Inhibition of Cronobacter sakazakii Virulence Factors by Citral

Cronobacter sakazakii is a foodborne pathogen associated with fatal forms of necrotizing enterocolitis, meningitis and sepsis in neonates and infants. The aim of this study was to determine whether citral, a major component of lemongrass oil, could suppress putative virulence factors of C. sakazakii that contribute to infection. Sub-inhibitory concentrations of citral significantly decreased motility, quorum sensing, biofilm formation and endotoxin production. Citral substantially reduced the adhesion and invasion of C. sakazakii to Caco-2 cells and decreased bacterial survival and replication within the RAW 264.7 macrophage cells. Citral also repressed the expression of eighteen genes involved in the virulence. These findings suggest that citral has potential to be developed as an alternative or supplemental agent to mitigate the infections caused by C. sakazakii.

Scientific RepoRts | 7:43243 | DOI: 10.1038/srep43243 Although most of previous studies focused on the antimicrobial properties of citral, a limited number of studies examined its anti-virulence potential, even less information is available regarding its effect on reducing pathogenic potential of C. sakazakii. The purpose of this study is to investigate the effect of citral at sub-inhibitory concentrations (SICs) on the reduction of the virulence properties of C. sakazakii. Changes in motility, specific biofilm formations, QS, endotoxin production, adhesion and invasion of Caco-2 cells and survival and replication in macrophage cells were analyzed. In addition, expression of different virulence genes were determined by reverse-transcriptase (RT) PCR.

Minimum Inhibitory Concentrations and Sub-Inhibitory Concentrations. Citral exhibited inhibitory
effects against three tested C. sakazakii strains. Complete growth inhibition was achieved after 24 hours by citral at 3600 μ M as measured by OD 600 (Fig. 1). Thus the minimum inhibitory concentration (MIC) of citral against C. sakazakii strains was considered to be 3600 μ M. Moreover, concentrations below 225 μ M (1/16 MIC) exhibited no inhibition against C. sakazakii ATCC 29544 (Fig. 1) and those concentrations were further chosen as SICs to study the effects of citral on C. sakazakii virulence. After determining that citral was equally effective against three C. sakazakii isolates (ATCC 29544, ATCC 29004 and 7-17), we selected strain ATCC 29544 for further experiments.
To examine the possible explanation for the effect of citral on motility of C. sakazakii ATCC 29544, we determined by electron transmission microscopy (TEM) whether there are changes in flagella after treatment with citral. The TEM images revealed that structurally intact flagellar filaments were observed on C. sakazakii ATCC 29544 cells obtained from the edge of swimming halos (Fig. 2C). C. sakazakii strains treated with citral exhibited less flagella than control cells (Fig. 2C).
Biofilm Formation. The anti-biofilm efficacy of citral was investigated on C. sakazakii ATCC 29544. Bacteria were grown at 12 °C and 25 °C for 24, 48 and 72 hours on microtiter plates. Compared with the control, cells treated with citral showed a significant and dose-dependent inhibition of biofilm formation of C. sakazakii. The biofilm formation was inhibited by 67.1%, 69.5% and 70.1% with 225 μ M citral after treatment at 25 °C for 24, 48 and 72 hours, respectively (Table 1). Quorum Sensing. The effects of citral on C. sakazakii QS were indirectly assessed using C. violaceum 12472.
This bacterium produces the pigment violacein in response to QS signal homologous to those produced by C. sakazakii. Citral showed no apparent antimicrobial activity against C. violaceum at concentrations used in this study (56.25 μ M, 112.5 μ M and 225 μ M) with a broth micro-dilution method (data not shown). As seen in Fig. 3, anti-QS activity was shown when citral was used at 56.25 μ M, 112.5 μ M and 225 μ M, as evidenced by a decreased production of violacein (about 94.44%, 83.33% and 69.44% of the control level, respectively).

