Pseudomonas putida is a Gram-negative, rod-shaped, saprophytic soil bacterium that belongs to the fluorescent group of Pseudomonas species . It is widely distributed in various ecological habitats, such as soil, water, plants, animals, and human beings. It has a versatile metabolism and can degrade a wide range of organic compounds, including aromatic hydrocarbons, alcohols, sugars, and amino acids. It can also produce different forms of fluorescent pigments, such as pyoverdine and pyoluteorin.

Pseudomonas putida is generally considered a non-pathogenic bacterium that rarely causes infections in humans . However, some strains of P. putida can act as opportunistic pathogens in immunocompromised individuals, such as newborns, cancer patients, and people with cystic fibrosis or AIDS . The common sites of infection include the bloodstream, skin, soft tissues, and lungs . The infections can be serious and life-threatening if not treated promptly and appropriately .

Pseudomonas putida was first described by Trevisan in 1889. The genus name `putida` is derived from the Latin term `putida`, meaning stinking or fetid, indicating the occurrence of the bacteria in spoiled food items and the aromatic odor on solid media. Pseudomonas putida is considered an evolutionary group that includes several other species based on the common 16S rRNA sequences. P. putida is further classified into two biovars; biovar A and biovar B.

Pseudomonas putida is one of the most studied and exploited bacteria in biotechnology and bioremediation. It has the ability to decompose various pollutants, such as oil spills, pesticides, herbicides, and heavy metals. It can also produce useful compounds, such as biodegradable plastics, antibiotics, enzymes, and biosurfactants. Moreover, it can be used as a biocontrol agent to protect plants from pathogenic microorganisms.

Pseudomonas putida is a remarkable bacterium that has a great potential for environmental and industrial applications. It is also a model organism for studying bacterial physiology, genetics, and evolution. In this article, we will provide an overview of the classification, habitat, morphology, cultural characteristics, biochemical characteristics, virulence factors, pathogenesis, clinical manifestation, lab diagnosis, treatment, prevention, and industrial uses of Pseudomonas putida.

The classification of bacteria into different orders, families, and genera is crucial in the fields of environmental microbiology and epidemiology. Traditionally, the classification was made on the basis of phenotypic and metabolic characteristics of the organisms like morphology, cultural characteristics, and biochemical testing. More recently, the basis of classification has been changed to the analysis of the nucleic acid components. The most common basis for the classification of bacteria includes DNA sequences via methods like DNA fragment analysis and DNA sequence analysis. At the genus and species level, however, 16S rRNA gene sequence analysis is adopted.

Pseudomonas putida belongs to the genus Pseudomonas, which is a large and diverse group of Gram-negative rod-shaped bacteria that are widely distributed in nature. The genus name Pseudomonas means "false unit" in Greek, indicating that the bacteria were initially mistaken for fungi due to their similar morphology. The genus Pseudomonas is divided into several groups based on phylogenetic analysis of 16S rRNA gene sequences. The groups are numbered from 1 to 16, with group 1 being the most closely related to the type species Pseudomonas aeruginosa.

Pseudomonas putida is considered an evolutionary group and includes several other species based on the common 16S rRNA sequences. These species are P. alkylphenolia, P. alloputida, P. monteilii, P. cremoricolorata, P. fulva, P. parafulva, P. entomophila, P. mosselii, P. plecoglossicida and several genomic species that are not yet validly defined as new species. P. putida is further classified into two biovars; biovar A and biovar B. There are about 103 strains in biovar A and about nine strains belonging to biovar B. The biovars differ in their ability to utilize certain carbon sources and their susceptibility to antibiotics.

The following is the taxonomical classification of P. putida:

• Domain: Bacteria
• Phylum: Proteobacteria
• Class: Gammaproteobacteria
• Order: Pseudomonadales
• Family: Pseudomonadaceae
• Genus: Pseudomonas
• Species: Pseudomonas putida

Pseudomonas putida is a saprophyte that can be found in various ecological habitats where there is oxygen, such as soil (mostly rhizosphere), drains, fresh and saltwater, and animate surfaces of plants, animals, and human beings . The ability of an organism to survive in different habitats depends on seasons and the adaptive mechanism adopted by the organism. These organisms have extraordinarily broad niches as a result of their metabolic and competitive capabilities. They can use a variety of metabolic compounds and can withstand large ranges of abiotic challenges.

