Genotypic and antimicrobial resistance gene analysis, and the development of rapid diagnostic tests, for ß-lactam-resistant Gram-negative bacteria

Bacterial resistance to antimicrobial agents is not new—for millions of years bacteria have evolved resistance mechanisms to counteract the antimicrobial agents produced by competing bacteria and fungi. The impact of antimicrobial resistance on human health, however, is fairly new and was first realized in the 1940s when bacterial resistance to the first clinically used antibiotic, penicillin, was discovered. Since then the antimicrobial and antimicrobial resistance arms race between scientists and bacteria has ensued, to the point where there is a resistance mechanism to virtually every clinically used antibacterial agent. The prevalence of antimicrobial resistance is projected to increase, such that scientists fear we are nearing the post-antibiotic era, where even simple bacterial infections will be impossible to treat with currently available drugs./nThe Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) strive to educate the community, scientists, medical professionals, and policy makers on the problems associated with antimicrobial resistance. In 2013, the CDC released a report—Antibiotic Resistance Threats in the United States—detailing the organisms and resistance mechanisms that require action now. More recently, in 2017, the WHO released a similar priority list for antibiotic-resistant bacteria. At the top of the CDC and WHO lists were Gram-negative bacteria (GNB) with carbapenem resistance, and also GNB producing extended-spectrum ß-lactamases (ESBLs). These organisms are GNB with resistance to broad-spectrum ß-lactam antibiotics, including resistance to the last-resort ß-lactam drugs, the carbapenems. In its report, the CDC described four core actions that should be taken to prevent or slow the spread or occurrence of antimicrobial resistance. These included: 1) preventing infections to reduce the occurrence and spread of resistance, 2) improving surveillance of antimicrobial-resistant infections, 3) improving antibiotic prescribing practices and promoting antimicrobial stewardship, and 4) developing new antimicrobial agents and new diagnostic tests for antimicrobial-resistant bacteria./nThe dissertation, herein, focuses specifically on issues relating to ß-lactam-resistant GNB. It addresses the need for better surveillance and understanding of the GNB responsible for ß-lactam-resistant infections, as well as the need for rapid diagnostic tests that detect ß-lactam-resistant GNB, specifically in regards to urinary tract infections (UTIs)./nIn the first chapter, we assess the lineages of uropathogenic E. coli (UPEC) causing ß-lactam-resistant and susceptible community-acquired urinary tract infections (CA-UTIs), to understand the contribution of specific lineages, drug-resistance genes, and plasmids to the prevalence and dissemination of ß-lactam-resistant CA-UTIs in a community. We analyzed 273 consecutively collected UPEC isolated from patients presenting to a university health center with symptoms of UTI. UPEC were subtyped by MLST and fimH typing to establish lineage, and were tested by PCR and sequencing for carriage of ß-lactamase genes and plasmid incompatibility/replicon groups. We found that the distribution of lineages/sublineages among ß-lactam-resistant and susceptible UPEC was different, indicating that selective pressure from ß-lactam drugs is not exerted equally on UPEC. A limited set of circulating UPEC sequence types, sublineages, ß-lactamase genes, and Inc type plasmids likely harboring these genes, contributed to a substantial proportion of ß-lactam-resistant infections in this community. Understanding factors that affect the prevalence and dissemination of antimicrobial resistance may provide insights into strategies to slow the spread of resistance, and inform better empirical treatment decisions. Additionally, population-based studies like these provide essential information for the development of diagnostic tests that target antimicrobial resistance mechanisms./nThe development of non-nucleic acid-based strategies for detecting antimicrobial resistance directly from urine specimens are the focus of the second and third chapters. In the second chapter, we describe our first milestone towards development of a lateral flow assay (LFA) for detecting ß-lactam resistance in GNB causing CA-UTIs. The aim of this project is to develop an LFA that detects clinically relevant ß-lactamases found in GNB causing CA-UTIs, to create a diagnostic test that would inform physicians whether their patients could safely receive narrow-spectrum ß-lactam drugs for treatment. LFAs are rapid antibody-based tests; as such, extensive antibody testing is an essential component in their creation. In this chapter, we describe the development of a sensitive and specific anti-CTX-M sandwich ELISA to universally detect the CTX-M ß-lactamases. The CTX-M ß-lactamases are a challenging target for antibody-based and nucleic acid-based approaches, since these ß-lactamases are a relatively sequence-diverse class of enzymes. Sandwich ELISAs represent the first major milestone in our antibody development workflow, and the CTX-M ß-lactamases represent one of several important ß-lactamase targets for our diagnostic test./nIn the third chapter, we investigate the utility of a biochemical-based strategy, DETECT, as a diagnostic tool to identify ß-lactamase activity in Gram-negative uropathogens. The aim of this project was to develop a rapid diagnostic test that detects the activity of a variety of clinically relevant ß-lactamases, to provide physicians with resistance information that would guide the selection of appropriate ß-lactam therapy for patients with suspected UTI. In this chapter, we stringently studied the first-generation DETECT system to define its capacity to detect different classes of ß-lactamases. We found that DETECT was able to identify the activity of expanded-spectrum beta-lactamases, namely CTX-M and CMY. Insights gained from this work are directing future iterations of the DETECT diagnostic system, to reach alternative ß-lactamase targets that could impact clinical decision-making.

Advisor: Riley, Lee W.