Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Sodium ascorbate as a quorum sensing inhibitor of Pseudomonas aeruginosa S.A. El-Mowafy, M.I. Shaaban and K.H. Abd El Galil Microbiology Department, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt

Keywords furanone derivative, Ps. aeruginosa, quorum sensing inhibitor, sodium ascorbate, virulence factors. Correspondence Mona I. Shaaban, Microbiology Department, Faculty of Pharmacy, Mansoura University, Mansoura, 35516, Egypt. E-mails: [email protected], [email protected] 2014/1113: received 29 May 2014, revised 2 August 2014 and accepted 22 August 2014 doi:10.1111/jam.12631

Abstract Aims: Quorum sensing circuits regulate virulence factors in Pseudomonas aeruginosa and coordinate bacterial pathogenicity. We are interested in exploring available medications for their antiquorum sensing activity. Methods and Results: First, we determined the MIC of ascorbate against Ps. aeruginosa strain PAO1, and all further experiments used concentrations below the MIC so that results could not be caused by reduced viability. Tests of subinhibitory concentrations of sodium ascorbate on cell signals were performed using a reporter strain assay. Sub-MICs of sodium ascorbate resulted in significant reduction of the signalling molecules C4-HSL and 3oxo-C12-HSL (P < 0
01). The influence of sub-MIC of sodium ascorbate on virulence factors was also determined and ascorbate treatment led to significant depression of elastase, protease and haemolysin activities. In addition, inhibition of pyocyanin production, attenuation of biofilm formation and alteration of Pseudomonas motility was observed. Analysis by RT-PCR tested the effect of ascorbate on the expression of QS regulatory genes. Expression of QS regulatory genes, lasI, lasR, rhlI, rhlR, pqsR and pqsA, was repressed compared to untreated Ps. aeruginosa PAO1, confirming that ascorbate QS inhibition works on gene expression at the molecular level. Conclusion: Sodium ascorbate, even at low concentrations, inhibited QS and related virulence factors of Ps. aeruginosa PAO1. Significance and Impact of the Study: This study demonstrated that sodium ascorbate could function as signal modulator and virulence inhibitor in Ps. aeruginosa.

Introduction Pseudomonas aeruginosa produces an arsenal of virulence factors and evades the immune system by a great variety of adaptive mechanisms. Treatment of Pseudomonas infection is complicated by the problem of multidrug resistance and by production of various pathogenic agents including elastase, proteases, pyocyanin, biofilm and toxins (Wagner and Iglewski 2008). These virulence factors are especially harmful in persistent infections such as cystic fibrosis, endocarditis, bacteraemia, wound and burn infections and urinary tract infections (Willcox et al. 2008). Bacterial communication via quorum sensing (QS) plays an important role in control of virulence factors 1388

(Siehnela et al. 2010), in antibiotic resistance (Fuqua and Greenberg 2002) and in biofilm development (Heydorn et al. 2002). Quorum sensing by cell to cell signalling in Ps. aeruginosa relies on small diffusible molecules: 4-quinolones (Pesci et al. 1999) and the N-acylhomoserine lactones (AHLs): C4-HSL and 3-oxo-C12-HSL (Fuqua et al. 1994). These signalling compounds are involved in production of exoenzymes and regulation of virulence factors, bacterial adhesion and biofilm formation (Mattmann and Blackwell 2010). Inhibition of quorum sensing is a promising approach to combat the pathogenicity of Ps. aeruginosa and eliminate infections (Hentzer and Givskov 2003; Umesha and Shivakumar 2013). Furanones, which are structurally similar to AHLs, have been shown to disrupt bacterial

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communication and control bacterial infections by interacting with AHLs and binding to LuxR-type receptors (Grossmann et al. 2003; Smith et al. 2003; Rasmussen et al. 2005). Furanone derivatives are found naturally as pheromones, flavour compounds and secondary metabolites (Martinelli et al. 2004). Naturally occurring furanones such as halogenated furanones from Delisea pulchra have been found to inhibit bacterial infection and biofilm formation by Ps. aeruginosa (Hentzer et al. 2002). Another natural 2-(5H)-furanone, ascorbic acid (vitamin C), has been identified as a quorum sensing analogue (Slaughter 1999). Ascorbic acid provides a degree of protection against adhesion and colonization by some uropathogens (Habash et al. 1999). Moreover, a combination of ascorbate and levofloxacin inhibits adherence and mature biofilm in uropathogenic infections (El-Gebaly et al. 2012). Also, ascorbic acid reduces quorum sensing and pathogenic potential of Clostridium perfringens (Novak and Fratamico 2004). In addition, supplementation with vitamin C improves human immune system function through increased activity of natural killers, chemotaxis and lymphocyte proliferation (Wintergerst et al. 2006). However, the effect of Vit C on QS signals of Ps. aeruginosa and related virulence factors has not yet been established. Therefore, this study investigated the influence of Vit C salt (sodium ascorbate) on quorum signalling and biofilm formation. Furthermore, this work evaluated the effect of different sodium ascorbate concentrations on numerous virulence factors such as elastase, total proteases and pyocyanin. The pathogenesis of Ps. aeruginosa is mediated in part by cellular mobility functions, like pili and flagella, so bacterial motility was also tested in the presence of sodium ascorbate.

