1. History and Chronology of Studies with Pyocyanin

The pigment was first described in 1860 by Fordos, when observing a bluish purulent sample, from an infection caused by P. aeruginosa. The name of the pigment was proposed by the combination of Greek words, used to designate pus and the colour blue ^[1][14]. Dr. Fordos also described different properties of the pigment, such as its solubility and the colours it exhibited at different pH levels. He proposed that the bacterium exhibits four different types of colours. This was later known as the chameleon effect ^[2][15].

The first isolation of the pigment from the “pyocyanic bacillus” occurred in 1882 when Gessard attempted to verify the parasitic origin of the phenomenon that gave the colour blue to pus and tissues close to an infection ^[3][16]. In addition, Jordan in 1899 identified pyocyanin spread throughout a common laboratory culture medium ^[4][17]. The pure compound was isolated only in 1924, becoming the first natural phenazine obtained and purified in a laboratory ^[5][18]. In 1929, Wrede and Straek proposed the chemical structure of pyocyanin, later corrected by Hillemann, in 1938 ^[6][19].

Waksman, in his last published manuscript, presented a retrospective on antibiotic therapy and reminded the reader that between late 1941 and early 1942, he coined the word “antibiotic”, in response to a request to create a word to designate compounds and preparations with a defined chemical structure, which produced a therapeutic effect against infectious diseases ^[7][20]. In this work, he cited pyocyanase as one of those compounds. Pyocyanase was the first formulation to use the potential of pyocyanin in therapy ^[8][21].

The term antibiosis was coined by Vuillemin in 1889 to designate the natural selection of one organism over another. Ten years later, Ward extended the term to define microbial antagonism. At the same time, it was understood that not only the presence of certain microbes prevented the growth of others, but that the phenomenon also occurred due to the action of substances produced by these organisms ^[9][22].

In the same year, Emmerich and Löw isolated pyocyanase from a macerate of P. aeruginosa cultures. The lysate was not initially identified as a molecule, being erroneously described as an enzyme mixture, reflected in the nomenclature used to designate the compound. Scientists also observed that the pyocyanase produced by the “pyocyanic bacillus” could be used to treat diphtheria and against meningococci. In addition, it served as a mouthwash and years later it was shown to be effective against anthrax. Pyocyanase in the form of eye drops, sprays and mouthwashes were the most common presentations because the systemic use proved to be very toxic. Thus, pyocyanase was probably the first antibiotic produced industrially and applied therapeutically in humans, decades before the discovery of penicillin and the era of antimicrobial chemotherapy ^[10][11][23,24].

In the early years of interest in this pigment, encouraged by the attraction for the colour, the most investigated function was its antimicrobial activity. In the early 20th century, pseudomonads were the largest group of non-differentiating microbes used to produce antibiotics. Three compounds reached the stage of clinical application: pyocyanin acids, pyrrolnitrin and pyocyanase ^[12][25]. With the advancement of research, pyocyanase demonstrated protection of experimental animals against rabies and the vaccinia viruses. From the beginning, however, the compound was identified as more effective against Gram-positive bacteria than Gram-negative bacteria and other organisms ^[13][26].

A hydroalcoholic solution containing 10,000 to 20,000 units/mg of pyocyanase was seen to provide antimicrobial activity when diluted 1:10 and 1:20. Antimicrobial activity was reported against staphylococci, streptococci, pneumococci, gonococci and V. cholerae, however, there was no consensus on the active principle of the lysate, given the fact that pyocyanase was not an enzyme. Between 1909 and 1928, much controversy was raised regarding the use of pyocyanase. The aqueous solution was unstable and lasted only a week [22]. On the other hand, the cell suspension or the lipid extract obtained from the cultures, containing a suspension of a crystalline material, appeared to be more effective, leading scientists to deduce that the active substance of pyocyanase was the lipids derived from P. aeruginosa. The clinical use of pyocyanase however became secondary due to significative side effects reported, including severe damage to tissues and mucosa ^[14][27]. In 1935 Kramer discovered that not all the strains of P. aeruginosa produced pyocyanase or lost it, for some unrevealed reason ^[15][28].

