ABSTRACT
Isolation and identification of Pseudomonas aeruginosa from Hospital environment for pyocyanin production was done. The isolates from Hospital bed and wound swab were identified as Pseudomonas aeruginosa. The virulence factor, antibiotic pigment, pyocyanin which is one of the characteristic feature of P. aeruginosa and which also confers on it growth advantage over competing bacteria occupying the same niche was also isolated and characterised. Quantitative assay of pyocyanin production was conducted which is based on the absorbance of pyocyanin at 520 nm in acidic solution. The concentration of pyocyanin was determined by multiplying the optical density at 520 nm (OD520) by 17.072. The result showed that the isolates produced pyocyanin with the total concentration of 13μg/ml, while the isolates from wound swab produced no pyocyanin but pyorubin. Pyocyanin produced was characterized by biochemical tests, its dissolution in chloroform and Hydrochloric acid (HCl) to give a blue and pink to red colour respectively confirms the presence of pyocyanin. The purification of pyocyanin was done by liquid-liquid extraction method using Na-borate buffer at the pH of 10 to neutralize the HCl and re extracted into chloroform and acid before storage. Since pyocyanin is labile to many conditions, the purified pyocyanin was stored in acid at freezing temperature in dark container to prevent decomposition. Since pyocyanin can be isolated successfully from P. aeruginosa, it can be used in vitro to carry out its antimicrobial activity on the growth of other microorganisms; also study on the mechanism of its virulence can be done using the isolated pyocyanin.
TABLE OF CONTENTS
TITLE………………………………………………………………………………………….i
CERTIFICATION…………………………………………………………………….………ii
DEDICATION………………………………………………………………...……..……….iii
ACKNOWLEGEMENTS………………………………………………………………..……..iv
TABLE OF
CONTENTS………………………………………………………………….....v-vi
ABSTRACT……………………………………………………………………...….…..……vii
CHAPTER
1
1.0 Introduction
………………………………………………………………………….....1-2 1.1
Objectives…..….…………………………………………………………………………….2 1.2 Literature
review…………………………………………………………………….……3-9
CHAPTER
2
2.0 Materials and
method……….....…………………………..………………………………10 2.1 Sampling.………………………………………………………………………….……….10 2.2 Isolation of the test
organism……………………………………………………………..10 2.3
Identification of test organism..…………………………………………………………..10 2.4 Media
preparation…………….……………………………………………………………10 2.5
Gram staining method……….…………………………………………………………….11 2.6 Biochemical
tests……….…..…………………………………………………………..11-12 2.7
Preparation of chemicals used in quantitative assay………………………………………12 2.8
Quantitative assay…..…….………………………………………………………….12-13 2.8.1 Pyocyanin production……………………………………………………………………..12
2.8.2 PB medium preparation……..…………………………………………………………12-13 2.8.3 Extraction of
pyocyanin…………………………………………………………………..13 2.8.4
Concentration of pyocyanin determination………………………………………………..14 2.9 Purification of pyocyanin………………………………………………………………14-15
CHAPTER
3
3.0
Results….………………………………………………………………………………16-21
CHAPTER
4
4.0
Discussion………………………………………………………………………………22-24 4.1
Conclusion….………………………………………………………………………………24 References
CHAPTER
1
1.0 INTRODUCTION
Pseudomonas
aeruginosa,
a common bacterium is widespread in the
environment (Nester et al., 2001) and present in soil, water, skin flora and in
most of the man made environments such as Hospital, Industry, Effluent
treatment plant. P. aeruginosa one of the most important
opportunistic pathogen which causes nosocomial infections to the patient in
Intensive care unit has become a major threat in the medical care. It also
exhibits multidrug resistance which has drawn the attention of the microbiologist.
The characteristic feature of Pseudomonas
aeruginosa is the production of soluble green pigment pyocyanin, or an
antibiotic substance which act as a bio-indicator for the identification of a
contaminant in hospital environment (Zourob et al., 2008;
Kurachi, 1958a). Strains of P. aeruginosa
produce variety of redox-active phenazine compounds, including pyocyanin,
phenazine-1-carboxamide (Budzikiewicz, 1993). Pyocyanin is the main phenazine
pigment associated with this particular organism. About 90% to 95% of P.
aeruginosa strains produce
pyocyanin (Smirnov and Kiprianova, 1990). Defined conditions for the reliable
production of phenazines have greatly aided by biosynthetic studies (MacDonald,
1967). The simultaneous accumulation of more than one phenazine by P.
aeruginosa had been reported (Korth et al., 1978) and culture
conditions which differentially affect the fractional composition of phenazine
pigments had also been described (Kanner et al., 1978).