inson and Company, Franklin Lakes, NJ). Ampicillin 100 lg ml1 and tetracycline 100 lg ml1 were added as required. Determination of minimal inhibitory concentration of sodium ascorbate and the effect of subinhibitory concentrations on bacterial growth The lowest concentration of sodium ascorbate that inhibited bacteria growth was determined by broth dilution test against Ps. aeruginosa PAO1 (CLSI 2006). Twofold serial dilutions of sodium ascorbate (400, 200, 100, 50, 25, 12
5, 6
25, 3
15 mg ml1) were performed in 1 ml LB broth (pH 7
2). To each concentration, 0.1 ml of culture containing 5 9 106 CFU ml1 of PAO1 was added to the prepared ascorbate dilutions and incubated at 37°C for 24 h. Minimal inhibitory concentration (MIC) was regarded as the lowest concentration that inhibited visible growth of bacteria. Viability of Ps. aeruginosa PAO1 was examined in the presence of subinhibitory concentrations of sodium ascorbate (1/8 and 1/20 MICs). Cells were diluted in LB broth by tenfold serial dilutions. Viability count was performed using the pour plate method (Standards Australia 1995), and surviving cells were counted on LB agar plates. The viable count of Ps. aeruginosa PAO1 treated with 1/8 MIC of ascorbate was compared to untreated cells grown under the same conditions. Growth inhibition analysis was also performed by inoculating LB containing sodium ascorbate (1/8 MIC) with overnight culture of PAO1 at 37°C for 24 h. Untreated PAO1 control was cultivated under the same conditions. Samples were taken every hour to measure OD600 nm for both treated and untreated cultures (Nalca et al. 2006).

Materials and methods Bacterial strains, media and growth conditions

Assessment of QS signals and virulence factors of Pseudomonas aeruginosa

Bacterial standard strains used in this study are listed in Table 1. Cultures were grown aerobically with shaking (150 rev min1) at 37°C for 16–18 h in Luria-Bertani broth medium (LB; Tryptone 10 g l1, Yeast extract 5 g l1 and NaCl 10 g l1) (Musthafa et al. 2011) or LB with 18 g l1 agar (Bacto-agar, BD Difco, Becton, Dick-

Overnight cultures of Ps. aeruginosa PAO1 (0
5 ml, 1 9 108 CFU ml1) were inoculated into 5 ml LB broth containing subinhibitory concentrations of sodium ascorbate 1/8 and 1/20 of MIC equivalent to 12
5 and 5 mg of ascorbate, respectively, and were incubated with shaking (150 rev min1) at 37°C for 16–18 h. Untreated PAO1

Table 1 Standard bacterial strains used in this study Strains

Genotype

Reference

Pseudomonas aeruginosa PAO1 Ps. aeruginosa PAO-JP2 Ps. aeruginosa pME3846 Escherichia coli MG4/pKDT17

Wild type Ps. aeruginosa (▵lasI::Tn10, Tcr; ▵rhlI::Tn501, Hgr) (rhlI-lacZ translational fusion; Tcr) (las reporter; lasB::lacZplac-lasR Apr)

Holloway and Morgan (1986) Pearson et al. (1997) Pessi et al. (2001) Pearson et al. (1994)

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cultures (positive control) and PAO-JP2 (negative control) were propagated in LB broth (Musthafa et al. 2011). Cultures were then centrifuged at 8500 g for 15 min at 4°C, and supernatants were filtered twice using 0
45-lm syringe filter. Cell free filtrates were stored at 20°C and used for measurement of AHL and Pseudomonas virulence factors (Gupta et al. 2011).

The assay tests proteolytic activity by the change in turbidity of skim milk. Culture supernatants of PAO1 (0
5 ml) treated or untreated with sodium ascorbate were added to 1 ml skim milk (12
5 g l1), incubated at 37°C for 30 min and turbidity was measured at 600 nm. Proteolytic activity was performed in triplicates. Haemolysis assay

