SpringerLink
Log in

Extraction, purification and characterization of phenazine from Pseudomonas aeruginosa isolate of wastewater sources: a panacea towards clinical pathogens

  • Original Article
  • Published:
Applied Nanoscience Aims and scope Submit manuscript

Abstract

Today with the help of advancement in the biotechnological processes, researchers are concentrating more on economical and versatile ways to recover value-added products from natural resources such as waste waters. Pseudomonas aeruginosa is known to produce a broad range of secondary metabolites during its active growth phase. In this study, P aeruginosa was densely isolated from the waste dum** zone of the highly polluted Cooum River (CR), Tamil Nadu, India. The strain was identified through biochemical analysis and molecular characterization by 16S rRNA sequencing and named as JAAKPA. Enzyme profiling of the strain revealed that it had potential lipase and protease activities. Antibacterial and antifungal activities of the strain against ten clinical pathogens were tested. A significant antibacterial activity against Gram-positive strains such as methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis, and moderate antifungal activity against Candida albicans, were observed. The culture supernatant of JAAKPA was subjected to thin-layer chromatography and column chromatography to extract and purify the antimicrobial compound (Phenazine). The purified compound was further subjected to gas chromatography–mass spectrometry (GC-MS), fourier transform infrared spectroscopy (FTIR), and nuclear magnetic resonance (NMR) studies. The results revealed phenazine to be the major secondary metabolite responsible for the antimicrobial activities.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  • Abken H-J, Tietze M, Brodersen J, Bäumer S, Beifuss U, Deppenmeier U (1998) Isolation and characterization of methanophenazine and function of phenazines in membrane-bound electron transport ofMethanosarcina mazei Gö1. J Bacteriol 180:2027–2032

    CAS  Google Scholar 

  • Antoran A, Aparicio-Fernandez L, Pellon A, Buldain I, Martin-Souto L, Rementeria A, Ghannoum MA, Fuchs BB, Mylonakis E, Hernando FL (2020) The monoclonal antibody Ca37, developed against Candida albicans alcohol dehydrogenase, inhibits the yeast in vitro and in vivo. Sci Rep 10:1–12

    Google Scholar 

  • Bean HD, Zhu J, Sengle JC, Hill JE (2014) Identifying methicillin-resistant Staphylococcus aureus (MRSA) lung infections in mice via breath analysis using secondary electrospray ionization-mass spectrometry (SESI-MS). J Breath Res 8:041001

    CAS  Google Scholar 

  • Bultel-Poncé V, Berge J-P, Debitus C, Nicolas J-L, Guyot M (1999) Metabolites from the sponge-associated bacterium Pseudomonas species. Mar Biotechnol 1:384–390

    Google Scholar 

  • Butiuc-Keul A, Carpa R, Podar D, Szekeres E, Muntean V, Iordache D, Farkas A (2021) Antibiotic resistance in Pseudomonas spp. through the urban water cycle. Curr Microbiol 78:1–11

    Google Scholar 

  • Chin-A-Woeng TF, Bloemberg GV, Lugtenberg BJ (2003) Phenazines and their role in biocontrol by Pseudomonas bacteria. New Phytol 157:503–523

    CAS  Google Scholar 

  • Cui Q, Lv H, Qi Z, Jiang B, **ao B, Liu L, Ge Y, Hu X (2016) Cross-Regulation between the phz1 and phz2 operons maintain a balanced level of phenazine biosynthesis in Pseudomonas aeruginosa PAO1. PLoS ONE 11:e0144447

    Google Scholar 

  • Datta R, Huang SS (2008) Risk of infection and death due to methicillin-resistant Staphylococcus aureus in long-term carriers. Clin Infect Dis 47:176–181

    Google Scholar 

  • de Meneses L, Pereira JR, Sevrin C, Grandfils C, Paiva A, Reis MA, Freitas F (2020) Pseudomonas chlororaphis as a multiproduct platform: conversion of glycerol into high-value biopolymers and phenazines. New Biotechnol 55:84–90

    Google Scholar 

  • Dietrich LE, Price-Whelan A, Petersen A, Whiteley M, Newman DK (2006) The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa. Mol Microbiol 61:1308–1321

    CAS  Google Scholar 

  • El-Baky RMA, Mandour SA, Ahmed EF, Hashem ZS, Sandle T, Mohamed DS (2020) Virulence profiles of some Pseudomonas aeruginosa clinical isolates and their association with the suppression of Candida growth in polymicrobial infections. PLoS ONE 15:e0243418

    Google Scholar 

  • Giridharan L, Venugopal T, Jayaprakash M (2008) Evaluation of the seasonal variation on the geochemical parameters and quality assessment of the groundwater in the proximity of River Cooum, Chennai, India. Environ Monit Assess 143:161–178