Adhesion and Invasion of Caco-2 cells.
The effect of citral on the adhesion and invasion of C. sakazakii ATCC 29544 in Caco-2 cells is depicted in Fig. 4. Citral at 56.25 μ M, 112.5 μ M and 225 μ M inhibited adhesion (P ≤ 0.01) of C. sakazakii to 82%, 72% and 50% of the control, respectively (Fig. 4A). Citral was also effective in inhibiting (P ≤ 0.01) the ability of C. sakazakii to invade Caco-2 cells (Fig. 4B). The invasiveness of C. sakazakii ATCC 29544 was reduced by 27%, 43% and 70% with 56.25 μ M, 112.5 μ M and 225 μ M of citral, respectively. Survival and Replication in RAW 264.7 cells. Macrophages were able to kill C. sakazakii as demonstrated by the decrease cell population in citral-untreated samples (Fig. 5). The results showed all three concentrations (56.25 μ M, 112.5 μ M and 225 μ M) of citral were able to significantly (P ≤ 0.01) decrease survival and replication of C. sakazakii in the macrophages in a dose-dependent manner during 48 hours (Fig. 5) when compared to the control medium.

Endotoxin.
A good linearity was established between the absorbance intensity at 585 nm and the concentration of endotoxin (y = 0.377x + 0.021; R 2 = 0.98). After the addition of citral to the cells, decreased endotoxin concentration was detected (Fig. 6). The original endotoxin concentration of C. sakazakii ATCC 29544 was 0.64 ± 0.03 EU/mL. Addition of citral at 112.5 μ M and 225 μ M caused a significant fall (P ≤ 0.01) in C. sakazakii endotoxin concentration to 0.49 ± 0.02 and 0.45 ± 0.01 EU/mL.

Virulence-associated Genes.
RT-qPCR results demonstrated that citral significantly decreased the expression of eighteen virulence-related genes in C. sakazakii (Table 2). Citral down-regulated the expression of fliD, flhD, and flgJ genes (critical for flagella regulation) to various degrees. Other down-regulated genes were ompA (outer membrane protein A), ompX (outer membrane protein X), uvrY (adherence and invasion), motA, motB   (flagellar motor protein), sod (survival in macrophages), bcsA (cellulose synthase catalytic subunit), bcsG (cell biosynthesis and biofilm formation), galE (colanic acid synthesis), kpsT (K-antigen synthesis), lpx, wzx (LPS biosynthesis), and luxR (LuxR family transcriptional regulator). Citral also down-regulated the expression of two putative virulent plasmid associated genes: iuc (iron transport and regulation) and eit (iron uptake and siderophore system). Citral at 225 μ M of citral was more effective than 112.5 μ M in suppressing the expression of virulence genes ( Table 2). Among the various virulence genes assayed, the expression of genes encoding C. sakazakii flagella, colanic acid, K-antigen and LPS were highly down-regulated in the presence of citral.

