Combating Pseudomonas aeruginosa Biofilms by Potential Biofilm Inhibitors

 

Hisham A. Abbas*, Fathy M. Serry, Eman M. EL-Masry

Department of Microbiology and Immunology-Faculty of Pharmacy-Zagazig University- Zagazig- Egypt

*Corresponding Author E-mail: h_abdelmonem@yahoo.com

 

ABSTRACT:

Ten potential antibiofilm agents (N-acetylcysteine (NAC), ambroxol, piroxicam, diclofenac sodium, ketoprofen, 4-nitropyrdidine-N-oxide (4NPO), sodium ascorbate, sucralose, xylitol and sorbitol) showed varied activity against pre-formed biofilms formed by twenty clinical isolates of Pseudomonas aeruginosa as demonstrated by minimum biofilm inhibitory concentration (MBIC). 4NPO was the most active; Diclofenac sodium, ketoprofen, N-acetylcysteine, ambroxol, sodium ascorbate and piroxicam showed moderate activity, while sucralose, xylitol, and sorbitol demonstrated weak activity.

 

KEYWORDS:Pseudomonas aeruginosa, biofilm inhibition, antibiofilm agents


 

INTRODUCTION:

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a major causative microorganism of nosocomial respiratory infections.1 It has emerged as a dominant pulmonary pathogen with biofilm-forming capability.2

 

Biofilms are defined as multicellular aggregates of sessile cells that are irreversibly attached to a substratum or interface or to each other, encased in a self-produced extracellular matrix of polysaccharides, proteins and nucleic acids and exhibit an altered phenotype in terms of growth rate and gene expression as compared with planktonic bacteria. 3,4,5,6

 

The formation of biofilms contributes to the high resistance of Pseudomonas aeruginosa to antibiotics making the treatment of biofilm infections more difficult. In addition, bacteria in biofilm were demonstrated to show elevated resistance to the host immune system clearance. 7,8

 

Factors which explain the high antimicrobial resistance of biofilms include decreased diffusion of antibiotics through the biofilm matrix, decreased oxygen and nutrient, decreased growth rates and metabolism9,10,11,12, quorum sensing and induction of a biofilm specific phenotype13,11,14,15,16 and formation of persister cells which contribute to tolerance. 11,17,12

               

This study investigated the in vitro antiplanktonic and antibiofilm activities of ten potential antibiofilm agents against established P. aeruginosa biofilms.

 

MATERIALS AND METHODS:

Bacterial strains

 20 isolates of Pseudomonas aeruginosa from intensive care unit patients with lower respiratory tract infections in Zagazig University Hospitals were obtained by endotracheal aspiration.

 

Qualitative assessment of biofilm by tube method

The isolates were tested for their ability to form biofilm according to Stepanovic et al.18 with some modifications. Test isolates, grown on tryptone soya agar plates for 24 h, were inoculated into 7 mL tryptone soya broth with 1% glucose (TSBglu) in Falcon tubes, and the turbidity was adjusted to match that of a 0.5 McFarland standard. Five mL of inoculated broth were transferred to new sterile Falcon tubes for quantitative assessment. Uninoculated TSBglu tubes were used as negative controls. Tubes were incubated for overnight at 37 şC. The content of each tube was carefully aspirated with a pipette, and the tubes were immediately stained with 2 mL of 0.25% safranin for          1 minute. The tubes were decanted and inverted without washing. After overnight standing at room temperature the tubes were examined for biofilm formation. The test was considered positive when there was a stained film or adherent layer on the inner surface of the tube. The biofilm formation was estimated as negative (0), weak (+), moderate (++), or strong (+++).

 

Quantitative assessment of biofilm by spectrophotometric method

Quantification of biofilm was based on the previously described method18. Five mL quantities of inoculated TSBglu with optical density matching that of a 0.5 McFarland standard were distributed in sterile Falcon tubes.  Uninoculated TSBglu tubes were used as negative controls.  The tubes were incubated for 24 hours at 37 şC; the content of each tube was aspirated, and washed three times with sterile physiological saline. The tubes were vigorously shaken to remove any non-adherent bacteria. For fixation of adherent bacteria, 99% methanol was added for 15 minutes. Then, the tubes were decanted, left to dry, and stained with 2% Hucker crystal violet for 5 minutes. Excess stain was rinsed off by running tap water. After the tubes were air dried, the dye bound to the adherent cells was resolubilized with 33% (v/v) glacial acetic acid The Optical density (OD) was measured at 570 nm using Spectrophotometer (UV-1800 Shimadzu, Japan). Based on the measured ODs of the bacterial biofilms, tested isolates were classified into four categories, non-biofilm forming (if OD ≤ ODc), weak biofilm forming (if OD > ODc, but ≤ 2x ODc), moderate biofilmforming (if OD>2x ODc, but ≤ 4x ODc), and strong biofilm forming (if OD> 4x ODc). The cut-off OD (ODc) was defined as equivalent to three times standard deviations above the mean OD of the negative control. The test was made in triplicates and repeated three times, and the data was then averaged.

