The objective of the work reported in this paper is one of a two-fold nature. The first objective is to develop a disk-plate sensitivity test that is more rapid than that of existing methods. The second requisite is that the materials, techniques, interpretation, and reporting of results be the sane as those required for the disk-plate method described in the Difco Manual.

Antibiotic resistance is a quickly emerging public health crisis, accounting for more than 700,000 annual global deaths. Global human antibiotic overuse and misuse has significantly expedited the rate at which bacteria become resistant to antibiotics. A renewed focus on discovering new antibiotics is one approach to addressing this crisis. However, it alone cannot solve the problem: historically, the introduction of a new antibiotic has consistently, and at times rapidly, been followed by the appearance and dissemination of resistant bacteria. It is thus crucial to develop strategies to improve how we select and deploy antibiotics so that we can control and prevent the emergence and transmission of antibiotic resistance. Current gold-standard antibiotic susceptibility tests measure bacterial growth, which can take up to 72 hours. However, bacteria exhibit more immediate measurable phenotypes of antibiotic susceptibility, including changes in transcription, after brief antibiotic exposure. In this dissertation I develop a framework for building a paper-based cell-free toehold sensor antibiotic susceptibility test that can detect differential mRNA expression. I also explore how long-term lab evolution experiments can be used to prospectively uncover transcriptional signatures of antibiotic susceptibility. Paper-based cell-free systems provide an opportunity for developing clinically tractable nucleic-acid based diagnostics that are low-cost, rapid, and sensitive. I develop a computational workflow to rapidly and easily design toehold switch sensors, amplification primers, and synthetic RNAs. I develop an experimental workflow, based on existing paper-based cell-free technology, for screening toehold sensors, amplifying bacterial mRNA, and deploying sensors for differential mRNA detection. I combine this work to introduce a paper-based cell-free toehold sensor antibiotic susceptibility test that can detect fluoroquinolone-susceptible E. coli. Next, I describe a methodology for long-term lab evolution and how it can be used to explore the relationship between a phenotype, such as gene expression, and antibiotic resistance acquisition. Using a set of E. coli strains evolved to acquire tetracycline resistance, I explore how each strain's transcriptome changes as resistance increases. Together, this work provides a set of computational and experimental methods that can be used to study the emergence of antibiotic resistance, and improve upon available methods for properly selecting and deploying antibiotics. / 2023-03-17T00:00:00Z

Negli ultimi decenni l'utilizzo degli antibiotici a scopo terapeutico o come promotori della crescita nell'allevamento animale ha portato alla comparsa e alla diffusione di microrganismi resistenti. In questo contesto, la presenza di Lattobacilli (LAB) antibiotico resistenti non rappresentano di per sé un rischio clinico. Tuttavia la possibilità che essi ma possono essere veicolo di geni codificanti l'antibiotico-resistenza verso batteri patogeni presenti negli alimenti o nel tratto gastro-intestinale umano (inclusi enterococchi, streptococchi e listeria), costituisce un possibile rischio per la salute umana che deve essere attentamente valutato. Obiettivo di questo lavoro è stato quello di valutare attraverso metodi di indagine fenotipica con le tecniche delle microdiluizioni in brodo, Etest e disc-diffusion, i livelli di antibiotico resistenza per le specie S. thermophilus e L. plantarum verso gli antibiotici tetraciclina, eritromicina, clindamicina, streptomicina, gentamicina, ampicillina. Ceppi atipici appartenenti alla specie S. thermophilus sono stati sottoposti ad analisi genetiche con lo scopo di caratterizzare e localizzare i geni responsabili della resistenza. E' stato inoltre testato il possibile trasferimento orizzontale dei geni di antibiotico resistenza nativi da S. thermophilus verso i batteri Gram-positivi E. faecalis e Listeria monocytogenes. In alcuni ceppi di S. thermophilus resistenti si sono infine osservati e studiati particolari caratteri fenotipici ( fitness ) correlati alla presenza delle determinanti genetiche di antibiotico resistenza nell'ospite batterico. / In the last decades, the use of antibiotics in human therapy or in animal husbandry as growth promoters has induced the development and the diffusion in antibiotic resistant micro-organisms. In this context antibiotic resistant Lactic Acid Bacteria (LAB) do not represent a clinical risk in themselves. However, the possibility that S. thermophilus cultures might transfer antibiotic resistance genes to pathogenic species either present in food or in the gastrointestinal tract (including enterococci, streptococci and listeria) represents a potential clinical risk that needs to be carefully evaluated. The aim of this study was to evaluate by means of phenotypic methods (microdilution, E-test, disc-diffusion) the levels of antibiotic resistance for S. thermophilus and L. plantarum species against the antibiotic tetracycline, erythromycin, clyndamicin, streptomycin, gentamycin and ampicillin. The atypical resistant S. thermophilus strains were subjected to genetic analyses in order to characterise and to localise the antibiotic resistance determinants. Furthermore the ability of the resistant S. thermophilus strains in transferring the antibiotic resistant determinant was assessed in mating experiments using as recipients the Gram-positive bacteria E. faecalis and Listeria monocytogenes. In same resistant S. thermophilus strains, special bacterial fitness related with the presence of the antibiotic resistance determinants in the bacterial hosts were observed and studied.

Antimicrobial resistance is considered by many prominent researcher and scientist as a profound global health crisis that us humans must face in the next decade. It is threatening the effectiveness of these once-reliable weapons against bacterial infections and leaving us susceptible to pathogenic agents. The indiscriminate overprescription of antibiotic in healthcare and animal husbandry, has led to an increased emergence of “super bugs”— a resistant strain of bacteria that were once susceptible to antibiotic—. The escalating creation of those resistant bacteria has been coupled with a proliferation of research papers that seek to explain the working mechanism of antibiotics and their efficacy on the bacterial pathogens, however these efforts often fall short of explaining the impact that antibiotics has on the bacterial metabolism. This project utilizes an established technology, specifically silicone nano-wire ion-selective field-effect transistor in an innovative approach to discern alteration in the metabolic pathways induced by various antibiotics. The methodology involves measuring extracellular acidity of the tested culture and converting it to an electrical signal to extract valuable information about the metabolic process of the bacteria, and how is altered in the presence of antibiotics. Empirical observations pertaining bacteriostatic antibiotics suggests comprehensive suppression of metabolic pathways, encompassing the efflux transition from acetyl-CoA to acetate, resulting an elevated pH level in cultures treated with bacteriostatic agents relative to their wild-type counterparts. Our experimental data also indicates a shift in bacterial metabolic and physiological responses to bactericidal antibiotic-induced stress which include an increased respiration rate, and a heightened activity of the TCA cycle in the test group with bactericidal antibiotics, causing acetate uptake from the medium and decelerating the acidification of the treated culture compared to the wild-type. The results clearly demonstrate a successful utilization of the chip to further study the effects that antibiotics have on bacteria and the interplay between bacterial metabolism and antibiotic efficacy.