Pseudomonas Aeruginosa: Characteristics And Associated Infections

Overview Of Pseudomonas Aeruginosa

A gram-negative bacteria that’s often found in moist environments but thrives in many settings, Pseudomonas aeruginosa is a multidrug resistant pathogen.

Associated with various illness and hospital-acquired infections, the bacterium, from the genus Pseudomonas, has increased antibiotic resistance.

The advanced resistance to antimicrobial treatments has made Pseudomonas aeruginosa a key area of study in understanding infection. 

Studying Pseudomonas Cultures

Pseudomonas aeruginosa is ubiquitous, although it is most commonly found in moist environments. It’s thought that extensive diversity and adaptation among the bacteria has led to its proliferation, as it exists in widespread ecological niches.

A rod-shaped bacterium, Pseudomonas aeruginosa is aerobic, but is also able to grow anaerobically when around nitrate.

It can also survive a wide temperature range – from 4 degrees Celsius to 42 degrees Celsius – although it grows best at 37 degrees Celsius. Undoubtedly, this versatility contributes to the increased range of the bacteria. 

For microbial cultures, the diverse adaptability of Pseudomonas aeruginosa allows for various growth media to be used successfully. Pseudomonas isolation agar, King A, and LB Broth (among others) can be used to grow and study the bacterium. 

For this guide, we’ll grow Pseudomonas aeruginosa in Pseudomonas isolation agar.

You will need:

• Glass plates
• Pseudomonas isolation agar
• Pseudomonas aeruginosa sample
• Sterile loop

1. Begin by cleaning and sterilizing the plates, and then warming them to room temperature.
2. Pour the agar into the plates, and leave it to dry. Pay close attention to the state of the agar. Once it has dried, the specimen will need to be inoculated as soon as possible after it has been collected.
3. Use a sterile loop to streak the specimen over the surface, covering roughly one third of the agar. It’s essential to use a sterile loop, or you risk contamination from other microorganisms.
4. Incubate the plates for 24 hours, aerobically, at 37 degrees Celsius. After a day, colonies of Pseudomonas aeruginosa should appear blue-green. If there are other colors present, this likely indicates other Pseudomonas or bacteria. However, if you are using another medium, then the color may be different. See below for further details.

Notes On Developing The Culture

Although in this experiment the Pseudomonas aeruginosa bacterium should appear blue-green, other pigments are possible. Yellow-green pyoverdine, or red-brown pyorubin are also common, and might appear depending on the medium.

Grown in Cetrimide agar, Pseudomonas aeruginosa has an irregular growth, is a green-blue color, and forms a medium-sized colony. 

As well as color, be aware of the smell. Pseudomonas aeruginosa that has been grown in media is often described as having a grape smell, or the odor of fresh tortilla.

Pseudomonas aeruginosa is a non-fermenter, typically producing acids in a culture, rather than the gasses that tend to come from fermenting bacteria.

Grown in Nutrient agar, Pseudomonas aeruginosa is strongly associated with several sweet and savory odors. 

It is possible for Pseudomonas aeruginosa to grow in an anaerobic environment if nitrate is present. It will require a higher temperature of roughly 42 degrees Celsius, and it is likely to grow slower.

Due to its metabolic diversity, Pseudomonas aeruginosa can be grown in various cultures, including MacConkey agar. Instead of using the lactose inherent in this medium, Pseudomonas aeruginosa relies on peptone.

Grown this way, Pseudomonas aeruginosa has been shown to form colonies. At around 2 or 3 mm in diameter, these colonies are flat and smooth, with regular margins.

They are also colorless when grown in this medium, which is thought to be due to the lack of fermenting lactose.

Pseudomonas Aeruginosa Gram Stain

Having grown Pseudomonas aeruginosa in a culture, gram staining allows researchers to better study the characteristics of the bacteria.

Through the simple gram stain procedure, we can identify the components of the cell wall, and determine if the bacteria is gram positive or gram negative. Gram stains also allow for the analysis of the structure of the colony. 

