Plant Extracts against Pseudomonas Aeroginosa - in Vitro & in Vovo Studies

Etude Scientifique , 2011 , 95 Pages , Note: None

Biologie - Maladies, Santé, Allimentation

Pseudomonas infections are very hard to treat in immunocompromised patients, in the post operational and other hospitalization related conditions and are responsible for high number of deaths. Thus Pseudomonas can be very notorious opportunistic pathogen. The major problem associated with chronic P. aeruginosa infections is the development of antibiotic resistance due to long continuous treatment durations in which the resistant mutants start to accumulate. Recent understandings regarding the development of antibiotic resistance have added a new dimension of hypermutator strains which have comparatively high rates of mutations then wild type strains. In Ayurveda, many plants and plant extracts have been widely used for treatment of diseases which may be due to bacterial, fungal or viral pathogen or due to imbalance in the body’s various systems. Modulation of immune response to alleviate disease has been one of the ways practiced in ayurveda including others such as re-establishing the imbalance in the body, lifestyle changes, and diet. The plants extracts used are seeds of Moringa oleifera, Nigella sativa, Vernonia anthelmintica, fruit of Terminalia chebula and root bark of Terminalia bellerica. These plant extracts have been shown to be effective as an antibacterial agent specifically against P. aeruginosa. The plant extracts used in this study are easily available and is being used in normal day-to-day life for various purpose and also have been proven to be effective medicine in Ayurveda.

Table of Content

Chapter 1: Introduction & Review of Literature
Characteristics
Pseudomonas aeruginosa as a Human pathogen
Virulence factors of P. aeruginosa
Quorum sensing: A Global Regulation System of P aeruginosa Extracellular
Virulence Factors Host Defenses
Epidemiology and Control of P.aeruginosa Infections
Toxinogenesis
Current Therapeutic Approaches
Novel Therapeutic Approaches
Probable Drawbacks
Role of Plant Extracts

Chapter 2: Materials & Methods
Phase I In vivo study
Phase II In vitro study

Chapter 3: RESULTS

Chapter 4: DISCUSSION

Chapter 5: CONCLUSION

Chapter 6: REFERENCES

CHAPTER 1 INTRODUCTION & REVIEW OF LITERATURE

P. aeruginosa is member of the Gamma Proteobacteria class of Bacteria. It is a Gram-negative, aerobic rod belonging to the bacterial family Pseudomonadaceae. P. aeruginosa is the type species of its group and it contains 12 other members (Kenneth, 2008). Like other members of the genus, P. aeruginosa is a free-living, gram negative rod shaped bacterium, commonly found in soil and water. It occurs regularly on the surfaces of plants and occasionally on the surfaces of animal body. They are one of the few groups of bacteria that are true pathogens of plants (Pitt, 1986; Robert and Dowling, 1998).

Pseudomonas can grow in many habitats including soil, surface waters, plants and various foods such as vegetables eaten by man (Neu, 1983; Robert and Dowling, 1998). P. aeruginosa has become increasingly recognized as an emerging opportunistic pathogen of clinical relevance. P. aeruginosa is a leading cause of nosocomial infections. The risk of emergence of antibiotic resistance may vary with different antibiotic treatments (Carmeli et al., 1997). As compared to hardcore bacterial pathogens, P. aeruginosa is only weakly pathogenic and in the host whose immune system is properly functioning, it is virtually non-virulent (Pitt, 1986). However Pseudomonas can cause severe form of infections which may be urinary tract, nosocomial or wound infections (Schaber et al., 2004). On the contrary, Pseudomonas infections are very hard to treat in immunocompromised patients, in the post operational and other hospitalization related conditions and are responsible for high number of deaths. Thus Pseudomonas can be very notorious opportunistic pathogen.

P. aeruginosa in nature is present as a saprophyte or pathogen of plants, insects and animal. In hospitals, P. aeruginosa can be found in sinks, respirators, humidifiers etc and is occasionally found on the hands of medical personnel (Neu, 1983; Pitt, 1986). P. aeruginosa is a leading cause of nosocomial infections, ranking second among the gram-negative pathogens reported to the National Nosocomial Infection Surveillance System (Harris et al., 2010).

The major problem associated with chronic P. aeruginosa infections is the development of antibiotic resistance due to long continuous treatment durations in which the resistant mutants start to accumulate (Livermore, 2002). Recent understandings regarding the development of antibiotic resistance have added a new dimension of hypermutator strains which have comparatively high rates of mutations then wild type strains (Oliver et al., 2000). These hypermutator variants of normal strains acquire resistance via mutations to almost every antibiotic which is currently used, making them impossible to eradicate (Bla'zquez, 2003; Macia' et al., 2005).

