Inhibition of Virulence of Pseudomonas aeruginosa: A Novel Role of Metronidazole Against Aerobic Bacteria

 

Hisham A. Abbas

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

*Corresponding Author E-mail: hishamabbas2008@mail.com

 

 

ABSTRACT:

Antibiotic resistance is one of the most dangerous challenges in modern medicine. Pseudomonas aeruginosa is a common nosocomial bacterium that shows high resistance to antibiotics. Anti-virulence agents are alternatives to antibiotics to combat the emergence of resistance. Quorum sensing controls the production of virulence factors. Metronidazole is an analogue of acylhomoserine lactones, the signaling molecules of quorum sensing in Gram-negative bacteria. This study aimed to investigate the inhibitory activity of metronidazole against virulence factors of Pseudomonas aeruginosa. The effect of sub-inhibitory concentration of metronidazole on twitching and swimming motilities in addition to biofilm formation, pyocyanin, pyoverdin, protease and hemolysin was investigated. As compared to the control, metronidazole showed significant inhibition of virulence factors. It reduced twitching motility by 47.57% and swimming motility by 64.81%. Pyocyanin and pyoverdin production was reduced by 43.79% and 82.82%, respectively.  Metronidazole also showed high inhibiting activity against protease, hemolysin and biofilm formation. Metronidazole inhibited protease by 60.22% and biofilm formation by 86.49%. Moreover, hemolytic activities in the presence of metronidazole was 7.16% as compared to the control (100% hemolytic activity). In conclusion, metronidazole is suggested as a potential anti-virulence agent for the treatment of Pseudomonas aeruginosa infection.

 

KEYWORDS: Pseudomonas aeruginosa, virulence factors, inhibition, metronidazole

 

 

INTRODUCTION:

Pseudomonas aeruginosa is a highly resistant common nosocomial pathogen that causes urinary tract infection. Biofilm formation greatly increases the resistance of Pseudomonas aeruginosa to antibiotics1.As a result, it is of great importance to find alternatives to antibiotic therapy. Anti-virulence agents that inhibit production represent a strategy to avoid the emergence of antibiotic resistance because they do not suppress the bacterial growth. Moreover, the use of virulence inhibitors can enable the immune system to eradicate the pathogens1,2. Quorum sensing (QS) regulates the expression of virulence factors and hence targeting quorum sensing can inhibit the production of virulence factors3, 4.

 

Quorum sensing is an intercellular communication mechanism used by bacterial cells to help them detect their numbers by secretion of signaling molecules. The concentration of signaling molecules is proportional to the bacterial cell density5. P. aeruginosa produces an arsenal of virulence factors including pyocyanin, hemolysin, alkaline protease and the siderophore pyoverdine. Motility and biofilm formation enhances the virulence6-9. The production of biofilms makes P. aeruginosa highly resistant to antibiotics and to the immune system10. The resistance of biofilm cells to antibiotics is 10-1000 times like that of planktonic cells11.

 

Metronidazole is a widely used drug for treatment of anaerobic bacterial infections12. Metronidazole was found to be an analogue of acyl homoserine lactone (AHL) signaling molecules; the signaling molecules of quorum sensing in P. aeruginosa13. As a result, it may interfere with quorum sensing and production of virulence factors.

 

This study aimed to investigate the inhibitory activities of metronidazole on virulence factors of Pseudomonas aeruginosa.

 

MATERIAL AND METHODS;

Media and Chemicals:

Tryptone soya broth and Mueller Hinton broth were purchased from Oxoid (Hampshire, UK). Luria-Bertani (LB) broth, LB agar, peptone and tryptone were obtained from Lab M Limited (Lancashire, United Kingdom). Azocasein was the product of Sigma (St. Louis, USA) and metronidazole was obtained from Sanofi-Aventis, Cairo, Egypt. Other chemicals were of pharmaceutical grade.

