Investigation of the role of oxidative stress in the evolution of Pseudomonas aeruginosa biofilms using a mutagenesis reporter

Abstract

Antibiotics commonly used against Pseudomonas aeruginosa biofilms are believed to elicit the production of reactive oxygen species (ROS) as part of their bactericidal mechanism. Depending on their concentration, ROS can either induce cell death by damaging proteins, lipids, and DNA or cause mutations that can lead to antibiotic resistance. Hence, antioxidant defenses, including catalase enzymes, are crucial for bacterial survival during antibiotic treatment. The absence of the major catalase KatA in P. aeruginosa may increase mutagenesis and accelerate the development of resistance when exposed to subinhibitory concentrations of antimicrobials.

This project aimed to establish different fluorescent transcriptional reporters for nfxB mutant detection in a ΔkatA P. aeruginosa background to investigate the role of oxidative stress in biofilm mutagenesis. An in-frame deletion of the katA gene encoding the catalase KatA was introduced into two strains via allelic exchange, facilitating the construction of chromosomal CFP-, GFP-, and YFP-based monitor strains. These reporters allowed us to follow the occurrence of nfxB mutants, which overexpress the MexCD-OprJ multidrug efflux pump, using confocal laser scanning microscopy (CLSM).

No significant differences in the percentage of nfxB mutants were observed between wild-type and ΔkatA monoculture flow-cell biofilms after 24 hours of exposure to sublethal ciprofloxacin levels. However, in co-culture flow-cell biofilms, nfxB mutants from a ΔkatA background dominated the resistant subpopulation under the same treatment. Automated CLSM tracking of the same microcolonies throughout treatment suggests that KatA deficiency influences nfxB mutagenesis earlier than anticipated.

In conclusion, this study indicates that oxidative stress may contribute to the development of resistance within the initial hours of low-dose antimicrobial exposure. Future research on novel therapeutic strategies could explore targeting ROS effects to mitigate the spread of antibiotic-resistant bacteria.

Popular Abstract

Breathing Oxygen Has Its Consequences

Pseudomonas aeruginosa is a tricky human pathogen that causes persistent infections by forming biofilms—clusters of cells that stick together to withstand the immune system and antibiotic attacks. Normally, bacteria produce small but harmful molecules called reactive oxygen species (ROS) in limited quantities as byproducts of their respiration. When antibiotics come into play, bacterial cells are disturbed in a way that makes them ‘breathe’ more and thus produce more ROS.

However, ROS can be a double-edged sword: at high levels, they kill bacteria, but at lower levels, they induce mutations that can lead to antibiotic resistance. Luckily for them, bacteria have a multi-layered armory to protect themselves against ROS damage. One of the most important defenses in P. aeruginosa is a catalase enzyme named KatA, which breaks down hydrogen peroxide into water and oxygen, preventing ROS formation.

We hypothesized that bacterial cells lacking KatA would be more prone to acquire mutations and become resistant when treated with low doses of ciprofloxacin, an antibiotic commonly used to treat P. aeruginosa biofilm infections. To test this, we removed the gene responsible for producing KatA in P. aeruginosa and modified the cells to distinguish between resistant (green, cyan, or yellow fluorescence) and sensitive (red fluorescence) bacteria.

Using a powerful microscope, we tracked these different colors in both mutant (lacking KatA) and wild-type (having KatA) biofilms exposed to subinhibitory concentrations of ciprofloxacin, which do not kill bacteria but slow down their growth. After 24 hours, there were no differences in the proportion of resistant cells. Yet, further experiments led us to believe that significant events were likely happening in the KatA-deficient mutants within the first few hours of antibiotic exposure.

Our findings suggest that ROS-derived stress (also known as oxidative stress) may be involved in antibiotic resistance during prolonged treatments of biofilm infections, where sublethal doses of antibiotics prevail. Identifying new antimicrobial targets, such as oxidative stress responses, is crucial in combating the rising threat to public health posed by multi-resistant bacteria.

Master’s Degree Project in Molecular Biology, 60 credits, 2024
Department of Biology, Lund University

Advisors: Oana Ciofu and Jens Bo Andersen
Costerton Biofilm Center, Department of Immunology and Microbiology, University of Copenhagen

• author: Otero Sande, Maria
• supervisor: Oana Ciofu; Jens Bo Andersen
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20232
• year: 2024
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9176233
• date added to LUP: 2024-10-07 14:57:26
• date last changed: 2024-10-07 14:57:26