The discovery of antibiotics have revolutionized modern medicine, facilitating the treatment of a variety of bacterial infections, enabled surgeries otherwise impossible to perform and increased life expectancy in all countries. However, the rapid development of resistance among microorganisms and the increasing numbers of clinical outbreaks caused by multiresistant bacteria have accelerated the need for new alternatives to antibiotics. Probiotic bacteria armed with defense systems have been studied as potential substitutes of antibiotics. These probiotic competitors can still contribute to the spread of resistance genes among microorganisms through horizontal gene transfer. The aim of this study was to investigate whether constructed CRISPR-Cas9 systems have the potential to protect probiotic bacteria against horizontal transfer of antibiotic resistance genes. Transformation, transduction and conjugation assays in strains carrying or not carrying a plasmid-bourne CRISPR-Cas9 system were performed in order to compare the frequencies of transfer of the most common resistance genes. The transformation and transduction assays demonstrated that the constructed CRISPR-Cas9 system entails a decrease in efficiency of transfer for targeted resistance genes. Moreover, it can be concluded that potentially increasing Cas9 levels by reducing its degradation results in increased prevention of horizontal gene transfer through transformation and transduction. Finally, we state that the CRISPRCas9 system does not result in protection against antibiotic resistance genes entering the cells through conjugation.

The evolution of antibiotic resistance provides a well-documented, rapid, and recent example of a selection driven process that has occurred in many bacterial species. An exhaustive collection of Moraxella catarrhalis that spans a transition to chromosomally encoded penicillin resistance was used to analyze genetic changes accompanying the transition. The population was characterized by high haplotypic diversity with 148 distinct haplotypes among 372 isolates tested at three genomic regions. The power of a temporally stratified sample from a single population was highlighted by the finding of high genetic diversity throughout the transition to resistance, population numbers that remained high over time, and no evidence of departures from neutrality in the allele frequency spectra throughout the transition. The direct temporal analysis documented the persistence, antibiotic status, and haplotypic identity of strains undergoing apparent clonal expansions. Several haplotypes that were β-lactamase nonproducers in early samples converted to producers in later years. Maintenance of genetic diversity and haplotype conversions from sensitive to resistant supported the hypothesis that penicillin resistance determinants spread to a diverse array of strains via horizontal exchange. Genetic differentiation between sample years, estimated by FST, was increasing at a rate that could cause complete haplotype turnover in less than 150 years. Widespread linkage disequilibrium among sites within one locus (copB) suggested recent mutation followed by clonal expansion. Nonrandom associations between haplotypes and resistance phenotypes provided further evidence of clonal expansion for some haplotypes. Nevertheless, the population structure was far from clonal as evidenced by a relatively low frequency of disequilibria both within sites at a second locus (M46) as well as between loci. The haplotype-antibiotic resistance association that was accompanied by gradual haplotype turnover is consistent with a hypothesis of genetic drift at marker loci with directional selection at the resistance locus.

Effective treatment of tuberculosis requires at least six months of combination therapy involving four antibiotics. Alterations in the physiological state of Mycobacterium tuberculosis during infection may reduce drug efficacy and prolong treatment, but these adaptations are incompletely defined. To investigate the mechanisms limiting antibiotic efficacy, I performed a comprehensive genetic study to identify M. tuberculosis genes and pathways important for bacterial survival during antibiotic treatment in vivo. First, I identified mutants in the glycerol kinase enzyme, GlpK, that promote survival under combination therapy. Similar glycerol catabolic mutants are enriched in extensively drug-resistant clinical isolates, indicating that these mutations may promote survival and the development of resistance in humans. A majority of these mutations are frameshifts within a homopolymeric region of the glpK gene, leading to the hypothesis that M. tuberculosis may reversibly produce drug-tolerant phenotypes through genetic variation introduced at homopolymer sites as a strategy for survival during antibiotic treatment. Second, I identified bacterial mutants with altered susceptibility to individual first-line anti-mycobacterial drugs. Many of these mutations did not have obvious effects in vitro, demonstrating that a wide variety of natural genetic variants can influence drug efficacy in vivo without altering standard drug-susceptibility tests. A number of these genes are enriched in drug-resistant clinical isolates, indicating that these genetic variants influence treatment outcome. Together, these data suggest new targets for improving therapy, as well as mechanisms of genetic adaptations that can reduce antibiotic efficacy and contribute to the evolution of resistance.