Equipment For Pseudomonas Aeruginosa Gram Stain

Having acquired your culture grown sample, very little is needed for Gram staining. We recommend preparing roughly 3 slides, for the best results. You will need:#

• Culture grown Pseudomonas aeruginosa sample (see above)
• Glass slides
• Sterile wire loop
• Water
• Bunsen burner
• Primary stain (crystal violet)
• Mordant (Gram’s iodine)
• Decolorizer (ethanol)
• Counterstain (Safranin)

What To Do

1. Place a drop of water onto the glass slide, to prepare it for the bacteria. Using a sterile wire loop, add your sample to the drop of water.
2. Using the same sterile wire loop, smear the sample across the center of the glass slide.
3. Leave the slide to dry, and allow for the water to evaporate.
4. Heat fix by passing the glass slide through a flame briefly. Be careful not to leave the slide in the flame, or it will overheat. Passing the slide through the flame three times should heat fix the sample, without causing the glass to overheat.
5. Flood the slide with the primary stain; crystal violet. Leave for 60 seconds, before gently rinsing with water.
6. Apply Gram’s iodine, the mordant, to the slide, and allow it to sit for 60 seconds. Gently rinse with water.
7. Apply the decolorizer to the slide. Only a few drops are necessary. Gently rinse with water.
8. Flood the slide with the counterstain, Safranin. Rinse the slide with water.
9. Finish by applying a drop of immersion oil to the slide. It’s now ready to be viewed under the microscope. 

Results

Pseudomonas aeruginosa is a gram-negative bacteria, and it should appear a reddish pink after Gram staining. This reveals that the bacteria has a thin peptidoglycan cell, and is unable to hold the crystal violet stain.

Pseudomonas aeruginosa is typically from 0.5 to 0.8 um in diameter, and roughly 1.5 to 3.0 um in length. 

When viewed under a microscope, Pseudomonas aeruginosa appears as a rod-like bacteria with a single polar flagellum.

This is used to enable movement, particularly in the moisture rich areas preferred by the cell. Some strains have been shown to have several polar flagella, which can develop as a result of a hostile environment. 

The surface of the bacteria is characterized by pili, appendages similar to hairs that protrude from the surface and attach to cells.

The pili helps to stabilize the bacteria when it comes into contact with a surface, and can also assist with movement and the production of biofilm. 

From just this simple observation, we’re able to learn a lot about Pseudomonas aeruginosa. We can also observe some of the key characteristics that have made Pseudomonas aeruginosa such an effective pathogen.

Pseudomonas Aeruginosa And Infections

Pseudomonas aeruginosa is often considered to be an opportunistic pathogen, taking advantage of existing conditions. It’s responsible for many hospital acquired infections, particularly among those suffering from cystic fibrosis or burns.

However, Pseudomonas aeruginosa can also affect the immunocompetent, and is responsible for conditions such as hot tub folliculitis. Pseudomonas aeruginosa has also been shown to colonize medical devices, and can spread via infected equipment.

Hospital Acquired Infections

Hospital acquired infections, otherwise known as nosocomial infections, are infections that occur in hospital and healthcare facilities. These are infections acquired post-admission. Pseudomonas aeruginosa is a common cause of nosocomial infection.

Traumatic Burn Wounds

Pseudomonas aeruginosa is one of the leading causes of infection and sepsis for those hospitalized with traumatic burns. Able to grow within the fluids of burn wound exudates, the bacteria can cause major delays to recovery, and even result in fatalities.

Developing within the wound, the pathogen is at times able to adapt and colonize the area, leading to sepsis.

The high antimicrobial properties of Pseudomonas aeruginosa, even compared to other bacteria such as Staphylococcus aureus, have made it a major concern for treating burns. 

Pneumonia And Cystic Fibrosis

Nosocomial pneumonia infections caused by Pseudomonas aeruginosa are a high risk to patients with cystic fibrosis, and those with non-CF bronchiectasis.

