World J Microbiol Biotechnol (2014) 30:1159–1168
DOI 10.1007/s11274-013-1552-5
REVIEW
Pyocyanin: production, applications, challenges and new insights
Sheeba Jayaseelan • Damotharan Ramaswamy •
Selvakumar Dharmaraj
Received: 22 May 2013 / Accepted: 31 October 2013 / Published online: 9 November 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract Pseudomonas aeruginosa is an opportunistic,
Gram-negative bacterium and is one of the most com-
mercially and biotechnologically valuable microorganisms.
Strains of P. aeruginosa secrete a variety of redox-active
phenazine compounds, the most well studied being pyo-
cyanin. Pyocyanin is responsible for the blue-green colour
characteristic of Pseudomonas spp. It is considered both as
a virulence factor and a quorum sensing signalling mole-
cule for P. aeruginosa. Pyocyanin is an electrochemically
active metabolite, involved in a variety of significant bio-
logical activities including gene expression, maintaining
fitness of bacterial cells and biofilm formation. It is also
recognised as an electron shuttle for bacterial respiration
and as an antibacterial and antifungal agent. This review
summarises recent advances of pyocyanin production from
P. aeruginosa with special attention to antagonistic prop-
erty and bio-control activity. The review also covers the
challenges and new insights into pyocyanin from P.
aeruginosa.
Keywords Antimicrobial effect
Bio-control agent

Pigment production
Pyocyanin
Pseudomonas
aeruginosa
S. Jayaseelan
S. Dharmaraj (&)
Dr. Sir A. L. Mudaliar Vocational Arts and Science College,
Vengal 601103, Tamil Nadu, India
e-mail: biochem_selva@yahoo.com
S. Jayaseelan
D. Ramaswamy
Department of Plant Biology and Biotechnology, Presidency
College, Chennai 600005, Tamil Nadu, India
Introduction
Pseudomonas aeruginosa is a member of the Gamma
Proteobacteria class of bacteria. Based on the revisionist
taxonomy done by analysis of conserved macromolecules
(e.g. 16S ribosomal RNA), the genus Pseudomonas was
included in the bacterial family Pseudomonadaceae
(Glazebrook et al. 1978). Other members in the genus
include P. alcaligenes, P. anguilliseptica, P. citronellolis,
P. flavescens, P. jinjuensis, P. mendocina, P. nitroredu-
cens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans
and P. straminae.
Pseudomonas aeruginosa is a Gram-negative, aerobic rod
shaped bacterium measuring 0.5–0.8 lm wide and
1.5–3.0 lm long. Almost all strains are motile by means of a
single polar flagellum (Palleroni 2005; Moore et al. 2006). It
thrives not only in normal atmospheric conditions but also in
hypoxic atmospheres, and has colonised many natural and
artificial surroundings (Green et al. 1974; Migula 1984;
Suthar et al. 2009). Its habitat is thus widespread and it is
found in soil, water and many other environments.
Pseudomonas aeruginosa attracts attention because of
its colour and pigment production. One of the most rec-
ognisable signs of an unknown colony being P. aeruginosa
is the characteristic fruity, grape-like odour derived from
the production of 2-aminoacetophenone by the organism.
On sheep blood agar plates, colonies of P. aeruginosa often
display beta-haemolysis and a greenish metallic sheen due
to its pigment production. No other species of Gram-neg-
ative non-fermenting bacteria produce pyocyanin, making
its presence helpful in identifying the organism. Pigments
secreted by P. aeruginosa include pyocyanin (blue-green),
pyoverdin (yellow, green and fluorescent), pyomelanin
(light-brown) and pyorubrin (red-brown) (Reyes et al.
1981; Meyer 2000).
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Nearly 90–95 % of all isolates of P. aeruginosa produce
the pigment pyocyanin, which is deep blue in colour and
referred to as ‘‘blue pus’’ (from pyocyaneus) (Fordos 1863;
Gessard 1984; Ran et al. 2003). Pyocyanin is produced
abundantly in media with low iron content and plays an
important role in iron metabolism. Pyocyanin appears to
participate in a reduction mechanism which is capable of
reducing and releasing the iron from transferrin. Iron is a
crucial requirement for the growth of P. aeruginosa (Cox
1986). Pyocyanin pigment is a phenazine which is a
nitrogen-containing heterocyclic compound. It is a redox
active secondary metabolite and is soluble in chloroform.
This metabolite, 1-hydroxy-N-methyl phenazine, contrib-
utes to bacterium survival. Little is known about the two
enzymes designated PhzM and PhzS that function in the
synthesis of pyocyanin from the precursor phenazine-1-
carboxylic acid. Phenazine compounds produced from the
rhizosphere of plants contribute to the biological activity of
P. aeruginosa against Fusarium (wilt of chickpea) and
Pythium (damping-off of bean).