Determination of C4-HSL and 3-oxo-C12-HSL The levels of AHLs in bacterial culture supernatants were quantified by determining b-galactosidase activity using the reporter strains Escherichia coli MG4 (pKDT17) for detection of C12-HSL (Pearson et al. 1994), and Ps. aeruginosa (pME3846) for C4-HSL (Pessi et al. 2001). Assay of 3-oxo-C12-HSL was performed for treated and untreated supernatant of Ps. aeruginosa PAO1 using E. coli MG4/pKDT17 lacZ reporter strain (Pearson et al. 1994; Schaefer et al. 2000). An overnight culture of E. coli MG4 was diluted to OD600 nm of 0
1. Pseudomonas aeruginosa supernatant (1 ml) was mixed with 0
5 ml of reporter E. coli MG4. Cells were propagated till OD600 nm of 0
3–0
4 and b-galactosidase activity was measured using Miller assay (Miller 1972). All assays were performed in triplicate. For detection of C4-HSL, the reporter strain Ps. aeruginosa pME3846, which harbours a translation rhlI0 –0 lacZ fusion, was grown overnight in LB medium (Pessi et al. 2001). The overnight culture was diluted to OD600 nm of 0
1, and 2 ml was incubated with 1 ml of culture supernatants from ascorbate treated or untreated PAO1 at 37°C with shaking at 150 rev min1 for 3 h. Cells were pelleted, and the level of b-galactosidase activity was determined as described previously (Miller 1972). Elastolytic assay Elastolytic activity of Ps. aeruginosa PAO1 propagated in various concentrations of sodium ascorbate was determined by the elastin Congo red (ECR) assay. Filtrates (0
5 ml) of Ps. aeruginosa PAO1 were added to tubes containing 10 mg of ECR (ECR; Sigma Chemicals, St. Louis, MO) and 0
5 ml of buffer (0
1 mol l1 Tris pH 7
2, 10 mol l1 CaCl2). Tubes were incubated for 6 h at 37°C with shaking (Ohman et al. 1980). Insoluble ECR was removed by centrifugation, and the OD495 nm was measured. Elastase level was calculated as a per cent decrease of elastin production compared to the untreated PAO1. Total protease production Proteolytic activity was determined using the skim milk assay (Skindersoe et al. 2008) with some modifications. 1390

Haemolysis was performed essentially as described by Dacheux et al. 2001. Sheep red blood cells (RBCs) were washed three times in physiological saline and resuspended in Tris-buffered saline (0
05 mol l1 Tris-HCl and 0
15 mol l1 NaCl, pH 7
4) to a final concentration of 2%(v/v) at 4°C. Haemolysis assays were started by mixing 700 ll of RBCs and 500 ll of bacterial supernatant, and the mixture was incubated at 37°C for 2 h. Release of haemoglobin from samples was measured at OD540 nm after centrifugation at 2500 g for 5 min at 4°C. Percentage lysis was calculated as follows: % = [(X-B)/ (T-B)]9100, where B is the negative control corresponding to RBCs incubated with LB broth, T is the positive control, corresponding to total RBCs lysis with 1 g l1 SDS and X is the analysed sample (Rossignol et al. 2008). Haemolysis by treated cultures was then expressed as % lysis of sheep RBCs compared to lysis by PAO1. Haemolysis assays were made in triplicates. Assay of pyocyanin Pyocyanin levels were determined in the presence and absence of different concentrations of sodium ascorbate using King A broth media (peptone 20 g l1, K2SO4 10 g l1 and MgCl2 1
4 g l1). Media were inoculated with overnight cultures of Ps. aeruginosa PAO1 adjusted to OD600 of 0
02. Cultures were grown at 37°C with shaking at 200 rev min1 for 48 h. Pyocyanin production was quantified by extracting a 5 ml of culture with 3 ml of chloroform followed by mixing with 1 ml of 0
2 mol l1 HCl. The absorbance of the upper red phase was measured at OD520 nm, and the concentration of pyocyanin was determined as lg ml1 = (OD520 917
072) (Essar et al. 1990; Ra’oof and Latif 2010). Pyocyanin concentration was calculated in triplicate. Quantification of biofilm formation Biofilm formation was quantified using a microtiter plate assay (Adonizio et al. 2008) by measuring OD495 of crystal violet stained adherent cells. Overnight cultures of Ps. aeruginosa PAO1 were subcultured at 37°C in LB medium with or without sodium ascorbate for 24 h in 96-well polystyrene microtiter plates using 100 ll of

Journal of Applied Microbiology 117, 1388--1399 © 2014 The Society for Applied Microbiology