It has been identified as a common agent in ventilator-associated pneumonia. The possibility of being infected by Pseudomonas aeruginosa has been shown to increase with the duration of time using a ventilator. 

In rare cases, Pseudomonas aeruginosa has also been found as an agent in community-acquired pneumonia, although this is significantly less common. 

The conditions of the respiratory tract allow Pseudomonas aeruginosa to grow, affecting the airways of both immunocompromised and immunocompetent patients.

Radial Keratotomy Surgery

Pseudomonas aeruginosa has been identified as a risk to those recovering from radial keratotomy surgery, which corrects nearsightedness. The favorable conditions of the eye allow for the growth of the bacteria.

Bloodstream Infections

Immunocompromised patients are at risk of contracting bloodstream infections such as bacteremia, with Pseudomonas aeruginosa being a common cause.

Patients contracting bacteremia due to Pseudomonas aeruginosa are shown to be at a high risk of mortality, due to the antibiotic resistance of the bacteria. 

Skin Infections

Roughly one tenth of all nosocomial infections are thought to be the result of Pseudomonas aeruginosa. However, it is also implicated in community-acquired infections.

Due to a preference for moist conditions, Pseudomonas aeruginosa is thought to be a common cause of dermatitis, and other skin rashes.

This is often the result of improper water monitoring, and a lack of quality water. These infections can typically be prevented with regular water treatment. 

Nosocomial Urinary Tract Infections

Improper cleaning of hospital equipment can lead to an increased risk of nosocomial Pseudomonas aeruginosa infections. This is a particular problem with catheters, leading to hospital-acquired urinary tract infections.

Pseudomonas aeruginosa is able to form a biofilm on the catheter surface, leading to an infection with a strong resistance to antibiotics. 

Increased Virulence Factor And Pathogenesis In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen, with several virulence factors increasing its ability to colonize the host.

Examples of Pseudomonas aeruginosa virulence factors include:

Lipopolysaccharide

Found in the outer membrane of Gram-negative bacteria such as Pseudomonas aeruginosa, lipopolysaccharides (LPS) protect the structure of the bacteria. LPS is able to induce an enhanced response from the immune system in animals.

LPS consists of three components: o-antigen, the core, and lipid A. The O-antigen is a target for the host’s antibodies, while lipid A is responsible for much of the toxicity in gram-negative bacteria, as it releases into circulation when the cell is broken down. 

Flagellum

The flagellum is a hairlike appendage that aids the mobility of the Pseudomonas aeruginosa bacteria. The polar flagellum allows the bacteria to swim in moist environments, as well as form attachments with the hosts’ epithelium.

In certain conditions, Pseudomonas aeruginosa has been observed to develop multiple flagellum, to better aid swarming. 

Pili

Pseudomonas aeruginosa has Type IV pili, hairlike structures that protrude from the surface to aid adherence to other cell surfaces. The pili are able to anchor and stabilize the bacteria, as well as assisting in movement.

Pili also plays a role in the development and formation of biofilm. Having attached to a surface, the biofilm is released, forming a much stronger seal.

Exotoxin A

Exotoxins are secreted by the bacteria, and able to damage cells. Exotoxin A is produced by Pseudomonas aeruginosa, and it inactivates eukaryotic elongation factor 2 in host cells.

Without elongation factor 2, the cells are unable to synthesize protein. This leads to tissue damage.

Proteases

Pseudomonas aeruginosa secretes proteases to act as an exotoxin, destroying extracellular structures.

Alginate

The layer of alginate produced by Pseudomonas aeruginosa allows the bacteria to grow and thrive under different conditions, adapting to both microaerobic and anaerobic environments.

The cell is able to adhere to, for example, the lungs, and grow with limited access to oxygen. When the bacteria has formed a biofilm, the immune system becomes overwhelmed. 

Antibiotic Resistance Of Pseudomonas Aeruginosa

The antibiotic resistance of Pseudomonas aeruginosa is a key consideration when assessing the number of nosocomial infections caused by the bacteria.