 

Determination of minimum inhibitory concentration (MIC)

The minimum inhibitory concentrations (MICs) of the test compounds were determined by the agar dilution method according to CLSI19. Bacterial inocula were prepared and standardized to match  a 0.5 McFarland standard. The bacterial suspensions were then diluted with sterile saline to have an approximate cell density of 107 CFU/mL. A standardized inoculum was delivered to the surface of Mueller-Hinton agar containing antibiotic dilutions and the control plates, so that the final inoculum on the agar contains approximately 104 CFU per spot. Antimicrobial-free plates were used as growth control. After standing at room temperature until the inoculums was absorbed into the agar, the agar plates were inverted and incubated at 35-37 °C for 16–20 h. The MIC was calculated as equivalent to the lowest concentration of antimicrobial agent that can completely inhibit growth.

Determination of minimum biofilm inhibitory concentration (MBIC)

The MBICs of the antibiotics and potential antibiofilm agents were determined according to Černohorská and Votava.20,21 The experiments were done in 96-wells polystyrene microtiter plates with round bottoms.  An overnight culture adjusted with TSB to achieve a turbidity equivalent to that of a 0.5 McFarland standard, then 75 μL aliquots of the inoculted media were added to the wells of microtiter plates.  The plates were incubated for 24 h at 37°C. The wells were washed three times with phosphate-buffered saline (PBS) under aseptic conditions to remove unattached bacteria and dried in an inverted position. Volumes of 100 μL of appropriate two-fold dilutions of the respective antimicrobial agents or the potential antibiofilm agents in Mueller–Hinton broth were transferred into the dried wells with established biofilms. The microtiter plates were incubated for 18–20 hours at 37 şC, and minimum Biofilm inhibitory concentration (MBIC) was determined, which corresponds to the lowest concentration of antibiotic which inhibits growth of biofilm cells as indicated by absence of visible growth in the wells. A positive control and a negative control were included in all experiments. The experiment was repeated three times.

 

RESULTS:

Assessment of biofilm formation

All isolates were biofilm forming using both the tube and spectrophotometric methods; sixteen isolates (80%) were strong biofilm forming; two isolates (10%) were moderate biofilm forming and two isolates (10%) were weak biofilm forming.

 

Susceptibility of planktonic and biofilm cells to antimicrobial agents

Biofilm cells demonstrated higher resistantance than planktonic cells to different antibiotics as demonstrated by the ratios of MBIC to MIC of antibiotics in Table 1. This ratio was lowest for meropenem (2-8) folds, followed by cefepime (1-32) folds, ofloxacin (1-64), ciprofloxacin and imipenem (2-64) folds, norfloxacin, amikacin and ceftriaxone (2-128) folds, tobramycin (1-256) folds, cefotaxime (2-256) folds, and the highest ratios were found to be for gentamicin and cefoperazone (16-1024) folds and ceftazidime (16-2048) folds.

 

susceptibility of Pseudomonas aeruginosa isolates to potential antibiofilm agents

Antibacterial activity was found against planktonic bacteria (Table 2). Moreover, antibiofilm effect was demonstrated (Table 3). Considering the growth inhibition and the biofilm inhibition, 4-nitropyridine-N-oxide was the most potent agent, followed by diclofenac, ketoprofen, N-acetylcysteine, ambroxol, piroxicam, and sodium ascorbate, while sucralose, xylitol, and sorbitol demonstrated the lowest antiplanktonic and antibiofilm effect.


 

                                                                                                                                    

Table 1. Ratio of MBIC to MIC of antibiotics against Pseudomonas aeruginosa isolates.

Isolate

No.