Discussion
Bacterial motility is a complex process that employs mechanisms such as swimming and swarming that contribute to the subsequent formation of biofilm and is instrumental in host-microbial interactions and pathogenesis 22 . Both motility systems are directly mediated by a surface appendage, flagella, which play a role in the initial phase of biofilm development 23 . In the present study, it was demonstrated that citral greatly reduced two important types of motility in C. sakazakii. Additionally, RT-qPCR data showed that citral substantially decreased the expression of C. sakazakii genes associated with flagella structure and biosynthesis (motA, motB, fliD, flhD, and flgJ). It was reported that the velocity at which a swarming colony of Serratia liquefaciens colonizes a surface correlates with the level of expression of the flhD master operon 24 . Houry et al. 25 reported that motA and motB form a bicistronic operon and encode two proteins that comprise the flagellar basal body. Therefore, the decreased motility caused by citral could be partially explained by inhibition of flagella biosynthesis or function. It was reported that trans-cinnamaldehyde significantly reduced the diameter of the motility zone of C. sakazakii and the expression of several genes linked to motility 17 . Burt et al. 26 demonstrated that addition of carvacrol results in an aflagellate and nonmotile phenotype in Escherichia coli O157:H7. A significant decrease in biofilm formation in the presence of citral at 12 °C and 25 °C was demonstarted. These two temperatures were selected by their relevance to the food industry (12 °C) 27 , and common room temperature in houses (25 °C). Moreover, citral exerted a stronger anti-biofilm ability at lower temperatures. Upadhyay et al. 28 reported that trans-cinnamaldehyde, carvacrol, thymol and eugenol inhibited Listeria monocytogenes biofilm at 37 °C, 25 °C, and 4 °C to varying degrees. In addition, trans-cinnamaldehyde inhibited C. sakazakii biofilm on all matrices tested at 24 °C and 12 °C 29 . The enhanced anti-biofilm effect of citral at 12 °C could be due to the lower metabolic and growth rates of C. sakazakii at this temperature, which potentially result in a weaker biofilm that is more sensitive to citral 30 . Cellulose was identified as one of the exopolysaccharide (EPS) components responsible for biofilm production by members of the Enterobacteriaceae family, which contribute to protect cells from various environmental stressors 31 . Our results revealed that citral significantly down-regulated the expression of bcsA and bcsG that encode the cellulose biosynthesis operon 32 . We assumed that citral may influence the biosynthesis of components associated with EPS and consequently change the structure of biofilm making it more susceptible.
Citral greatly inhibited the QS-signaling system of C. violaceum 12472, which produced several types of N-acyl-L -homoserine lactones (AHLs), and substantially down-regulated the expression of luxR in C. sakazakii that encodes a transcriptional regulator associated with AHL type QS system 33 . As the central regulator of virulence factors, the QS system of Gram-negative bacteria produces AHLs as cell-cell signaling molecules that mediate several pathogenic processes including swarming, biofilm development and LPS release 34 . AHLs regulate bioluminescence, pigment and antibiotic production in various bacteria in response to cell density 33 . C. sakazakii isolates were previously reported to produce two different AHL molecules (3-oxo-C6-HSL and 3-oxo-C8-HSL) 35 . It is postulated that citral suppressed the QS by inhibiting the synthesis of the LuxR protein and interfering with bacterial cell-to-cell communication. Olivero-Verbel et al. 36 reported that essential oils from Lippia alba, including geranial and neral, two isomeric acyclic monoterpene aldehydes of citral, had the capacity to significantly inhibit QS as observed by the reduction of violacein production in a C. violaceum CV026 bioassay while exhibiting insignificant effects on cell growth. Additionally, Zhang et al. 37 demonstrated that citral could reduce the synthesis of AI-2, an additional QS-signaling molecule that functions in both Gram-positive and Gram-negative bacteria. Altogether, these results suggest that citral could play an important role in reducing virulence in C. sakazakii through QS inhibition.
Adherence to host surfaces, such as to intestinal epithelial layer, and survival in the intestinal wall with subsequent entrance into the bloodstream are both necessary for a microbe to establish infection 38 . Previous studies demonstrated that adhesion and invasion of Salmonella enteritidis to chicken oviduct epithelial cells were significantly reduced by carvacrol, thymol and eugenol 39 . Inamuco et al. 40 reported that carvacrol did not affect adhesion but significantly reduced invasion of Salmonella to intestinal epithelial cells. In this study, we demonstrated that citral significantly suppressed attachment and invasion of Caco-2 cells by C. sakazakii and inhibited the expression of ompA, ompX and uvrY, which largely contributed to bacterial attachment and invasion of host cells 17 . Previously, outer membrane protein A (OmpA) was shown to be necessary for the colonization of C. sakazakii in Virulence studies showed that C. sakazakii strains were able to survive in macrophage cells, the major constituent of the innate immune system 3 . This bacterium survives and multiplies within phagocytic cells, enhancing its ability to avoid the host immune response and cause bacteremia, which could be advantageous to migrate through the blood-brain barrier endothelium 43 . Citral was found to significantly decrease the ability of C. sakazakii to survive and reproduce intracellularly and decreased the expression of sod. This gene was reported to be responsible for superoxide dismutase production, which protects bacteria from the oxidative stress generated by macrophages 44 . In a previous study, C. sakazakii pre-treated with trans-cinnamaldehyde was found to have difficulty surviving in human macrophages U937 17 . According to this study, citral inhibited the survival and replication of C. sakazakii within macrophages, diminishing its ability to withstand bactericidal activity and to evade the host immune response.
Previous studies showed that powdered infant formula is frequently contaminated with high levels of LPS, which is a major virulence factor and a key contributor to the initial adhesion of bacterial cells to a surface or    45 . Our results demonstrated that citral significantly reduced the production of LPS in C. sakazakii and significantly down-regulated lpxB and wzx, which encode lipid A disaccharide synthase and O-antigen flippase, respectively 46 . In accordance with our findings, Amalaradjou et al. 17 reported that 750 μ M trans-cinnamaldehyde decreased endotoxin synthesis by 50% in C. sakazakii. Furthermore, Song et al. 47 found that treatment of human umbilical vein endothelial cells with citral significantly inhibited TNF-α and IL-8 expression induced by LPS. The two dominant domains of LPS: lipid A and O-antigen play a major role in virulence mediating inflammatory response-induced endotoxicity and being responsible for surface attachment thus eliciting a strong antibody response from the infected host 48 . Therefore, we hypothesize that citral damages the structure of LPS and reduces its deleterious effects on the intestinal barrier integrity.
Recently, anti-virulence strategy is considered to be a promising avenue to combat bacterial infection, which was thought to impose less selective pressure on bacteria to reduce the chance of selecting resistant strains 49 . Anti-adhesion, anti-biofilm and anti-QS have all been considered promising novel therapeutics to deal with multi-resistant bacterial infections 50 . In this study, we demonstrated that SICs of citral decreased crucial virulence factors and reduced the synthesis of flagella and biofilms, and interfered with cell-to-cell signaling, all of which would contribute to diminished virulence of C. sakazakii. It is worth mentioning that researchers recently showed that 10-day consecutive selection with citral at SIC induces genotypic resistant E. coli strains 51 and exposure to citral may enhance bacterial virulence of Listeria spp. in a Caenorhabditis elegans model 52 . Although resistant mutant was not observed in our trial experiments, further research is warranted to test this possibility to determine the appropriate application of the citral, either being developed as an alternative/supplementary strategy to control C. sakazakii infections or as a novel hurdle in food preservation in combination with other preservative technologies.