Characteristics:

P. aeruginosa is a Gram-negative rod measuring 0.5 to 0.8 pm by 1.5 to 3.0 pm (Putty, 2007). Almost all strains are motile by means of a single polar flagellum. The bacterium is ubiquitous in soil and water, and on surfaces in contact with soil or water. Its metabolism is respiratory and never fermentative, but it will grow in the absence of O2 if NO3 is available as a respiratory electron acceptor. The typical Pseudomonas bacterium in nature might be found in a biofilm, attached to some surface or substrate, or in a planktonic form, as a unicellular organism, actively swimming by means of its flagellum. Pseudomonas is one of the most vigorous, fast­swimming bacteria seen in hay infusions and pond water samples (Kenneth, 2008).

P. aeruginosa has very simple nutritional requirements. It is often observed "growing in distilled water", which is evidence of its minimal nutritional needs (Putty, 2007). In the laboratory, the simplest medium for growth of P. aeruginosa consists of acetate as a source of carbon and ammonium sulphate as a source of nitrogen. It is tolerant to a wide variety of physical conditions. P.aeruginosa is capable of growth in diesel and jet fuel, where it is known as hydrocarbon utilizing microorganism (or “HUM bug”) causing microbial corrosion. It is resistant to high concentration of salts and dyes, weak antiseptics, and many commonly used antibiotics (Murray et al., 1995). Its optimum temperature for growth is 37 degrees, and it is able to grow at temperatures as high as 42 degrees (Baltch et al., 1994). It is tolerant to a wide variety of physical conditions, including temperature. It is resistant to high concentrations of salts and dyes, weak antiseptics, and many commonly used antibiotics. P. aeruginosa has a predilection for growth in moist environments, which is probably a reflection of its natural existence in soil and water. These natural properties of the bacterium undoubtedly contribute to its ecological success as an opportunistic pathogen. They also help explain the ubiquitous nature of the organism and its prominence as a nosocomial pathogen (Kenneth, 2008).

P. aeruginosa isolates may produce three colony types. Natural isolates from soil or water typically produce a small, rough colony. Clinical samples, in general, yield one or another of two smooth colony types. One type has a fried-egg appearance which is large, smooth, with flat edges and an elevated appearance. Another type, frequently obtained from respiratory and urinary tract secretions, has a mucoid appearance, which is attributed to the production of alginate slime. The smooth and mucoid colonies are presumed to play a role in colonization and virulence (Kenneth, 2008).

P. aeruginosa strains produce two types of soluble pigments, the fluorescent pigment pyoverdin and the blue pigment pyocyanin. The latter is produced abundantly in media of low-iron content and functions in iron metabolism in the bacterium. Pyocyanin (from "pyocyaneus") refers to "blue pus", which is a characteristic of supportive infections caused by P.aeruginosa (Byng et al., 1979; Wilson et al., 1987; Robert and Dowling, 1998).

Fig 1 : P. aeruginosa colonies on agar (Kenneth, 2008)

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Fig 2: The soluble blue pigment pyocyanin is produced by many, but not all, strains of P. aeruginosa (Kenneth, 2008)

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Pseudomonas aeruginosa as a Human pathogen

Pseudomonas aeruginosa is the most common Gram-negative bacterium found in nosocomial infections. P. aeruginosa is responsible for 16% of nosocomial pneumonia cases (Wiblin, 1997), 12% of hospital-acquired urinary tract infections (Pollack, 1995), 8% of surgical wound infections (Kluytmans, 1997) and 10% of bloodstream infections (Gordon et al., 1998). Immunocompromised patients, such as neutropenic cancer and bone marrow transplant patients are particularly susceptible to opportunistic infections. In this group of patients, P. aeruginosa is attributable for pneumonia and septicemia with deaths reaching 30% (Fergie et al., 1994; Bergen and Shelhamer, 1996).

P. aeruginosa is also one of the most common and lethal pathogens responsible for ventilator-associated pneumonia in intubated patients (Dunn and Wunderink., 1995), with directly attributable death rates reaching 38% (Brewer et al., 1996). In burn patients, P. aeruginosa bacteremia has declined as a result of better wound treatment and dietary changes (Kluytmans, 1997). However, P. aeruginosa outbreaks in burn units are still associated with high around 60% death rates (Richard et al., 1994). In the expanding AIDS population, P. aeruginosa bacteremia is associated with 50% of deaths (Mendelson et al., 1994). Cystic fibrosis (CF) patients are characteristically susceptible to chronic infection by P. aeruginosa, which is responsible for high rates of illness and death in this population (Govan and Deretic, 1996). Along with CF patients, many of the bronchiectasis and patients with chronic obstructive pulmonary disease also get infected chronically by P. aeruginosa (Nicotra et ah, 1995; Hill et al., 2000). It is also responsible of keratitis in eyes which is spread via contaminated contact lenses (Donshik, 1997; Cheng et al., 1999; Zhu et al., 2002).

It is virtually impossible to completely eradicate a chronic Pseudomonas aeruginosa acquired respiratory infection which is frequently observed to accompany respiratory diseases such as cystic fibrosis (Govan and Deretic, 1996; Lyczak et al., 2002; Gibson et al., 2003; Smith et al., 2006), bronchiectasis (Nicotra et al., 1995; Evans et al., 1996) and chronic obstructive pulmonary disease (COPD) (Hill et al., 2000). Dual antibiotic therapy usually with a ß-lactam and an aminoglycoside is given to patients to control infections and maintain lung functions (Hoiby, 2002).