Ceftazidime

 

Cefoperazone

 

Cefepime

Cefotaxime

Ceftriaxone

Ofloxacin

Ciprofloxacin

Norfloxacin

Amikacin

Gentamicin

Tobramycin

Meropenem

Imipenem

PA1

PA2

PA3

PA4

PA5

PA6

PA7

PA8

PA9

PA10

PA11

PA12

PA13

PA14

PA15

PA16

PA17

PA18

PA19

PA20

512

512

16

256

256

256

32

32

32

64

16

256

256

64

64

32

2048

512

64

512

256

512

64

256

128

64

256

256

256

64

64

256

256

16

64

128

64

16

64

1024

2

2

2

2

2

2

2

2

2

2

2

1

1

1

4

16

16

32

4

16

32

256

8

32

8

8

8

8

8

16

8

16

32

8

4

16

4

2

2

256

128

128

8

128

16

2

16

8

16

8

8

8

16

16

4

16

2

2

8

128

4

16

8

4

2

2

2

2

64

16

8

1

1

2

2

2

16

2

4

2

8

16

64

2

2

4

2

2

64

32

64

2

2

2

2

2

32

2

16

4

2

4

128

8

2

32

8

8

16

2

64

2

2

64

2

2

64

2

2

16

8

8

16

16

4

8

128

16

4

8

32

2

2

128

4

16

16

16

32

16

512

256

8

256

2

128

4

8

128

64

8

2

2

32

64

4

512

512

64

256

128

64

4

32

1

2

4

4

4

4

4

1

2

32

4

4

256

128

2

256

2

2

2

2

2

2

2

4

2

2

2

2

2

2

2

2

4

8

4

4

2

4

32

16

32

32

4

32

32

64

64

4

4

4

2

4

16

16

64

4

 


 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 2. Minimum inhibitory concentrations and minimum biofilm inhibitory concentrations of potential antibiofilm agents against Pseudomonas aeruginosa isolates.

 

 

Isolate No.

N-acetyl cysteine

Ambroxol hydrochloride

Sodium ascorbate

Xylitol

Sucralose

MIC

MBIC

MIC

MBIC

MIC

MBIC

MIC

MBIC

MIC

MBIC

PA1

PA2

PA3

PA4

PA5

PA6

PA7

PA8

PA9

PA10

PA11

PA12

PA13

PA14

PA15

PA16

PA17

PA18

PA19

PA20

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

5.0

5.0

5.0

5.0

5.0

5.0

5.0

5.0

5.0

5.0

2.5

5.0

5.0

5.0

5.0

10

5.0

5.0

5.0

5.0

5.0

3.75

3.75

3.75

3.75

3.75

3.75

3.75

3.75

3.75

3.75

3.75

3.75

3.75

3.75

3.75

3.75

3.75

3.75

1.875

3.75

7.5

7.5

7.5

7.5

7.5

7.5

7.5

7.5

7.5

7.5

3.75

7.5

7.5

7.5

7.5

7.5

7.5

7.5

7.5

7.5

5.0

5.0

10.0

5.0

5.0

5.0

5.0

5.0

10.0

10.0

5.0

5.0

5.0

10.0

5.0

10.0

10.0

20.0

10.0

5.0

10

10

20

20

20

5.0

5.0

10

20

10

5.0

5.0

5.0

20

20

20

20

20

20

10

200

200

200

200

200

200

200

200

200

200

200

400

200

400

200

200

400

400

200

200

200

200

200

200

200

200

200

200

200

200

200

200

200

200

200

200

200

200

200

200

200

200

100

100

100

200

100

100

100

200

200

200

200

200

200

200

200

200

200

200

100

50

100

100

50

50

100

100

100

50

50

100

50

50

100

100

50

50

100

50

 

 


DISCUSSION:

Different techniques have been used to study the susceptibility of biofilm to antimicrobial agents. To determine the biofilm susceptibility to the tested agents, an assay similar to but more economic than that based on the Calgary Biofilm Device (CBD)22 was used. It was proposed by Černohorská and Votava20 and involves the direct formation of biofilms on the wells of the microtiter plates.

 

In this study, the resistance of biofilm cells to antibiotics was higher than that of planktonic cells, as measured by MBIC/MIC ratio. Maximum increase in biofilm resistance was shown with few isolates. The MBIC/MIC ratio increased for 5% of isolates by 2048 folds with ceftazidime, 1024 folds with cefoperazone, 128 folds with norfloxacin, 64 folds with ofloxacin, 32 folds with cefepime and 8 folds with meropenem.

 

Little effect on biofilm susceptibility (2 folds) was shown to amikacin and imipenem in 10% of isolates, to gentamicin in 13% of isolates, to cefotaxime, tobramycin and ceftriaxone in 15% of isolates, to norfloxacin and ofloxacin in 45% of isolates, to ciprofloxacin in 50% of isolates, to cefepime in 55% of isolates and to meropenem in 75% of isolates.