 

Bacterial Strains:

The strain used in this study is a clinical uropathogenic Pseudomonas aeruginosa isolate. It was identified by Gram-staining, growth on Mac Conkey agar, oxidase test, green pigmentation on nutrient agar, motility and growth at 42°C according to Koneman et al.14

 

Quantitative assessment of biofilm by the spectrophotometric method:

The ability of P. aeruginosa strain to form biofilm was assessed according to Stepanovic et al.15The bacterial strain was grown overnight on TSB and the culture was diluted with TSB to have an approximate inoculum density of 1 × 106 CFU/ml. Aliquots of 100 μl of the prepared suspension were added to the wells of 96-well sterile microtiter plates with rounded bottom and the plates were incubated for 24 h at 37°C. The planktonic cells were gently removed by aspiration and the wells were washed 3 times with sterile phosphate buffered saline (PBS, pH 7.2). The remaining adherent bacterial cells were fixed with 99% methanol for 20 minutes after which it was removed and the wells were stained with crystal violet (1%) for 20 minutes. The excess dye was washed off with distilled water, the plates were air-dried and the bound dye was eluted by 33% glacial acetic acid. The optical densities were measured with a spectrofluorimeter (Biotek, USA) at 590 nm. Measurements were performed in triplicate and repeated 3 times. To determine the extent of biofilm formation, the cut-off optical density (ODc) was calculated as three times standard deviations above the mean OD of the negative control. The tested strain was considered non-biofilm producer (OD ≤ ODc), weak biofilm producer (OD > ODc, but ≤ 2x ODc), moderate biofilm producer (OD>2x ODc, but ≤ 4x ODc), and strong biofilm producer (OD> 4x ODc). To test the inhibitory effect of metronidazole on biofilm formation, the same procedure was followed with the addition of sub-MIC of metronidazole in TSB before incubation of the microtiter plates.

 

Swimming and Twitching Motilities Assay:

To assay the effect of metronidazole on swimming and twitching motilities, the method of Rashid and Kornberg16 was used. Swimming agar plates (1% tryptone, 0.5% sodium chloride and 0.3% agar) with sub-inhibitory concentration of metronidazole were prepared. Control plates were prepared in the same way but without the addition of metronidazole. Five µl of a diluted overnight culture in tryptone broth were used for surface inoculation of the plates in the center and the plates were incubated for 24h at 37°C. The diameters of swimming zones were measured. To assay the twitching inhbition, LB agar plates (1%) were stab-inoculated with 2µl of the prepared suspension and incubated at 37°C for 48h. The twitching zones were measured after removing the agar leaving the plates to dry. The experiment was repeated three times and the results were averaged.

 

Assay of virulence factors:

The tested strain was grown overnight in LB broth with and without sub-MIC of the tested agent at 37°C. To separate the cell free supernatant, the suspension was centrifuged at 8500 g for 15 minutes and the supernatant was used for assay of virulence facors18.

 

Protease assay

The proteolytic activity in the presence and absence of metronidazole was estimated by using the azocasein assay according to Kessler et al.18. To 150 µl of the prepared cell free culture supernatant, 1 ml of azocasein (0.3%) in 0.05M Tris HCl and 0.5M CaCl2 (pH 7.5) was added, and the mixture was incubated for 15 minutes at 37°C. to stop the reaction, Trichloroacetic acid (10%, 0.5 ml) was added followed by centrifugation to remove the precipitated azocasein. The absorbance of the supernatants was measured at 400 nm using Biotek Spectrofluorimeter (Biotek, USA).

 

Pyocyanin assay:

The method of Essar et al.19 and Ra'oof and Latif20 was used for pyocyanin assay. The tested strain was grown overnight in LB broth and diluted to have OD600 of 0.02. King A media (peptone 2%, K2SO4 1% and MgCl2 0.14%) with and without sub-inhibitory concentration of metronidazole was inoculated with the diluted culture. The cultures were incubated at 37°C for 48h and the absorbance of pyocyanin was measured at 520 nm using Biotek Spectrofluorimeter (Biotek, USA).The concentration of pyocyanin was calculated from the formula (pyocyanin concentration in µg/ml = OD520x 17.072). The experiment was performed in triplicate and the average concentration was calculated.

 

Hemolysin assay

To detect the effect of metronidazole on hemolysin, the modified method of Rossignol et al.21 was used. Fresh 2% v/v erythrocytes suspension in saline (0.7 ml) was added to cell free supernatant (0.5 ml) and incubated at 37°C for 2h. The hemoglobin release was measured at 540 nm after centrifugation at 2500 g for 5 minutes at 4°C. Positive control (erythrocytes completely lysed with 0.1% SDS) and negative control (erythrocytes incubated in LB broth) were used for comparison. Percentage hemolysis was calculated from the formula: % hemolysis= [X-B/T-B] x 100, where X is the treated and untreated samples; B is the negative control and T is the positive control. The hemolysis by treated cultures was expressed as % compared to hemolysis by untreated culture. The experiment was made in triplicate.