    CAS  Google Scholar 

  • Guttenberger N, Blankenfeldt W, Breinbauer R (2017) Recent developments in the isolation, biological function, biosynthesis, and synthesis of phenazine natural products. Bioorg Med Chem 25:6149–6166

    CAS  Google Scholar 

  • Hancock RE (1998) Resistance mechanisms in Pseudomonas aeruginosa and other nonfermentative gram-negative bacteria. Clin Infect Dis 27:S93–S99

    CAS  Google Scholar 

  • Holtm JG, Sneath PH (1986) Bergey’s manual of systematic bacteriology. Williams & Wilkins

    Google Scholar 

  • Hou L, Zhang L, Li F, Huang S, Yang J, Ma C, Zhang D, Yu C-P, Hu A (2021) Urban ponds as hotspots of antibiotic resistome in the urban environment. J Hazard Mater 403:124008

    CAS  Google Scholar 

  • Ibrahim S, El-Liethy MA, Elwakeel KZ, Hasan MAE-G, Al Zanaty AM, Kamel MM (2020) Role of identified bacterial consortium in treatment of Quhafa Wastewater Treatment Plant influent in Fayuom, Egypt. Environ Monit Assess 192:161

    CAS  Google Scholar 

  • Janda JM, Bottone EJ (1981) Pseudomonas aeruginosa enzyme profiling: predictor of potential invasiveness and use as an epidemiological tool. J Clin Microbiol 14:55–60

    CAS  Google Scholar 

  • Laxminarayan R, Duse A, Wattal C, Zaidi AK, Wertheim HF, Sumpradit N, Vlieghe E, Hara GL, Gould IM, Goossens H (2013) Antibiotic resistance—the need for global solutions. Lancet Infect Dis 13:1057–1098

    Google Scholar 

  • Lee H-J, Kim JS, Park S-Y, Suh M-E, Kim HJ, Seo E-K, Lee C-O (2004) Synthesis and cytotoxicity evaluation of 6, 11-dihydro-pyridazo-and 6, 11-dihydro-pyrido [2, 3-b] phenazine-6, 11-diones. Bioorg Med Chem 12:1623–1628

    CAS  Google Scholar 

  • Lee K, Omar D, Cheng G, Nasehi A, Wong M (2018) Characterization of phenazine and phenazine-1-carboxylic acid isolated from Pseudomonas aeruginosa UPMP3 and their antifungal activities against Ganoderma boninense. Pertanika J Trop Agric Sci 41:1795–1809

    Google Scholar 

  • Liu P (1952) Utilization of carbohydrates by Pseudomonas aeruginosa. J Bacteriol 64:773

    CAS  Google Scholar 

  • Liu T, Ye F, Pang C, Yong T, Tang W, **ao J, Shang C, Lu Z (2020) Isolation and identification of bioactive substance 1-hydroxyphenazine from Pseudomonas aeruginosa and its antimicrobial activity. Lett Appl Microbiol 71:303–310

    Google Scholar 

  • Mavrodi DV, Bonsall RF, Delaney SM, Soule MJ, Phillips G, Thomashow LS (2001) Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. J Bacteriol 183:6454–6465

    CAS  Google Scholar 

  • Mavrodi DV, Blankenfeldt W, Thomashow LS (2006) Phenazine compounds in fluorescent Pseudomonas spp. biosynthesis and regulation. Annu Rev Phytopathol 44:417–445

    CAS  Google Scholar 

  • Mazzola M, Cook R, Thomashow L, Weller D, Pierson L (1992) Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats. Appl Environ Microbiol 58:2616–2624

    CAS  Google Scholar 

  • Morales DK, Grahl N, Okegbe C, Dietrich LE, Jacobs NJ, Hogan DA (2013) Control of Candida albicans metabolism and biofilm formation by Pseudomonas aeruginosa phenazines. Mbio 4:e00526

    CAS  Google Scholar 

  • Oggioni MR, Pozzi G, Valensin PE, Galieni P, Bigazzi C (1998) Recurrent septicemia in an immunocompromised patient due to probiotic strains of Bacillus subtilis. J Clin Microbiol 36:325–326

    CAS  Google Scholar 

  • Peng J-M, Du B, Qin H-Y, Wang Q, Shi Y (2021) Metagenomic next-generation sequencing for the diagnosis of suspected pneumonia in immunocompromised patients. J Infect 82:22–27

    CAS  Google Scholar 

  • Price-Whelan A, Dietrich LE, Newman DK (2006) Rethinking ‘secondary’ metabolism: physiological roles for phenazine antibiotics. Nat Chem Biol 2:71–78

