When visiting a hospital, be sure to wash your hands. Within healthcare settings, patients are at risk for healthcare-associated infections (HAIs). This is a danger particularly for patients on ventilators, those with wounds from surgery, and those with catheters. These HAIs can be deadly and it is vital to practice good hygiene in healthcare settings to prevent the spread of these infections.
Figure 1: A medical professional washing their hands to protect patient health. Credit: Defense Visual Information Distribution Service (2017).
One of the most common HAIs is an infection by Pseudomonas aeruginosa. P. aeruginosa is a gram-negative, rod-shaped bacterium that can travel using a single flagellum. To get energy, it primarily breaks down organic compounds using respiration. It prefers to use oxygen as an electron acceptor during respiration, but it can use nitrate instead if no oxygen is available. According to the CDC, P. aeruginosa bacteria can be found commonly in the environment around us, particularly in soil and water. In healthcare settings it can be spread by contaminated hands and equipment. The CDC estimates that in 2017, multi-drug resistant P. aeruginosa caused 32,600 infections in hospitalized patients and 2,700 deaths.
P. aeruginosa is an opportunistic pathogen, meaning it usually doesn’t infect healthy individuals but it can cause disease if a person has a weakened immune system. You can imagine this as ants trying to build a colony in your house. Normally it may be difficult for the ants to get and stay inside your house, but if your windows won’t close and you run out of ant traps, you lose important defenses and it becomes much easier. Due to lowered immune defenses, hospital patients are commonly at higher risk for opportunistic infections.
Figure 2: P. aeruginosa bacteria. Credit: Center for Disease Control (2019).
P. aeruginosa is particularly common and dangerous for those with cystic fibrosis. Cystic fibrosis (CF) is a genetic disorder which results in a thick, sticky mucus building up in organs like the lungs and pancreas. This mucus can provide a hospitable place for bacteria to grow, causing serious infections. One of the most common bacteria that causes infections in people with CF is Pseudomonas. The cystic fibrosis foundation estimates that of all adults with CF, about 60% have a Pseudomonas infection.
Figure 3: The airways of a healthy lung compared to a lung with cystic fibrosis. There is a much thicker layer of mucus which allows biofilms to form more easily. Credit: Hollman, Perkins, & Walsh. British Society for Immunology.
P. aeruginosa can be treated with antibiotics, however this is made more difficult by two obstacles: antibiotic resistance and the formation of biofilms. Strains of bacteria which develop resistance to antibiotics will continue to grow and spread, making bacterial infections much more difficult to treat. Many bacteria, including P. aeruginosa, will also form multicellular community structures called biofilms. One common biofilm you’re probably familiar with is plaque; if you don’t brush your teeth this biofilm can build up, trapping bacteria in your mouth. By clustering together and forming a biofilm, bacterial cells gain protection from many different stressors, including antibiotics.
In order to treat patients with P. aeruginosa infections, we must first be able to study it effectively. According to a hypertextbook on biofilms from Montana State University, many antibiotics and other products used against bacteria have been developed by studying planktonic cells, which float around as individuals and are not part of a colony. As a result, these products were not as effective as may have been expected when they were applied to biofilms.
We will explore one study that sought to investigate a better model to study P. aeruginosa: “Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In Vivo-Like Characteristics”. Previous models had not been able to produce small biofilm aggregates which do not attach to epithelial cell surfaces. These types of aggregates are found in wound beds and in the lungs of CF patients.
In order to produce similar aggregates in an in vitro model, this study used alginate beads.
Figure 4: An alginate bead used for this study. A microsensor is being used to measure oxygen concentration. Credit: Sønderholm et al. (2017). Appl Environ Microbiol.
A good in vitro model allows researchers to conduct studies outside a living organism by simulating the in vivo conditions found in a living organism. These beads provided a steep oxygen gradient, which imitates the oxygen concentration gradient found in in vivo infections. When P. aeruginosa was grown in these beads, it formed small aggregates with no surface attachment, similar to the dense aggregates seen in the lungs of CF patients.
Figure 5: (A) Alginate-encapsulated P. aeruginosa aggregates. (B) P. aeruginosa aggregates from the lung of a CF patient. Credit: Sønderholm et al. (2017). Appl Environ Microbiol.