Even though P. putida and P. fluorescens tend to be present in similar habitats, the occurrence of P. putida is more in household sites which is due to the occurrence of P. putida in sewage and wastewater. P. putida is not isolated from most high-temperature sites that can harbor P. fluorescens and P. aeruginosa due to the inability of P. putida to survive at higher temperatures like 42°C. Most strains of P. putida have a broad global distribution with some species being isolated from soil samples in and around lakes in Antarctica. However, there are some strains that have a more restricted distribution as a result of a less versatile metabolism.

In animals and humans, P. putida occurs as non-pathogenic species, but some strains can act as opportunistic pathogens in immunocompromised individuals and newborns. P. putida are obligate aerobes and thus, require oxygen-rich environments for growth and development. Some members of the P. putida group belong to the normal inhabitants of plant parts like roots and leaves.

Some possible additional sentences are:

• P. putida can also be found in some food products like cheese, milk, meat, and vegetables where it can cause spoilage or produce off-flavors.
• P. putida can also colonize medical devices like catheters, respirators, and implants where it can cause nosocomial infections or biofilm formation .
• P. putida can also degrade organic pollutants like hydrocarbons, pesticides, aromatic compounds, and caffeine in soil and water environments where it can be used for bioremediation or biotransformation purposes .

Pseudomonas putida is a rod-shaped, non-spore-forming, Gram-negative bacterium that has a size of 0.5 to 1.0 µm in length . The length of the cells might vary depending on the age of the cell on culture media as young cells tend to be longer. Some cells might also have a variable width to length ratio depending on the culture conditions. Some cells are longer and broader and might look filamentous.

Pseudomonas putida is a motile bacterium that has multiple polar flagella on its outer surface . The flagella have a waveform that is usually 2 to 3 wavelengths long. The flagella are driven by a motor present at the base which moves the filament in a helical way. The flagella enable the movement of bacteria through swimming motion.

In addition to the flagella, Pseudomonas putida also has fimbriae or pili on its outer surface, which help in movement as well as attachment. The pili facilitate the binding of bacteria to the host cell surface or other surfaces. Both of these structures are crucial in the movement and colonization of different habitats by P. putida.

The outermost covering of Pseudomonas putida is the cell envelope, which is composed of three distinct layers; an outer membrane, a peptidoglycan layer, and a cell membrane. The peptidoglycan layer is responsible for protecting the bacteria against other microbes and antibacterial agents. The cell membrane is made up of a lipid bilayer, which can change its fluidity according to the outer environmental conditions.

The number of proteins in Pseudomonas putida ranges between 3748-6780, whereas the GC content of the genome is about 60%. Pseudomonas putida belongs to the fluorescent group of Pseudomonas species and produces a fluorescent pigment pyoverdine . Pyoverdine is produced by Pseudomonads to collect iron from the environment. Pseudomonas putida does not, however, produce phenazine pigments.

Pseudomonas putida can grow on various artificial culture media as it has minimal nutritional requirements. The optimum temperature for growth is between 30-35°C, and the optimum pH is between 7-8. The bacteria are obligate aerobes and require oxygen for growth. P. putida produces a fluorescent pigment called pyoverdine, which gives a greenish color to the colonies under UV light. However, it does not produce phenazine pigments or denitrify nitrate.

The following are some cultural characteristics of P. putida on different culture media:

• Nutrient Agar: P. putida forms smooth white colonies with entire edges and convex shapes on nutrient agar. The colonies are circular, butyrous, glistening, nearly opaque, and pale green with darkened centers. The colonies may produce a greenish color on the media under sunlight or UV radiation due to the diffusion of pyoverdine pigment.
• Cetrimide Agar: Cetrimide agar is a selective medium for Pseudomonas species as it contains cetrimide, which inhibits the growth of other bacteria. P. putida grows well on cetrimide agar and forms dry and flat colonies with a circular circumference and lobular edges. The colonies are opaque and greenish due to the diffusion of pyoverdine pigment on the media.
• MacConkey Agar: MacConkey agar is a differential medium that distinguishes lactose-fermenting and non-fermenting bacteria based on the color change of the pH indicator neutral red. P. putida is a lactose non-fermenter and does not change the color of the media. It forms round and flat colonies that are colorless or pale pink. However, the colonies may produce a greenish color around them due to the diffusion of pyoverdine pigment on the media.

