Purification and characterization of antifungal phenazines from a fluorescent Pseudomonas strain FPO4 against medically important fungi

doi:10.1016/j.mycmed.2014.02.003

Journal de Mycologie Médicale

Volume 24, Issue 3, September 2014, Pages 185-192

https://doi.org/10.1016/j.mycmed.2014.02.003 Get rights and content

Summary

The strain FPO4 was isolated from the rhizoplane of rice plant root and identified as a fluorescent Pseudomonas aeruginosa on the basis of 16S rDNA sequences and BLAST analysis. The extracellular metabolites produced by this strain were purified by silica gel column chromatography and isolated four pure compounds. Based on the spectral data the four compounds were identified as phenazin-1-ol, phenazine-1-carboxylic acid (PCA), 2-heptyl-3-hydroxyl-4(1H)-quinolone (PQS), and phenazine-1-carboxamide (PCN), respectively. Phenazin-1-ol and PCA were active against all the eight fungi tested. The highest activity of 4 μg/mL by PCA was recorded against Trichophyton rubrum, a human pathogen responsible for causing athlete's foot, jock itch, ringworm and fingernail fungus infections, followed by Candida albicans and Candida tropicalis. The activity of phenazin-1-ol, PCA against Candida spp. was found to be better than the standard antifungal agent amphotericin B. Furthermore, the present study reports the antimicrobial activity of the purified phenazines on major human pathogen, T. rubrum for the first time.

Résumé

La souche FPO4 a été isolée à partir de rhizome de racine de riz et identifiée en tant que Pseudomonas aeruginosa fluorescent sur la base des séquences d’ADNr 16S et de l’analyse BLAST. Les métabolites extracellulaires produits par cette souche ont été purifiés par chromatographie sur colonne de gel de silice et quatre composés purs ont été isolés. Sur la base des données spectrales les quatre composés ont été identifiés comme phénazine-1-ol, l’acide phénazine-1-carboxylique (APC), le 2-heptyl-3-hydroxy-4 (1H)-quinolone (PQS) et phénazine-1-carboxamide (PCN), respectivement. La phénazine-1-ol et l’APC étaient actifs contre tous les huit champignons testés. L’activité la plus forte de 4 μg/mL pour l’APC a été enregistré contre Trichophyton rubrum, un agent pathogène humain responsable de pied d’athlète, d’intertrigo inguinal, d’herpès circiné et d’infections fongiques des ongles, suivi par Candida albicans et Candida tropicalis. L’activité de la phénazine-1-ol, et APC contre Candida spp. a été jugée meilleure que celle de l’agent antifongique amphotéricine B. En outre, la présente étude rapporte l’activité antimicrobienne des phénazines purifiés sur un pathogène humain majeur, T. rubrum pour la première fois.

Introduction

Resistance to antifungal agents is widely recognized [29] requiring continuous development of new antifungal agents [35]. Diseases due to pathogenic fungi represent a critical problem to human health and they are one of the main causes of morbidity and mortality worldwide. The emergence of pathogens resistant to antibiotics as a result of their excessive use in clinical and veterinary applications represents a serious public health concern [20]. In the last three decades, drug resistant fungi, particularly Candida strains, have caused major health problems, especially in women throughout the world [28]. Azoles and amphotericin B has been a predominant therapy drug for fungal infections for a long time now. Unfortunately, the resistance of fungi to these drugs is a global health problem. The search for novel microorganisms with antifungal activity has gained increasing importance in recent years due to the development of drug resistance.

Fluorescent pseudomonads are considered to be the most promising group of plant growth promoting rhizobacteria involved in biocontrol of plant diseases [13]. Fluorescent pseudomonad species such as Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas putida and Pseudomonas pyrrocinia have been demonstrated to show antifungal activity with varying degrees of antagonism [10]. Some of the fluorescent pseudomonads have currently received world-wide attention due to the production of a wide range of antifungal compounds viz, fluorescent pigments [26]; siderophores; volatile compounds such as HCN [11], antibiotics such as phenazine-1-carboxylic acid [17]; pyoluteorin [16]; viscosinamide and tesin [3]; 2,4-diacetylphloroglucinol [32] and lytic enzymes.

In the present study, we described the characterization of major secondary metabolites produced by a fluorescent Pseudomonas strain FPO4 and its antifungal activity against some medically important fungi.

Section snippets

Chemicals and media

All the chemicals used for extraction and column chromatography were of analytical grade and was purchased from Merck Limited, Mumbai, India. Silica gel (230–400 mesh) used for column chromatography and precoated silica gel 60 GF₂₅₄ plates used for Thin Layer Chromatography (TLC) were purchased from Merck Limited, Germany. All microbiological media were purchased from Hi-Media Laboratories Limited, Mumbai, India. All other reagents were of analytical grade and other chemicals used in this study

Results

The strain FPO4 was identified as P. aeruginosa by IMTECH based on morphological and classical biochemical test. The strain was currently deposited in IMTECH (Institute of Microbial Technology, Chandigarh; India) and the MTCC number of this strain was P. aeruginosa MTCC 7277. Final confirmation of this strain was done based on molecular biology tool. The bacteria isolate (P. aeruginosa strain) was identified based on 16S rDNA gene sequencing. PCR amplification yielded ∼1500 bp amplicon. Blast