In this review, production, purification, antagonistic
property, bio-control activity, challenges and new insights
of pyocyanin from P. aeruginosa are explained in detail.
Pyocyanin production and factors that influence
production
Previously, pyocyanin production was carried out using
King’s Medium (20 g peptone, 1.4 g magnesium chloride,
10 g potassium sulphate, 15 g agar in 1 l of distilled water,
pH 7.0 ± 0.2, 25 °C) as described by King et al. in 1954.
Based on King’s Medium, Pseudomonas agar medium was
formulated which is recommended for pyocyanin prodcu-
tion by Pseudomonas species. This medium enhances the
elaboration of pyocyanin but inhibits the formation of other
pigments. There are many reports which determined the
cultural characteristics and conditions which favour the
production of pyocyanin (Burton et al. 1948; Karpagam
et al. 2013; Sudhakar et al. 2013). Combination of amino
acids, notably alanine and leucine, in the culture medium
resulted in a more effective pigment production. There
were several works on designing the medium for pigment
production. Media formulations that produce optimal levels
of pyocyanin contain glycerol, alanine, sulphur, and iron.
Presence of alanine and glycerol as joint substrates was
highly effective and acted as a precursor for pyocyanin
production. This joint substrate medium is the Frank and
De Moss medium, recommended for the rapid diagnosis of
P. aeruginosa and demonstration of pyocyanin (Frank and
De Moss 1958; Wahba and Darrel 1965; Subramaniam and
Shriniwas 1985). A modified formulation of King’s Med-
ium for pyocyanin production was developed which
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contained glycerol. The addition of nalidixic acid to this
medium, with decreased concentration of cetrimide,
inhibited contaminating flora and thus allowed for better
recovery of P. aeruginosa with a concomitant increase in
pyocyanin production (Brown and Lowbury 1965; Lilly
and Lowbury 1972).
Recently there was a report on production of pyocyanin
from hundred strains of P. aeruginosa isolated from clin-
ical samples using various modified medium (Sheeba
2007). Seven liquid media [Frank’s medium (FM) ? 1 %
yeast extract, FM ? 0.5 % tryptone, FM ? 0.1 % KNO3,
FM ? 1 % bacto-peptone, FM ? 0.1 % glucose, peptone,
nutrient broth] and six solid media (Tryptone glucose yeast
agar, Seller’s differential medium, Nutrient agar, FNA
medium, Cetrimide agar and Pseudomonas agar) were used
for pigment production. Frank’s medium [FM] ? 1 %
yeast extract showed highest production of pyocyanin than
the other liquid media. Among the six solid medium used,
cetrimide agar showed maximal production of pyocyanin.
The production of bacterial pigment makes its identifica-
tion more rapid from clinical samples due to the antibiotic
activity of the pigment.
There are some factors that influence pigment produc-
tion when using various media. Production of pyocyanin
from Pseudomonas wild type and mutant strains was
investigated, and from the study it was concluded that
phosphate depletion in the culture medium enhances pig-
ment production (Ingledew and Campbell 1969; Byng et al.
1979; Porter 2009). In another report, there is evidence that
depletion of iron in the medium favours pyocyanin pro-
duction (Cox 1986). Depending on the cell density, the
organism produces pigment and when there is a depletion
of some nutrients, there is more production. It has been
reported that a satisfactory yield of pyocyanin depends
mainly on the composition of the media (Chang and
Blackwood 1969; Reyes et al. 1981).
Pigment extraction and purification
Formerly, liquid glycerol–peptone–phosphate medium was
used for enhancing pyocyanin production from P. aeru-
ginosa. The pyocyanin pigment which is diffusible into the
medium can be solvent-extracted by chloroform. The
extracts were purified using a column of aluminium oxide
with elution solvent mixture of chloroform-ethanol and
crystallised (El-Samerraie et al. 1997). Later in another
report, the pigment was isolated by various biochemical
profiling and the peptide was purified by ammonium sul-
phate precipitation, gel filtration and ion-exchange chro-
matography (Fontoura et al. 2009). O’Malley et al. (2003)
extracted pyocyanin using 10 mM hydrochloride followed
by neutral water instead of chloroform and this was
World J Microbiol Biotechnol (2014) 30:1159–1168
confirmed by high pressure liquid chromatography
(HPLC). There is a report on extraction of the pyocyanin
pigment using nine different solvents and among them,
chloroform showed the highest extractability of the pig-
ment. The purity of the extracted pigment was checked by
thin layer chromatography. In this study, pyocyanin was
extracted with 3 ml of chloroform and re-extracted with
1 ml of 0.2 N HCl (Hassanein et al. 2009; Raoof and Latif
2010).