Pyocyanin a biologically and
chemically active blue-pigmented phenazine derivative, an important secondary
metabolite (Idiolyte) produced by the human pathogen Pseudomonas aeruginosa
(Xu et al., 2005), endows P.
aeruginosa with a competitive growth advantage in colonized tissue and in
the environment. It is also known to be a
virulence factor in diseases such as cystic fibrosis and AIDS where patients
are commonly infected by pathogenic Pseudomonads due to their immunocompromised
state. Pyocyanin contributes to the unusual persistence of P. aeruginosa infections (Lau et
al., 2004). Pyocyanin is also a
chemically interesting compound and toxic largely due to its unusual
oxidation-reduction activity that deplete cells of NADH, glutathioxide and other antioxidants
(Reszka et al., 2006). It has
antibiotic activity against bacteria (Caltrider, 1967), fungi (Costa and
Cusmano, 1975), and protozoa (Dive, 1973), but is of little therapeutic value
because it is quite toxic to eucaryotic cells (Stewart-Tull and Armstrong,
1972). Also Stephen and Hawkey, (2006) indicated that pyocyanin has antibiotic
activity against other bacteria and fungi, it is bactericidal for many species
including Escherichia coli, Staphylococcus aureus and Candida
albicans. This
killing is observed on agar plates as clear zones on lawns of sensitive
bacteria. This antibiotic activity of pyocyanin may allow P. aeruginosa an
advantage over competing bacteria occupying the same niche. Furthermore,
pyocyanin has a variety of pharmacological effects on eukaryotic and
prokaryotic cells (Saha et al., 2008).
1.1 OBJECTIVES
Because
pyocyanin endows Pseudomonas aeruginosa with
a competitive growth advantage in the environment and colonised tissues, this
study was conducted with the following objectives;
·
Isolation and identification of Pseudomonas aeruginosa from any source.
·
Detection of pigment (pyocyanin) production.
·
Isolation (Extraction), confirmation and purification of
pyocyanin.
1.2 LITERATURE REVIEW
1.2.1 Characteristics
The
antibiotic, Pyocyanin (PCN-) is one of the many toxins (extracellular
pigment) produced and secreted by certain strains of the Gram negative bacterium Pseudomonas aeruginosa
when grown on certain media. Pyocyanin is a blue, secondary metabolite with the ability
to oxidise and reduce other molecules (Hassan and Fridovich, 1980) and
therefore can kill microbes competing against P. aeruginosa as well as
mammalian cells of the lungs which P. aeruginosa has infected during cystic
fibrosis. Since pyocyanin is a zwitterion
at blood pH, it is
easily able to cross the cell membrane. There are three different states in
which pyocyanin can exist; oxidised, monovalently reduced or divalently
reduced. Mitochondria play a huge role in the cycling of pyocyanin between its
redox states. Due to its redox-active properties, pyocyanin generates reactive oxygen species. It was first
synthesized chemically by Wrede and Strack (1929) who proposed a bimolecular
structure for it. Further evidence, summarized by Hillemann (1938), indicated
that the correct structure is as is shown in the figure 1 below. Pyocyanin
dissolves in water at neutral or basic pH to give a blue solution, and it is
extracted from such solutions with chloroform to give a blue solution.
Pyocyanin dissolves in aqueous acid to give a red solution, and it is not
extracted from such solutions by chloroform. These properties are commonly used
to determine if pyocyanin is present in cultures and are used in purification
of the compound. Kurachi (1958b) has described, in detail, procedures that can
be used for the isolation and purification, and for its spectrophotometric
analysis in aqueous solutions. The value E11%
cm = 205 at 690mμ can be used for the analysis of pyocyanin in
neutral or alkaline aqueous solution, and the value E11
% cm = 117 at 520mμ for its
analysis in aqueous solution (Kurachi, 1958b).
Figure 1.Structure of pyocyanin
1.2.2 Production of Pyocyanin by cells grown in
culture media
Pyocyanin is produced by the organism Pseudomonas aeruginosa also called Pseudomonas pyocyanea and Bacillus pyocyaneus in the literature
(Mac Donald, 1967). In
order to decide a suitable cultural condition for pyocyanin formation and to
find out a clue for the
mechanism of biosynthesis of pyocyanin, various experiments were carried out.