Discussion

Phenazines are natural products found in Pseudomonas spp., Streptomyces and a few other genera from soil or marine habitats. Phenazines are large family of colorful, nitrogen-containing tricyclic molecules with antibiotic, antitumor, and antiparasitic activities [23]. Phenazines isolated from Pseudomonas species (e.g., P. aeruginosa, P. aureofaciens, P. fluorescens and P. cepacia) are mostly simple hydroxyl- and carboxyl-substituted structures. Pyocyanin (5-N-methyl-1-hydroxyphenazine),

Disclosure of interest

The authors declare that they have no conflicts of interest concerning this article.

Acknowledgements

The authors are thankful to Dr. Suresh Das, Director National Institute for Interdisciplinary Science and Technology (CSIR), Thiruvananthapuram and Kerala State Council for Science, Technology and Environment (KSCSTE) (No. 001-06/PDF/2013/KSCSTE) for providing the necessary facility and fund to carry out the present work.

References (37)

B. Cezairliyan et al.
Identification of Pseudomonas aeruginosa Phenazines that kill Caenorhabditis elegans
PLOS Pathogens
(2013)
T.C. Charles et al.
A chromosomally sensory transduction system is required for tumefaciens
J Bacteriol
(1993)
T.F.D. Chin-A-Woeng et al.
Phenazine and their role in biocontrol by Pseudomonas bacteria
New Phytol
(2003)
E.J. Choi et al.
6-Hydroxymethyl-1-phenazinecarboxamide and 1,6-phenazinedimethanol from a marine bacterium, Brevibacterium sp. KMD 003, associated with marine purple vase sponge antibacterial phenazines from Brevibacterium sp. KMD 003
J Antibiot
(2009)
Clinical and Laboratory Standards Institute (CLSI)
M44-A2
Method for antifungal disk diffusion susceptibility testing of yeasts; approved guideline
(2009)
Clinical and Laboratory Standards Institute (CLSI)
Performance standards for antifungal disk diffusion susceptibility testing of non-dermatophyte filamentous fungi; Informational supplement – First edition. CLSI document M51-A
(2010)
Clinical and Laboratory Standards Institute (CLSI)
Reference method for broth dilution antifungal susceptibility testing of filamentous fungi, as the document is M38-A2
(2008)
Clinical and Laboratory Standards Institute (CLSI).
Reference method for broth dilution antifungal susceptibility testing of yeasts, as the document is M27-S4
(2012)
M. Conda-Sheridan et al.
Potential chemopreventive agents based on the structure of the lead compound 2-Bromo-1-hydroxyphenazine, isolated from Streptomyces species, strain CNS284J
Med Chem
(2010)
L.A. de Weger et al.
Siderophores and outer membrane proteins of antagonistic, plant-growth-stimulating, root-colonizing Pseudomonas spp
J Bacteriol
(1986)

G. Defago et al.

Pseudomonads as antagonists of soil borne plant pathogens: mode of action and genetic analysis

Soil Biochem

(1990)

B.S. Dileep Kumar et al.

Plant growth promotion and fungal pest control through an antibiotic and siderophore producing fluorescent Pseudomonas strain from tea (Camellia sinensis (L) O. Kuntze) plantations

Indian J Exp Biol

(1997)

J.M. Gardner et al.

Growth promotion and inhibition by antibiotics producing fluorescent Pseudomonads on citrus root

Plant Soil

(1984)

J.A. Hindler

Special antimicrobial susceptibility tests

J.T. Hodgkinson et al.

Microwave and flow syntheses of Pseudomonas quinolone signal (PQS) and analogues

Org Biomol Chem

(2011)

H.B. Hu et al.

Isolation and characterization of a new Pseudomonas strain produced both phenazine 1-carboxylic acid and pyoluteorin

J Microbiol Biotech

(2005)

J. Huang et al.

Temperature-dependent expression of phzM and its regulatory genes lasI and ptsP in rhizosphere isolate Pseudomonas sp. strain M185

Appl Environ Microbiol

(2009)

G.S. Jayatilake et al.

Metabolites from an Antarctic sponge-associated bacterium, Pseudomonas aeruginosa

J Nat Prod

(1996)

Cited by (21)

