To Örebro University

Biocides are crucial in industrial applications to minimize microbial growth and prevent product spoilage. Water-based construction coatings are susceptible to microbial contamination during manufacturing and storage and this adversely impacts product properties, reduces shelf-life and leads to substantial commercial losses. The future trend to lower the biocide concentrations in water-based coatings raises concerns about the emergence of biocide-resistant microbes. This study aims to identify and characterize the biocide-resistant microbe isolated from construction water-based coating materials to better understand its mechanisms of resistance. A total of 63 samples were collected from spoiled products, raw materials, and water from a manufacturing facility, and Pseudomonas oleovorans P4A were identified in all biocides-treated samples. A comparison between a P. oleovorans reference strain, 1045, and the P4A isolate revealed distinct colony morphology, growth rate and sensitivity to biocides and antibiotics. The P4A isolate was 3-fold more resistant to 5-chloro-2-methyl-isothiazolin-3-one (CMIT) and 1.5-fold more resistant to benzothiazolinone (BIT) compared to the reference strain. Conversely, it was 1.4-fold more sensitive to methylisothiazolinone (MIT) compared to the reference strain. No cross-resistance to antibiotics was observed. Metabolomic analysis using liquid chromatography combined with high-resolution mass spectrometry of lipids and polar metabolites showed that P4A had a relatively higher amount of lipids, while 1045 had a relatively higher amount of polar metabolites identified. A significant difference in lipid composition, specifically in diacylglycerol, phosphatidic acid, phosphatidylcholine, and phosphatidylserine was observed between P. oleovorans strains 1045 and P4A. These distinctions highlight increased lipid metabolism in P. oleovorans P4A and this may contribute to its adaptation to biocides. Microbial resistance can directly affect the effectiveness of these products, leading to an increased need for frequent maintenance and replacement, safety concerns, and environmental implications.

The thesis investigates the resistance mechanisms of Pseudomonas oleovorans strains, with an overarching goal of exploring biocide-free alternatives in construction materials and developing efficient formulations with minimal biocide content. The research focuses on the industrially significant P4A isolate, offering a detailed analysis of its unique characteristics compared to the reference strain 1045. Key findings reveal the superior resistance and tolerance of P4A to standard biocides commonly used in industrial coatings, such as BIT and CMIT, as demonstrated through minimum inhibitory concentration (MIC) analysis.

Whole genome sequencing (WGS) was employed to explore the genetic basis of biocide resistance. While no specific resistance genes or mutations aligned with existing databases, notable genomic differences suggest potential genetic contributors to antimicrobial resistance. The study also highlights the role of Serine/Threonine protein kinases in bacterial adaptation, with mutations in these kinases influencing survival and metabolic pathways.

Metabolic profiling provided insights into the effects of biocide exposure on bacterial metabolism, identifying changes in key metabolites such as tetradecenoic acid, 5-Hydroxyindole-3-acetic acid (5-HIAA), and amino acids. Pathway analysis revealed significant alterations in stress response, energy generation, and protein synthesis pathways, with a specific focus on peptidoglycan biosynthesis in the biocide-resistant strain P4A.

Additionally, the thesis examines the emissions of volatile organic compounds (VOCs) from industrial products treated with biocides and biocide-free alternatives. The findings demonstrate that both biocide-free and traditionally biocide-preserved products effectively protect against microbial contamination. However, while traditional biocide-treated products may exhibit slightly higher VOC emissions, these emissions are more significantly influenced by raw materials, such as solvents and plasticizers, rather than biocides alone.

Biocides were shown to contribute substantially to the long-term durability and microbial protection of products. By selecting low-VOC biocides and optimizing formulations, manufacturers can achieve a balance between microbial efficacy and environmental impact. The results indicate that biocide-free products offer notable environmental advantages, while biocide-preserved products remain crucial for ensuring microbial safety in industrial applications.

Biocide resistance poses a significant challenge in industrial processes, with bacteria like Pseudomonas oleovorans exhibiting intrinsic resistance to traditional antimicrobial agents. In this study, the impact of biocide exposure on the metabolome of two P. oleovorans strains, namely, P. oleovorans P4A, isolated from contaminated coating material, and P. oleovorans 1045 reference strain, were investigated. The strains were exposed to 2-Methylisothiazol-3(2H)-one (MI) MIT, 1,2-Benzisothiazol-3(2H)-one (BIT), and 5-chloro-2-methyl-isothiazol-3-one (CMIT) at two different sub-inhibitory concentrations and the lipids and polar and semipolar metabolites were analyzed by ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry UPLC-Q-TOF/MS. Exposure to the BIT biocide induced significant metabolic modifications in P. oleovorans. Notable changes were observed in lipid and metabolite profiles, particularly in phospholipids, amino acid metabolism, and pathways related to stress response and adaptation. The 1045 strain showed more pronounced metabolic alterations than the P4A strain, suggesting potential implications for lipid, amino acid metabolism, energy metabolism, and stress adaptation. Improving our understanding of how different substances interact with bacteria is crucial for making antimicrobial chemicals more effective and addressing the challenges of resistance. We observed that different biocides trigged significantly different metabolic responses in these strains. Our study shows that metabolomics can be used as a tool for the investigation of metabolic mechanisms underlying biocide resistance, and thus in the development of targeted biocides. This in turn can have implications in combating biocide resistance in bacteria such as P. oleovorans.