It has been observed that pyocyanin formation was easily affected by various
cultural conditions such as the
composition of culture medium, especially the kind or the concentration of
carbon source, the pH of the medium, the incubation temperature, the aeration
and frequency of shakes (Kurachi, 1958a). It has been noted that in the organic
medium prepared from natural materials such as bouillon, yeast or malt extract
and a certain commercial peptone, pyocyanin formation was hardly revealed owing
to the existence of the substance considered to inhibit the enzyme action in
pyocyanin synthesis system (Kurachi, 1958a). In the cultured solution of the
bacteria, besides pyocyanin, some amounts of yellow pigment, α-hydroxyphenazine
and other green fluorescent pigment which is insoluble in any organic solvents
are usually detected. α-Hydroxyphenazine is derived from pyocyanin by its
destruction, and this substance somewhat resembles pyocyanin in its behavior
toward organic solvents, so that on the extraction of cultured solution, this
may ordinarily be accompanied with pyocyanin fraction. However their
distribution coefficients between solvent and water are different from each
other according to the pH of their solution. α-hydroxyphenazine is recognized
in the aged cultured solution and is obtained by heating pyocyanin in alkaline
solution (Kurachi, 1958b). Some strains of P.
aeruginosa produce pyocyanin, even on rather complex media. Other strains
produce it on complex media only, and still other strains will produce it on
simple synthetic media. Some strains of P.
aeruginosa produce pyocyanin when first isolated but lose this ability when
subcultured in the laboratory (Mac Donald, 1967). With any strain that will
produce pyocyanin, media generally have been found on which the organism will
grow but not produce pyocyanin. Pyocyanin production, therefore does not
necessarily accompany growth, and some evidence shows that pyocyanin occurs
after the logarithmic phase of growth of the organism (Mac Donald, 1967).
Pyocyanin is produced in aerobic cultures, and the amount synthesized is small
compared with the amount of the organic substrates added to the medium (Mac
Donald, 1967). Investigations reviewed below illustrates these generations.
Jordan (1899) tested seven cultures of Bacillus
pyocyaneus qualitatively for their ability to produce pyocyanin. Six of
these produced pyocyanin on nutrient gelatin. Three of six produced pyocyanin
on media containing salts and a single organic compound (asparagines, ammonium
succinate, ammonium lactate, ammonium acetate or ammonium citrate) but did not
produce pyocyanin on an ammonium tartrate salt medium, although they grew well
on this medium. Jordan (1899) cited examples to show that the pyocyanogenic
powers of some cultures can decrease on cultivation in the laboratory. His
isolation of a non-pyocyanogenic mutant of one strain suggests that this may be
caused by mutation. More recently, Valette et
al. (1964) grew P. aeruginosa strain
A237, on media containing salts, NH4cl as the nitrogen source, and
single organic acids as the sole source of carbon. They found that the acids
from which pyocyanin could be synthesized were more or less related to acids of
the tricarboxylic acid cycle or krebs. Jordan (1899) and Valette et al., 1964 showed that certain organic
compounds could support growth and pyocyanin production. Such information gives
clues as to the immediate precursors of pyocyanin. It is evident that when a
culture is growing on a single carbon source, the cells are synthesizing a
great number of organic compounds, some of which may be much closer precursors
of pyocyanin than the compound added to the medium. In addition, it is also
possible that a compound which is an immediate precursor of pyocyanin might not
be a good substrate for growth of the organism. Azuma and Witter (1964) were
able, by a rather complex procedure, to obtain pyocyanin production in some
normally apyocyanogenic strains of P.
aeruginosa. The strains were first grown on a medium containing salts,
glucose, DL-alanine and carbobenzoxy-DL-alanine. The presence of the
carbobenzoxy-amino acid in this medium caused a modification of the cells so
that they subsequently produced pyocyanin for three transfers on Klinge’s agar
medium (Klinge, 1959). Strains affected in this way were a psychrophilic mutant
isolated by the authors, and strains NRRL-B 241 and B275. The authors suggested
that further study of this effect of carbobenzoxy amino acids might be of value
in defining the mechanism of pyocyanin formation. Certainly, an explanation of
this effect would be of interest. Harris (1950), found that in a medium
containing nutrient broth and glycerol, the production of pyocyanin by P. aeruginosa did not parallel growth,
but started after the logarithmic phase of growth and was still increasing
between 60 and 120 hours of growth,
during which period the turbidity of the culture was decreasing. Harris
therefore classified pyocyanin as a waste product of metabolism. His finding
that pyocyanin is produced after the logarithmic phase of growth has been
confirmed by other workers ( Kurachi, 1958a; Frank and Demoss, 1959). Ra’oof
and Latif, (2010) grew different strains of P.
aeruginosa which produced pyocyanin on Pseudomonas
Broth (PB) medium which they prepared in the laboratory. Thus, the
production of pyocyanin appears to confer a growth and/or survival advantage in
mixed culture settings.