Saosoong et al. (2009) reported pyocyanin production
from P. aeruginosa. The pigment was purified by passing
the supernatant in Amberlite XAD-4 resin column and
concentrating the pigment with a silica gel column. It was
re-chromatographed by HPLC and purity was checked by
both UV/visible and IR spectroscopy. Porter (2009) also
reported the production of pyocyanin from P. aeruginosa
and it was extracted using chloroform. The pigment was
found to be produced by the inner part of the cells which
was lysed by addition of chloroform. The purity was con-
firmed by passing the aqueous solution into a Sephadex
G-10 column.
Pyocyanin structure and involvement of genes
The pigment pyocyanin is composed of 2 subunits of N-
methyl-1-hydroxyphenazine. Pyocyanin has been crystal-
lized in pure form and classified as a phenazine-type of
molecule (Norman et al. 2004). The biosynthesis of
phenazine has been extensively studied in Pseudomonas
(Hollstein and Marshall 1972; Hollstein and McCamey
1973; Herbert et al. 1974, 1976). Phenazine is a hetero-
cyclic compound that is produced naturally as the deep red
5-methly-7-amino-1-carboxyphenazium betaine, which is
then converted to the lemon yellow coloured phenazine-1-
carboxylic acid (PCA), and finally to the bright blue
1-hydroxy-5-methyl phenazine (pyocyanin) (Gohain et al.
2006).
The shikimic acid pathway was found to be the primary
metabolic pathway which branched into phenazine bio-
synthesis. Shikimic acid pathway was the precursor for
phenazine which gives a branching point for chorismic acid
for the synthesis of phenazine (Hollstein and Marshall
1972; Herbert et al. 1976; Millican 1962). In the synthesis
of pyocyanin by P. aeruginosa seven genes have been
identified, namely phz C, D, E, F, G, M and S. The phen-
azine biosynthetic loci were found in all Pseudomonas
species (Mavrodi et al. 2001; Ahuja 2006; Gohain et al.
2006). Of these genes, phzM and phzS were the main genes
responsible for converting phenazine-1-carboxylic acid to
pyocyanin (Fig. 1). Two steps are suggested to be involved
in the synthesis of pyocyanin from PCA, which is the
common precursor for many different species-specific
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phenazines. In the first step, it is catalyzed by the enzyme
PhzM, an S-adenosylmethionine (SAM) dependent meth-
yltransferase, in which PCA is converted to 5-methylphe-
nazine-1-carboxylic acid betaine by transfer of a methyl
group to the nitrogen atom of phenazine-ring moiety. The
second step is catalyzed by the enzyme PhzS, a FAD-
dependent monooxygenase, and involves the hydroxylative
decarboxylation of 5-methylphenazine-1-carboxylic acid
betaine to pyocyanin (Parsons et al. 2007).
Anti-bacterial activity of pyocyanin
Pyocyanin is a good example of a secondary metabolite,
which has antibiotic activities and also able to co-ordinate
the response of microbial communities to changes in the
environment. Little attempts have been made to determine
the relation between pyocyanin and its inhibitory action.
Anti-bacterial activity of pyocyanin has been reported
since the year 1940 (Waksman and Woodruff 1940). The
pigment inhibited the growth of Escherichia coli and was
named as Colicin. The protein fraction was liberated upon
lysis of bacteria which exhibited the properties of pyocy-
anin (Young 1947). The purified form of pyocyanin
showed antibacterial activity and depending on the con-
centration of pyocyanin, the bactericidal effect varied
(Baron and Rowe 1981). The mechanism by which pyo-
cyanin inhibits bacterial growth was investigated and it was
concluded that, pyocyanin interacts with the cell membrane
respiratory chain resulting in the inability of the bacterial
cells to perform their active metabolic transport process
(Baron et al. 1989). Exposure of E. coli cultures to pyo-
cyanin causes depletion of oxygen supply to the cells,
produces H2O2 and also diverts the electron flow, causing
toxicity to E. coli cells (Hassan and Fridorich 1980). Py-
ocyanin negatively influences the active transport mecha-
nism of several organisms (Baron et al. 1989).
There are reports on anti-staphylococcal activity by P.
aeruginosa. P. aeruginosa present in the sputum of cystic
fibrosis (CF) patients inhibited the growth of Staphylo-
coccus aureus. The antagonistic effects were proved by
cross-streak test, well plate assay and growth of mixed
culture (Machan et al. 1990).
The antibiotic action of pyocyanin from P. aeruginosa
isolated from the marine environment was studied by
Angell et al. (2006). Marine P. aeruginosa were isolated
from ocean sediments collected in Hawaiian Islands and
screened for antimicrobial activity. A blue metabolite,
pyocyanin was identified in P. aeruginosa Pup14B culture.