One of the researchers’ main focuses was investigating metabolic activity and respiration in the beads. They found that deeper within the bead, P. aeruginosa growth decreased, likely because there is less oxygen available. In vivo, host immune cells gather around bacterial aggregates and consume large amounts of oxygen, however P. aeruginosa can perform anaerobic respiration using a different electron acceptor, for example nitrate. You can think of nitrate as a slightly leaky cup. You probably want a cup that doesn’t leak, but if a slightly leaky cup is all that you have you could still use it, though you might lose a little bit of your drink. Similarly, using nitrate as an electron acceptor does not yield as much energy as oxygen, but it still yields some energy, which the cell can use to grow and reproduce.
Since nitrate is present in the human body, researchers added nitrate to the alginate model. In beads with added nitrate, the difference in the growth of P. aeruginosa close to the surface vs deeper in the bead was eliminated. They found that after 48 hours, average aggregate volume was greater in nitrate-supplemented beads, and total biomass was greater in nitrate-supplemented beads after 24 hours. In beads without nitrate, there was more growth at the surface, while in beads with nitrate the aggregates were able to form much deeper in the bead. They also found that P. aeruginosa cells growing in alginate had a lower respiration rate than free-floating planktonic cells.
Figure 6: Alginate-encapsulated P. aeruginosa aggregates growth (A) without nitrate (B) with nitrate. Credit: Sønderholm et al. (2017). Appl Environ Microbiol.
In addition to measuring the rates of respiration and the size and location of the aggregates, researchers compared gene expression between planktonic and alginate-encapsulated P. aeruginosa, as well as between nitrate supplemented beads and beads without nitrate. In order to do this, they mapped which genes are ‘on’ for each of these groups. They found that many genes were expressed differently between the groups. For instance, they found that some denitrification genes were mildly upregulated in P. aeruginosa in the nitrate-supplemented beads. When comparing the bacterial cells in the alginate model to planktonic cells, they found that genes for translation, post translational processes, degradation, ribosomal proteins, and iron regulation were downregulated in the beads. This makes sense because many of these genes are associated with cell growth. Overall, when compared to free-floating cells, cells that formed aggregates in the alginate beads demonstrated lower respiration rates and downregulation of genes which suggests less metabolic activity. This is a result of the hypoxia stress, or stress from low oxygen conditions, that the P. aeruginosa in the alginate are undergoing. By producing a steep oxygen gradient and providing nitrate as an alternative electron acceptor, nitrate supplemented alginate beads mimic the conditions that P. aeruginosa face in human patients.
In addition to looking at growth and metabolic activity, the researchers investigated antibiotic resistance. They found that as long as the bacteria was allowed to grow for a while first, the bacteria aggregates in the alginate bead were less susceptible to antibiotics than the planktonic bacteria. Planktonic P. aeruginosa and P. aeruginosa that were newly embedded in alginate were susceptible to antibiotics, but over time, the P. aeruginosa embedded in alginate became more resistant to antibiotics.
Overall, this study presented a new model to study small, dense aggregates of P. aeruginosa bacteria, like those seen in wound beds and the lungs of CF patients. They found that there was lower metabolic activity, less respiration, and increased antibiotic resistance in the aggregates than in planktonic bacteria. They also found that adding nitrate allowed for growth deep within the beads despite the oxygen concentration gradient. Future studies could use this model to examine the formation of aggregates and the use of anaerobic respiration in P. aeruginosa. Testing antibiotics against P. aeruginosa in this model may also be more effective than testing it against planktonic bacteria in demonstrating which antibiotics are actually strong enough to kill bacteria in biofilms. One topic that could have been expanded on in this article was the limitations of the model and areas for future improvement. Further studies could build on this model and try to simulate in vivo conditions even more closely. By determining which antibiotics can effectively kill P. aeruginosa in biofilms similar to those formed under in vivo conditions, researchers can develop more effective treatments to fight this opportunistic pathogen and protect at-risk patients.
About the Author:
Marlena Klein ‘23 is a Biological Sciences and Computer Science double major with a nexus in Data Science. They are interested in studying disease and public health.
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