The biochemical characteristics of P. putida are based on the metabolic activities and enzymatic reactions of the bacteria. These characteristics help in the differentiation of P. putida from other Pseudomonas species and other Gram-negative bacteria. The biochemical characteristics of P. putida can be tabulated as follows:

One of the important biochemical tests to distinguish P. putida from other fluorescent Pseudomonas species is the gelatin hydrolysis test, which is negative for P. putida but positive for P. aeruginosa and P. fluorescens. Another important test is the ability to grow at 41°C, which is negative for P. putida but positive for P. aeruginosa and P. fluorescens.

Pseudomonas putida is a non-pathogenic bacterium that is found in environmental sources like air, water, and soil. The organism colonizes different parts of the animal body as an indigenous bacteria, but can rarely cause nosocomial infections in immunocompromised individuals and newborns. The most important attribute is the ability of P. putida to cause infections is its ability to survive at 37°C. It can also withstand a low temperature of 4°C and thus can survive in blood and other clinical samples stored for further use. There are different attributes and structures that support the mechanism of disease production by P. putida. P. putida is not as virulent as other fluorescent Pseudomonas species like P. aeruginosa and P. fluorescens, but it does, however, has some common factors like antibiotic resistance. The exact mechanism of disease or pathogenesis of P. putida is not yet understood, but it is known that antibiotic resistance and biofilm formation play an important part in the process. Some of the common virulence factors associated with infections caused by P. putida can be described as follows:

• Flagella and pili: Flagella and pili represent the structural component of P. putida involved in the attachment and movement of bacteria through the tissues. P. putida has more than one flagella that enable the movement of bacteria via swimming motion. The flagella are driven by a motor present at the base which moves the filament in a helical way. Similarly, the pili present on the outer surface of the bacteria help in the binding of bacteria to the host cell surface. Both of these structures are crucial in the movement and colonization of the host tissue surface by P. putida. In addition to helping in colonization, flagella and pili are also important in the formation of biofilm by enabling the binding of several cells to one another.

• Quorum Sensing and Biofilm: Quorum sensing is an important mechanism of regulating various cells of the Pseudomonas population to assist the process of disease progression. Quorum sensing is responsible for the production of alginates that facilitate the accumulation of extracellular material and cells to form biofilms on animate and inanimate surfaces. One of the predisposing factors of biofilm formation is the presence of medical devices like catheters and respirators. The formation of biofilm helps in the protection of bacteria against the host immune system as well as antibiotic components.

• Secondary metabolites: Pseudomonas putida is among the few fluorescent species of Pseudomonas that are capable of producing secondary metabolites with antibacterial properties. These products include compounds like hydrogen cyanide and rhizoxin, which help the bacteria compete with other microbial agents. Hydrogen cyanide is crucial to fight against plant pathogens as it disrupts the intake of water and minerals by the competing microorganism. In the case of mammalian cells, the production of hydrogen cyanide by P. putida is regulated by hcnABC genes which are upregulated in the absence of oxygen.

• Lipopolysaccharides: The outer cell envelope of P. putida is composed of lipopolysaccharides that protect the bacteria against phagocytosis and lysis. Besides, the lipid A present on the membrane also induces cytotoxic activity and further assists protection against phagocytosis. The lipopolysaccharides also cause excessive stimulation of the immune system, resulting in the release of factors like IL-8 and TNF-α. It has been discovered that excessive inflammatory response is the primary cause of disease production in different organs.

• Proteases: The production of proteases like elastases and phospholipases are also involved in tissue damage and facilitating the dissemination of bacteria to different areas. Phospholipase is involved in the degradation of phospholipids which form an important constituent of the lipid component of the body. One of the products of phospholipid degradation is diacylglycerol which has toxigenic activity against host cells by inducing the production of metabolites like protein kinase and arachidonic acid. These metabolites further induce an inflammatory response and cause tissue damage.