    CAS  Google Scholar 

  • Puyol D, Batstone DJ, Hülsen T, Astals S, Peces M, Krömer JO (2017) Resource recovery from wastewater by biological technologies: opportunities, challenges, and prospects. Front Microbiol 7:2106

    Google Scholar 

  • Rajan S, Geethu V, Sampath S, Chakraborty P (2019) Occurrences of polycyclic aromatic hydrocarbon from Adayar and Cooum Riverine Sediment in Chennai city, India. Int J Environ Sci Technol 16:7695–7704

    CAS  Google Scholar 

  • Römer A (1983) 13C NMR spectra of substituted phenazines: substituent effects on carbon-13 chemical shifts and the use of 13C–15N coupling constants for the assignment of the aromatic carbons. Org Magn Reson 21:130–136

    Google Scholar 

  • Sarker SD, Nahar L, Kumarasamy Y (2007) Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods 42:321–324

    CAS  Google Scholar 

  • Shahid I, Han J, Hardie D, Baig DN, Malik KA, Borchers CH, Mehnaz S (2021) Profiling of antimicrobial metabolites of plant growth promoting Pseudomonas spp. isolated from different plant hosts. 3 Biotech 11:1–14

    Google Scholar 

  • Tanguturu K, Mondal M, Banik A, Raman G, Sakthivel N (2020) Metabolites of fluorescent Pseudomonads and their antimicrobial and anticancer potentials. Bioactive natural products in drug discovery. Springer, pp 355–377

    Google Scholar 

  • Valgas C, de Souza SM, Smânia EF, Smânia A Jr (2007) Screening methods to determine antibacterial activity of natural products. Braz J Microbiol 38:369–380

    Google Scholar 

  • Vilaplana L, Marco M-P (2020) Phenazines as potential biomarkers of Pseudomonas aeruginosa infections: synthesis regulation, pathogenesis and analytical methods for their detection. Anal Bioanal Chem 412:5897–5912

    CAS  Google Scholar 

  • Wang K, Kai L, Zhang K, Hao M, Yu Y, Xu X, Yu Z, Chen L, Chi X, Ge Y (2020) Overexpression of phzM contributes to much more production of pyocyanin converted from phenazine-1-carboxylic acid in the absence of RpoS in Pseudomonas aeruginosa. Arch Microbiol 202:1507–1515

    CAS  Google Scholar 

  • Wang S-Y, Shi X-C, Chen X, Laborda P, Zhao Y-Y, Liu F-Q, Laborda P (2021) Biocontrol ability of phenazine-producing strains for the management of fungal plant pathogens: a review. Biol Control 155:104548

    CAS  Google Scholar 

  • Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703

    CAS  Google Scholar 

  • Westerhoff P, Lee S, Yang Y, Gordon GW, Hristovski K, Halden RU, Herckes P (2015) Characterization, recovery opportunities, and valuation of metals in municipal sludges from US wastewater treatment plants nationwide. Environ Sci Technol 49:9479–9488

    CAS  Google Scholar 

  • Yang H, Abouelhassan Y, Burch GM, Kallifidas D, Huang G, Yousaf H, ** S, Luesch H, Huigens RW (2017) A highly potent class of halogenated phenazine antibacterial and biofilm-eradicating agents accessed through a modular Wohl-Aue synthesis. Sci Rep 7:1–16

    Google Scholar 

Download references

Acknowledgements

The authors wish to thank M. Karthick, Pondicherry University, Puducherry, India for his help with NMR spectroscopy. We are grateful to Dr. Dilipkumar Thacharodi for his comments on an earlier version of this manuscript.

Author information

Authors and Affiliations

  1. Department of Research and Development, Dr. Thacharodi’s Laboratories, No. 24, 5th Cross, Thanthaiperiyar Nagar, Ellapillaichavadi, Puducherry, 605005, India

    Aswin Thacharodi, R. Priyadharshini, G. Karthikeyan & Akhila. P. Reghu

  2. Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, 620024, India

    C. Jeganathan

  3. School of Renewable Energy, Maejo University, Chiang Mai, 50290, Thailand

    Arivalagan Pugazhendhi

  4. College of Medical and Health Science, Asia University, Taichung, Taiwan

    Arivalagan Pugazhendhi

Authors
  1. Aswin Thacharodi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. Priyadharshini
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. G. Karthikeyan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C. Jeganathan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Akhila. P. Reghu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Arivalagan Pugazhendhi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Arivalagan Pugazhendhi.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

Not required.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Thacharodi, A., Priyadharshini, R., Karthikeyan, G. et al. Extraction, purification and characterization of phenazine from Pseudomonas aeruginosa isolate of wastewater sources: a panacea towards clinical pathogens. Appl Nanosci 13, 2365–2378 (2023). https://doi.org/10.1007/s13204-021-01944-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13204-021-01944-y

Keywords

Search

Navigation