Full characterisation of pyocyanin were done in the strain,
using X-ray analysis and 2D-NMR and corrections made in
1
H and 13C-NMR assignments of the molecule misreported
in the chemical literature and yeast transcriptome analysis.
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Fig. 1 Biosynthesis of pyocyanin from phenazine-1-carboxylic acid, a derivative of shikimic acid pathway
The transcriptional effect were consistent with the com-
pound purported role as an inducer of oxidative stress and
damage, and illustrates the overall potential of the method
to reveal the primary biological/cellular effects of a natural
product.
Nearly 90–95 % of antimicrobial inhibitions of P. aeru-
ginosa strains were due to production of the water soluble
secondary metabolite pyocyanin. It showed antagonistic
activity against pathogenic bacteria like Salmonella paraty-
phi, E. coli and Klebsiella pneumonia (Saha et al. 2008). Py-
ocyanin isolated from P. aeruginosa 4B strain showed
antibiotic activities against various pathogens and food
spoilage bacteria like Listeria monocytogens and Bacillus
cereus. The secondary metabolite along with various enzymes
like haemolysin and hydrolytic enzymes played a key role for
their antimicrobial activities (Fontoura et al. 2009).
The development of simple and low cost micro-bioreactor
with high bio-processing for the production of pyocyanin
from P. aeruginosa DSO-129 has been reported (Rahman
et al. 2009). The strain was inoculated in the bioreactor with a
modified nutrient broth. The organisms produced the sec-
ondary metabolite pyocyanin in the medium which was fil-
tered, and demonstrated for antimicrobial effect on
organisms like S. aureus, Staphylococcus epidermis, Bacil-
lus subtilis, Micrococcus luteus and Saccharomyces cerevi-
siae. There has been another report related to the antibiotic
activity of pyocyanin against different pathogens. The pig-
ment produced by the strain showed very effective activity
against organisms like E. coli, Acinetobacter, S. aureus and
Streptococcus pneumonia (Sweden 2010).
Anti-fungal activity of pyocyanin
Pyocyanin isolated from P. aeruginosa also inhibited the
growth of fungi like Aspergillus fumigatus and Candida
albicans isolated from the sputum of CF patients (Kerr
et al. 1999). There was a clinical evidence that pyocyanin
suppressed the growth of different species of C. albicans in
patients with lung infection. Reoccurring of C. albicans
was noticed after the suppression of pyocyanin (Kerr
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1994). Substances like pyocyanin, pyrrolnitrin and
pseudomonic acid produced by P. aeruginosa showed
antibiotic actions in vivo on Candida species grown on
Sabroud’s Dextrose Agar (Pal and Revathi 1998; Kaleli
et al. 2007). Signal mediated interactions between P.
aeruginosa and C. albicans in CF patients was found. The
presence of N-acyl homoserine lactones (HSLS) produced
by P. aeruginosa affected the morphology of C. albicans.
In the same way C. albicans inhibited the swarming
motility of P. aeruginosa. When P. aeruginosa was co-
cultured with C. albicans, the former synthesised large
amounts of pyocyanin and even the growth of C. albicans
was inhibited (Hogan and Kolter 2002; Mc Alester et al.
2007; Gibson et al. 2009; Hassanein et al. 2009).
Sudhakar et al. (2013) reported the production of pyo-
cyanin from P. aeruginosa WS1 and its antagonistic
activity against commonly encountered phytopathogens.
The pyocyanin compound was extracted and purified using
gas chromatography–mass spectrometer (GC–MS) and
found to have a molecular weight of 210.23 kDa with an Rf
value 11.94. The MIC of pyocyanin was further analysed
against phytopathogens and was found to be 64 lg/ml
against Aspergillus flavus and Aspergillus fumigates, and
128 lg/ml against Candida species.
Bio-control activity of pyocyanin
Biological control of plant diseases has been considered as
a valuable alternative method to manage various plant
diseases (Cook 1993). In plant pathology, the term bio-
control applies to the use of microbial antagonists to sup-
press plant diseases as well as the use of host specific
pathogens to control weed populations. The microbial
agent that suppresses the pathogen is referred to as the
biological control agent (BCA). In the hostile and nutrient-
limiting soil environment, plant roots are the places for
both beneficial and deleterious soil borne microbes. Ben-
eficial Pseudomonas species reach root surfaces chemo-
tactically by flagella motility and colonise them. It was
found that Pseudomonas also promotes the plant growth.
World J Microbiol Biotechnol (2014) 30:1159–1168
The root and rhizosphere offer an ecological niche (Bais
et al. 2006).