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culture per well. Planktonic cells were removed, and biofilms adherent to the wells were washed twice with physiological saline (9 g l1 NaCl) and fixed with 150 ll of 99% (v/v) methanol. Bound cells were stained using 5 g l1 crystal violet for 10 min at room temperature, washed three times with water and dried. Crystal violet was solubilized by 150 ll of glacial acetic (33% v/v) for 20 min, and absorbance was measured at 495 nm. Each data point was run four times (four wells), and the mean absorbance was calculated from two reads of each well. Motility assays Bacterial twitching was measured according to Chow et al. (2011). Diluted Pseudomonas culture (2 ll) at OD600 nm 0
4–0
5 was stabbed into LB plates with 10 g l1 agar containing various concentrations of sodium ascorbate and incubated overnight at 37°C for 48 h. The diameter of the twitching zone at the plate– agar interface was measured. Bacterial migration along the plastic surface was detected by crystal violet (10 g l1) staining after removing the agar from the plate. Twitching motility was determined by measuring the diameter of the stained areas (Murray et al. 2010). Swimming motility was measured according to Murray et al. (2010). Diluted PAO1 was point-inoculated onto plates containing 10 g l1 tryptone, 5 g l1 NaCl, 5 or 12
5 mg ml1 of sodium ascorbate and 5 g l1 agar. The plates were incubated for 18 h at 37°C. The distance of colony migration around the inoculation site was measured and compared to Ps. aeruginosa inoculated onto plates without sodium ascorbate. A swarming assay was performed on 5 g l1 agar plates contained 5 g l1 peptone, 2 g l1 yeast extract and 10 g l1 glucose (Kinscherf and Willis 1999) supplemented

with sub-MIC of sodium ascorbate. Treated and untreated plates were inoculated with 2 ll of the diluted PAO1 culture and incubated at 37°C for 16 h (Krishnan et al. 2012). Extraction of RNA and preparation of cDNA Total RNA was extracted at the middle of the exponential growth phase from Ps. aeruginosa PAO1 cultivated in the presence of 1/8 MIC of sodium ascorbate (12
5 mg ml1). Pseudomonas aeruginosa PAO1 and Ps. aeruginosa PAO-JP2 were analysed after growth under the same condition. Extraction was carried out using the TRIzol reagent (Sigma Chemicals) according to the manufacturer’s instructions. The concentration and purity for each RNA sample were determined spectrophotometrically at 260 and 260/280 nm, respectively, using a NanoDrop Spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE). RNA was then reverse transcribed into complementary DNA (cDNA) using QuantiTect Reverse Transcription kit, (QIAGEN, Hilden, Germany) and genomic DNA was removed using gDNA wipeout buffer according to the manufacturer’s instructions. The cDNA samples were then used for real-time PCR as described below. Quantitative real-time PCR Real-time PCR was used to measure the effect of sodium ascorbate on expression of quorum sensing circuit genes lasIR/rhlIR and PQS in PAO1 using the primers listed in Table 2. Amplification and expression were performed using 59 FIREPol EvaGreen, qPCR Mix, ROX Dye (Solis BioDyne, Tartu, Estonia), according to the manufacturer’s instructions. The reaction mixture was 4 ll of FIREPolâEvaGreenâqPCR Mix, 0
1 lmol l1 of forward

Table 2 Primers utilized in RT-PCR Gene name RopD PA0576 LasI PA1432 LasR PA1430 RhlI PA3476 RhlR PA3477 PqsA PA0996 PqsR PA0964

Type Fw Rev Fw Rev Fw Rev Fw Rev Fw Rev Fw Rev Fw Rev

Primer Sequence 0

0

5 -CGAACTGCTTGCCGACTT-3 50 -GCGAGAGCCTCAAGGATAC-30 50 -CGCACATCTGGGAACTCA-30 50 -CGGCACGGATCATCATCT-30 50 -CTGTGGATGCTCAAGGACTAC-30 50 -AACTGGTCTTGCCGATGG-30 50 -GTAGCGGGTTTGCGGATG-30 50 -CGGCATCAGGTCTTCATCG-30 50 -GCCAGCGTCTTGTTCGG-30 50 -CGGTCTGCCTGAGCCATC-30 50 -GACCGGCTGTATTCGATTC-30 50 -GCTGAACCAGGGAAAGAAC-30 50 -CTGATCTGCCGGTAATTGG-30 50 -ATCGACGAGGAACTGAAGA-30

Journal of Applied Microbiology 117, 1388--1399 © 2014 The Society for Applied Microbiology

Annealing temp.

Amplicon size (bp)

56°C

131

56°C

176

55°C

133

58°C

101

58°C

160

55°C

74

55°C

142

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found to be 100 mg ml1 using the broth dilution method. When cultured with 1/8 MIC of ascorbate (12
5 mg ml1), Ps. aeruginosa PAO1 showed the same bacterial count (154 9 106 CFU ml1) as that of untreated cultures (161 9 106 CFU ml1). Furthermore, ascorbate at 1/8 MIC had no effect on growth rate, and stationary phase was reached at approx. 8 h in both treated and untreated cultures. Therefore, in further experiments 1/8 MIC of sodium ascorbate was used in the assessment of QS signal molecules and other virulence factors. Moreover, the influence of other lower concentration of sodium ascorbate (1/20 MIC) on quorum sensing and virulence factors was performed to investigate possible concentration dependence (Fig. 1a).

and reverse primers and 12 ll of RNase-free water. An aliquot of 18 ll of this mix was distributed to each tube. Next, 2 ll (100 ng) of the template cDNA (sample tubes) or 2 ll of RNase-free water as a no template control (NTC) was added and mixed well to give a final volume of 20 ll. Expression of the target genes was measured relative to the standard sample. The expression of the target genes was normalized to the expression of reference gene rpoD. Statistical analysis The Excel data analysis package was used to calculate mean, standard deviation of the mean. Data were analysed using the GraphPad Instate software package (version 3
05) according to the Tukey–Kramer multiple-comparison test at a P value < 0
05 or P < 0
01. All the results were calculated from the mean of three replicate samples for each data point.