Materials and Methods
Reagents. Citral (CAS:5392-40-5) was obtained from Chengdu Must Bio-technology Co., Ltd. (Chengdu, Sichuan, China) at a HPLC purity of at least 99.2%. Citral solution were prepared in 0.1% DMSO before use. All other chemicals were of analytical grade and were unaltered.

Bacterial Strains and Growth Conditions. C. sakazakii strains ATCC 29544, ATCC 29004 (ATCC,
Manassas, USA) and 7-17 (our laboratory strain collection) were used in the present study. C. sakazakii 7-17 was originally isolated from infant formula in China and confirmed before by species-specific PCR 53 . All of the C. sakazakii isolates were used in MIC and SIC assays, and only ATCC 29544 was used for further experiments because it is commonly used in C. sakazakii virulence studies and it contains phenotypic and genotypic characteristics tested in the following experiments. The QS indicator strain Chromobacterium violaceum ATCC 12472 (ATCC) was used for QS inhibition assays 54 . All C. sakazakii strains were grown and prepared as described before 55 . Bacteria was cultured at 37 °C for 24 hours, and cell growth was monitored at 600 nm as described in a previous study 56 . The MIC was defined as the lowest concentration (μ M) of citral in the presence of which C. sakazakii failed to grow. In addition, the three highest concentrations of citral that did not inhibit bacterial growth were selected as SICs for the following assays.