However, years of intensive antibiotic therapy to control the negative outcomes of the chronic colonization of patients results in the development of drug resistance in the infecting bacterial population and ultimately leads to treatment failure (Fish et al., 1995; Carmeli et al., 1999; Gibson et al., 2003; Juan et al., 2005). This happens due to simultaneous or sequential selection of mutant alleles that give birth to antibiotic resistant decedents of sensitive parents and this is a critical factor in the management of chronic infections in cystic fibrosis CF) patients. Ability of P. aeruginosa to escape the effects of antibiotics is attributed by its capability of growing in microaerophilic environment (Hassett et al., 2002; Worlitzsch et al., 2002; Hill et al., 2005) as biofilms (Hoiby, 2002; Moskowitz et al., 2004; Hill et al., 2005) both of which reduce the efficiency of many antibiotics. Resistance towards antimicrobial agents to survive in the lungs during frequent and prolonged treatments also requires great adaptive skills.

The capacity of P. aeruginosa to cause many types of infections can be attributed to its ability of producing several intracellular and extracellular virulence factors that affect different components of innate as well as acquired defenses of the host and allow the organism to establish infection (VanDelden and Iglewski, 1998). Resistance towards many different types of antibiotics at concentrations that are achievable in vivo provide the organism an extra protective shield that renders it insensitive to antibiotic therapies continued for even several years and most importantly the ability to communicate intercellularly to synchronize the production of virulence factors after certain cell density is reached allows the organism evade the host immune system during early phase of colonization and then modulate the host immune responses to establish the infection and survive in the host (Ramphal and Pyle, 1983; Pearson et al., 1997; VanDelden and Iglewski, 1998; Erickson et ah, 2002; Favre-Bonté et al., 2007).

For an opportunistic pathogen such as P. aeruginosa, the disease process begins with some alteration or circumvention of normal host defenses. The pathogenesis of P .aeruginosa infections is multifactorial (Kenneth, 2008).

Bacteremia and septicemia

P. aeruginosa causes bacteremia primarily in immunocompromised patients (Fergie et al., 1994; Bergen and Shelhamer, 1996). Predisposing conditions include hematologic malignancies, immunodeficiency relating to AIDS, neutropenia, diabetes mellitus, and severe burns. Most P.aeruginosa bacteremia is acquired in hospitals and nursing homes. P. aeruginosa accounts for about 25 percent of all hospital acquired Gram-negative bacteremias (Kenneth, 2008).

Central nervous system infections

P. aeruginosa causes meningitis and brain abscesses. The organism invades the CNS from a contiguous structure such as the inner ear or paranasal sinus, or is inoculated directly by means of head trauma, surgery or invasive diagnostic procedures, or spreads from a distant site of infection such as the urinary tract (Kenneth, 2008).

Endocarditis

P. aeruginosa infects heart valves of IV drug users and prosthetic heart valves. The organism establishes itself on the endocardium by direct invasion from the blood stream (Kenneth, 2008).

Respiratory infections

Respiratory infections caused by P. aeruginosa occur almost exclusively in individuals with a compromised lower respiratory tract or a compromised systemic defense mechanism. Primary pneumonia occurs in patients with chronic lung disease and congestive heart failure. Bacteremic pneumonia commonly occurs in neutropenic cancer patients undergoing chemotherapy. Lower respiratory tract colonization of cystic fibrosis patients by mucoid strains of P .aeruginosa is common and difficult, if not impossible, to eradicate (Kenneth, 2008).

Ear infections including external otitis

P. aeruginosa is the predominant bacterial pathogen in some cases of external otitis, including "swimmer's ear". The bacterium is infrequently found in the normal ear, but often inhabits the external auditory canal in association with injury, maceration, inflammation, or simply wet and humid conditions (Kenneth, 2008).

Eye infections

P. aeruginosa can cause devastating infections in the human eye. It is one of the most common causes of bacterial keratitis, and has been isolated as the etiologic agent of neonatal ophthalmia. P. aeruginosa can colonize the ocular epithelium by means of a fimbrial attachment to sialic acid receptors. If the defenses of the environment are compromised in any way, the bacterium can proliferate rapidly through the production of enzymes such as elastase, alkaline protease and exotoxin A, and cause a rapidly destructive infection that can lead to loss of the entire eye (Kenneth, 2008).

Bone and joint infections

P. aeruginosa infections of bones and joints result from direct inoculation of the bacteria or the hematogenous spread of the bacteria from other primary sites of infection. Blood-borne infections are most often seen in IV drug users and in conjunction with urinary tract or pelvic infections. P. aeruginosa has a particular tropism for fibrocartilagenous joints of the axial skeleton. P. aeruginosa causes chronic contiguous osteomyelitis, usually resulting from direct inoculation of bone and is the most common pathogen implicated in osteochondritis after puncture wounds of the foot (Kenneth, 2008).