Pseudomonas putida is a non-pathogenic bacterium that is commonly found in soil, water, and plant surfaces. However, it can occasionally cause opportunistic infections in immunocompromised individuals, especially in hospital settings. The pathogenesis of P. putida infections is not well understood, but it involves several factors that facilitate the entry, colonization, tissue damage, and biofilm formation of the bacteria.

Entry and Colonization

The entry of P. putida into the human body can occur through different routes, such as:

• Contaminated blood transfusion or intravenous fluids
• Wounds, burns, or surgical sites
• Medical devices, such as catheters, respirators, or bronchoscopes
• Inhalation of aerosols or aspiration of contaminated water

Once inside the body, P. putida can attach to various host surfaces, such as epithelial cells, mucosal membranes, or extracellular matrix components. The attachment is mediated by surface structures, such as flagella, pili, and lipopolysaccharides (LPS). Flagella and pili are also involved in the motility and chemotaxis of the bacteria, which enable them to reach their preferred niches. LPS is a component of the outer membrane that protects the bacteria from phagocytosis and complement-mediated lysis.

Tissue Damage

After colonizing the host tissues, P. putida can cause tissue damage by producing various enzymes and toxins that degrade the host cell components or induce inflammatory responses. Some of these factors are:

• Proteases: P. putida can produce elastase and alkaline protease that degrade elastin, collagen, fibrinogen, and immunoglobulins. These enzymes can also facilitate the dissemination of bacteria to neighboring tissues or the bloodstream.
• Phospholipase C: This enzyme hydrolyzes phospholipids in the host cell membrane, resulting in cell lysis and release of inflammatory mediators.
• Hydrogen cyanide: This toxin inhibits cellular respiration and causes cytotoxicity and apoptosis. It also interferes with the oxidative burst of phagocytes and reduces their killing activity.
• Pyoverdine: This is a siderophore that chelates iron from the host and transports it to the bacteria for their growth. Iron deprivation can impair the host immune system and increase susceptibility to infections.

Biofilm Formation

P. putida can also form biofilms on various surfaces, such as medical devices, wounds, or mucosal membranes. Biofilms are complex communities of bacteria embedded in a matrix of extracellular polymeric substances (EPS) that provide protection from environmental stresses, such as antibiotics, host defenses, or nutrient limitation. The EPS of P. putida biofilms consists mainly of polysaccharides (such as alginate, Pel, and Psl), extracellular DNA (eDNA), and proteins (such as lectins). The formation of biofilms is regulated by quorum sensing (QS), a mechanism of cell-to-cell communication that coordinates gene expression in response to population density. QS also modulates the production of virulence factors and antibiotic resistance in P. putida biofilms.

Pseudomonas putida is a non-pathogenic bacterium that rarely causes infections in humans. However, some strains of P. putida can act as opportunistic pathogens in immunocompromised individuals, such as newborns, cancer patients, and people with HIV infection or cystic fibrosis. The clinical manifestation of P. putida infections depends on the site and severity of the infection, as well as the underlying condition of the patient. Some of the common clinical manifestations of P. putida infections are:

• Bacteremia: This is a serious and potentially life-threatening condition where P. putida enters the bloodstream and causes systemic infection. Bacteremia can occur as a complication of other infections, such as soft-tissue infections, respiratory infections, or urinary tract infections, or due to the transfusion of contaminated blood or fluids. Bacteremia can cause symptoms such as fever, chills, hypotension, shock, and multiorgan failure. Bacteremia caused by P. putida is often resistant to many antibiotics and requires prompt and aggressive treatment.
• Soft-tissue infections: P. putida can cause skin and soft-tissue infections, such as cellulitis, abscesses, ulcers, necrotizing fasciitis, and wound infections . These infections can occur due to trauma, burns, surgery, or medical devices that breach the skin barrier and allow the entry of bacteria. Soft-tissue infections caused by P. putida can cause symptoms such as pain, swelling, redness, pus discharge, and tissue necrosis. These infections can also spread to the bloodstream and cause bacteremia.