The interaction of P. aeruginosa with plants as a ben-
eficial association was found to be quite common. Recent
studies have provided a sight into this complex regulatory
network. P. aeruginosa, produces pyocyanin which is
present in the rhizosphere soil and other sources. In soil it
promotes direct plant growth and protects plants from the
phytopathogens (Cook 1988; Glick 1995; Bashan and
Holguin 1998). The interaction between plants and bene-
ficial Pseudomonas spp. exploiting bacterial traits for crop
protection has been well explained. The acquisition of
information concerning the biosynthesis of the phenazine
molecules has almost exclusively been done through pyo-
cyanin produced by P. aeruginosa (Mercado-Blanco and
Bakker 2007).
Ali Siddiqui et al. (2001) reported the use of rhizobac-
teria in the control of root rot and root knot disease com-
plex of mungbean. The organism isolated from the
rhizosphere was found to be P. aeruginosa which acted as a
bio-control agent in inhibiting the growth of Macropho-
mina phaseolina, Fusarium solani and Rhizoctonia solani.
Pseudomonas also showed nematicidal activity in killing
the second stage larvae of Meloidogyne javanica. The
isolates from the soil were designated as P. aeruginosa IE-
6 and P. aeruginosa Pa-7, -3, -4 and -33. Out of 33 isolates,
17 of them exhibited antagonism against M. phaseolina, 14
were effective against F. solani and 18 inhibited the radial
growth of R. solani. In greenhouse the effect of suppression
treatment of R. solani was observed, and the strain Pa-7
which was used in seed dressing inhibited R. solani to the
maximum level. P. aeruginosa IE-6 was the most effective
in reducing nematode penetration. P. aeruginosa Pa-7
resulted in maximum plant height as well. Thus, P. aeru-
ginosa not only acted as bio-control agents but also
increased plant growth rate.
The role of pyocyanin from P. aeruginosa 7NSK2
strain, in inducing resistance to Botrytis cinerea that causes
infection in tomato and grapevine was demonstrated by
Audenaert et al. (2002). In their work, pyocyanin negative
mutant strain and pyocyanin wild strain were used. In the
mutant strain PHZ l was coded by phzM gene. PHZ l was
unable to induce resistance and was co-inoculated with P.
aeruginosa 7NSK2 which restored resistance because of
pyocyanin production by the strain. Anjaiah et al. (2003)
studied the use of pyocyanin from Pseudomonas species
isolated from rhizosphere soil which controls Fusarium,
the causative agent of wilt of chick pea and Pythium
damping of bean.
Reports on production of PCA produced by particular
strains of P. aeruginosa strain PUPa3 showed bio-control
activity against a wide range of phytopathogenic fungi that
infect rice, groundnut, tobacco, chilli, mango, sugarcane,
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tea, cotton and banana crops (Sunish kumar et al. 2004).
The minimal inhibitory concentration of PCA was found to
be 29 lg/ml for Sclerotium rolfsii NCM1084, which also
inhibited the growth of phytopathogens such as Aspergillus
niger NCIM 1025, Fusarium oxysporum NCIM 1008, S.
rolfsii NCIM1084 and Colletotricum falcatum (Rane et al.
2007). Pseudomonas isolated from soyabean plant pro-
duced anti-fungal metabolites which inhibit the germina-
tion of mycelia of Sclerotina scletorum (Dilantha Fernando
et al. 2005).
De Vleesschauwer et al. (2006) reported the use P.
aeruginosa 7NSK2 strain as a bio-control agent against the
leaf blast (Magnaporthe grisea) and sheath blight (R. so-
lani) in the monocot model rice plant. P. aeruginosa
7NSK2 was treated in root which protected rice against leaf
blast leaving unprotected from sheath blight. The pyocya-
nin produced by 7NSK2 enhanced the production of H2O2
in roots and leaves, which degraded the toxic enzyme from
the plants. Onbasli and Aslim (2008) found that pyocyanin
from Pseudomonas inhibits the E. coli isolates from sugar
beet molasses.
There are few cases in which the relative importance of
antibiotic production by bio-control bacteria like P. aeru-
ginosa has been demonstrated, where one or more genes
responsible for biosynthesis of the antibiotics have been
manipulated. For example, mutant strains incapable of
producing phenazines (pyocyanin) or phloroglucinols have
been shown to be equally capable of colonizing the rhi-
zosphere but much less capable of suppressing soil borne
root diseases than the corresponding wild-type and com-
plemented mutant strains. Bio-control strains are known to
produce multiple antibiotics which can suppress one or
more pathogens (Pal and Gardner 2006).
Phenazines have been shown to influence the growth
and to elicit induced systemic resistance (ISR) in plants
(Pierson and Pierson 2010). Pseudomonas strains isolated
from the rhizosphere plant were used to treat against var-
ious species of Fusarium, Ralstonia and Meloidogyne
which cause wilting disease in coleus and ashwagandha
species (Mallesh 2008). The mechanism involved was
studied and it was suggested that siderophore, HCN, indole
acetic acid, pyocyanin and other volatile metabolites syn-
thesised by Pseudomonas strain exhibits the bio-control
effect and they can also act as plant growth enhancers. Al
Hinai et al. (2010) isolated umpteen number of P. aeru-
ginosa from greenhouse soils and all exhibited antagonistic
effects against Pythium aphanidermatum which causes
damping off of cucumber.