Skin and soft tissue infections, including wound infections, pyoderma and dermatitis

P. aeruginosa can cause a variety of skin infections, both localized and diffuse. The common predisposing factors are breakdown of the integument which may result from burns, trauma or dermatitis; high moisture conditions such as those found in the ear of swimmers and the toe webs of athletes, hikers and combat troops, in the perineal region and under diapers of infants, and on the skin of whirlpool and hot tub users. Individuals with AIDS are easily infected. Pseudomonas has also been implicated in folliculitis and unmanageable forms of acne vulgaris (Kenneth, 2008).

Gastrointestinal infections

P. aeruginosa can produce disease in any part of the gastrointestinal tract from the oropharynx to the rectum. As in other forms of Pseudomonas disease, those involving the GI tract occur primarily in immunocompromised individuals. The organism ha]s been implicated in perirectal infections, pediatric diarrhea, typical gastroenteritis, and necrotizing enterocolitis. The GI tract is also an important portal of entry in Pseudomonas septicemia and bacteremia (Kenneth, 2008).

Urinary tract infections

Urinary tract infections (UTI) caused by P.aeruginosa are usually hospital-acquired and related to urinary tract catheterization, instrumentation or surgery. P. aeruginosa is the third leading cause of hospital-acquired UTIs, accounting for about 12 percent of all infections of this type. The bacterium appears to be among the most adherent of common urinary pathogens to the bladder uroepithelium. As in the case of E. coli, urinary tract infection can occur via an ascending or descending route. In addition, P.aeruginosa can invade the bloodstream from the urinary tract, and this is the source of nearly 40 percent of P. aeruginosa bacteremias (Kenneth, 2008).

Virulence factors of P. aeruginosa

Inability of P. aeruginosa to infect normal individuals suggests that a single virulence factor is not so potent to allow the bacterium to invade the host to establish infection. Yet a wide array of potential virulence factors have been described which contribute to its pathogenicity in the compromised patients from which some of the major players are described briefly as under.

- Mucoid Exo-polysaccharide (alginate) (MEP)

The mucoid form of P. aeruginosa produces large amounts of an extracellular polysaccharide called Mucoid Exo-Polysaccharide (MEP), which is often referred to as alginate because of its chemical similarity to a polysaccharide normally found in seaweed algae. Alginate is composed of highly organized linear strands of polysaccharides radiating outwards from the cell surface that mediates attachment to respiratory epithelium. Mucoid forms account for up to 90% of isolates from patients with cystic fibrosis (Pitt, 1986). Typically, if P. aeruginosa is isolated for the first time, it is a non-mucoid type but after a variable period, after one or two years, it becomes mucoid and patients infected by mucoid strains tend to have detoriating lung function and nutritional state. Bacterial microcolonies are entrapped in alginate layer and protected from attack by host defenses (Pitt, 1986). It is known to generate hyperimmune conditions in the lungs leading to inflammatory actions. The major functions of alginate are adherence to epithelium, inhibition of phagocytosis and resistance towards antibiotics, protection of cells from opsonization and neutralization by antibody and complement binding (Ramphal and Pyle, 1983; Ramphal and Pier, 1985; Wilson et al., 1987; Pedersen et al., 1992; Wiblin, 1997; Coleman et al., 2003).

- Protease enzymes

Several proteases are produced by P. aeruginosa including LasB elastase and LasA elastase (Morihara et al., 1965; Pitt, 1986; Toder et al., 1994). Elastin is a major part of human lung tissue which is responsible for lung expansion and contraction. Elastin is also present in blood vessels (Galloway, 1991). The concerted activity of two enzymes, LasB elastase and LasA elastase, is responsible for elastolytic activity (VanDelden and Iglewski, 1998). The ability of P. aeruginosa to destroy the protein elastin is a major virulence determinant during acute infection. LasB elastase degrades not only elastin but also fibrin and collagen. It can inactivate substances such as human immunoglobulins G and A, airway lysozyme, complements components (C3b receptors) and substances involved in protecting the respiratory tract against proteases such as a 1-proteinase inhibitor and bronchial mucus proteinase inhibitor. Therefore, LasB elastase not only destroys tissue components but also interferes with host defense mechanisms (Pitt, 1986; VanDelden and Iglewski, 1998).