• Respiratory infections: P. putida can cause respiratory infections, such as pneumonia, bronchitis, tracheobronchitis, and lung abscesses . These infections can occur due to inhalation of contaminated aerosols or aspiration of oral secretions or gastric contents. Respiratory infections caused by P. putida can also occur in hospital settings due to the use of contaminated bronchoscopes or ventilators. Respiratory infections caused by P. putida can cause symptoms such as cough, sputum production, dyspnea, chest pain, and fever. These infections can also lead to respiratory failure or bacteremia.

  Lab Diagnosis of Pseudomonas putida

The laboratory diagnosis of Pseudomonas putida infections involves the isolation and identification of the bacteria from the clinical samples, such as blood, pus, or tissue. The identification is based on the morphological, biochemical, immunological, and molecular characteristics of the bacteria. The following are the detailed methods of laboratory diagnosis of P. putida from clinical samples:

Preliminary identification

The methods of preliminary identification of P. putida involve the isolation of bacteria from the clinical samples by growing them on artificial culture media. The colony morphology produced by the bacteria provides some information about their identity. For example, P. putida produces smooth white colonies with entire edges and convex shapes on nutrient agar, and opaque and greenish colonies on cetrimide agar. The colonies also produce a greenish fluorescence under UV light due to the production of pyoverdine pigment.

The isolation is followed by the microscopic observation of the organism to observe its morphology. P. putida is a Gram-negative rod-shaped bacterium with one or more flagella and pili on its surface.

In order to confirm the presence of P. putida in the clinical sample, biochemical tests specific to P. putida are performed. Some of the important biochemical tests that differentiate P. putida from other Pseudomonas species are:

• Gelatin hydrolysis test: P. putida is unable to hydrolyze gelatin, whereas P. aeruginosa and P. fluorescens can.
• Growth at 41°C: P. putida cannot grow at 41°C, whereas P. aeruginosa and P. fluorescens can.

Immunological methods

Immunological methods are based on the detection of proteins and enzymes that are specific to the bacteria by using techniques like enzyme-linked immunosorbent assay (ELISA) and Western blotting. These techniques use antibodies that bind to the antigens present on the bacterial surface or in their secretions.

Besides, other rapid tests can also be performed to identify the bacteria based on the presence or absence of antigens and other similar structures. For example, a latex agglutination test can be used to detect the presence of pyoverdine antigen in the bacterial culture.

Molecular methods

Molecular methods are based on the analysis of nucleic acid components, such as DNA or RNA, that are unique to the bacteria by using techniques like polymerase chain reaction (PCR) and DNA sequencing. These techniques amplify and sequence specific regions of DNA or RNA that can be used to identify the bacteria.

Molecular methods are more reliable and accurate than preliminary and immunological methods as they can detect even small amounts of bacteria in the sample and can differentiate between closely related species.

One of the common molecular methods used for the identification of P. putida is 16S rRNA gene sequence analysis, which compares the sequence of a part of the ribosomal RNA gene with a database of known sequences from different bacteria.

Another molecular method that can be used for the identification of P. putida is DNA fragment analysis, which compares the size and pattern of DNA fragments obtained by digesting the bacterial DNA with restriction enzymes with a reference standard.

Molecular methods can also be used to detect antibiotic resistance genes or plasmids in P. putida by using specific primers or probes that target these genes or plasmids.

Treatment of Pseudomonas putida

Pseudomonas putida infections are usually treated with antibiotics, but the choice of antibiotics depends on the site and severity of the infection, as well as the susceptibility of the bacteria. Antibiotic resistance is a common problem with P. putida, as the bacteria can acquire plasmids that encode resistance genes. Therefore, antibiotic susceptibility testing should be performed before initiating therapy.

Some of the antibiotics that are effective against P. putida include:

In addition to antibiotic therapy, some other measures that may help in the treatment of P. putida infections are:

• Removal of infected devices: If the infection is associated with medical devices such as catheters, respirators, or implants, they should be removed as soon as possible to prevent further spread of bacteria.
• Supportive care: Depending on the condition of the patient, supportive care such as fluid replacement, oxygen therapy, blood transfusion, or organ support may be required.

• Infection control: To prevent transmission of P. putida in hospital settings, infection control practices such as hand hygiene, use of gloves and gowns, disinfection of equipment, and isolation of infected patients should be followed.