Effect of sodium ascorbate on QS signals The production of AHL molecules was determined using a reporter strain assay. Positive controls of untreated PAO1 cultures yielded the highest levels of 3-oxo-C12HSL (3125 Miller units) and C4HSL(325
5 Miller units) (Fig. 1). In contrast, the Ps. aeruginosa double mutant PAO-JP2 did not produce any measurable signalling molecules. When PAO1 cultures were grown in the presence of 1/8 and 1/20 MIC of ascorbate the level of 3-oxo-C12HSL was reduced by 66 and 58%, respectively, a significant value relative to control (P < 0
01, Fig. 1b). A significant decrease in the level of C4-HSL (P < 0
01) was also observed, with 88 and 83% reduction at 1/8 and 1/ 20 MIC of sodium ascorbate, respectively (Fig. 1c).

Results Determination of subinhibitory concentrations of sodium ascorbate and viability of PAO1 The viability of PAO1 in the presence of sub-MIC of ascorbate is important to determine whether any effects of ascorbate are caused by modification of cell function as opposed to bacteriostatic or bacteriocidal effects. The MIC of sodium ascorbate for Ps. aeruginosa PAO1 was

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Figure 1 (a) Growth of PAO1 treated with 12
5 mg ml1 sodium ascorbate compared to the untreated culture. Assay of quorum sensing signals C12-HSL (b) and C4-HSL (c) by detection of b-galactosidase activity in the presence of a sub-MICs of 1/8 and 1/20 sodium ascorbate compared to the untreated PAO1 (**, highly significant, P < 0
01). PAO1, PAO1/Sod ascorbate.

1392

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ascorbate caused significant decrease (46–55%) in pyocyanin with P < 0
01 (Fig. 2b) compared to the untreated PAO1.

Sodium ascorbate inhibited the production of elastase, total proteases and haemolysin Pseudomonas aeruginosa is known for its ability to secrete several hydrolytic enzymes including elastase, haemolysin and other proteases (Gupta et al. 2011) that assist in bacterial dissemination and interfere with host defence mechanisms. In this study, we investigated the effect of sub-MICs of sodium ascorbate on the production of elastase, haemolysin and total proteolytic activities of Ps. aeruginosa PAO1 compared to untreated cultures (Fig. 2a). There was a significant decrease (74–78%) in elastase activity observed in PAO1 treated with sodium ascorbate. Sodium ascorbate reduced elastase activity in PAO1 almost to the level of the double mutant (Fig. 2a). Also, sodium ascorbate caused significant reduction (P < 0
01) in total protease activity at both 1/8 and 1/20 MICs reaching 85 and 80% compared to untreated PAO1 (P < 0
01, Fig. 2a). Moreover, haemolytic activity was significantly decreased with subinhibitory concentrations of sodium ascorbate (76–86% decrease) with P < 0
01 (Fig. 2a).

Sodium ascorbate reduced biofilm formation Biofilm formation by Ps. aeruginosa is intimately linked to interbacterial communication. The effect of sodium ascorbate on biofilm formation is shown in Fig. 2c. In general, a significant decrease in biofilm formation was observed when Ps. aeruginosa PAO1 was grown with 1/8 and 1/20 MIC of sodium ascorbate (P < 0
01, Fig. 2c). The inhibitory effect was concentration dependent. At 12
5 mg ml1, ascorbate caused a 64% reduction in the biofilm formation and lower concentration (5 mg ml1) showed 55% decrease in biofilm production. Effect on Pseudomonas motility The effect of sodium ascorbate on twitching, swimming and swarming motility of Ps. aeruginosa was determined by inoculating overnight cultures of PAO1 onto motility plates (Murray et al. 2010). All three motility functions decreased in Ps. aeruginosa PAO1 treated with ascorbate. Sodium ascorbate exhibited a significant decrease in both twitching and swimming (Fig. 3a) ranging from 65 to 72% and 60 to 70% with 1/8 and 1/20 MIC of sodium ascorbate, respectively. Swarming motility was also affected by sodium ascorbate with variable pattern of cell growth and morphology compared to untreated PAO1 (Fig. 3b).