- Pyocyanin superfamily

Pyocyanin and other phenazines produced by P. aeruginosa are blue coloured pigments that serve multiple functions and also have broad range of effect on different types of cells. Targets include cell cycle, electron transport and respiration, epidermal cell growth, protein sorting, vesicle transport and the vacuolar ATPase whose inactivation may negatively impact the lung function of cystic fibrosis patients (Ran et al., 2003; Hoffmann et al., 2005). Pyocyanin oxidizes NAD and releases oxygen-free radicals, in the absence of enzyme action, through the electron transport system (Hassant and Fridovich, 1980; Pitt, 1986). It Inhibits ciliary beat in the lung air-way epithelium (Kanthakumar et al., 1993), acts as a siderophore to remove iron from transferrin (Cox, 1986), enhances oxidative metabolism of neutrophils and induces apoptosis, inhibits lymphocyte proliferation and prostacylin release from endothelial cells (Muller et al., 1989; Ras et al., 1990; Kamath et al., 1995) and inactivates a1 Protease Inhibitor (Morihara et al., 1979). It is also toxic to other bacterial species such as S. aureus, B. licheniformnis, B. subtilis and Acinetobacter (Baron and Rowe, 1981) and eukaryotic cells such as fungi (Kerr et al., 1999). However even Pesudomonads that are not able to produce pyocyanin are tolerant to high concentration of pyocyanin (Hassett et al., 1992).

Other virulence factors from P. aeruginosa include Phospholipase C which is responsible for haemolysis, tissue damage and destroys surfactant (Pitt, 1986).

Rhamnolipids cause haemolysis, inhibit ciliary beat, stimulate mucus secretion and affect ion transport across epithelium (Pitt, 1986). Pili and exoenzyme S help in adherence to epithelium and lipase causes tissue damage (Ramphal et al., 1984; Pitt, 1986). Histamine impairs epithelial integrity and Leukocidin is cytotoxic to neutrophils and lymphocytes (Pitt, 1986).

Quorum sensing: A Global Regulation System of P. aeruginosa Extracellular Virulence Factors

Cell-to-Cell Signalling Systems

P. aeruginosa appears to control the production of many of its extracellular virulence factors by a mechanism that monitors bacterial cell density and allows communication between bacteria by cell-to-cell signaling. Bacteria are able to sense their environment, process information, and react appropriately; however, their ability to sense their own cell density, to communicate with each other, and to behave as a population instead of individual cells has only recently been understood. This phenomenon, called quorum-sensing or cell-to-cell signaling, is a generic phenomenon described in many gram-negative and gram-positive bacteria. At least two complete quorum-sensing systems, las and rhl, are present in the opportunistic human pathogen P. aeruginosa. These systems are known to control the expression of a number of virulence genes in response to bacterial cell density (Fuqua et al., 1996), but their specific effect on each other has not been studied. The las and rhl systems each contain homologs of the LuxR and LuxI proteins of the prototypic lux quorum-sensing system from Vibrio fischeri (Fuqua, W C et al., 1996). The las system consists of the transcriptional activator protein LasR and of LasI, which directs the synthesis of the autoinducer PAI-1 [Ж-(3- oxododecanoyl)-L-homoserine lactone] (Gambello et al., 1991, Passador et al., 1993 and Pearson et al., 1994). This system has been shown to activate the expression of lasI, lasB, lasA, apr, and toxA (Todar et al, 1991; Gambello et al., 1993; Passador et al., 1993; seed et al., 1995).

Similarly, the rhl system consists of the transcriptional activator protein RhlR and RhlI, which directs the synthesis of the autoinducer PAI-2 (A-butyryl-L-homoserine lactone; formerly known as factor 2) (Ochsner et al., 1994; Ochner et al., 1995 and Pearson et al., 1995). This system controls the expression of rhll and rhlAB, which codes for a rhamnosyltransferase required for rhamnolipid (heat-stabile hemolysin) production (Ochsner et al., 1994; Ochsner et al., 1995; Latifi et al., 1996 ;). It has also been reported that rhl quorum sensing activates the expression of rpoS, a stationary-phase sigma factor that controls numerous genes (Latifi et al., 1996).

The general model for quorum sensing (Fuqua et al., 1996) begins with the autoinducer, which is a diffusible molecule, being produced at a basal level at low cell densities. The autoinducer concentration then increases with cell density until a threshold concentration is reached. At this concentration, the autoinducer binds to its specific target protein (i.e., LasR or RhlR), and the autoinducer-protein complex activates genes that it controls.

Experiments on the interchange ability of the las and rhl system components showed that they were not compatible, in that PAI-2 does not activate LasR nor does PAI-1 activate RhlR in Escherichia coli. However, it was apparent that these two systems were not completely independent of one another. It was first indicated that the las and rhl systems may be linked when showed that PAI-2 was poorly expressed in a P. aeruginosa lasR strain, which led to the conclusion that LasR may control PAI-2 production (Pearson et al., 1995). After this, it was reported that the rhl system was working in tandem with the las system to control the production of elastase in P. aeruginosa (Brint et al., 1995 and Seed et al., 1995). The rhlR transcription was controlled by LasR-PAI-1 (Laitifi et al., 1996). The las quorum­sensing system controls RhlR, the transcriptional activator of the rhl system, at both the transcriptional and posttranslational levels. In addition, lasR and rhlR are expressed in a growth-dependent manner, with each gene being activated during the second half of log-phase growth. The resulting increase in expression of these genes can reach 1,000-fold. The autoinducer, therefore, allows the bacteria to communicate with each other (cell-to-cell signaling), to sense their own density (quorum-sensing), and together with a transcriptional activator to express specific genes as a population instead of individual cells (Kenneth, 2008).

