Defense Date

6-4-2024

Document Type

Thesis

Degree Type

Master of Science

Degree Name

Biological Sciences

First Advisor

Robert Smith

Second Advisor

Aarti raja

Third Advisor

Julie torruellas Garcia

Keywords

inoculum effect, metabolism, growth rate, β-lactamase, antibiotic resistance, Escherichia coli

Abstract

2.8 million Americans are infected with antibiotic-resistant bacteria on an annual basis, leading to increased mortality and morbidity. With the incidence of infection due to antibiotic-resistant bacteria on the rise, it is critical to understand the mechanisms bacteria use to resist antibiotics. The inoculum effect (IE) is a phenomenon that has been observed for many antibiotics and bacterial species. In IE, the density of the bacterial population determines the minimum inhibitory concentration of the antibiotic (MIC) of an antibiotic; as the density of the bacterial population increases, the MIC increases. IE is highly prevalent in bacteria that express β-lactamases where higher-density populations can collectively degrade β -lactam antibiotics. Indeed, clinical studies have shown that β-lactamases expressing bacteria can cause IE in humans, leading to antibiotic failure. Despite the widespread nature of IE and β-lactamases expressing bacteria, a mechanism to account for IE has yet to be discovered. It was recently revealed that, for bacteria that do not have a resistance mechanism, growth productivity- which is the relationship between [ATP] and growth rate- can account for IE, where increasing growth productivity can eliminate IE. The expression and catalytic activity of β-lactamases alters [ATP] and growth, however, we do not know if growth productivity can account for IE in β-lactamase expressing bacteria. To address this question, we used a combination of ATP assays, growth rate assays, and MIC assays using Escherichia colicontaining a plasmid-borne copy of NDM-1, a drug resistance gene for the production of β-lactamases. When using glucose as a carbon source, we found that increasing the expression rate of β-lactamases, increased the strength of IE. However, counterintuitively, it also increased log[ATP]/growth rate, which we used as a surrogate of growth productivity. Interestingly, over a range of log[ATP]/growth rate values, we found that β-lactamase expression determines the relationship between log[ATP]/growth rate and the strength of IE. When β-lactamase was expressed at low levels, the strength of IE peaked at intermediate values of log[ATP]/growth rate, Increasing the expression of β-lactamase served to increase the linearity of the relationship between log[ATP]/growth rate and the strength of IE; increasing log[ATP]/growth rate reduced the strength of IE. Our results indicate that an ATP-altering adjuvant co-administered along with antibiotics may reduce or eliminate IE in the clinic by increasing antibiotic susceptibility in the high-density bacterial population. Which would lower the risk of driving new antibiotic-resistant strains and improve the treatment outcomes of patients.

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