Natural microbial communities are the ultimate drivers of change in any ecosystem. Through chemical contamination of natural environments, these communities are exposed to many different types of chemical stressors; however, research on whole genome responses to this contaminant stress is limited. This research examined the stress response of a common soil bacterium, <i>Pseudomonas aeruginosa</i>, to a common environmental pollutant, pentachlorophenol (PCP). In the first part of the research, it was revealed that nutrient-limited <i>P. aeruginosa</i> is able to respond to PCP with minimal physiological damage due to the upregulation of multidrug efflux pumps. Further study of this PCP-mediated induction of efflux pumps revealed a simultaneous increase in antibiotic resistance. It was discovered that the resistance nodulation-cell division (RND) efflux pump, MexAB-OprM, in particular is responsible for the PCP-induced increase in antibiotic resistance. Both whole cell physiological indicators and whole genome analysis were used to examine the stress response of <i>P. aeruginosa</i> to PCP. Cells were grown in a chemostat at a low growth rate to simulate nutrient-limiting growth in the natural environment. Whole cell acetate uptake rates (WAUR) and viable cell counts as colony forming units (CFU) were determined as cells were exposed to increasing concentration of PCP. At the same time, changes in gene expression were examined by Affymetrix microarray technology. Results showed little change in whole-cell physiology, with no difference in WAUR and only a slight reduction in CFU. However, the microarrays revealed that over 100 genes either increased or decreased expression greater than two-fold due to the PCP exposure. In particular, multiple multidrug efflux genes were upregulated in response to the PCP. The results were validated by real time reverse transcription polymerase chain reaction (RT-PCR) for one of these genes. Further analysis of the effects of MexAB-OprM showed that this particular efflux pump is essential for the response of <i>P. aeruginosa</i> to the toxin PCP. Induction of multidrug efflux pumps is responsible for the development of antibiotic resistance in strains of <i>P. aeruginosa</i>. Therefore, it was investigated whether PCP might induce resistance to a variety of antibiotics. The research was further extended to examine the effect of a variety of organic contaminants on MexAB-OprM efflux and antibiotic resistance development. PCP, 2,4-dinitrophenol, benzoate and Roundup® all induced antibiotic resistance. However, although MexAB-OprM is required for optimal growth in the presence of all chemicals, this particular efflux pump is only involved in increased resistance with PCP. This was confirmed using RT-PCR as <i>mexB</i> expression was induced by PCP, but not by the other three chemicals. A long term generational study on the effects of PCP did not result in a stable antibiotic-resistant phenotype; however, RT-PCR showed that <i>mexB</i> induction is a direct result of PCP exposure and can be reversed by removal of PCP. Together, these results demonstrate the necessity to understand functional responses to contaminant stress. Discovery of direct induction of multidrug efflux pumps and the resulting increase in antibiotic resistance has significant implications for environmental microbiology and public health. This research suggests that organic contamination may result in antibiotic resistance and that antibiotic resistant strains may have a survival advantage in contaminated environments. / Ph. D.

Elevated expression of proteins, such as those involved in native antibiotic resistance pathways or introduced to enable biosynthesis of a metabolic engineering target, frequently leads to increased fitness cost. This can result in reduced growth and places selective pressure on cells. In conditions where there is diversity in expression within the population, this can result in cells with higher fitness out-competing their low-fitness counterparts. In the antibiotic resistance context, differential fitness costs caused by antibiotic resistance machinery can be exploited to select against resistant bacteria. However, in biotechnology applications, introducing burdensome synthetic constructs often requires additional engineering to increase genetic stability and maintain production. In this thesis, we investigate the origin of fitness costs and strategies for either exploiting or reducing it, focusing on specific examples related to antibiotic resistance and metabolic engineering. In the resistance work, we study the multiple antibiotic resistance activator MarA and related proteins in Escherichia coli. We quantify the differential fitness cost impacts of salicylate on E. coli antibiotic resistance variants. We demonstrate that salicylate, the natural inducer of MarA, imposes a higher fitness cost on resistant cells compared to the susceptible counterparts, making it possible to bias bacterial population membership towards those cells that are susceptible. In a second study, we focus on the role of salicylate in antibiotic tolerant persister cell formation, finding that salicylate induces reactive oxygen species and consequently persistence. In the metabolic engineering parts of the thesis we first review the mechanisms of fitness cost and existing strategies to ameliorate cost and cell-to-cell variation. Next, we present a technique for reducing fitness cost while maintaining production that takes advantage of transcription factor decoy sites to regulate biosynthesis in E. coli. Using arginine production as a model system, the transcription factor decoy is able to increase production by 16-fold without detectable growth differences. Together, the thesis provides an understanding of the origins and mechanisms of fitness cost in the context of antibiotic resistance and metabolic engineering. It also introduces strategies to exploit fitness costs to select against resistant bacteria and engineering strategies to ameliorate cost while increasing production and genetic stability.