The las Cell-to-Cell Signaling System of P. aeruginosa

The first cell-to-cell signaling system described in P. aeruginosa was shown to regulate expression of LasB elastase and was therefore named the las system. The las cell-to-cell signaling system is composed of lasI, the autoinducer synthase gene responsible for the synthesis of 3-oxo-C12-HSL (^-[3-oxododecanoyl]-L-homoserine lactone, previously named PAI-1 or OdDHL), and the lasR gene that codes for a transcriptional activator protein. Cell-to-cell signaling system regulates lasB expression and is required for optimal production of other extracellular virulence factors such as LasA protease and exotoxin A. LasI is the most sensitive gene to activation by LasR/3-oxo-C12-HSL. The preference for the lasI promoter allows an initial rapid rise in autoinducer synthesis, which increases the amount of 3-oxo- C12-HSL available to bind to LasR. This autoinduction hierarchy is responsible for a dramatic increase of expression of virulence genes (such as lasB) once a critical cell density has been reached. Recently, the las system has also been shown to activate the xcpP and xcpR genes that encode proteins of the P. aeruginosa secretory pathway. 3-oxo-C12-HSL alone has been suggested to contribute to the virulence of P. aeruginosa because it has some immunomodulatory activity. The las cell-to-cell signaling system is positively controlled by GacA, as well as by Vfr, which is required for the transcription of lasR. An inhibitor, RsaL, that represses the transcription of lasI, has also been described (Kievit et al., unpub. data). The multiple regulatory levels of the las cell-to-cell signaling system and the various genes under its control highlight the importance of this system for P. aeruginosa.

The rhl Cell-to-Cell Signaling System of P. aeruginosa

P. aeruginosa has a second cell-to-cell signaling system, named the rhl system because of its ability to control the production of rhamnolipid. This system is composed of rhlI, the C4-HSL (Ж-butyrylhomoserine lactone, previously named PAI-2 or BHL) autoinducer synthase gene, and the rhlR gene encoding a transcriptional activator protein. This system regulates the expression of the rhlAB operon that encodes a rhamnosyltransferase required for rhamnolipid production. The rhl system is also necessary for optimal production of LasB elastase, LasA protease, pyocyanin, cyanide, and alkaline protease. Therefore, like the las cell-to-cell signaling system, the rhl system, sometimes referred to as vsm (virulence secondary metabolites), regulates the expression of various extracellular virulence factors of P. aeruginosa. Interestingly, the rhl system also regulates the expression of rpoS, which encodes a stationary sigma factor (dS) involved in the regulation of various stress- response genes.

The Cell-to-Cell Signalling Hierarchy in P. aeruginosa

Recent data have shown that the las and rhl cell-to-cell signaling systems of P. aeruginosa interact. Both systems are highly specific in that their respective autoinducers are unable to activate the transcriptional activator protein of the other system (i.e., 3-oxo-C12-HSL cannot activate RhlR, and C4-HSL cannot activate LasR). It has also been shown that the R-protein/autoinducer complexes prefer certain promoters that they will activate; LasR/3-oxo-C12-HSL preferentially activates lasB over rhlA, and RhlR/C4-HSL preferentially activates rhlA over lasB. However, neither system is completely independent of the other. The LasR/3-oxo- C12-HSL complex activates the expression of rhlR placing the las system in a cell-to- cell signaling hierarchy above the rhl system. Moreover, 3-oxo-C12-HSL can bind to RhlR, blocking the binding of C4-HSL to its transcriptional activator rhlR. The las system therefore controls the rhl system at both a transcriptional and posttranslational level. Another yet unidentified regulatory mechanism directly influencing the expression of rhlRI has been suggested. Rhl system regulation of such important genes as rpoS could explain why multiple levels of controls are required for its tight regulation (Everett et al., 1997).

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Fig: Model of the P. aeruginosa quorum-sensing circuitry, LasR, RhlR, PAI1 and PAI2 are symbolized by circles. Plus symbols indicate transcriptional activation of the gene(s) at the end of an arrow. The effect of the LasR-PAI-2 complex on lasR is unclear, so this was indicated by “+/-?” The minus symbol by the arrow extending from PAI-1 to the arrow between PAI-2 and RhlR indicates blocking of the association between PAI-2 and RhlR. To begin the quorum-sensing cascade, LasR and PAI-1 are both produced at a basal level. As culture density increases, lasR is activated by Vfr, and PAI-1 reaches a threshold concentration and binds to LasR. At low culture density, the PAI-1 concentration is in the excess of PAI-2 concentration, allowing PAI-1 to block the interaction of RhlR and PAI-2. The auto induction of lasI by LasR- PAI-1 could keep the PAI-1 concentration well below that of PAI-2 until enough RhlR and/or PAI-2 is produced to overcome the blocking effect of PAI-1. Once RhlR associates with PAI-2, autoinduction of rhU occurs and the remainder of the RhlR-PAI-2 controlled genes is activated.