Sodium ascorbate decreased pyocyanin production Pyocyanin is a redox-active secondary metabolite that is produced by Ps. aeruginosa. Pyocyanin mediates tissue damage and necrosis during lung infections (Lau et al. 2004). Pyocyanin was quantified using King A media according to Essar et al. (1990). The findings presented in Fig. 2b showed that 1/8 and 1/20 MICs of sodium

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Figure 2 Effect of subinhibitory concentrations of sodium ascorbate on levels of (a) protease ■, elastase duction and (c) biofilm formation in Pseudomonas aeruginosa (**, highly significant, P < 0
01).

Journal of Applied Microbiology 117, 1388--1399 © 2014 The Society for Applied Microbiology

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(b) 10

8 7 6 PAO1

5

PAO-JP2

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**

**

** Figure 3 Effect of sodium ascorbate on the motility of Pseudomonas aeruginosa PAO1 (a) swimming □, twitching ■ and (b) swarming motilities of PAO1 in the absence and presence of sodium ascorbate (**, highly significant, P < 0
01).

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Expression of QS-regulated genes

Discussion Pseudomonas aeruginosa is an opportunistic human pathogen that causes pathogenic nosocomial infections and is resistant to many antibiotics (Rossolini and Mantengoli 2005). Pseudomonas aeruginosa utilizes its quorum sensing regulatory system to control virulence traits and to 1394

1

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0·8

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Expression of QS-regulated genes was performed for treated and untreated PAO1 using the PCR primers listed in Table 2. As a control, gene expression was performed for the double mutant strain PAO-JP2. The relative expression of QS-regulated genes, lasI, lasR, rhlI, rhlR, pqsR and pqsA, was determined from calculated Ct values. Average relative amounts of tested genes were then normalized to the average relative amount of the RopD reference gene in the same sample. The standard curve of the ropD gene showed that all the tested samples were on the same line with R2 value of 0
99643. Furthermore, all the standard curves for the expressed genes revealed R2 values ranged 0
99–0
97. Melting curves obtained with ropD, lasI, lasR, rhlI, rhlI, pqsR and pqsA indicated that Ps. aeruginosa standards and samples had the same melting profile with the formation of pure amplicons of these genes and no primer dimer. The relative expression levels from cultures grown in the presence of sodium ascorbate were compared with those from untreated cultures, and the data were analysed using the 2DDCt method (Livak and Schmittgen 2001). Changes in expression were reported in Fig 4. Nongrowth-inhibitory concentrations of sodium ascorbate significantly repressed the expression of lasI, lasR, rhlI, rhlR, pqsA and pqsR by 60, 80, 90, 80, 82 and 66%, respectively.

2

0

JP

**

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O-

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SA

Motility diameter (cm)

9

Figure 4 Sodium ascorbate inhibited QS regulatory circuits of Pseudomonas aeruginosa PAO1; relative expression of ■ lasI, lasR, □ rhlI, rhlR, pqsA, pqsR in the presence of 1/8 MIC sodium ascorbate (SA) related to untreated PAO1 (**, highly significant, P < 0
01).

establish and maintain chronic infections. Quorum sensing regulates virulence phenotypes such as bacterial adhesion and biofilm formation, as well as production of elastase, proteases, haemolysin, pyocyanin, rhamnolipids and secondary metabolites. Small chemical and natural compounds have been investigated for activity against quorum sensing, with the aim of developing alternative antibacterial strategies (Bjarnsholt et al. 2010; Jakobsen et al. 2013; Tan et al. 2013). Natural and synthetic

Journal of Applied Microbiology 117, 1388--1399 © 2014 The Society for Applied Microbiology

