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Antimicrobial Effectiveness of Bioactive Phenazine Against Clinical Isolates.

Pseudomonas aeruginosa, motile gram-negative bacterium grow in normal atmospheric conditions and hypoxic atmospheric conditions also facilitate its growth (Bettina et al., 2012). Different bacterial species produce over 100 phenazine derivatives (Gibson et al., 2009) but P. aeruginosa is the only non-lactose fermentor that produce pyocyanin (Meyer, 2000; Reyes et al., 1981) that may alter the microbial community by inhibiting the growth of sensitive microorganisms (Norman et al., 2004). Phenazine is a heterocyclic naturally occuring deep red compound (5-methyl-7-amino-1-carboxymethylphenazinium betaine) converted to lemon yellow coloured phenazine-1-carboxylic acid (PCA) and finally to the bright blue pyocyacnin 1-hydroxy-5 methylphenazine (Gohain et al., 2006).

Pyocyanin regulates the redox mechanism and initiate the production of reactive oxygen species which in turns inhibits cellular respiration, reduction in level of ATP and cAMP thus chloride ion channel could be effected that is controlled by ATP (Winstanley and Fothergill, 2008). The purified form of pyocyanin showed antibacterial effect that varied with the concentration of pyocyanin (Fouly et al., 2015; Kanthakumar et al., 1993; Baron and Rowe, 1981). Bacterial strains generates increase resistance towards antibiotics, germicides and disinfectants in this regards phenazines have been of great importance from last decades to pharmaceutical and clinical researchers in treating the antibiotic-resistant and susceptible infections (Shouny et al., 2011).

Previous studies revealed that in lung patients the growth of different species of Candida sp. has been suppressed by pyocyanin and recurrence of the Candida sp. with the suppression of pyocyanin (Kerr, 1994), as in this study zone of inhibition against Candida sp. by pyocyanin showed some inhibitory action. The available antifungals like azoles and echinocandins are costly and associated with some side effects so there is a need of some other compounds that have better anti-Candida effects (Bhattacharyya et al., 2013).

Isolation of P. aeruginosa. Different strains of P. aeruginosa were isolated from clinical samples obtained from pathological laboratories of tertiary care hospital of Karachi, Pakistan and identified by conventional method by performing gram's staining and standard biochemical test (Kathleen and Christopher, 2014).

Pyocyanin extraction and optimization. Soluble pigment of P aeruginosa was extracted by inoculating samples on Pseudomonas Cetrimide agar (Fig. 1). They were incubated at 37[degrees]C for 24 h and were observed for colour change. The pigment was extracted using chloroform solvent system as described by Sweedan, (2010). Extraction of pyocyanin was confirmed with pinkish-red colour production when exposed to 0.2N HC1 as shown in Fig. 2-3 (Sudhakar et al., 2015).

Antimicrobial assay. Antibacterial activity ofpyocyanin was performed with some modifications of the protocol proposed by Sweedan (2010). The tryptic soya broth was inoculated with tests strains which are clinical isolates such as E. coli, S. aureus, Proteus sp., Klebsiella sp., Bacillus sp. and Candida sp. for 3 h at 37[degrees]C and the turbidity was checked using 0.5 McFarland standard. Then 100 [micro]L of bacterial suspension was spread by glass rod, 20 [micro]L of purified pyocyanin was added to the prepared wells in the same plate and then incubated at 37[degrees]C for 24 h and the diameter of zone was measured and the results were recorded.

The extracted pyocyanin was tested against different pathogenic microorganisms to identify the inhibitory effect as shown in Table 1. Maximum combatant activity was observed against Candida sp. showed 17 mm zone of inhibition (Fig. 4) in terms of diameter followed by Proteus sp. which was 16 mm as shown in (Fig. 5). The minimum activity was observed against E.coli i.e 10 mm.

In some diseases there is co-infection of Pseudomonas in that case our results showed the inhibition of microorganism by pyocyanin for e.g. in Cystic fibrosis patients there is a co-infection with Staphylococcus aureus (Abdulrudha, 2011) so the pyocyanin could be the inhibitory molecule for S. aureus as in this study zone of inhibition against S. aureus was 13.5 mm (Fig. 6). Our study indicates that pathogenic microorganism become susceptible with the action of pyocyanin.

Acknowledgement

The authors would like to thank Department of Microbiology, Jinnah University for Women for their cooperation throughout this study. Thank are also due to the administration of Jinnah University for Women for the approval and funding of this research.

Conflict of Interest. The authors declare no conflict of interest.

References

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Baron, S.S., Rowe, J.J. 1981. Antibiotic action of pyocyanin. Antimicrobial Agents and Chemotherapy, 20: 814-820.

Bettina, S., Cormac, T., Taylor, T., Kirsten, S. 2012. Hypoxia Increases antibiotic resistance in Pseudomonas aeruginosa through altering the composition of multidrug efflux pumps. Antimicrobial Agents and Chemotherapy, 56: 2114-2118.