Biofilm and QS

Populations of surface-attached microorganisms comprising either single or multiple species are commonly referred to as biofilms (O’Toole et al., 1998). Naturally, clinically and in industrial waste bacteria found predominantly in biofilms, not in planktonic form. Prominent feature of biofilm is thought to producing a large quantity of exopolysacharides and another feature of biofilm is its antibiotic resistance (Costerton et al., 1995). It was believed that QS regulates the formation of biofilm. Because QS requires sufficient density of bacteria, none of the QS signals would be expected to participate in the earlier stage of biofilm formation, attachment and proliferation. It has been proved that dominantly lasI is regulating the biofilm differentiation, not RhlI (Davies et al., 1998). So, QS targeting may decrease the biofilm formation.

Diagnosis

Diagnosis of P.aeruginosa infection depends upon isolation and laboratory identification of the bacterium. It grows well on most laboratory media and commonly is isolated on blood agar or eosin-methylthionine blue agar. It is identified on the basis of its Gram morphology, inability to ferment lactose, a positive oxidase reaction, its fruity odour, and its ability to grow at 42°C (Colwell, 2007). Fluorescence under ultraviolet light is helpful in early identification of P. aeruginosa colonies. Fluorescence is also used to suggest the presence of P. aeruginosa in wounds.

Host Defenses

Most strains of P. aeruginosa are resistant in serum alone, but the addition of polymorphonuclear leukocytes results in bacterial killing. Killing is most efficient in the presence of type-specific opsonizing antibodies, directed primarily at the antigenic determinants of LPS. This suggests that phagocytosis is an important defense and that opsonizing antibody is the principal functional antibody in protecting from P. aeruginosa infections. Once P. aeruginosa infection is established, other antibodies, such as antitoxin, may be important in controlling disease. The observation that patients with diminished antibody responses (caused by underlying disease or associated therapy) have more frequent and more serious P. aeruginosa infections underscores the importance of antibody-mediated immunity in controlling Pseudomonas infections, unfortunately, Cystic fibrosis is the exception. Most cystic fibrosis patients have high levels of circulating antibodies to bacterial antigens, but are unable to clear P. aeruginosa efficiently from their lungs. Cell-mediated immunity does not seem to play a major role in resistance or defense against Pseudomonas infections (Kenneth, 2008).

Epidemiology and Control of P.aeruginosa Infections

P. aeruginosa is a common inhabitant of soil, water, and vegetation. It is found on the skin of some healthy persons and has been isolated from the throat (5 percent) and stool (3 percent) of nonhospitalized patients. In some studies, gastrointestinal carriage rates increased in hospitalized patients to 20 percent within 72 hours of admission. Within the hospital, P. aeruginosa finds numerous reservoirs: disinfectants, respiratory equipment, food, sinks taps, toilets, showers and mops. Furthermore, it is constantly reintroduced into the hospital environment on fruits, plants, vegetables, as well by visitors and patients transferred from other facilities. Spread occurs from patient to patient on the hands of hospital personnel, by direct patient contact with contaminated reservoirs, and by the ingestion of contaminated foods and water.

The spread of P. aeruginosa can best be controlled by observing proper isolation procedures, aseptic technique, and careful cleaning and monitoring of respirators, catheters, and other instruments. Topical therapy of burn wounds with antibacterial agents such as silver sulfadiazine, coupled with surgical debridement, dramatically reduces the incidence of P. aeruginosa sepsis in burn patients.

P. aeruginosa is frequently resistant to many commonly used antibiotics. Although many strains are susceptible to gentamicin, tobramycin, colistin, and fluoroquinolins, resistant forms have developed. The combination of gentamicin and carbenicillin is frequently used to treat severe Pseudomonas infections. Several types of vaccines are being tested, but none is currently available for general use. Multiple diverse determinants of virulence are expected in the wide range of diseases caused, which include septicemia, urinary tract infections, pneumonia, chronic lung infections, endocarditis, dermatitis, and osteochondritis.

Most Pseudomonas infections are both invasive and toxinogenic. The ultimate Pseudomonas infection may be seen as composed of three distinct stages: (1) bacterial attachment and colonization; (2) local invasion; (3) disseminated systemic disease. However, the disease process may stop at any stage. Particular bacterial determinants of virulence mediate each of these stages and are ultimately responsible for the characteristic syndromes that accompany the disease (Kenneth, 2008).

Colonization

Although colonization usually proceedes infections by P. aeruginosa, the exact source and mode of transmission of the pathogen are often unclear because of its ubiquitous presence in the environment. It is sometimes present as part of the normal flora of humans, although the prevalence of colonization of healthy individuals outside the hospital is relatively low (estimates range from 0 to 24 percent depending on the anatomical locale) (Kenneth, 2008).