Biocontrol ability of phenazine-producing strains for the management of fungal plant pathogens: A review
2021, Biological Control
Citation Excerpt :
In general, it can be observed that PCA showed higher inhibitory activity in comparison with other reported phenazines. PCA inhibited the mycelial growth of Gaeumannomyces gramini by 91% at only 1 µg/mL (Mazzola et al., 1995), and its MIC against Penicillium expansum was only 16 µg/mL (Gorantla et al. 2014). In contrast, 4-hydroxyphencomycin did not show any antifungal activity below 128 µg/mL (Han et al., 2014), and PCN did not show any inhibitory activity against Fusarium oxysporum at less than 1000 µg/mL.
The potential of phenazine-producing strains for the management of fungal and oomycete plant pathogens has been reviewed for the first time. Isolated natural phenazines showed similar, or even higher, antifungal activity in comparison with commercial fungicides. The occurrence and concentration of phenazines strongly depended on the studied strains and the antifungal activity of the strains was found to be dependent on the biosynthesis of phenazines. Except for phenazine-1-carboxylic acid (PCA), most phenazines were found in the secretions below their minimum inhibitory concentrations (MIC) and, in general, most strains showed low in vivo inhibitory activities, observing the highest biocontrol ability when using PCA-producing Pseudomonas chlororaphis and P. fluorescens. Most reports focused on the study of Pseudomonas, reducing the structural diversity, and limited the biocontrol screenings to root diseases. The conclusions achieved in this work help to understand the current state of the research field, and reveal new insights on the development of efficient biocontrol strategies using phenazine-producing strains.
Pyocyanin as anti-tyrosinase and anti tinea corporis: A novel treatment study
2016, Microbial Pathogenesis
Citation Excerpt :
Pyocyanin, one of the phenazines, was reported earlier as antibacterial and antifungal against human pathogenic C. albicans yeast [36]. Recently, few effective antimicrobial agents against T. rubrum are recorded due to their high costs and severe side effects [37]. Tyrosinase inhibitors were used in the treatment of many different skin diseases such as hyperpigmentation and melasma.
The aim of this study was to evaluate the efficiency of pyocyanin pigment as a novel compound active against tyrosinase with its depigmentation efficiency for combating Trichophyton rubrum which could be a major causative agent of tinea corporis.
Fifty swabs of fungal tinea corporis infections were collected and identified. Five MDRPA isolates were tested for their levels of pyocyanin production. The purified extracted pyocyanin was characterized by UV spectrum and FT-IR analysis. Pyocyanin activity against tyrosinase was determined by dopachrome micro-plate. In addition, the antidermatophytic activity of pyocyanin against T. rubrum was detected by radial growth technique. In vivo novel trial was conducted to evaluate the efficiency and safety of pyocyanin as an alternative natural therapeutic compound against T. rubrum causing tinea corporis.
Purified pyocyanin showed highly significant inhibitory activity against tyrosinase and T. rubrum. In vivo topical treatments with pyocyanin ointment revealed the efficiency of pyocyanin (MIC 2000 μg/ml) to cure tinea corporis compared to fluconazole, which showed a partial curing at a higher concentration (MIC 3500 μg/ml) after two weeks of treatment. In addition, the results revealed complete healing and disappear of hyperpigmentation by testing the safety of pyocyanin ointment and its histopathological efficiency in the skin treatment without any significant toxic effect.
Pyocyanin pigment could be a promising anti-tyrosinase and a new active compound against T. rubrum, which could be a major causative agent of tinea corporis. In fact, if pyocyanin secondary metabolite is going to be used in practical medication, it will support the continuous demand of novel antimycotic natural agents against troublesome fungal infections.
Interaction between Pseudomonas aeruginosa and dermatophyte fungi: Repercussions on the clinical course and microbiological diagnosis of tinea pedis
2016, Actas Dermo-Sifiliograficas
Genome analysis of Pseudomonas strain 4B with broad antagonistic activity against toxigenic fungi
2024, Brazilian Journal of Microbiology
Phenazine 1-carboxylic acid Producing Seed Harbored Endophytic Bacteria from Cultivated Rice Variety of Kerala and Its Broad Range Antagonism to Diverse Plant Pathogens
2023, Probiotics and Antimicrobial Proteins
Electro-Oxidative Synthesis of Phenazines
2023, Organic Letters

View all citing articles on Scopus

View full text

Summary

Résumé

Introduction

Section snippets

Chemicals and media

Results

Discussion

Disclosure of interest

Acknowledgements

Identification of Pseudomonas aeruginosa Phenazines that kill Caenorhabditis elegans

PLOS Pathogens

A chromosomally sensory transduction system is required for tumefaciens

J Bacteriol

Phenazine and their role in biocontrol by Pseudomonas bacteria

New Phytol

6-Hydroxymethyl-1-phenazinecarboxamide and 1,6-phenazinedimethanol from a marine bacterium, Brevibacterium sp. KMD 003, associated with marine purple vase sponge antibacterial phenazines from Brevibacterium sp. KMD 003

J Antibiot

M44-A2

Method for antifungal disk diffusion susceptibility testing of yeasts; approved guideline

Performance standards for antifungal disk diffusion susceptibility testing of non-dermatophyte filamentous fungi; Informational supplement – First edition. CLSI document M51-A

Reference method for broth dilution antifungal susceptibility testing of filamentous fungi, as the document is M38-A2

Reference method for broth dilution antifungal susceptibility testing of yeasts, as the document is M27-S4

Potential chemopreventive agents based on the structure of the lead compound 2-Bromo-1-hydroxyphenazine, isolated from Streptomyces species, strain CNS284J

Med Chem

Siderophores and outer membrane proteins of antagonistic, plant-growth-stimulating, root-colonizing Pseudomonas spp