Over the passage of time, pyocyanin fell into disuse. The 1950s, however, marked a decade of studies interested in the development of culture media for obtaining the pigment. The production of pyocyanin by traditional means, as commonly used in the routine of a microbiology laboratory, is based on the energetic state of the bacterium. This is reduced to a low concentration of nutrients, resulting in decreased growth rate and increased pigment concentration ^[16][29]. Nutritional scarcity, especially related to PO₄⁻³ and Ca⁺², forces the pyocyanin strains to develop the pigment that propagates through the medium ^[17][30]. This production generally starts at the beginning of the stationary phase, which is dependent on the generating time of the strain. The bacterium under these cultivation conditions tends to exhibit a generation time ranging between 3–6 h ^[18][31], resulting in the blue-green colour that appears between 48–72 h after the beginning of the incubation.

Chorismic acid as a precursor molecule of pyocyanin was discovered in the 1960s, a decade dedicated to understanding the biochemical pathways of P. aeruginosa in pigment synthesis. The following two decades explored the physiological role of pyocyanin for the bacterium. The mechanisms of action of pyocyanin began to be understood, opening up old questions and revealing the metabolic and ecological advantages of P. aeruginosa, compared to other organisms ^[19][32]. Competition is a natural process and occurs when one organism produces a substance with an inhibitory effect on the growth of another; this relationship ensures the balance of species in coexistence, as well as the entire ecosystem ^[20][33]. These substances may be of various natures, such as pigments, enzymes, organic acids and antibiotics. In addition, variations in temperature, pH, nutrient and oxygen levels, as well as population concentration are extrinsic factors that influence the pyocyanin synthesis. In this context, P. aeruginosa naturally has an advantage over other microbes ^{[21][22][23][24]}[34–37].

Between the 1990s and 2010s, the focus of studies involving pyocyanin tried to elucidate the genetic, molecular and biochemical basis of the regulation of phenazine synthesis, including pyocyanin ^[24][25][26][37–39]. It was observed that the locus responsible for phenazine biosynthesis is highly conserved in Pseudomonas spp. The production varies according to the specie and is strongly influenced by nutritional factors or genes. The expression of their regulatory systems is dependent on temperature ^[27][40]. From this epoch on, pyocyanin gained new status and different applications were tested and proposed, as discussed below.

2. Mechanism of Action of Pyocyanin

P. aeruginosa was the first microbe studied in terms of the ability to inhibit other organisms ^[28][94]. Pyocyanin acts by causing oxidative stress in susceptible prokaryotes and eukaryote cells, through the flow of electrons and the accumulation of ROS, especially O²⁻ and H₂O₂, after reaction with molecular oxygen ^[29][19][11,32]. The lethal concentration of pyocyanin against bacteria, filamentous fungi, yeasts, protozoa, algae and small animals varies significantly from study to study, depending on the model organism evaluated, and may occur from minute concentrations up to about 2000 µg/mL ^[30][31][95,96]. In Table 1, some organisms susceptible to the action of pyocyanin are summarized. The virulence mechanism used by aeruginosa is evolutionarily conserved and applied to different susceptible organisms ^[32][97].

As a planar molecule with hydrophobic and hydrophilic properties, pyocyanin interacts easily with the membrane of several organisms ^[33][98]. The formation of intracellular ROS in host cells after exposure to pyocyanin results in oxidative damage to the components of the cell cycle, depletion of NAD(P)H and enzymatic inhibition ^[34][68], in addition to specific damage to DNA ^[35][99]. The reduction of NAD(P)H and subsequent generation of ROS is irreversible possibly involving ring cleavage of the pyocyanin skeleton; however, pyocyanin can be oxidized by H₂O₂, formed in the oxidation of NAD(P)H by pyocyanin itself and catalysed by microperoxidases. This may be a relevant strategy for P. aeruginosa in in vivo conditions ^[36][100].

Initial investigations have already shown that oxygen is essential for the activity of pyocyanin against its competitors, and that the bactericidal effect depends on the concentration of the pigment. This may result in reductions ranging from one to 8 log unit cells/mL of the sensitive organism ^[37][101].

In eukaryotes, on the other hand, the interaction of pyocyanin can occur at the level of the cell wall or membrane, as well as in the respiratory chain of the mitochondria ^[38][102]. This interaction results in the release of mitochondrial ROS, accelerating the process of senescence and apoptosis ^[39][103]. In addition, concentrations less than 5 µg/mL of the pigment can disturb the vegetative state of certain filamentous fungi, promoting significant inhibition of the growth of the vegetative mycelium and the development of reproductive mycelia ^[40][10].

