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Date of Award

Summer 8-15-2015

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Multidrug-resistant (MDR) human pathogens represent a growing threat to human health. This threat is compounded by dissemination of antibiotic resistance genes from diverse microbial reservoirs. The selection of current antibiotic drugs that can clear bacterial infections with minimal human side effects is limited, and bacteria can rapidly evolve or acquire new resistance to these drugs on the order of weeks. Compounding this issue is the existence of an “innovation gap”, where drug-discovery efforts of pharmaceutical companies to screen massive libraries of natural and synthetic compounds have reached practical limits. Concurrently with drug-discovery, synthetic tailoring methods with existing drug scaffolds, cycling of existing drugs to exploit collateral sensitivity, and lower-order combination therapies have slowed, but not stopped this rise. These strategies have, in the past, been successfully employed against the major MDR Gram-negative and Gram-positive human isolates, also known as ESKAPE pathogens, comprised of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and ESBL-producing Enterobacteriaceae species. These six ESKAPE strains are collectively responsible for a substantial percentage of nosocomial infections in the modern hospital and represent the vast majority of isolates whose increasing resistance to antibiotic agents presents serious therapeutic dilemmas for physicians, not to mention the considerable economic and social burdens placed. Attenuating this increase in multidrug resistance is crucial, as the pipeline of novel antibiotic compounds is rapidly drying up.

In this work, I explore three aspects of antibiotic resistance, with particular focus on multidrug-resistant (MDR) human pathogens: 1) how antibiotic resistance genes have been disseminated in the clinic and the environment by Pseudomonas aeruginosa, a bacterial strain that can serve as a reservoir and vector of antibiotic resistance; 2) how existing antibiotic drugs can be combined to generate increased potency through synergy, and can also suppress the emergence of higher resistance in MRSA through collateral sensitivities of the components; and 3) how a novel bifunctional antibiotic resistance enzyme, AAC(6’)-Ib-cr, can overcome fitness costs incurred by its acquired resistance function against ciprofloxacin, through mutational analysis of its variants in a diverse genomic library.

We determined that the pseudomonads isolated from the clinical niche library were significantly enriched for all resistance gene functions in general, and all beta-lactamases in particular. Strikingly, these resistance genes were found on contigs with collinear resistance genes conferring resistance to multiple drug classes, and adjacent to mobilizable genomic elements like transposons and integrons. Also notable, many of the resistance genes have highest nucleotide and amino-acid identity to non-Pseudomonas species, indicating signatures of recent horizontal gene transfer (HGT). Collectively, these findings strongly suggest Pseudomonas aeruginosa to be a reservoir species and possible vector for the further dissemination of mobilizable antibiotic resistance genes.

We identified multiple triple combinations of antibiotics with high synergy against P. aeruginosa DK2. This was not unexpected in this case, as prior work suggested that drugs targeting maximally orthogonal systems in bacteria would increase the likelihood of inducing a fragile state, where the bacterium is no longer capable of performing basic metabolic functions and is killed. In contrast to orthogonal drug components composing maximally synergistic combinations in Pseudomonas, we identified a new potential therapy against MRSA N315 consisting of a combination of clinically approved drugs from three distinct generations and subclasses of β-lactam antibiotics, all targeting cell-wall synthesis: meropenem, piperacillin, and tazobactam (ME/PI/TZ). Because MRSA strains are highly resistant to most beta-lactam drugs, the remarkable synergy present in this triple combination was unexpected, and we found the synergy to derive from the differential targeting of multiple constituents of the cell wall synthesis system in MRSA, especially the allosteric triggering of the PBP2a enzyme by meropenem to open the active site for inhibition by the beta-lactams in the combination. The efficacy of the ME/PI/TZ combination in completely clearing aggressive MRSA infection in mice was also unexpected, as use of beta-lactams is not currently suggested for treating MRSA in the clinic because of the resistance conferred against beta-lactams given singly by the PBP2a enzyme.

After generating libraries of barcoded, wild-type and mutant variants of the aac(6’)-Ib-cr gene in an E. coli host, and exposing the libraries to high concentrations of kanamycin and ciprofloxacin, we found several clones that displayed increases in fitness toward both kanamycin and ciprofloxacin, thus refuting our hypothesis of increases in fitness toward one drug being anti-correlated with fitness to the other. However, we successfully generated libraries of the aac(6’)-Ib-cr gene with unique barcodes for each clone and with endogenous ribosome binding sites for proper expression of the gene variants under arabinose-inducible expression in plasmid pBAD24. Initial high-throughput sequencing runs of libraries with MiSeq were of low efficiency, likely due to the large size of the gene construct and necessary adapter sequences for hybridization to the Illumina flow cells. Our attempts to subclone the construct, in order to generate sub-libraries more amenable to high-throughput sequencing with MiSeq, were unsuccessful. But, we successfully generated circularized aac(6’)-Ib-cr constructs, bringing the necessary F and R barcodes in close proximity for short, direct sequencing with MiSeq.

In sum, we have surveyed the dissemination of antibiotic resistance across global ecological niches in Pseudomonas aeruginosa, which we may consider a reservoir and vector of antibiotic resistance genes. We have sought out new ways to treat highly MDR human pathogens and discovered triple antibiotic drug combinations that are highly synergistic against Pseudomonas and MRSA strains, and especially an unexpected triple combination of beta-lactam drugs that strongly synergize to confer high potency against MRSA infections in vitro and in vivo, and also suppress emergence of higher antibiotic resistance through reciprocal collateral sensitivities of the components. Finally, we have focused on the potential of one novel gene conferring bifunctional resistance against aminoglycoside and fluoroquinolone antibiotics, aac(6’)-Ib-cr, to assess whether this gene can acquire more mutations to become even more robust at resisting these drug classes in the clinic.

Language

English (en)

Chair and Committee

Gautam Dantas

Committee Members

Barak Cohen, Justin Fay, James Havranek, Robi Mitra, Ting Wang,

Comments

Permanent URL: https://doi.org/10.7936/K78K778Q

Available for download on Thursday, August 15, 2115

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