Ascorbate is a signal modulator of Ps. aeruginosa

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halogenated furanones interfere with AHL-mediated quorum sensing in Ps. aeruginosa, reducing the production of important virulence factors and affecting biofilm architecture (Hentzer et al. 2002). Because sodium ascorbate is well tolerated in humans, we studied the influence of this natural furanone analogue on quorum signalling and related virulence factors of Ps. aeruginosa PAO1. In contrast to classical antimicrobials, quorum quenchers inhibit virulence rather than bacterial growth, minimizing the chance of generating resistance (Bjarnsholt et al. 2010). In our study, treated PAO1 with 12.5 mg ml1 of ascorbate showed no significant change in bacterial count or growth rate when compared to untreated PAO1. Therefore, low concentrations of sodium ascorbate, 12
5 and 5 mg ml1, were utilized in this study (Fig. 1a). Sodium ascorbate at concentrations below MIC significantly reduced the level of the important homoserine lactones 3-oxo-C12-HSL and C4-HSL (Fig. 1). Similarly, some antibiotics, such as azithromycin, ceftazidime and ciprofloxacin (Skindersoe et al. 2008; Bala et al. 2011) at concentrations below MIC inhibit QS, by changing membrane permeability to inhibit flux of 3-oxo-C12-HSL. Streptomycin inhibits gene activation that is dependent on quorum sensing in Acinetobacter baumannii and also acts as a 3-oxo-C12-HSL antagonist (Saroj and Rather 2013). Furthermore, natural furanones and furanone derivatives (Givskov et al. 1996; Martinelli et al. 2004; Wu et al. 2004) significantly inhibit QS signalling in Ps. aeruginosa. Structural similarity of ascorbic acid to furanones (Slaughter 1999) may thus explain its inhibition of quorum sensing in Ps. aeruginosa. The las and rhl systems are key elements controlling a variety of physiological processes including biofilm formation and production of many virulence factors. Inhibition of las and rhl signals has been shown to attenuate pathogenicity of Ps. aeruginosa (Whitehead et al. 2001; Adonizio et al. 2008). In the present study, we reported significant reduction by sodium ascorbate of QS-related virulence factors such as elastase, total protease, cell free haemolysin, pyocyanin and also biofilm formation (Fig. 2). Similarly, the quorum quenching agent furanone C-30 inhibits associated virulence factors (Mattmann and Blackwell 2010) and eliminates exoprotease activity in PAO1 (Hentzer et al. 2003). A different molecule, phenylacetic acid, also decreases elastase and protease in PAO1 and inhibits AHL production (Musthafa et al. 2012). Another important aspect of bacterial virulence is biofilm formation which is associated with incomplete penetration of antibiotics and development of bacterial resistance. QS plays a key role in Ps. aeruginosa biofilm formation and maintenance, which is required for bacterial adhesion (O’Loughlin et al. 2013). The first evidence

that QS influences biofilm formation in Ps. aeruginosa was the finding that a lasI mutant produces a thinner biofilm that was more susceptible to disruption by detergents (Zegans et al. 2012). Clinically, chemical interference with the lasI system by sodium ascorbate reduced the number of the viable adherent bacteria on catheters treated with 80 and 100 mg ml1 vitamin C by up to 92% (El-Gebaly et al. 2012). Ascorbic acid and sodium ascorbate inhibited quorum sensing in Cl. perfringens (Novak and Fratamico 2004). Furthermore, sodium ascorbate at 5–20 mg ml1 inhibited biofilm production in isolates of Ps. aeruginosa (Abbas et al. 2012). These and our current studies show that sodium ascorbate inhibits Ps. aeruginosa PAO1 biofilm formation by repressing the QS signal cascade. Inhibition of QS has been shown to promote the eradication of biofilms by antimicrobial therapy and make biofilms more susceptible to phagocytosis (Hentzer et al. 2002; Rasmussen et al. 2005). Normal motility is important in bacterial adhesion and biofilm formation as QS-deficient strains lacking motilities form thin and disperse biofilms (Heydorn et al. 2002). Bacterial motility is positively regulated by both QS signals las and rhl and loss of any QS component affects bacterial motility (Glessner et al. 1999). Natural furanones are capable of inhibiting swarming motility in Proteus mirabilis and E. coli at concentrations that do not affect bacterial growth (Ren et al. 2001). Extending these findings, our data showed that motility of Ps. aeruginosa PAO1 treated with sodium ascorbate was significantly impaired relative to untreated PAO1 (Fig. 3). Our data expand this knowledge by demonstrating that expression of the QS-regulated genes lasI, lasR, rhlI, rhlR, pqsR and pqsA is reduced by sodium ascorbate treatment. RT-PCR analysis showed that the lasI relative expression level was significantly decreased in the presence of sodium ascorbate, by 60% of the untreated level. Sodium ascorbate showed strong specificity for the lasR gene, inhibiting its expression by 80%. We tested multiple genes and found that sodium ascorbate lowered the expression of the rhlI gene by 90%, the rhlR gene by 80%, the pqsA gene by 82% and the pqsR gene by 66% (Fig. 4). Sodium ascorbate reduced related virulence factors. In addition, sodium ascorbate may act as AHL analogue, inhibiting the binding to the lasR receptor site. The lasI/R system regulates the transcription of rhlI/R. The three systems, lasI/R, rhlI/R and pqsA/R, are intimately connected (H€aussler and Becker 2008). The Pseudomonas quinolone signal affects biofilm formation and plays a significant role in transcription of rhl-dependent Ps. aeruginosa virulence genes, including those for elastase, pyocyanin, rhamnolipid and lectin (Deziel et al. 2004).

