease in the swimming motility of C. sakazakii compared with the untreated control, with the observed decreases in motility occurring in a dosedependent manner. Similarly, the swarming motility of C. sakazakii decreased compared with that of the control in response to increasing CoQ 0 concentration (1.6-6.4 μg/mL) (Fig. 5B).

Adhesion to and invasion of HT-29 cells
We next examined the effects of CoQ 0 on the ability of C. sakazakii ATCC 29544 to adhere to and invade HT-29 cells. As shown in Fig. 6A

RT-PCR analyses
RT-PCR analyses showed that following treatment with CoQ 0 at 3.2 μg/mL, the expression of uvrY (adherence and invasion) in C. sakazakii cells was signi cantly (P<0.05) down-regulated compared with the untreated control (Fig. 8). Similarly, the mRNA levels of gJ and iD ( agellar assembly), motA and motB ( agellar motor proteins), bcsG (cell biosynthesis and bio lm formation), bcsA (cellulose synthase operon), and ompX and lpxB (LPS biosynthesis) were signi cantly (P<0.01) lower than those in the control. However, no differences in the levels of transcription of gJ, uvrY, and motB compared with the control were observed at CoQ 0 concentrations ≤1.6 μg/mL.

Discussion
C. sakazakii, with its array of virulence factors, poses a signi cant threat to the health of infants and young children because of its ability to form bio lms on surfaces in facilities that process milk and dairy products [39]. With the global increase in antibiotic resistance and safety concerns surrounding the use of chlorine-based disinfectants, the antibacterial properties of plant-derived compounds are increasingly being investigated [40,41]. CoQ 0 , derived from A. cinnamomea, has attracted attention for its potential as a natural food preservative and as a therapeutic antibiotic [28]. Based on growth curve analysis, we identi ed appropriate SICs of CoQ 0 that had no effect on growth for use in subsequent assays. These experiments showed that at SICs, CoQ 0 has signi cant anti-bio lm and antibacterial activities that cannot be attributed to inhibition of bacterial growth, indicating that low concentrations of CoQ 0 affect bacterial virulence factors instead.
Bio lm formation contributes to the survival of C. sakazakii in sub-optimal environments, is involved in evading and circumventing the host immune system, and provides protection against antibiotics and disinfectants [42]. Therefore, identifying potential anti-bio lm strategies has been a major focus for controlling the growth of C. sakazakii [12]. In the current study, the effects of CoQ 0 on the formation of C.
sakazakii bio lms was rst evaluated via crystal violet staining. The results showed that at SICs, CoQ 0 signi cantly reduced C. sakazakii bio lm biomass at 25°C (normal room temperature) and bio lm formation at 37°C (the optimal temperature for C. sakazakii growth) (Fig. 2). In addition, CoQ 0 signi cantly reduced the expression of genes related to bio lm formation, including bcsA and bcsG (Fig.  8). bcsA encodes the catalytic subunit of cellulose synthase, while bcsG encodes a conserved hypothetical protein involved in cellulose biosynthesis. Cellulose is an important component of the extracellular matrix of C. sakazakii bio lms and is essential for bio lm formation [43]. Previous studies have shown that cell-free supernatants derived from goat milk-origin lactobacilli cultures can prevent the formation of C. sakazakii bio lms [44], while pomegranate and rosemary extracts were shown to have a synergistic effect in combination with traditional antibiotics against bio lm formation by Pseudomonas aeruginosa [45]. Shi et al. (2018) reported that thymequinone signi cantly inhibited the bio lm formation of C. sakazakii by reducing the production of cellulose [33]. Therefore, our results suggest that CoQ 0 reduces the transcription of genes required for cellulose biosynthesis, ultimately inhibiting C. sakazakii bio lm formation.
With increasing CoQ 0 concentration, C. sakazakii bio lms contained fewer cells overall and a higher percentage of damaged and dead cells compared with the control (Fig. 3). These SEM-and CLSM-based ndings supported the results of quantitative crystal violet staining analysis, con rming that CoQ 0 inhibits C. sakazakii bio lm formation. Similarly, Yang et al. (2019) used SEM analysis to show that shikimic acid (0.3125 mg/mL) effectively inhibits the formation of Salmonella Typhimurium bio lms, while CLSM observations by Nair et al. (2018) demonstrated that selenium damages Escherichia coli O157:H7 bio lms and decreases live cell counts [32,46].