The pili of P. aeruginosa will adhere to the epithelial cells of the upper respiratory tract and, by inference, to other epithelial cells as well. These adhesins appear to bind to specific galactose or mannose or sialic acid receptors on epithelial cells. Colonization of the respiratory tract by Pseudomonas requires pili adherence and may be aided by production of a protease enzyme that degrades fibronectin in order to expose the underlying pilus receptors on the epithelial cell surface. Tissue injury may also play a role in colonization of the respiratory tract, since P. aeruginosa will adhere to tracheal epithelial cells of mice infected with influenza virus but not to normal tracheal epithelium. This has been called opportunistic adherence, and it may be an important step in P.aeruginosa causing keratitis and urinary tract infections, as well as infections of the respiratory tract.

The receptor on tracheal epithelial cells for P. aeruginosa pili is probably sialic acid (N-acetylneuraminic acid). Mucoid strains, which produce an exopolysaccharide (alginate), have an additional or alternative adhesin which attaches to the tracheobronchial mucin (N-acetylglucosamine). Besides pili and the mucoid polysaccharide, there are possibly other cell surface adhesins utilized by P. aeruginosa to colonize the respiratory epithelium or mucin (Pitt, 1986). It is also possible that surface-bound exoenzyme S could serve as an adhesin for glycolipids on respiratory cells.

The mucoid exopolysaccharide produced by P. aeruginosa is a repeating polymer of mannuronic and glucuronic acid referred to as alginate. Alginate slime forms the matrix of the P. aeruginosa biofilm which anchors the cells to their environment and in medical situations, it protects the bacteria from the host defenses such as lymphocytes, phagocytes, the ciliary action of the respiratory tract, antibodies and complement (Ramphal and Pyle, 1983; Ramphal and Pier, 1985; Wilson et al., 1987; Pedersen et al., 1992; Wiblin, 1997; Coleman et al., 2003). Biofilm mucoid strains of Pseudomonas are also less susceptible to antibiotics than their planktonic counterparts. Mucoid strains of P. aeruginosa are most often isolated from patients with cystic fibrosis and they are usually found in lung tissues from such individuals (Kenneth, 2008).

Invasion

The ability of P. aeruginosa to invade tissues depends upon production of extracellular enzymes and toxins that break down physical barriers and damage host cells, as well as resistance to phagocytosis and the host immune defenses. As mentioned above, the bacterial capsule or slime layer effectively protects cells from opsonization by antibodies, complement deposition, and phagocyte engulfment (Kenneth, 2008).

Two extracellular proteases have been associated with virulence that exerts their activity at the invasive stage: elastase and alkaline protease. Elastase has several activities that relate to virulence. The enzyme cleaves collagen, IgG, IgA, and complement. It also lyses fibronectin to expose receptors for bacterial attachment on the mucosa of the lung. Elastase disrupts the respiratory epithelium and interferes with ciliary function. Alkaline protease interferes with fibrin formation and will lyse fibrin. Together, elastase and alkaline protease destroy the ground substance of the cornea and other supporting structures composed of fibrin and elastin. Elastase and alkaline protease together are also reported to cause the inactivation of gamma interferon (IFN) and tumor necrosis factor (TNF) (Kenneth, 2008).

P. aeruginosa produces three other soluble proteins involved in invasion: a cytotoxin (mw 25 kDa) and two hemolysins. The cytotoxin is a pore-forming protein. It was originally named leukocidin because of its effect on neutrophils, but it appears to be cytotoxic for most eukaryotic cells. Of the two hemolysins, one is a phospholipase and the other is a lecithinase. They appear to act synergistically to break down lipids and lecithin. The cytotoxin and hemolysins contribute to invasion through their cytotoxic effects on neutrophils, lymphocytes and other eucaryotic cells (Kenneth, 2008).

One Pseudomonas pigment is probably a determinant of virulence for the pathogen. The blue pigment, pyocyanin, impairs the normal function of human nasal cilia, disrupts the respiratory epithelium, and exerts a proinflammatory effect on phagocytes. A derivative of pyocyanin, pyochelin, is a siderophore that is produced under low-iron conditions to sequester iron from the environment for growth of the pathogen. It could play a role in invasion if it extracts iron from the host to permit bacterial growth in a relatively iron-limited environment. No role in virulence is known for the fluorescent pigments (Kenneth, 2008).

Dissemination

Blood stream invasion and dissemination of Pseudomonas from local sites of infection is probably mediated by the same cell-associated and extracellular products responsible for the localized disease, although it is not entirely clear how the bacterium produces systemic illness. P. aeruginosa is resistant to phagocytosis and the serum bactericidal response due to its mucoid capsule and possibly LPS.

[...]

• Cours: Department of Biochemistry & Biotechnology
• Note: None
• Auteur: Sriram Seshadri (Auteur)
• Année de publication: 2011
• Pages: 95
• N° de catalogue: V188467
• ISBN (ebook): 9783656123804
• ISBN (Livre): 9783656124344
• Langue: anglais
• mots-clé: plant, extracts, pseudomonas, aeroginosa, vitro, vovo, studies