Table 2. Continued

 

Isolate No.

Sorbitol

4-Nitropyridine-N-Oxide

Diclofenac sodium

Ketoprofen

Piroxicam

MIC

MBIC

MIC

MBIC

MIC

MBIC

MBIC

MIC

MIC

MBIC

PA1

PA2

PA3

PA4

PA5

PA6

PA7

PA8

PA9

PA10

PA11

PA12

PA13

PA14

PA15

PA16

PA17

PA18

PA19

PA20

 

400

400

400

400

400

400

400

400

400

400

400

400

400

400

400

400

400

400

400

400

 

400

200

100

200

200

100

400

200

400

200

100

200

200

200

400

200

100

400

400

400

 

0.064

0.032

0.032

0.064

0.032

0.032

0.032

0.032

0.032

0.032

0.032

0.032

0.032

0.032

0.032

0.032

0.032

0.032

0.032

0.032

 

0.064

0.064

0.064

0.064

0.064

0.032

0.032

0.032

0.064

0.032

0.032

0.032

0.032

0.064

0.032

0.064

0.064

0.032

0.032

0.064

 

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

 

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

1.56

1.56

3.125

3.125

3.125

3.125

3.125

3.125

3.125

 

6.25

6.25

6.25

6.25

6.25

6.25

6.25

6.25

6.25

6.25

6.25

6.25

6.25

6.25

6.25

6.25

6.25

6.25

6.25

6.25

 

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

3.125

 

5.0

5.0

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

5.0

2.5

2.5

 

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

 


 

No effect on biofilm susceptibility was observed with 10% of isolates for each of ofloxacin and tobramycin and in 15% of isolates for cefepime.

 

To make the judgement concerning the impact of biofilm on resistance to individual antibiotics more objective, the magnitude of the effect of biofilm on resistance to individual antibiotics expressed as the ratio of MBIC/MIC expressed by ≥ 50% of the tested isolates was used. Thus, such ratio was high for ceftazidime, cefoperazone and gentamicin (≤ 64 folds) reflecting high effect of biofilm on antibiotic resistance. This ratio was lowest for meropenem, cefepime and ciprofloxacin (≤ 2 folds) reflecting low effect of biofilm on resistance. Intermediate effect was found with ofloxacin, tobramycin and norfloxacin (≤ 4 folds), cefotaxime and ceftriaxone (≤ 8 folds) and amikacin, imipenem (16 folds). High resistance of biofilms to antimicrobial agents was reported by other studies.22, 23,24,25

 

In order to combat the high resistance of biofilm to antimicrobial agents, several potential antibiofilm agents were used to inhibit the biofilm cells. These agents include N-acetylcysteine, ambroxol, sodium ascorbate, ketoprofen, diclofenac sodium, piroxicam, Xylitol, sucralose, sorbitol and 4-nitropyridine-N-oxide. N-acetylcysteine (NAC) is a non-antibiotic and a thiol-containing antioxidant drug that has antibacterial properties. NAC exerts its antimicrobial potential by competitively inhibiting cysteine utilization in bacteria or by reaction of its sulfhydryl group with bacterial cell proteins.26 NAC can disperse the biofilms formed by Pseudomonas aeruginosa.27 NAC decreases extracellular polysaccharides production and promotes disruption of mature biofilms.28,29 Moreover, NAC was shown not only to reduce bacterial adhesion but also to detach bacteria adherent on surfaces.30  

 

NAC was found to inhibit the planktonic growth of all Pseudomonas aeruginosa isolates at 2.5-5 mg/mL. Our results were lower than that reported by Zhao and Liu26 who found that the minimum inhibitory concentrations of NAC for 18 out of 20 P. aeruginosa isolates studied were 10 to 40 mg/mL and Roberts and Cole31 who reported that concentrations of 2%-5% of NAC were bactericidal against P. aeruginosa. Whereas, lower results were found with Parry and Neu32 who found that MICs of NAC against P. aeruginosa were 2-20 μg/mL.