1.2.3 Targets
Pyocyanin is able to target a wide
range of cellular components and pathways. Pathways which are affected by
pyocyanin include the electron transport chain, vesicular
transport, caspase 3-like proteases which can then go
on to initiate apoptosis and necrosis
and cell growth. An
enhanced susceptibility to pyocyanin is seen in cells which have mutation in
certain proteins or complexes (Ho et al.,
1993).
1.2.4 Importance Of Pyocyanin In Pseudomonas aeruginosa
Infected Airways
In
P. aeruginosa infected Cystic Fibrosis airways, there is an abundant
neutrophilic inflammatory response stimulated by both host and bacterial factors.
Dysregulated inflammatory responses lead to high levels of cytotoxic
phagocyte-derived reactive oxygen species (ROS). ROS are also produced by the redox-cycling
activity of pyocyanin, a blue-colored tricyclic phenazine, that is produced in
concentrations up to 100 μmol/L by P. aeruginosa in CF airways (Wilson et al.,1988). Notably, pyocyanin-mediated
ROS inhibit catalase activity, deplete cellular antioxidant reduced
glutathione, and increase the oxidized reduced glutathione in the bronchiolar
epithelial cells (Lau et al., 2004;
Lau et al., 2005). Excessive and
continuous production of ROS and inhibition of antioxidant mechanisms overwhelm
the antioxidant capacity, leading to tissue damage. As an immunomodulator,
pyocyanin inhibits ciliary beating of airway epithelial cells,(Wilson et al., 1988) nitric oxide production by
macrophages macrophages and endothelial cells, (Shellito et al., 1992) prostacyclin production by endothelial cells, (Kamath
et al., 1995) oxidation of
leukotriene B4 by neutrophils,(Muller and Sorrell, 1992) and eicosanoid
metabolism by platelets (Muller et al.,
1994). Pyocyanin also enhances superoxide production, (Muller and Sorrell,
1997) increases apoptosis in neutrophils, (Allen et al., 2005) and inactivates α1-protease Inhibitor (Britigan et al., 1999). In addition, pyocyanin
increases calcium signaling in human airway epithelial cells, stimulates
interleukin (IL)-8 release, and inhibits regulated on activation normal T cell
expressed and secreted and monocyte chemoattractant protein-1 release in human
epithelial cells (Look et al., 2005).
Pyocyanin
furthermore inhibits the expression of IL-2 and its receptor (Nutman et al., 1987). In animal models,
pyocyanin stimulates IL-8 release, neutrophil influx, and bronchoconstriction
in sheep and decreases tracheal mucus velocity in sheep, guinea pigs, and
baboons (Lauredo et al., 1998). Recently,
we provided direct evidence that pyocyanin participates in P. aeruginosa virulence
using pyocyanin-deficient mutants that were found to be attenuated in their
ability to infect mouse lungs in an acute pneumonia model of infection when
compared with isogenic wild-type bacteria (Lau et al., 2004). These mutants also were less competitive than isogenic
parental wild-type bacteria during competitive mixed infection using the agar
bead model of chronic lung infections (Lau et
al., 2004).
1.2.5 Importance Of
Pyocyanin In Burns Wound
The
dressings of burns infected with Pseudomonas
pyocyanea often have a bluegreen colour, which is due to the presence of
pyocyanin and other pigments in the exudate. There is evidence that P. pyocyanea may cause the failure of
skin grafts on burns and delay healing (Jackson et al.,1951). To find whether pyocyanin may play a part in causing
these adverse results, Cruickshank and Lowbury (1953), studied the effect of
different concentrations of the compound on human epithelial cells and
leucocytes, and have attempted to assess the concentration of pyocyanin in
green exudate from burns. They showed that pyocyanin is toxic to human
epidermis cultivated in vitro, at concentrations above 0-0024 mg./ml. Schoental
(1941) demonstrated that pyocyanin inhibited the growth of chick embryo
fibroblasts at concentrations above
0-025 mg./ml.
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