Minimum Inhibitory Concentration and Sub-Inhibitory Concentration Determinations. The
Swimming and Swarming Assay. Swimming and swarming were evaluated in LB containing different agar concentrations, as previously described 57 . For analysis of swimming ability, a medium containing 20 mL of LB broth and 0.3% (wt/vol) of agar was used. Citral was added to the warm medium (45 °C) to obtain final concentrations of 225 μ M and 112.5 μ M. Subsequently, the plates were allowed to dry for 1 hour at 25 °C before use. Five microliters of each C. sakazakii culture (~6.0 log CFU) were inoculated at the center of this semisolid medium and the plates were incubated at 37 °C for 7 hours. After this, the diameter of the bacterial halo was recorded. The medium without citral was used as a control.
For analysis of bacterial swarming ability, a medium containing 20 mL of LB broth, 0.5% (wt/vol) agar and 0.5% (wt/vol) glucose was used. Five microliters of C. sakazakii culture (~6.0 log CFU) were stabbed into the semisolid medium without and with citral at 225 μ M and 112.5 μ M. The plates were incubated upside down at 37 °C for 20 hours. The size of the swarm area in the presence or absence of citral was calculated using AutoCAD.

Visualization of Flagella by Electron Microscopy.
Transmission electron microscopy was used to examine the morphology of flagella. C. sakazakii strain ATCC 29544 cells were taken from swimming plates at different citral concentrations (0, 112.5 μ M and 225 μ M) and suspended in sterile PBS. One drop of culture was placed on a Formvar-coated grid (Electron Microscopy Sciences) for 20 min. Excess liquid was wiped off and the grid was stained with 1% sodium phosphotungstic acid (pH 6.8) for 1 min and washed three times with distilled water. All specimens were examined with a transmission electron microscope (HT7700, Hitachi, Tokyo, Japan).

