Understanding the Impact of Environmental Structure and Sub-inhibitory Antibiotic Concentrations on the Emergence of Resistance

Abstract

Antibiotic resistance represents one of the most pressing challenges in global health, threatening to render many of our most potent antimicrobial agents ineffective and lead to a resurgence of infections that are difficult to treat. The significance of this issue is compounded by the widespread presence of antibiotics at sub-inhibitory concentrations. In the environment, this is due to agricultural runoff, pharmaceutical waste, and insufficient wastewater treatment. Additionally, in clinical settings, incomplete dosing regimens and suboptimal antibiotic penetration can leave bacteria exposed to sub-inhibitory levels of drugs. Despite their critical role in the development of antibiotic resistance, the effects of sub-inhibitory antibiotic concentrations on bacterial populations remain relatively understudied. This thesis presents a comprehensive investigation into the dynamics of bacterial resistance development in Escherichia coli against ciprofloxacin at sub-inhibitory concentrations. I focus on understanding how environmental conditions, exposure concentrations, and bacterial phenotypic adaptations contribute to the emergence and proliferation of antibiotic resistance. The primary purpose of this study was to elucidate the multifaceted mechanisms by which bacteria adapt to sub-inhibitory antibiotic stress in structured and well-mixed environments, thereby informing more effective strategies for antibiotic use and resistance management. The research employed an integrative approach combining microfluidic experiments, genetic sequencing, and phenotypic analyses to explore bacterial growth dynamics, resistance patterns, and adaptive responses of E. coli under varying conditions of ciprofloxacin exposure. Exposure to sub-inhibitory levels of ciprofloxacin led to increased resistance, underscoring the risk posed by low-level antibiotic concentrations in natural and clinical settings. Cross-resistance to other antibiotics was observed, particularly in the well-mixed environment mutants, with genetic sequencing revealing prevalent resistance mechanisms. Bacteria in structured environments exhibited varied resistance mechanisms and mutation patterns compared to those in well-mixed environments, highlighting the impact of environmental structure on resistance development. Distinct phenotypic responses were identified, including persister cell formation in structured environments and population-level tolerance in well-mixed environments. Unusual, small cell phenotypes were also observed under high antibiotic stress, indicating novel bacterial survival strategies. The findings from this study highlight the complexity of bacterial resistance development, influenced by a multitude of factors including environmental conditions, antibiotic exposure concentration, genetic mutations, and phenotypic adaptations. The observed diversity in resistance mechanisms and adaptive responses underscores the need for tailored antibiotic treatment strategies that account for the specific environmental context and bacterial phenotypes involved in infections.