There have been studies on dual activity of pyocyanin
from P. aeruginosa TO3 as antibiotic against phytopatho-
gen M. phaseolina and as a signalling molecule for
development of biofilm by rhizobial strain Ca2. The
antagonistic activity of purified pyocyanin was determined
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by dry mass method which showed inhibition of M.
phaseolina and also biofilm formation by strain Ca2 was
performed by crystal violet assay. There was an increase in
biofilm development by Ca2 with an increase in pyocyanin
concentration up to 0.12 nmol/l. Using well-diffusion
method, the effect of pyocyanin on disease suppression and
biofilm formation by strain Ca2 on radicles of groundnut
(Arachis hypogaea L.) was studied. A field study in soil
infested with M. phaseolina showed that a co-inoculant of
P. aeruginosa TO3 and rhizobial strain Ca2 enhanced
nodule mass and nitrogenase activity over that of the
control (Khare and Arora 2011).
Despite their importance in biological control, little is
known about the genes involved in bio-control activity.
There has been a study in identifying the location of the
antagonistic genes in P. aeruginosa FP6 towards phyto-
pathogens like R. solani and Colletotrichum gloeosporio-
ides using PCR-based approach. A new bacterial strain,
designated as FP6, was isolated from rhizospheric soil and
identified as a member of P. aeruginosa based on 16S
rRNA analysis. The secondary metabolites produced by
this strain have shown broad-spectrum bio-control activity
against R. solani and C. gloeosporioides. The anti-metab-
olites of a non-enzymatic nature were found to be
responsible for the antagonism. A chromosomal gene was
found to be involved in the production of antagonistic
compound which was demonstrated by the gel elution
technique using antibiotic gene-specific primers. The study
speculates that anti-metabolites play an important role in
the control of phytopathogens (Bakthavatchalu et al. 2013).
It was found that P. aeruginosa act as very good bio-
pesticides against soyabean stunt virus (SSV). P. aeru-
ginosa has been isolated from rhizospheres of soyabean
and formulated in various forms such as liquid formulation,
together with polyacrylamide hydrogel. The formulations
were effective in plant growth and increased resistance
against SSV also increases the yield, chlorophyll content
and peroxidase activity. The study concluded that the
application of P. aeruginosa formulation was effective
against SSV (Khamdan and Suprapta 2011).
Challenges and new insights into pyocyanin
Pseudomonas aeruginosa is an opportunistic, Gram-nega-
tive bacterium that causes infections in immune-compro-
mised hosts, burn victims, individuals in intensive care and
patients with CF. The lungs of nearly all CF patients are
chronically colonised by P. aeruginosa, which significantly
reduces life expectancy and it is the leading cause of
morbidity and mortality for CF patients. The effectiveness
of P. aeruginosa as a pathogen can be attributed to its
arsenal of virulence mechanisms and its large metabolic
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capacity, including the ability to intrinsically resist antibi-
otics owing to its impermeable outer membrane, efflux
capabilities, tendency to colonise surfaces in a biofilm form
and ability to acquire and maintain antibiotic plasmids
(Driscoll et al. 2007). Novel approaches for the treatment
of P. aeruginosa infection are urgently needed.
The ability of P. aeruginosa to cause disease is depen-
dent upon the production of agents termed ‘virulence fac-
tors’, such as toxins and adhesion molecules, that actively
cause damage to host tissues. The concept of targeting
virulence using anti-infective or anti-virulence drugs that
can ‘disarm’ the pathogenic bacteria rather than kill them
has garnered increasing attention in recent years. It is
believed that such an approach places less selective pres-
sure on bacteria to evolve new strategies for survival,
thereby being more specific to pathogenic bacteria and
subject to less selection for drug resistance (Clatworthy
et al. 2007). In an analysis, it has been identified that
pathogen-associated proteins have homologues only with
pathogenic bacteria and not with non-pathogens (Ho Sui
et al. 2009). Such proteins are more likely to have viru-
lence-related functions. The list of pathogen-associated
proteins identified included components of the phenazine
biosynthesis pathway. Strains of P. aeruginosa produce
and secrete a variety of redox-active phenazine com-
pounds, the most well studied being pyocyanin.