Bacterial invasion of host cells requires motility, which is an important factor in the early stages of infection. Swimming motility allows bacteria to move through liquid media, while swarming and clustering are observed in broth/semi-solid media and on solid surfaces/agar, respectively [14]. Our ndings demonstrated that both the swimming and swarming motilities of C. sakazakii were decreased in a concentration-depended manner compared with the control as a result of CoQ 0 treatment (Fig. 5).
Consistent with this observation, motility-associated genes motA, motB, gJ, and iD were signi cantly down-regulated compared with the control following CoQ 0 treatment (Fig. 8). Other researchers have shown that the swimming and swarming motility of Listeria monocytogenes can be signi cantly reduced compared with morin-treated controls following treatment with a Bi dobacterium-derived bacteriocin [36] while the inhibition of agellum biosynthesis and function as a result of citral treatment helped reduce the motility of C. sakazakii [18]. Flagella are the main structures responsible for bacterial motility and are essential for maximum virulence [47]. iD and gJ encode the agellum capping protein and a muramidase involved in agella rod assembly, respectively. Hu et al. (2015) reported that motA and motB form a bicistronic operon encoding two proteins that form the agellum matrix [48]. Therefore, we predict that SICs of CoQ 0 inhibit C. sakazakii agellum biosynthesis, reducing its mobility and capacity to invade host cells.
The ability of C. sakazakii to adhere to and invade host cells, including various epithelial cell lines and brain endothelial cells, is an important virulence factor in the establishment of infection [49]. In C. sakazakii, outer membrane protein OmpX also contributes to host cell invasion [50]. In the current study, we found that CoQ 0 signi cantly inhibited the adhesion and invasion of C. sakazakii in HT-29 cells, likely by downregulating the transcription of ompX and uvrY, two genes that are positively correlated with bacterial adhesion/invasion of epithelial cells (Fig. 8). Similarly, CoQ 0 treatment downregulated the expression of adhesion/invasion-associated genes in L. monocytogenes, resulting in a signi cant decrease in the adherence to and invasion of Caco-2 cells [31]. Pediococcus acidilactici K10, isolated from kimchi, was also shown to reduce the adherence of S. Typhimurium KCTC 1925 and E. coli O157:H7 ATCC 35150 to HT-29 cells [51]. In light of these ndings, it is likely that CoQ 0 plays a signi cant role in reducing C. sakazakii adhesion to and invasion of enterocytes by regulating associated gene expression.
Clinical C. sakazakii isolates have been observed to continuously reproduce in macrophages and microglia [52]. Similar studies have reported that C. sakazakii tolerates the intracellular environment of macrophages, hiding inside these cells to evade the immune response and, ultimately, invade other organs [49,53]. In the present study, SICs of CoQ 0 prominently inhibited the survival and replication of C. sakazakii inside macrophages (Fig. 7), supporting ndings by Yang et al. (2019) who reported that the ability of S. Typhimurium to survive and reproduce intracellularly was impacted by CoQ 0 treatment [32].
Based on these ndings, we speculate that CoQ 0 can reduce the ability of C. sakazakii to overcome host barriers and evade the immune response.
In summary, the current study con rms that CoQ 0 is an ideal antibio lm and anti-virulence agent. We demonstrated that SICs of CoQ 0 effectively inhibited the initial formation of bio lms at 25°C and 37°C, prevented the adherence of the bio lm to glass slides, and caused signi cant collapse of the bio lm structure. In addition, CoQ 0 inhibited the swimming and swarming motilities of C. sakazakii cells and prevented their adhesion to and invasion of HT-29 cells. As well as decreasing the intracellular survival and replication of C. sakazakii in RAW 264.7 macrophages, SICs of CoQ 0 down-regulated the transcription of virulence-associated genes. Together, these activities signi cantly impacted the virulence of C. sakazakii. Our ndings suggest that CoQ 0 is a promising broad-spectrum anti-virulence therapeutic agent that could be used to control C. sakazakii bio lm pollution and provide new avenues for the prevention and control of infections.