EXTRACTION AND PURIFICATION OF PYOCYANIN FROM PSEUDOMONAS AERUGINOSA ISOLATED FROM HOSPITAL ENVIRONMENT

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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