The increasing bacterial resistance to available antibiotics requires the search for new antibacterial compounds to be broadened. This study investigated the antimicrobial properties of two secondary metabolites from fluorescent pseudo monads -- syringopeptin 25A, a lipodepsipeptide produced by Pseudomonas syringae pv. syringae, and a rhamnolipid mixture produced by Pseudomonas aeruginosa. The rate of antimicrobial action was determined by monitoring the rate of uptake of propidium iodide during exposure to the compounds. Inhibition was also confirmed by the microbroth dilution method to determine the MI Cs. Both the compounds inhibited growth of Gram-positive organisms, including Mycobacterium smegmatis, staphylococci, and listeria. Inhibition of spore germination was also notable. SP 25A inhibited two multiple antibiotic strains of Staphylococcus aureus subsp. aureus and Enterococcus faecalis, while RLs failed to do so, even at 60 μg/ml. Addition of the compounds together showed a synergistic activity against Listeria monocytogenes. Neither compound was toxic to human cells in vitro at 8 μg/ml. It is postulated that both compounds exert their antimicrobial effect by forming pores in the bacterial cell membrane, but we did not observe a relation between membrane permeabilization and inhibition of growth in each case. At sub-MIC concentrations RLs did cause pores in the membrane of L. monocytogenes, while SP 25A did not. However, RLs did not inhibit cell growth, while SP 25A completely inhibited cell growth. To investigate these effects gene expression was monitored just before treating the cells with the antimicrobials, 30 min after treatment and 120 min after treatment. The gene expression profile was distinct when cells were treated with both the antimicrobials. SP 25A repressed genes related to cell division, intermediary metabolism, transcription, translation, and virulence genes. These effects were not produced when cells were treated with RLs, hence giving indications that even though both the antimicrobials may act on the same site (i.e. the cell membrane), the cellular response was different, which led to different phenotypes for growth. This work indicates that SP 25A holds promise for further development as a therapeutic agent and provides evidence that the proposed pore-forming model alone does not suffice to explain the mode of action of SP 25A.

No description available.

This study is a part of a project at Q-linea that aims to present a rapid diagnostic instrument to speed up the process of identification of pathogens and determination of MIC-values (Minimal Inhibitory Concentration) of antibiotic needed to treat patients with sepsis. Specifically, this report is aimed to describe the development and implementation of algorithms that examine susceptibility profiles ofsepsis related pathogens where the bacteria have been exposed to different antibiotics and by different lapse of concentrations. The developed algorithms are based on a clustering technique that identify inhibited growth and present the lowest concentration needed to slow down the growth of the pathogen. The implemented solution was tested on sepsis related pathogens and the determined MIC values were compared to MIC values generated with a method commonly used in healthcare today. Approximately 90% of instances were correctly classified based on data from six hours long tests which is significantly faster than the reference method which takes 16-24 hours to complete. Furthermore, each result comes with a set of quality measures for validation of the algorithm results. Although, further studies are necessary to increase the performance at the four-hour target time, and more data is needed to validate the developed quality measures.

Antimicrobial susceptibility profiles were assembled for isolates of Moraxella catarrhalis collected from the Mountain Home Veteran's Affairs Medical Center (VAMC) clinical laboratory in Johnson City, Tennessee. The goal of the study was to identify isolates for genetic characterization using comparisons of susceptibility profiles. Isolates of Moraxella catarrhalis collected from July 1984 through 1994 were analyzed for β-lactamase production using a Cefinase disk assay. A multi-drug profile consisting of 11 β-lactam antibiotics was performed on the 41 M. catarrhalis isolates. Kirby Bauer disk assays were performed for 7 cephalosporin and 4 non-cephalosporin antibiotics. In summary, 2 observations implicate more complex resistance determinants than the 2 known forms of the BRO β-lactamase. First, there was overlap in the ranges of inhibition zones. Second, several isolates had antibiotic-specific deviations from typical profiles. These data suggest either more variation in the M. catarrhalis BRO β-lactamase than described or contributions to resistance from undescribed determinants.

To combat widespread infections caused by Staphylococcus aureus, mupirocin was introduced at the Veterans Affairs Medical Center, Mountain Home, Tennessee. Soon after introduction, high-level mupirocin-resistance emerged. The rapid emergence was hypothesized to be due to conjugative transfer of the mupA resistance gene from S. epidermidis to S. aureus. Results have shown that transfer of high-level mupirocin-resistance from S. aureus donors commonly occurs. However, transfer from naturally-occurring S. epidermidis donors was not attainable. Staphylococcus epidermidis transconjugants, however, were capable of serving as donors. Further examination of non-transmissibility included PCR analysis of conjugative transfer genes (tra genes) in capable and non-capable donors. Results confirmed that capable donors possess full-length copies of selected transfer genes. Non-capable donors varied in the presence/absence of full-length copies of transfer genes, but none had all three genes. The genetic differences among non-capable donors suggest that non-transmissibility has arisen independently in different strains via gene deletions and recombinations.