 

In the present study, the biofilm cells were inhibited at concentrations of 2.5 to 10 mg/mL. In agreement with these results, Zhao and Liu26 found that NAC at 0.5 mg/mL could detach mature P. aeruginosa biofilms, at 10 mg/mL completely disrupted biofilms. At 0.5 mg/mL and 1 mg/mL, NAC decreased extracellular polysaccharides (EPS) production by P. aeruginosa by 27.64% and 44.59%, respectively, while Olofsson et al.33 found that NAC had anti-microbial effect on biofilm-associated P. aeruginosa isolated from a paper mill at 2.5 mg/mL. Moreover, El-Feky et al.34 demonstrated the inhibitory effect of NAC on adherence and biofilm formation in two P. aeruginosa isolates by using the viable cell counting method. It was found that NAC reduced biofilm formation by 64-72.7% at 2 mg/mL and by 84-94% at 4 mg/mL. Moreover, NAC reduced viable cell counts of pre-formed biofilms by 68% and (84-90%) at 2 and 4 mg/mL, respectively.

 

Ambroxol was found to be a strong antiadhesion agent.35 In addition to its antiahesive effects, ambroxol interferes with biofilm formation by interference with quorum sensing and decreasing the matrix production in P. aeruginosa biofilms.36,37

 

The MICs of ambroxol against P. aeruginosa isolates were found to be 1.875-3.75 mg/mL. The MBIC values were 1.875 to 7.5 mg/mL. In agreement with the current study, Lu et al.37 reported that ambroxol, at concentrations of 1.875 mg/mL and 3.75 mg/mL, interfered with quorum sensing in P. aeruginosa, decreased viability of biofilms, showed antiadherent activity to abiotic surfaces and interfered with the maturation of biofilm in a dose-dependent manner. Moreover, Li et al.37 demonstrated that ambroxol at 3.75 mg/mL disrupted the biofilms. Furthermore, Li et al.38 found that ambroxol could destroy the structure of Pseudomonas aeruginosa biofilm on the endotracheal tube in a rat model and decrease the viable count of the biofilm bacteria.

 

Non-steroidal anti-inflammatory drugs are among the non-antibiotic drugs that have antimicrobial effect.39 Diclofenac sodium was found to exhibit broad-spectrum in vitro and in vivo antimicrobial activity against a variety of Gram-positive and Gram-negative bacteria.40,41 Mechanism of antimicrobial action may be attributed to the inhibition of DNA synthesis in bacterial cells.42

 

In addition to its antimicrobial properties, diclofenac has been discovered to inhibit microbial biofilms. Alem and Douglas43 found that diclofenac had a marked biofilm inhibitory on growing and fully mature biofilms of Candida albicans effect. The inhibition of prostaglandin synthesis was suggested to be NSAIDs’ mechanism of inhibiting biofilm formation.43 Diclofenac sodium was found to significantly decrease colonization of contact lenses pre-colonized with Pseudomonas aeruginosa.44

 

In the current study, diclofenac sodium and piroxicam showed comparable MIC values of 3.125 mg/mL for diclofenac sodium, 2.5-5 mg/mL for piroxicam, while all isolates were inhibited by 6.25 mg/mL of ketoprofen. Umaru et al.45 found that MIC of diclofenac sodium against Pseudomonas aeruginosa was 0.5 mg/mL. On the other hand, the action on biofilm cells was somewhat different. Ketoprofen and diclofenac could inhibit biofilm cells at lower concentrations than piroxicam. The MBICs for diclofenac sodium were 1.56 and 3.125 mg/mL for 8.75% and 91.3% of isolates respectively. MBIC of 3.125 mg/mL for Ketoprofen and MBIC of 10 mg/mL for piroxicam were reported in all isolates. It is noteworthy that biofilm inhibition was achieved by sub-MICs of ketoprofen in all isolates and diclofenac sodium in 10% of isolates only.The current results were higher than those reported by previous findings. Alem and Douglas43 reported strong biofilm inhibiting activity of diclofenac sodium against Candida albicans at concentration of 0.32 mg/mL. A weaker action by ibuprofen at 0.21 mg/mL was observed, while piroxicam at 0.33 mg/mL produced no significant inhbition of biofilm formation, but it reduced the viability of both planktonic and biofilm cells by about 40%. Naves et al.46 showed that ibuprofen significantly reduced biofilm development by five out of seven strains of E. coli with reductions ranging from 37.2% to 44.8% with MBIC values 0.002–0.125 mg/mL, the most potent inhibition being at 0.125 mg/mL.