 

Pyoverdin assay:

The cell free supernatants were diluted 1:10 in Tris-HCl buffer (pH 7.4) and aliquots of 100 μl were added to 96-well microtiter plates on ice. The Pyoverdin concentration was calculated by measuring the fluorescence of the test supernatant at an excitation wavelength of 405 nm and an emission wavelength of 465 nm using Biotek Spectrofluorimeter22.

Statistical analysis:

The effects of Metronidazole on virulence factors of P. aeruginosa were compared by One Way ANOVA followed by Bonforroni’s Multiple Comparison, Graph Pad Prism 5. P values <0.05 or were considered statistically significant.

 

RESULTS:

Identification of Pseudomonas aeruginosa:

The tested isolate was identified as Pseudomonas aeruginosa. It appeared as Gram-negative bacilli under the microscope. It was oxidase positive, motile, produced green pigmentation on nutrient agar and could grow at 42°C.

 

Inhibition of twitching and swimming motilities:

The inhibitory effect of ¼ MIC of metronidazole on twitching and swimming motilities was significant as compared to the control (P< 0.05). Metronidazole reduced twitching motility by 47.57% and swimming motility by 64.81% (Figures 1-3).

 

 

Figure 1. Inhibition of twitching motility by sub-MICs of metronidazole; A (Control), B (Metronidazole).

 

Figure 2. Inhibition of swimming motility by sub-MICs of metronidazole; A (Control), B (Metronidazole).

 

 

 

Figure 3. Inhibition of twitching and swimming motilities by sub-MICs of metronidazole. The results are expressed as mean ± SD of three independent experiments.

 

 

Inhibition of biofilm formation

According to the criteria of Stepanovic et al.16, the tested strain was found to be strong biofilm forming. The inhibition of biofilm by ¼ MIC of metronidazole was statistically significant (86.49%) as compared to the control (Figure 4).

 

 

Figure 4. Inhibition of virulence factors (pyocyanin, pyoverdin, protease, hemolysin and biofilm)  by sub-MIC of metronidazole. The results are expressed as mean ±SD of three independent experiments.

 

Inhibition of pyocyanin pyoverdin, protease and hemolysin

Metronidazole at ¼ MIC showed significant inhibitory activities against the tested virulence factors as compared to the control (P<0.05) (Figure 4). Pyocyanin production was reduced by 43.79%. On the other hand, metronidazole inhibition of pyoverdin was 82.82 %. Metronidazole inhibited protease by 60.22%. Similarly, hemolytic activities in the presence of metronidazole was 7.16%.

 

DISCUSSION:

Anti-virulence therapy is a promising trend as an alternative option to antibiotics to avoid the emergence of resistance. Such therapy makes no growth pressure that leads to mutations and resistance23. Quorum sensing regulates many virulence factors of Pseudomonas aeruginosa including protease, hemolysin, pyocyanin in addition to biofilm formation23,24. This makes quorum sensing inhibition a valuable tool in the battle against Pseudomonas aeruginosa. Metronidazole was found to inhibit quorum sensing in E. coli harboring plasmid pSB406 or pSB1075 that contain the QS regulatory systems of P. aeruginosa as the recognition elements and the lux CDABE gene cassette as the reporter by acting as a structural analogue of acyl homoserine lactone (AHL) signaling molecules25.

 

In this study, the inhibitory effect of sub-inhibitory concentration of anti-quorum sensing compound metronidazole against virulence factors was investigated.

Interference with bacterial motility impairs bacterial adhesion and biofilm formation26. In this study, metronidazole caused a significant decrease in swimmimg and twitching motilities and biofilm formation. Moreover, Metronidazole showed significant inhibitory activities against pyocyanin, pyoverdin, protease and hemolysin.  The use of quorum sensing inhibitors to interfere with virulence has been previously reported. Thus aspirin and sodium ascorbate; two quorum sensing inhibitors were found to inhibit the virulence factors production of Pseudomonas aeruginosa27,28. This study suggests the use of metronidazole as anti-virulence compound against P. aeruginosa to avoid the emergence of resistance to antibiotics.

 

CONFLICT OF INTEREST:

There is no conflict of interest.

 

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Received on 19.10.2015                              Modified on 15.11.2015

Accepted on 21.11.2015                             © RJPT All right reserved

Research J. Pharm. and Tech. 8(12): Dec., 2015; Page 1640-1644

DOI: 10.5958/0974-360X.2015.00295.4