Pyocyanin is considered both a virulence factor and a
quorum sensing (QS) signalling molecule for P. aerugin-
osa (Lau et al. 2004; Karatuna and Yagci 2010). QS-reg-
ulated virulence factors, and pyocyanin in particular, are
active in lung infections associated with CF and produce
numerous effects that are relevant to CF. Pyocyanin
modulates redox cycling and generates reactive oxygen
species capable of causing significant oxidative stress,
which in turn affects calcium homeostasis. It inhibits cel-
lular respiration, depletes intracellular cAMP and ATP
levels, and thus affects chloride ion channels that are
controlled by ATP-driven conformational changes (Win-
stanley and Fothergill 2008). Pyocyanin inhibits prostacy-
clin release and can inactivate human V-ATPases
(involved in receptor-mediated endocytosis), a1-protease
inhibitor (which modulates serine protease activity,
including neutrophil elastase) and nitric oxide (which
influences blood flow, blood pressure and immune func-
tions). Pyocyanin also alters the host immune response in
several ways to aid evasion of the immune system and
establish chronic infection. High concentrations of pyocy-
anin in the sputum of CF patients suggest that this com-
pound plays a key role in pulmonary tissue damage
observed with chronic lung infections, and early (in the
growth curve) overexpression of QS regulated virulence
factors such as LasA, elastase and pyocyanin. These were
common among populations of a highly successful CF
World J Microbiol Biotechnol (2014) 30:1159–1168
epidemic strain (Fothergill et al. 2007; Rada and Leto
2013; Rada et al. 2013). Additional evidence demonstrates
that pyocyanin significantly contributes to lung destruction
during chronic P. aeruginosa infection of bronchiectasis
airways in a mouse model and supports the hypothesis that
pyocyanin contributes to the accelerated decline in lung
function of CF and other bronchiectasis patients once they
are infected with P. aeruginosa (Caldwell et al. 2009).
Pyocyanin biosynthesis is therefore an attractive target for
anti-infective drug intervention.
Previously there has been investigative study on the
regulation of the phzA1B1C1D1E1F1G1 operon (phzA1).
In an study conducted by Liang et al. (2011) screening of
nearly 5,000 transposon mutants which revealed 14 inter-
rupted genes with altered phzA1 expression, including
PA2593 (QteE), which has been identified as a novel reg-
ulator of the QS system. Overexpression of qteE in P.
aeruginosa significantly reduced the accumulation of
homoserine lactone signals and affected the QS-controlled
phenotypes such as the production of pyocyanin, rhamn-
olipids, LasA protease and swarming motility. Indeed,
overexpression of qteE in P. aeruginosa attenuated its
pathogenicity in the potato and fruit fly infection models.
These findings suggest that qteE plays an important role in
P. aeruginosa pathogenicity and is part of the regulatory
networks controlling phenazine production.
Hassani et al. (2012) reported nearly eighty-two mutants
isolated from wild type strain of P. aeruginosa PHA-1
using various concentrations of cefotaxime. Wild type
PHA-1 and mutants were examined for production of py-
ocyanin. Remarkably, the mutant strain named S300-8 was
distinguished in productivity in comparison with wild type
strain PHA-1. In addition, pyocyanin produced by mutant
strain S300-8 revealed a potent efficacy against growth of
cancer cell line RD; the low concentration of this pigment
caused 65 % of dead cells after 72 h of incubation whereas
the cytotoxicity was improved by increasing the concen-
tration of pigment with period of exposure time.
Pseudomonas aeruginosa regulates pyocyanin produc-
tion using an intercellular communication mechanism
mediated by small signalling molecules termed as auto-
inducers. One native auto-inducer is N-(3-oxododecanoyl)-
L-homoserine lactone (OdDHL). There are reports about
the synthesis of collection of abiotic OdDHL-mimics. A
number of novel compounds capable of competing with the
endogenous OdDHL and consequently, inhibiting the pro-
duction of pyocyanin in cultures of wild type P. aeruginosa
were identified. The evidence suggest that the compounds
of this general structural type act as direct antagonists of
QS in P. aeruginosa and as such may find value as
molecular tools for the study and manipulation of this
signalling pathway. Direct quantitative comparison of the
pyocyanin suppressive activities of the most active
1165
OdDHL-mimics with some previously-reported inhibitors
(based around different general structural frameworks) of
QS from the literature, were also studied (Morkunas et al.
2012).
There has been growing interest in disrupting bacterial
virulence mechanisms as a form of infectious disease
control through the use of anti-infective drugs. There is a
report on analysis of P. aeruginosa PAO1 which deduced
proteome to identify pathogen-associated proteins. A
computational screening approach was used to discover
drug repurposing opportunities, i.e. identifying approved
drugs that bind and potentially disrupt the pathogen-asso-
ciated protein targets. The selective oestrogen receptor
modulator raloxifene, a drug currently used in the pre-
vention of osteoporosis and/or invasive breast cancer in
post-menopausal women, was predicted from this screen to
bind P. aeruginosa PhzB2. PhzB2 is involved in produc-
tion of the pyocyanin. Raloxifene was found to strongly
attenuate P. aeruginosa virulence in a Caenorhabditis
elegans model of infection. Treatment of P. aeruginosa
wild-type strains PAO1 and PA14 with raloxifene resulted
in a dose-dependent reduction in pyocyanin production
in vitro; pyocyanin production and virulence were also
reduced for a phzB2 insertion mutant. The results suggest
that raloxifene may be suitable anti-infective or anti-viru-
lence agents for further development as a therapeutic for P.
aeruginosa infection (Ho Sui et al. 2012).