 

The sugar alcohol xylitol showed antimicrobial activity against Streptococcus pneumoniae and Streptococcus mutans.47,48 Xylitol exerted anti-adherent action on many bacteria. A potential mechanism of action of xylitol includes metabolic inhibition; it accumulates as the non metabolisable xylitol phosphate, a toxic agent that inhibits bacterial growth.49 Xylitol was reported to inhibit Streptococcus mutans biofilm formation.50 Xylitol has been shown to suppress bacterial biofilm formation in S. aureus by inhibiting the formation of glycocalyx 51 and was found to inhibit biofilms formed by P. aeruginosa PAO1, Enterococcus faecalis and S. aureus in a chronic wound biofilm model.52 Xylitol treatment disrupted the biofilm structure of a clinical wound isolate of P. aeruginosa grown as a biofilm.53

 

Sucralose is a halogenated sucrose derivative54, which inhibits the activity of both fructosyl- and glucosyltransferases55 which results in inhibiting the formation of glucan and fructan polymers, which have a role in dental caries, a biofilm-dependent oral disease.56 Mah et al.57 found that glucosyltransferase inhibitors have antibiofilm activities. The ndvB gene, coding for a glucosyltransferase, is a genetic determinant of antibiotic resistance in P. aeruginosa. Sorbitol is a sugar alcohol that the human body metabolizes slowly. It is a sugar substitute that prevents the formation of glucan and dental plaque.58,59,60

 

The inhibitory activity against Pseudomonas aeruginosa isolates was somewhat higher in sucralose and xylitol than in sorbitol. The planktonic growth was inhibited at 100-200 mg/mL of sucralose, at 200-400 mg/mL of Xylitol and at 400 mg/mL of sorbitol. Sucralose also was more active as antibiofilm agent than xylitol, which in turn, showed higher antibiofilm potential than sorbitol. The MBIC values were 50-100 mg/mL, 200 mg/mL and 100-400 mg/mL for sucralose, Xylitol and sorbitol, respectively. Moreover, sub-MICs of sucralose, Xylitol and sorbitol could inhibit biofilm in 75%, 20% and 65% of isolates, respectively. These results are comparable with those of Dowd et al.52 who reported that xylitol had an increasing inhibitory effect on P. aeruginosa biofilms at 20 mg/mL, 100 mg/mL and 200 mg/mL. Xylitol at concentration of 200 mg/mL could completely inhibit biofilm formation of P. aeruginosa. Badet et al.61 studies the effect of xylitol, at concentrations of 10 and 30 mg/mL, on experimental in vitro model of oral biofilms and a clear inhibitory effect of xylitol on biofilm formation was observed. Sucralose at concentrations of 100 mg/mL and 200 mg/mL approximately showed an inhibiting activity against Streptococcus mutans glucosyltransferases, either free or adsorbed to saliva-coated beads.62

 

4-nitropyridine-N-oxide (4NPO) is an active quorum sensing inhibitor.63 Moreover, 4-nitropyridine-N-oxide can inhibit biofilm formation by decreasing attachment of bacteria to solid surfaces via its quorum sensing inhibiting activity and by changing the physicochemical properties of both bacterial and solid surfaces.64 In the present study, 4-nitropyridine-N-oxide was found to inhibit the growth of all Pseudomonas aeruginosa isolates at concentrations of 32 to 64 µg/mL. 4-nitropyridine-N-oxide could inhibit biofilm cells at the same concentrations.

 

Ascorbic acid and sodium ascorbate inhibited quorum sensing in Clostridium perfringens.65 Moreover, ascorbic acid was reported as efflux pump inhibitor in E. coli.66 Therefore, the antibiofilm mechanisms of ascorbic acid may be due to quorum sensing inhibition and efflux pump inhibition. Sodium ascorbate was inhibitory to planktonic growth of Pseudomonas aeruginosa isolates at 5-20 mg/mL. Sodium ascorbate showed biofilm inhibiting activity at the same concentrations.

 

The antimicrobial activity of ascorbic acid was reported in several studies. Ascorbic acid at pH 7.1 was found to have a strong antibacterial action on Pseudomonas aeruginosa.67 Zhang et al.68 found that the MICs of ascorbic acid at pH 7.4 against some Gram-negative bacteria were between 2.048 mg/mL and > 16,384 mg/mL. Furthermore, Sendamangalam 69 found that the MIC of ascorbic acid against Streptococcus mutans was 2 mg/mL, and ascorbic acid at 2 mg/mL could reduce biofilm formation by Streptococcus mutans by 75.7%.

 

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Received on 14.04.2012          Accepted on 20.05.2012        

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Asian J. Res. Pharm. Sci. 2(2): April-June 2012; Page 66-72