  Prevention of Pseudomonas putida

Pseudomonas putida is an opportunistic pathogen that can cause infections in immunocompromised individuals and hospital settings. Therefore, prevention of P. putida infections requires proper hygiene and infection control measures, both in the community and in the health care facilities. Some of the preventive actions and ways to reduce the risk of P. putida infections are:

• Wash your hands often. This is the best way to avoid getting pseudomonas and other germs. Use soap and water or an alcohol-based hand sanitizer, especially before and after touching wounds, catheters, or other medical devices .
• Rinse fruits and vegetables before eating. Even salad greens should be given a good wash to remove any possible contamination by P. putida or other bacteria.
• Clean your water bottles. Sterilize with boiling water between each use or use disposable bottles to prevent the growth of P. putida or other bacteria.
• Avoid unclean pools and hot tubs. P. putida can cause skin infections like folliculitis or ear infections like otitis externa if you swim in contaminated water . Check the water quality and chlorine levels before entering a pool or a hot tub, and shower with soap after getting out of the water. You should also remove swimming garments and dry your ears well after swimming.
• Ask questions about your medical care. If you are hospitalized or undergoing any medical procedure, ask your health care providers about the steps they take to prevent infections by P. putida or other bacteria. Make sure they wash their hands, wear gloves and coats, and use sterile equipment . If you have a wound, a catheter, or a respirator, ask how to care for them and when they should be changed.
• Take care of your health. If you have a chronic condition like cystic fibrosis, HIV/AIDS, diabetes, or cancer, follow your treatment plan and monitor your symptoms closely . If you have a weakened immune system, avoid contact with people who are sick or have skin infections. If you develop any signs of infection, such as fever, chills, pain, redness, swelling, pus, or foul-smelling discharge, seek medical attention promptly.

• Follow the guidelines for biotechnology workers. If you work with P. putida strains in a laboratory or an industrial setting, follow the biosafety protocols and wear appropriate personal protective equipment. Avoid contact with contaminated materials or waste, and dispose of them properly. Report any accidental exposure or spillage to your supervisor and seek medical advice if needed.

  Industrial Uses / Applications of Pseudomonas putida

Pseudomonas putida is a versatile bacterium that can be found in various ecological habitats and can utilize a wide range of organic compounds as carbon and energy sources. Due to its remarkable metabolic and adaptive capabilities, P. putida has been exploited for various industrial applications, ranging from bioremediation to biocatalysis. Some of the common industrial uses of P. putida are:

• Bioremediation: P. putida can degrade various environmental pollutants, such as aromatic hydrocarbons, naphthalene, styrene, toluene, and phenol . The bacteria can also convert styrene oil into the biodegradable plastic PHA, which can be further degraded by other microorganisms. P. putida has been used as a soil inoculant to reduce the naphthalene contamination in soil and as a biosensor to detect the presence of toxic compounds.
• Biocatalysis: P. putida can produce various enzymes and metabolites that have industrial value, such as hydrogen cyanide, rhizoxin, phenazine, rhamnolipids, terpenoids, polyketides, and non-ribosomal peptides . These compounds have antibacterial, antifungal, anticancer, or surfactant properties and can be used as biocontrol agents, pharmaceuticals, or biosurfactants. P. putida can also catalyze the synthesis of chiral compounds, such as amino acids and alcohols, which are important for the production of fine chemicals and drugs.
• Bioeconomy: P. putida can produce various bio-based chemicals from renewable feedstocks or waste streams, such as lignin, glycerol, glucose, or fatty acids . Some of the chemicals produced by P. putida include succinate, 2-hydroxyacid, 3-hydroxypropionic acid, adipic acid, muconic acid, and polyhydroxyalkanoates (PHAs) . These chemicals can be used as building blocks for the synthesis of bioplastics, biopolymers, or biofuels.

Pseudomonas putida is a promising industrial cell factory that can be genetically engineered and optimized for the production of desired products. Novel tools and techniques in systems biology, synthetic biology, and metabolic engineering have enabled the system-wide understanding and streamlined genomic engineering of this bacterium . The applications of P. putida are expected to expand in the future as more metabolic pathways and regulatory networks are discovered and manipulated.

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