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It is quite possible that sodium ascorbate exerts its anti-QS action by targeting the Las system. The subsequent cascade involving the rhl and PQS systems would then be inhibited and in turn las, rhl and PQS-dependant virulence factor production would be reduced. Thus sodium ascorbate would reduce lasB elastase, protease, haemolysin, pyocyanin, motility and biofilm development. Reduction of these virulence factors in Ps. aeruginosa PAO1 treated with sodium ascorbate was demonstrated in our data. In addition, high affinity binding of sodium ascorbate to its receptor could be deduced from similar activities of 12
5 and 5 mg ml1 of ascorbate on QS signals (Fig. 1) and related virulence factors (Figs 2 and 3). Sodium ascorbate is structurally similar to furanones. Many natural furanones are similar to N-acylhomoserine lactones from Streptomyces species such as butenolides (2 (5H)-furanones) (Mukku et al. 2000). Other furanones are produced by marine algae, sponges, fungi and ascidians (Amagata et al. 1998; Faulkner 2001; Krishnan et al. 2012). Natural QS inhibitors of gram-negative bacteria have been characterized, including cyclic sulphur compounds from garlic (Persson et al. 2005) and patulin from by Penicillium sp. (Rasmussen et al. 2005). It is clear that furanones prevent AHLs from binding to luxR homologs, resulting in a rapid turnover of these proteins (Manefield et al. 2002). Protein modelling studies on luxR and lasR receptors (Koch et al. 2005; Bottomley et al. 2007) suggest that furanones bind to lasR in the same position as the lactone ring of 3-oxo-C12-HSL (Fernandez-Pi~ nar et al. 2012). Several natural compounds decrease virulence, antibiotic resistance and biofilm formation of Ps. aeruginosa in vitro. However, clinical application of these compounds has been hindered due to low solubility or high toxicity. The present study addresses the use of a Vit C salt to control the virulence of Ps. aeruginosa at concentrations well below MIC, as low as 5 mg ml1. Sodium ascorbate is soluble and well tolerated. Furthermore, it is able to inhibit cell motility, production of virulence factors and production of biofilms. Because of these properties, sodium acetate is an intriguing candidate for further study on control of Ps. aeruginosa infections in vivo. Acknowledgements We would like to thank Prof. Martin Schuster, Department of Microbiology, Nash Hall, Oregon State University, Corvallis, OR 97331, for providing the E. coli biosensor strain MG4/pKDT17 and Ps. aeruginosa PAO-JP2. We also thank Prof. Paul Williams, Department of Molecular Medical Science Center for Biomolecular Science, University of 1396

Nottingham, UK for providing reporter strain Ps. aeruginosa/pME3846 used in the present study. All our work was performed at Microbiology Department, Faculty of Pharmacy, Mansoura University, Egypt. Conflict of Interest Authors declare no conflict of interest. References Abbas, H.A., Serry, F.M. and EL-Masry, E.M. (2012) Combating Pseudomonas aeruginosa biofilms by potential biofilm inhibitors. Asian J Res Pharm Sci 2, 66–72. Adonizio, A., Kong, K.F. and Mathee, K. (2008) Inhibition of quorum sensing-controlled virulence factor production in P. aeruginosa by South Florida plant extracts. Antimicrob Agents Chemother 52, 198–203. Amagata, T., Usami, Y., Minoura, K., Ito, T. and Numata, A. (1998) Cytotoxic substances produced by a fungal strain from a sponge: physicochemical properties and structures. J Antibiot (Tokyo) 51, 33–40. Bala, A., Kumar, R. and Harjai, K. (2011) Inhibition of quorum sensing in Pseudomonas aeruginosa by azithromycin and its effectiveness in urinary tract infections. J Med Microbiol 60, 300–306. Bjarnsholt, T., Jensen, O.P., Jakobsen, T.H., Phipps, R., Nielsen, A.K., Rybtke, M.T., Nielsen, T.T., Givskov, M. et al. (2010) Quorum sensing and virulence of P. aeruginosa during lung infection of cystic fibrosis patients. PLoS One 5, e10115. Bottomley, M.J., Muraglia, E., Bazzo, R. and Carfi, A. (2007) Molecular insights into quorum sensing in the human pathogen Pseudomonas aeruginosa from the structure of the virulence regulator LasR bound to its autoinducer. J Biol Chem 282, 13592–13600. Chow, S., Gu, K., Jiang, L. and Nassour, A. (2011) Salicylic acid affects swimming, twitching and swarming motility in P. aeruginosa, resulting in decreased biofilm formation. J Exp Microbiol Immunol 15, 22–29. CLSI (2006) Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard. 7th edn. CLSI Document M7-A7. Wayne, PA: CLSI. Dacheux, D., Goure, J., Chabert, J., Usson, Y. and Attree, I. (2001) Pore-forming activity of type III system-secreted proteins leads to oncosis of Pseudomonas aeruginosainfected macrophages. Mol Microbiol 40, 76–85. Deziel, E., Lepine, F., Milot, S., He, J., Mindrinos, M.N., Tompkins, R.G. and Rahme, L.G. (2004) Analysis of P. aeruginosa 4-hydroxy-2-alkylquinolines (HAQs) reveals a role for 4-hydroxy-2-heptylquinoline in cell-to-cell communication. Proc Natl Acad Sci USA 101, 1339–1344. El-Gebaly, E., Essam, T., Hashem, S. and El-Baky, R.A. (2012) Effect of levofloxacin and vitamin c on bacterial adherence

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