Bacterial adhesion and biofilm formation are both
dependent on the production of extracellular polymeric
substances (EPS) mainly composed of polysaccharides,
proteins, lipids, and extracellular DNA (eDNA). eDNA
promotes biofilm establishment in a wide range of bacterial
species. In P. aeruginosa, eDNA is major component of
biofilms and is essential for the formation and stability. Das
and Manefield (2012) in their study reported that produc-
tion of pyocyanin in P. aeruginosa PAO1 and PA14 batch
cultures is responsible for promotion of eDNA release. A
phzSH mutant of P. aeruginosa PAO1 that overproduces
pyocyanin displayed enhanced hydrogen peroxide (H2O2)
generation, cell lysis, and eDNA release in comparison to
its wildtype strain. A DphzA-G mutant of P. aeruginosa
PA14 deficient in pyocyanin production generated negli-
gible amounts of H2O2 and released less eDNA in com-
parison to its wildtype counterpart. Pyocyanin intercalation
with eDNA promotes cell-to-cell interactions in P. aeru-
ginosa cells by influencing their cell surface properties and
physico-chemical interactions (Das et al. 2013).
Using the combination of microarray and metabolite
analyses, Lundgren et al. (2013) demonstrated about the
assimilation of glycine as a carbon source and the bio-
synthesis of pyocyanin in P. aeruginosa PAO1; both are
dependent on the PA2449 gene. The inactivation of the
PA2449 gene was found to influence the set of core genes
123
1166
of transcription-encoding glycine cleavage system, serine
hydroxymethyltransferase, and serine dehydratase. PA2449
also affected the transcription of several genes that are
integral in cell signaling and pyocyanin biosynthesis in P.
aeruginosa PAO1.
Natural products have recently become a promising
source for deriving molecules that can potentially inhibit
QS related anti-virulent activities. Reports about screening
of the QS inhibitory properties of 16 high quality ayurvedic
medicinal plants derived from Indian sub-continent,
understand their mechanism of action and investigate their
effect on the expression of QS regulated virulence factors
in P. aeruginosa PAO1. QS Inhibition of sub-lethal con-
centrations (SLC) of plant extracts were measured in vio-
lacein producing Chromobacterium violaceum bioassay
model. Effect of these extracts on PAO1 virulent factors—
pyocyanin, elastase and total protease—were quantified by
standard protocols. Results indicated that the extracts
reduced violacein auto-inducers, pyocyanin synthesis, and
inhibited the activity of elastase and other proteolytic
enzymes significantly (Bryant et al. 2008).
There has been demonstrated work on clove extract
(Syzygium aromaticum) inhibiting the QS-regulated phe-
notypes in P. aeruginosa PA01, including expression of
lecA::lux (by hexane extract), swarming (maximum inhi-
bition by methanol extract), pyocyanin (maximum inhibi-
tion by hexane extract). The study proved that the presence
of natural compounds in the clove extracts that exhibit anti-
QS activity may be useful as the lead of anti-infective
drugs (Krishnan et al. 2012).
Conclusions
Pseudomonas aeruginosa has been found in all sources of
environment and recognised by its production of the pig-
ment pyocyanin. The pigment has antibiotic property
controlling other microbes and can be used as a bio-control
agent. Pyocyanin can be processed technically by using
various parameters such as an appropriate media for pig-
ment production from various environmental sources. The
primary screening of this metabolite is done by chloroform
extracts from the media and it is then purified by combined
techniques like HPLC, TLC and column chromatography.
The metabolite structural profiling can be done by NMR
and mass spectroscopy. Furthermore, genes responsible for
pigment production can be induced or inserted using
molecular techniques to enhance the production of the
metabolite. Several techniques have been used for pyocy-
anin production. Pyocyanin is a natural product which has
the ability to act as a bio-control agent, thus helping to
create an eco-friendly solution for the replacement of
123
World J Microbiol Biotechnol (2014) 30:1159–1168
chemical pesticides. Further new insights about pyocyanin
are building up in the field for new research opportunities.
Acknowledgments The authors are thankful to all the researchers
whose papers have been used for this review. Greatest gratitude goes
to Luigi Lucini, Ph.D., Università Cattolica del Sacro Cuore, Institute
of Environmental and Agricultural Chemistry.
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