BLT Stages

Abstract Bachelor Project FBT 2020-2021: What is the influence of commensal bacteria on the immune response, induced by Pseudomonas aeruginosa in epithelial cells of the lung?

Chronic lung diseases, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease (COPD) have a high incidence worldwide. Chronic inflammation plays an important clinical role in these respiratory conditions. Overactive inflammatory responses on infections with pathogenic bacteria are a prominent factor in this matter.
Conventional anti-inflammatory treatments are often not efficient enough and cause numerous side effects with long-term use. This shows the importance of finding new alternative treatment options for chronic respiratory inflammation. 
In this report we investigate what role several nonpathogenic commensal bacteria from the lung microbiome can have in fighting chronic respiratory inflammation. For this, 3 commensals from the respiratory tract are used and earlier investigations already showed the anti-inflammatory properties of some of these commensals (Rigauts, 2020). It is also investigated which bacterial metabolites or postbiotics cause this desirable inflammatory effect. Furthermore, synergetic effects between combined commensals are considered. In this way the anti-inflammatory effect can possibly be achieved by using a smaller number of bacteria, to keep the bacterial load in the lungs as small as possible.
During the experiment we use 3D models of A549 alveolar epithelial cells to mimic the in vivo situation of the human lungs. These cells are transfected with a luciferase reporter gene, that causes them to emit light when the NFkB inflammatory pathway is activated. The cells are infected with Pseudomonas aeruginosa and subsequently with the commensals or combinations of them. After incubation, it is possible to determine the anti-inflammatory influence of the commensals, based on the amount of emitted luminescence.
Results showed significant reductions of NFkB activation in lung cells infected with both
P. aeruginosa and mono-cultures of the commensals or combinations of them. This proves the anti-inflammatory potential of the used commensals and allows further investigation and even development of innovative treatments for patients with chronic respiratory diseases, who are often chronically infected with pathogens such as
P. aeruginosa.  

 

Abstract Bachelor Project FBT 2018-2019: Therapeutic potential of the human microbiome for acne vulgaris: study on the inhibition of the pro-inflammatory response of keratinocytes

A recent study at the Laboratory of Pharmaceutical Microbiology demonstrated that certain commensal Gram-positive bacteria have an anti-inflammatory effect against inflammation in lung epithelial cells. This research will evaluate whether this also applies to skin inflammation, such as in the context of acne vulgaris. There are two candidate anti-inflammatory bacteria used in this study. Both bacteria are part of the human microbiome. This will be determined by evaluating whether the candidate anti-inflammatory bacteria are able to dampen in vitro inflammation of keratinocytes (HaCaT cell line) induced by Pseudomonas aeruginosa lipopolysaccharide (LPS).

Following exposure of the keratinocytes to LPS for 4 of 24h, the supernatant is used to quantify the pro-inflammatory cytokines (IL-8 and IL-1α). This will reflect whether the candidate anti- inflammatory bacteria influence inflammation of the keratinocytes. But since false negative or false positive results can be obtained, the following control experiments are performed: determination of bacterial adhesion and human cell death. Via a bacterial adhesion assay, it is investigated whether the lipopolysaccharide has an inhibitory, stimulating or no effect on the adhesion of the bacterium to keratinocytes.

Next, human cell death is examined by measuring the concentration of released lactate dehydrogenase. A healthy monolayer must not have more than 20% human cell death. If this is higher, this can again give a distorted image. LPS has no effect on the adhesion of the bacteria and no high values of lactate dehydrogenase release have been observed. These results are important so that no false results are obtained when quantifying the cytokines. After 4 or 24 hours, the bacteria significantly diminished IL-8 production by the keratinocytes in response to LPS. However, no production of IL-1α was observed after four hours.

Because there is no production of IL-1 α, it is necessary to incubate longer in the future (48 hours). But it is also advisable to examine other skin cell lines, such as sebocytes. Furthermore, another more relevant pro-inflammatory stimulus must be used. This is because Propionibacterium acnes, which affects the pathology of acne vulgaris, does not contain LPS. It is opted to use the bacterium in its entirety for future experiments.

 

 

Cystic fibrosis (CF) is a genetic disease in which bacterial lung infections have a major impact on the life expectancy of the patients. The lungs of CF patients are colonized by a collection of microorganisms, called the CF lung microbiome that contains known pathogens and commensal bacteria. One of the most important life threatening bacteria is Pseudomonas aeruginosa (P. aeruginosa), which causes chronic lung infections. P. aeruginosa can form biofilms, which decrease its antibiotic susceptibility. In addition, upon initial colonization of the CF lungs by P. aeruginosa, this bacterium becomes gradually dominant which leads to a decreased bacterial diversity and richness and contributes to a poor disease outcome.

The aim of this study is to gain novel insights into the invasion/ dominance behavior of P. aeruginosa in an artificial CF lung microbiome by assessing if the invasion/dominance is strain-dependent. The two main goals of this study are:

• To investigate the biofilm formation of different P. aeruginosa strains in the presence of an artificial CF lung microbiome
• To determine the invasion of different P. aeruginosa strains in an established multispecies biofilm of an artificial CF lung microbiome.

Biofilm formation of six different P. aeruginosa strains in the presence of an artificial CF lung microbiome was evaluated through plating on selective media at different time points. The artificial lung microbiome contained five commonly co-isolated bacteria (Staphylococcus aureus, Streptococcus anginosus, Achromobacter xylosoxidans, Rothia mucilaginosa, Gemella haemolysans). Then, using statistical analysis, it was determined which P. aeruginosa strain became dominant at each time point and if differences in abundance were observed between the different P. aeruginosa strains.

It was noted the P. aeruginosa strain AA2, an early CF lung isolate, was more dominant, invasive and abundant in an artificial lung microbiome compared to other strains.

This study demonstrates that the dominance and invasion of P. aeruginosa is strain dependent. An early CF isolate (AA2) was more dominant and invasive than late CF isolates, and it will be of interest for future research to potentially confirm this for other isolates. Hence, bacterial factors that contribute to the dominance of P. aeruginosa could be identified which may be a new starting point for therapeutic intervention.