Specific Biofilm Formation (SBF) Inhibition
Assay. Biofilm formation assays were performed according to the method described by Naves, et al. 58 with minor modifications. Overnight cultures of C. sakazakii strains ATCC 29544 were centrifuged (4 °C, 5000 × g, 10 min) and re-suspended in TSB. Then, 250 μ L of the cell suspension (OD 600 = 1) were inoculated in sterile 96-well microtiter plates, and citral was added to each of the wells to obtain a final concentration of 0, 112.5 μ M and 225 μ M. Non-inoculated TSB was used as a control. Six samples were included for each treatment. The plates were incubated at 25 °C or 12 °C for 24, 48 and 72 hours without agitation. At each time points, the optical densities (ODs) of cell growth were measured at 630 nm using a microplate Scientific RepoRts | 7:43243 | DOI: 10.1038/srep43243 spectrophotometer (Model 680; Bio-Rad, Hercules, CA, U.S.A.). The suspension was then removed and the wells were rinsed once with 350 μ L of distilled water. After being air dried for 30 minutes, the wells were stained with 250 μ L of 1% crystal violet (wt/vol) (Tianjin Kermel Chemical Regent Co., Ltd, Tianjin, China) for 20 minutes at room temperature. To remove the non-conjugated colorant, the wells were rinsed three times with 350 μ L of distilled water. After being air-dried for 30 minutes, the adhered colorant was solubilized in 250 μ L of 33% (vol/vol) glacial acetic acid and incubated for 20 minutes at room temperature. Then the OD of each well was measured at 570 nm. The SBF was calculated by correcting the OD 570 with the OD 630 .
Quantitative QS Inhibition Assay. The effect of citral on the QS inhibitory activity was measured by quantifying the violacein production with the indicator strain C. violaceum ATCC 12472 59 . First, the inhibitory effect of citral on the growth of C. violaceum was studied to determine the SIC to be used in further experiments. The two highest concentrations of citral that did not inhibit C. violaceum growth after 24 hours of incubation were selected as SICs for this study. Duplicate wells were included for each citral concentration and the experiment was repeated three times.
A flask incubation assay was used to quantify the QS-inhibitory activity of citral. An overnight culture of C. violaceum was diluted to an OD 600 of 0.5. Volumes (30 mL) of LB broth that contained different concentrations of citral were placed into separate flasks. Each flask was inoculated with 100 μ L of culture. The flasks were incubated at 30 °C for 24 hours with 150 rpm. The violacein extraction and quantitation were carried out as previously described by Choo et al. 60 with minor modifications. Briefly, 5 mL of the C. violaceum culture was centrifuged (5000 × g, 5 min, 4 °C) to precipitate insoluble violacein and the culture supernatant was discarded. Then, 1 mL of DMSO was added to the pellet and the solution was vortexed vigorously for 1 min to completely solubilize violacein. The solution was then centrifuged at 5000 × g for 10 min to remove the cells. Two hundred microlitres of the violacein-containing supernatants were added to 96-well microtiter plates and the absorbance of violacein-containing supernatants was read with a microplate spectrophotometer ( Adhesion and Invasion Assay. The effect of citral on the adhesion and invasion of C. sakazakii was investigated by using Caco-2 cells, as described in a previous study 17 . Trypsin-treated cells were seeded in 24-well tissue culture plates (Nunc) containing supplemented DMEM (10 5 cells per well) and incubated for 24 hours. C. sakazakii was grown to its mid-log phase with and without SICs of citral (56.25 μ M, 112.5 μ M and 225 μ M), then centrifuged and re-suspended in cell culture media without antibiotics. Then, the Caco-2 cells were rinsed with PBS and inoculated in the medium with 10 6 CFU (10 MOI) of the C. sakazakii suspension. The tissue culture trays were centrifuged at 600 × g for 5 minutes and incubated at 37 °C in a humidified, 5% CO 2 incubator.
For the adhesion assay, the infected cell monolayers were rinsed three times in PBS after 1 hour of incubation, and lysed with 0.1% Triton X-100 (Amresco, Solon, OH, U.S.A.). The number of viable adherent C. sakazakii was determined by the serial dilution and plating on TSA agar plates and incubated at 37 °C for 24 hours before counting. For the invasion assay, the cell monolayers were incubated for 1 hour following infection, rinsed three times in PBS, and incubated for another 30 minutes in whole media containing gentamicin (100 μ g/mL; Amresco) to kill the extracellular bacteria. Finally, the wells were washed with PBS three times. The numbers of internalized C. sakazakii were determined as described in the adhesion assay. The numbers of bacteria in the treatments were expressed as a percentage relative to that of the control medium.
Effect of Citral on Intracellular Survival and Replication of C. sakazakii in RAW 264.7 Cells. The murine macrophage cell line RAW 264.7 cells were maintained in RPMI 1640 medium with 10% FBS. Twenty-four hours prior to infection, activated cells were seeded in 24-well tissue plates (10 5 cells per well) and cultured at 37 °C under 5% CO 2 . C. sakazakii was grown to its mid-log phase at various concentrations of citral (0, 56.25 μ M, 112.5 μ M and 225 μ M). Then the RAW 264.7 cells were washed gently with PBS and infected with 10 6 CFU (MOI = 10) of C. sakazakii. The plates were incubated for 45 minutes at 37 °C with 5% CO 2 . After incubation, RAW 264.7 cells were re-suspended in RPMI 1640 containing 1% FBS with gentamicin (100 μ g/mL) and incubated at 37 °C with 5% CO 2 for 30 minutes.
For intracellular survival assays, the cells were then washed three times with PBS and lysed with 0.1% Triton. After dilution (PBS 1:10), the samples were enumerated on TSA plates. The results were presented as the number of intracellular C. sakazakii cells after citral treatment. For replication assays, each well containing bacterial cells was replenished with RPMI 1640 containing 1% FBS with gentamicin (10 μ g/mL) and incubated at 37 °C with 5% CO 2 for either 24 or 48 hours. Cell washing, lysis and plating procedures were identical to those used in the analysis of bacterial survival. All assays were conducted in triplicate and repeated at least three times on different days. C. sakazakii Endotoxin Assay. ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript, Piscataway, NJ, U.S.A.) was used according to the method of Amalaradjou, et al. 17 . Overnight cultures of C. sakazakii strains ATCC 29544 was centrifuged (4 °C, 5000 × g, 10 min) and re-suspended in TSB. Then, 50 μ L of the cell suspension (OD 600 = 0.5) and citral solution was added in 30 mL TSB to obtain a final concentration of 0, 112.5 μ M and 225 μ M at 37 °C to its mid-log phase. Non-inoculated TSB was used as a control. Samples