The formation of O²⁻ is the primary mechanism of the antimicrobial effect of pyocyanin. The ion interacts with the membrane, resulting in inhibition of respiration and active transport of solutes from the sensitive cell. There is no specific site in the respiratory chain where pyocyanin can interact; but the fact that the pigment also promotes cyanide inhibition suggests that the binding site occurs before the action of cytochrome oxidase ^[41][92].

Additionally, pyocyanin alters the redox state of the cell preferentially depleting NADPH, but it can also act on glutathione, a fact that is advantageous for P. aeruginosa. Concentrations from 130 µM of glutathione result in the formation of H₂O₂, 30 times less when compared to the NADPH-Pyocyanin system^[42] [67]. In addition, glutathione inhibits the toxicity of pyocyanin in the host cell given that the molecule is an important antioxidant that can prevent the oxidative stress through the removal of ROS ^[43][104].

Table 1.

Summary of some susceptible organisms to pyocyanin.

Most microbes, however, can synthesize some metabolites with an inhibitory action that can affect P. aeruginosa. These compounds are secreted to inhibit, but not to kill potential competitors ^[38][102]. Negative ecological interactions between coexisting microbial species play a crucial role and thus maintain the balance between populations in a given microsystem. In this way, none of them is expected to become dominant, avoiding the collapse of the entire trophic chain involved ^[45][116]. In this context, the antimicrobial activity of pyocyanin can also be reduced by some organisms sensitive to it, for which they experience amensalism as the most obvious response strategy ^[46][117]. Amensalism is an ecological relationship in which the production and secretion of metabolites occurs to promote inhibition of a potential competitor, without favouring the antagonistic microbe, except to remain in that environment, coexisting with its competitor ^[50][118].

The relationship between Escherichia coli and P. aeruginosa is a good example of amensalism. E. coli can exhibit a diversity of metabolites to restrain the physiological advantages of P. aeruginosa. Some of them have been exploited as in the production of indole and acetate. Especially indole in concentrations between 0.5–2.0 mM can reduce the production of pyocyanin, as well as the formation of biofilm by P. aeruginosa ^[59][109]. In addition, the response to oxidative stress caused by pyocyanin may result from the expression of Mn-superoxide dismutase (Mn-SDO) and other SDO ^[19][32].

Enzyme expression is the preferred mechanism of many microbes against phenazine action, for example S. aureus which involves peroxidases in addition to persistence such as small colony variants (SCV) ^[60][119]. Bacillus subtilis produces NO, stimulating the synthesis of SDO ^[61][9]. On the other hand, given the need for oxygen to enhance the effect of pyocyanin, facultative microbes and strict anaerobes may naturally be more resistant to pyocyanin ^[62][50].

3. Mechanism of Resistance of Pseudomonas aeruginosa to Pyocyanin

Pyocyanin is toxic, but P. aeruginosa has a resistance to oxidative stress resulting from pigment activity. The activation of this mechanism is associated with the concentration of intracellular phosphate. Briefly, the expression of catalase, SDO and oxidoreductases occurs, which promotes a redox cycle mediated by NADPH, neutralizing the pyocyanin from its protonated state and thereby balancing the intracellular medium, keeping the cell stable in the environment and prolonging its stationary state ^[63][134].

There is also a mechanism to avoid the toxic effects of cyanide (CN¯). The bacterium appears to make use of active mechanisms of detoxification and synthesis of the respiratory chain, in which oxidases are insensitive to CN¯ ^[64][135]. Secondary metabolites may also be associated with inhibition or reduction of the rate of cell growth ^[65][136]. Pyocyanin may also modulate the growth of P. aeruginosa in the final stages of the logarithmic phase and at the beginning of the stationary phase. This hypothesis is supported by results of previous studies ^[66][67][24][37,137,138]. It is suggested that the phenomenon is directly related to the formation of the ion transport system, as well as the generation of CN¯, a product of glycine metabolism ^[68][139].

The role of the cyanide ion for the bacterium is not yet clear. As a virulence factor, CN¯ is extremely toxic and binds irreversibly to the terminal of the oxidases in the respiratory chain, inhibiting aerobic respiration ^[69][70][140,141]. In addition, pyocyanin also promotes increased expression of genes and operons that regulate redox transport and control, as well as decrease the expression of genes and operons involved in the acquisition of Fe³⁺. In contrast, the reduction in the expression of pyocyanin also allows cells to not be physiologically disturbed ^[71][69].