ANTIMICROBIAL, ANTIVIRULENCE, AND ANTIBIOTIC POTENTIATION PROPERTIES OF OHELO BERRY (VACCINIUM CALYCINUM) AGAINST SALMONELLA TYPHIMURIUM
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2023
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Abstract
Food contamination caused by pathogenic bacteria remains a significant concern in ensuring food safety. Salmonella is an important foodborne pathogen that can cause gastroenteritis in both humans and animals, posing a substantial threat to life and health. Such contamination leads to declining quantity and quality of food products and may adversely affect consumer health. The emergence and dissemination of antibiotic-resistant bacteria, fueled by the excessive and inappropriate use of antibiotics, further complicates the issue. Consequently, there is an increasing demand for the development of novel and natural antimicrobial agents, especially as the risks posed by drug-resistant bacteria seriously endanger human health. Ohelo berry (Vaccinium calycinum), an endemic and culturally significant fruit in Hawaii, is characterized by its high content of phenolic compounds. These compounds have demonstrated potent antimicrobial properties against pathogenic bacteria. This dissertation aims (1) to evaluate the antimicrobial activity and modes of action of ohelo berry against Salmonella Typhimurium; (2) to investigate the antivirulence effects of ohelo berry against S. Typhimurium; and (3) to explore the antibiotic potentiation property of ohelo berry against multi-drug resistant S. Typhimurium. In the first part, fresh ohelo berry fruits were collected from the island of Hawaii, and their bioactive compounds were extracted using an organic solvent (F0). These compounds were further separated into sugars plus organic acids (F1), monomeric phenolics (F2), anthocyanins (F3), and proanthocyanidins (F4). The minimum inhibitory concentrations (MIC) of ohelo berry F0, F1, F2, F3, and F4 against S. Typhimurium were determined as 3.125%, 12.5%, 25%, 50%, and 25%, respectively. Ohelo berry F0 significantly inhibited the physicochemical properties and biofilm formation of S. Typhimurium. The antimicrobial mode of action experiments revealed that F0 could disrupt cell membrane integrity, alter membrane potential, cause protein leakage, and interact with bacterial genomic DNA. Furthermore, all fractions induced significant damage to S. Typhimurium in varying degrees, with motility being inhibited by F1 and F4, and biofilm formation being primarily inhibited by F1. The decrease in hydrophobicity and auto-aggregation was mainly attributed to F2 and F4, respectively. Additionally, the fractions exhibited different bactericidal effects on S. Typhimurium, with membrane damage, protein leakage, membrane depolarization, and DNA binding being primarily attributed to F3, F4, F2, and F1, respectively. The findings from this part increased our understanding of S. Typhimurium’s susceptibility to ohelo berry constituents, providing a theoretical basis for applying ohelo berry in controlling S. Typhimurium infections.
The second part of this dissertation evaluated the pathogenicity and virulence gene expression of S. Typhimurium in the presence of sublethal concentrations of ohelo berry constituents. The sub-inhibitory concentrations of ohelo berry constituents showed no effects on the viability of human intestinal epithelial Caco-2 cells. The effects of ohelo berry constituents on bacterial adhesion and invasion and inflammatory response following S. Typhimurium infections under three scenarios: pre-incubation of ohelo berry with Caco-2 cells, pre-incubation of ohelo berry with bacteria, and co-incubation of ohelo berry with Caco-2 cells and bacteria. These three scenarios were applied to replicate possible conditions of bacterial infection. Results showed that the number of the adhered S. Typhimurium was significantly reduced by pre-incubation of bacteria with ohelo berry F0, F1, and F2. The invaded bacteria were significantly decreased by ohelo berry constituents to different degrees. The production of pro-inflammatory interleukin-8 by S. Typhimurium infected Caco-2 cells was significantly reduced in the presence of ohelo berry. Furthermore, viable bacterial counts were reduced to below the limit of detection (< 1 log CFU/mL) after the gastric phase in the presence of F0, and this reduction persisted through the intestinal phase. Ohelo berry F1 reduced viable bacterial counts to an undetectable level after gastric and intestinal phases (< 1 log CFU/mL). Ohelo berry F2 and F4 caused a significant reduction in viable bacterial counts (2 log CFU/mL) compared to the control after the intestinal phase. The relative expression of virulence genes (hilA/C/D, invA/E/H, and prgH/K) was significantly downregulated in the presence of sublethal concentrations of F0, F2, and F4. These findings demonstrated the potential of ohelo berry in exerting protective effects against Salmonella infection, suggesting gut health benefits.
In the last part, the interactions of ohelo berry with antibiotics against multi-drug resistant S. Typhimurium DT104 were investigated. The checkerboard microdilution assay was performed and showed that ohelo berry F4 (proanthocyanidins) exhibited the most potent synergistic effects with chloramphenicol and tetracycline, reducing MICs of antibiotics by 4- to 8-fold. Ohelo berry proanthocyanidins were able to restore the susceptibility of S. Typhimurium DT104 to antibiotics by preventing the evolution of resistance, suppressing the reactivation of antibiotic-exposed bacteria, disrupting membrane integrity, and inhibiting biofilm formation. The observed antibiotic potentiation properties of ohelo berry proanthocyanidins were due to increased outer and inner membrane permeability and inhibition of efflux pump activity. Overall, this research provided a foundation for novel therapy development using ohelo berry bioactive compounds in combination with currently available antibiotics against multi-drug resistant bacterial infections.
This study was the first to explore the impacts of ohelo berry on pathogenic bacteria. The findings increase our understanding of the antimicrobial, antivirulence, and antibiotic potentiation properties of ohelo berry against S. Typhimurium. Future research endeavors, including food product development, pharmacological analysis, clinical trials, and ecological impact assessments, are essential to fully elucidate the multifaceted role of ohelo berries. Such comprehensive studies hold the promise of enhancing our knowledge of the potential contributions of ohelo berries, particularly in the areas of nutrition, health, cultural significance, and conservation.
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Food science, Nutrition, antibiotic potentiation, antimicrobial activity, bacterial infection, ohelo berry, Salmonella Typhimurium
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214 pages
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