Bhattacharyya, S., Gupta, P., Banerjee, G., Jain, A., Singh, M. 2013. Inhibition of Candida biofilms by pyocyanin: An in-vitro study. International Journal of CurrentResearch, 05: 31-36.

El-Fouly, M.Z., Sharaf, A.M., Shahin, A.A.M., Heba, A., El-Bialy, A., Omara, A.M.A. 2015. Biosynthesis of pyocyanin pigment by Pseudomonas aeruginosa. Journal of Radiation Research and Applied Sciences, 8: 36-48.

Gohain, N., Linda, S.T., Mavrodi, D.V., Wulf, B. 2006. The purification, crystallization and preliminary structural characterization of PhzM, a phenazinemodifying methyltransferase from Pseudomonas aeruginosa. Acta Crystallographica Section F Structural Biology and Crystallization Communications, 62: 887-890.

Gibson, J., Sood, A., Hogan, D.A. 2009. Pseudomonas aeruginosa-Candida albicans interactions: Localization and fungal toxicity of a phenazine derivative. Applied and Environmental Microbiology, 75: 504-513.

Kanthakumar, K., Taylor, G., Tsang, K.W.T., Cundell, D.R., Rutman, A., Smith, S., Jeffery, P.K., Cole, P.J., Wilson, R. 1993. Mechanisms of action of Pseudomonas aeruginosa Pyocyanin on human ciliary beat in-vitro. Infection and Immunity, 61: 2848-2853.

Kathleen, S., Christopher, E. 2014. Pseudomonas isolation and identification: An introduction to the challenges of polyphasic taxonomy. Journal of Microbiology and Biology Education, 15: 287-291.

Kerr, J.R. 1994. Suppression of fungal growth exhibited by Pseudomonas aeruginosa. Journal of Clinical Microbiology, 32: 525-527.

Meyer, J.M. 2000. Pyoverdines: pigments, siderophores and potential taxonomic markers of fluorescent Pseudomonas species. Archives of Microbiology, 174: 135-142.

Norman, R.S., Moeller, P., McDonald, T.J., Morris, P.J. 2004. Effect of pyocyanin on a crude-oil-degrading microbial community. Applied and Environmental Microbiology, 70: 4004-4011.

Reyes, E.A., Bale, M.J., Cannon, W.H., Matsen, J.M. 1981. Identification of Pseudomonas aeruginosa by pyocyanin production on tech agar. Journal of Clinical Microbiology, 13: 456-458.

Shouny, W.A., Al-Baidani, A.R.H., Hamza, W.T. 2011. Antimicrobial activity of pyocyanin produced by Pseudomonas aeruginosa isolated from Surgical Wound-Infections. International Journal of Pharmacy and Medical Sciences, 1: 01-07.

Sudhakar, T., Karpagam, K.S., Premkumar, J. 2015. Biosynthesis, antibacterial activity of pyocyanin pigment produced by Pseudomonas aeruginosa SU1. Journal of Chemical and Pharmaceutical Research, 7: 921-924.

Sweedan, E.G. 2010. Study the effect of antibiotics on pyocyanin production from Pseudomonas aeruginosa and pyocyanin as antibiotic against different pathogenic bacteria. Journal of University of Anbarfor PureScience, 4: ISSN: 1991-8941.

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Fasiha Saeed (a) *, Fariha Ahmed (a), Farkhanda Afaque (a), Kholud Mehmood (a) and Syeda Ghufrana Nadeem (b)

(a) Department of Microbiology, Jinnah University for Women, Karachi-74600, Pakistan (b) Medical Mycology Research & Reference Laboratory, Department of Microbiology, Jinnah University for Women, Karachi -74600, Pakistan

(received September 25, 2016; revised September 11, 2017; accepted September 15, 2018)

* Author for correspondence;

E-mail: fasihasaeed37@gmail.com

Caption: Fig. 1. Growth of Pseudomonas on Cetrimide agar.

Caption: Fig. 2. Extracted Pyocyanin in TSB broth.

Caption: Fig. 3. Pink colour confirms presence of pyocyanin.

Caption: Fig. 4. Antibacterial effect against Candida sp.

Caption: Fig. 5. Antibacterial effect against Proteus sp.

Caption: Fig. 6. Antibacterial effect against S. aureus.
Table 1. The zone of inhibition (zone diameter) by phenazine
against clinical isolates by agar well diffusion

Pyocyanin   Pathogenic               Zone of inhibitions (mm)
            microorganism

20 pL       Escherichia coli         10
            Proteus sp.              16
            Staphylococcus aureus    13.5
            Klebsilla sp.            13
            Bacillus sp.             12.6
            Candida sp.              17
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Title Annotation:Short Communication
Author:Saeed, Fasiha; Ahmed, Fariha; Afaque, Farkhanda; Mehmood, Kholud; Nadeem, Syeda Ghufrana
Publication:Pakistan Journal of Scientific and Industrial Research Series B: Biological Sciences
Geographic Code:9PAKI
Date:Mar 1, 2019
Words:1334
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