Pseudomonas aeruginosa has shown to be resistant to many types of antimicrobials, making an infection incredibly difficult to treat.

This resistance is largely intrinsic. The structural characteristics of the Pseudomonas aeruginosa provide it with functions that are highly advantageous when confronted with antimicrobials.

An intrinsic resistance can be observed in several functions of the Pseudomonas aeruginosa.

Outer Membrane

The low permeability of the outer membrane of the Pseudomonas aeruginosa contributes to the antibiotic resistance. With an asymmetric bilayer formed of phospholipids and lipopolysaccharides, the antibiotics have limited opportunity to penetrate the surface.

The lipopolysaccharides form an outer layer, lined with the phospholipid inner face. While this won’t completely prevent antimicrobials from penetrating the surface, it effectively slows them down. 

The outer membrane of the Pseudomonas aeruginosa also contains porins; diffusion channels in the surface. These might be a potential source of entry for targeted antibiotics, although research into this is ongoing.

Efflux Pumps

The multidrug efflux pumps are thought to be one of the key defenses against antibiotics found in Pseudomonas aeruginosa. Efflux pumps are a system that work to remove compounds out of the cell.

The multidrug efflux pumps found in Pseudomonas aeruginosa bacteria are encoded with antibiotic resistance genes. With this, the pumps are able to expel antimicrobials that are able to enter the cell.

As the pumps become overexpressed, Pseudomonas aeruginosa can develop a resistance.

As the outer membrane and efflux pumps work together, the Pseudomonas aeruginosa bacteria is able to prevent large numbers of antimicrobials from entering the cell. Any that do enter can then be effectively removed. 

Enzyme Production

A further crucial component to Pseudomonas aeruginosa antibiotic resistance is the enzyme production. These enzymes are able to degrade or inactivate antibiotics.

These enzymes, including extended spectrum β-lactamases and AmpC cephalosporinases, can render an antibiotic completely ineffective. 

Developed Antibiotic Resistance

There is more than just intrinsic resistance to the Pseudomonas aeruginosa bacteria ability to resist treatment. Acquired antibiotic resistance further allows the bacteria to evade antibiotic treatments, and to continually respond to new antibiotics.

Mutation

Mutations allow Pseudomonas aeruginosa to adapt and avoid antibiotics without causing damage to the bacteria itself. By mutating, the bacteria changes the target of the antimicrobial, avoiding its intended action.

Acquisition of various mutations can enable multidrug resistance, possibly through overexpression of efflux pumps.

Horizontal Gene Transfer

Horizontal gene transfer enables bacteria to receive genes that determine antibiotic resistance. Through this method, Pseudomonas aeruginosa bacteria are able to acquire resistance to a number of antibodies.

Horizontal gene transfer is able to spread from one species of bacteria to another, arming against an antimicrobial as it goes. 

Biofilm Antibiotic Resistance

The final mechanism that ensures that Pseudomonas aeruginosa can successfully resist most antibiotics is the production of biofilm. This is considered to be adaptive antibiotic resistance. 

Biofilms are adherent microorganisms that allow cells to stick to surfaces. Initially, the cell produces pili, which are able to form an anchor.

Over time, Pseudomonas aeruginosa will begin to produce an adhesive matrix, forming a much stronger connection. Having surrounded the bacteria cell, a biofilm can also prevent the bacteria from any damaging environmental factors, such as antibiotics. 

Testing For Antibiotic Resistance

The enhanced antibiotic resistance of Pseudomonas aeruginosa has made it an important area of research. It’s resistant to many common antibiotics that are regularly employed as a first line of treatment. 

Antimicrobial Susceptibility Testing is used to determine if a strain has developed resistance to a common antibiotic. During this testing, the Pseudomonas aeruginosa bacteria is placed into contact with an antibiotic. It is then monitored, to see if the bacteria will grow. 

In hospitals, this testing can be used to develop a treatment plan. This is considered to be a better method than treating the infection empirically. By using Antimicrobial Susceptibility Testing, it’s possible to guide the treatment. 

We have provided a method for using Mueller-Hinton agar and disk diffusion to test Pseudomonas aeruginosa.

You will need:

• Mueller-Hinton agar
• Purified water
• Autoclave
• Petri dishes
• Sterile forceps
• Antimicrobial disks
• Inoculated bacteria sample

1. Suspend and mix 38 grams of Mueller-Hinton agar in a liter of purified water. 
2. Heat the mixture for roughly 60 seconds, using frequent agitation to ensure the content is thoroughly mixed.
3. Place the mixture into an autoclave heated to 121 degrees Celsius for 15 minutes.
4. Leave the agar to cool until it reaches 45 degrees Celsius.
5. Remove the cooled agar, and pour into your Petri dishes to a depth of roughly 4 mm.
6. Leave the plates to solidify at room temperature. The pH of the plates should remain at 7.3.11 when at room temperature of 25 degrees Celsius.
7. Using your sterile forceps, place the antimicrobial disks on the inoculated bacterium. Invert the plates, and allow them to incubate for 16 to 18 hours at a temperature of 38 degrees Celsius.

This method can be used to test a variety of antibiotics. If the antibiotics are still effective against the strain of Pseudomonas aeruginosa, then the bacteria will not be able to grow.

Repetition of this method using other forms of antibiotic can create a picture of the total resistance level of the strain, as well as help devise a treatment plan. 

In hospital settings, probiotic prophylaxis can be used to prevent or delay an infection. Phage therapy is also being investigated as a potential treatment against Pseudomonas aeruginosa.

Phages are created in a sterile liquid, and are a type of virus that will attach to the walls of a cell, and inject a genome.

This replaces the information inside the bacteria, stopping the infection as the cell can no longer reproduce. Phages have been used to treat ear infections caused by Pseudomonas aeruginosa. 

Tackling Pseudomonas Aeruginosa Through Water Treatment

The opportunistic pathogen can be found in both artificial and natural environments, but it is particularly drawn to moisture.

Standing bodies of water are an area that pose a risk for Pseudomonas aeruginosa infections, particularly those that aren’t monitored for a high water quality.

To prevent the proliferation of the bacteria, bodies of water need to be regularly cleaned, replaced, or disinfected. Regular water treatment is vital, stopping Pseudomonas aeruginosa before it has an opportunity to colonize.

Due to the high antibiotic resistance of the pathogen, water treatment isn’t always simple. Instead, antimicrobial susceptibility testing should be used to determine which disinfectants are likely to have the best effect.

But it’s important to find the balance, as the treatment must also be suitable for use around humans. Disinfectants that have shown potential include ozone and iodine, as well as chloramines and chlorine. 

As well as standing bodies of water, equipment that comes into regular contact with water should be disinfected routinely. This includes hospital equipment (such as catheters), and equipment at home (such as contact lenses).

Regular handwashing is also recommended. However, the high level of Pseudomonas aeruginosa in the natural environment has meant that even practicing good hygiene can’t guarantee protection against infection. 

The structure of Pseudomonas aeruginosa enables it to become an opportunistic pathogen. Able to exist in a range of environments and temperatures, and with reduced oxygen levels, Pseudomonas aeruginosa is incredibly common.

Polar flagella enable travel, while the protruding pili helps the cell to attach to different surfaces. Although Pseudomonas aeruginosa may prefer moisture, it can make itself at home in many places.

Scientific interest in Pseudomonas aeruginosa regularly focuses on its impressive antibiotic resistance. Extensive research into these advanced properties, and how they may be negotiated, is helping us to gain a better understanding of the bacteria.

Gram staining is a quick method for determining some of the obvious features of Pseudomonas aeruginosa, while antimicrobial susceptibility testing helps create a treatment plan. 

https://www.youtube.com/watch?v=3TD9yRnFg18