Browsing by Subject "Antibiotic resistance"

Now showing 1 - 7 of 7
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    The biodegradation and microbiological impacts of micropollutants in methanogenic communities
    (2012-12) McNamara, Patrick Joseph
    Pervasive usage of chemicals generates micropollutants throughout the environment. Anaerobic environments in particular accumulate high levels of hydrophobic micropollutants, and it is estimated that over 200 metric tons of micropollutants are discharged with biosolids each year. It is important to understand how treatment processes impact the fate of micropollutants as well as understand how micropollutants impact microbiological communities so that environmental risks can be minimized. This research elucidated the impact of an emerging treatment process, thermal-hydrolysis coupled to mesophilic anaerobic digestion (TH-MAD), on the fate of nonylphenol ethoxylates as well as the impacts of triclosan and perfluorooctane sulfonate (PFOS) on methanogenic community structure and function. The TH-MAD process inhibited biodegradation of nonylphenol ethoxylates to nonylphenol relative to MAD with no pretreatment. Indeed, the ratio of nonylphenol to the sum of nonylphenol ethoxylates + nonylphenol only increased by 24.6±3.1% in TH-MAD reactors compared to a 56% increase following MAD treatment. While post-aerobic treatment did reduce the sum of nonylphenol ethoxylates + nonylphenol, and concomitantly reduced estrogenicity, this research implied that source control is likely the most efficient option for removing these micropollutants. Triclosan is another wide-spread micropollutant that is persistent under anaerobic conditions. Triclosan is an antimicrobial agent that could therefore impact environmental systems that rely on healthy functioning of microorganisms. Methanogenic communities with no previous exposure to triclosan were able to adapt to triclosan at environmentally relevant levels and maintain function. When previously-exposed communities were exposed to triclosan at 4x current detected environmental levels, community structure shifted and methane production was inhibited. These levels of triclosan also selected for mexB, a gene that confers multidrug resistance, in previously unexposed communities. Lastly, PFOS was found to directly impact methanogenic communities and augment the impacts of triclosan in long-term exposure studies (140 days), but not in short-term (14 day) exposure studies.
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    A bistable genetic switch controls antibiotic resistance transfer in Enterococcus faecalis.
    (2011-08) Chatterjee, Anushree
    The recent rise in microbial drug resistance is a growing challenge for future therapy of bacterial infections. Increased prevalence of antibiotic resistance in bacteria is an outcome of evolution via natural selection. However, the built-in design feature of bacteria to transfer DNA containing antibiotic resistance both within the same species and across species is the main culprit for the spread of drug resistance. One of the main factors driving the rise of drug resistant microbes is the transfer of antibiotic resistance genes present on mobile plasmids between donor and recipient cells via the mechanism of conjugation. In order to combat microbial drug resistance, novel strategies need to be developed to block such transmission of antibiotic resistance. In this work, the gene regulatory components involved in transfer of tetracycline resistance confers plasmid pCF10 between plasmid-carrying donor cells and plasmid-deficient recipient cells in bacterium Enterococcus faecalis is investigated. In the native state the donor cell exists in an OFF or conjugation-incompetent state. A pheromone released by the recipient cells serves as the chemical trigger for switching the donor cell from OFF to an ON or conjugation-competent state. The onset of conjugation is tightly regulated via multi-layered regulation offered by two-key genes prgQ and prgX present on pCF10 in response to the pheromone secreted by recipient cells. Using mathematical modeling and experimentation, we describe a novel mechanism of gene-regulation due to transcriptional interference and sense-antisense RNA interaction as a result of convergent transcription in the prgX/prgQ operon. We demonstrate that such a multi-layered gene-regulatory mechanism confers the system a bistable genetic switch controlling conjugative gene transfer between donor and recipient cells. A similar regulatory advantage offered by convergent transcription in attributing a bistable switch-like behavior in the scbA-scbR operon controlling antibiotic production in S.coelicolor is also investigated. Both mathematical model and experiments demonstrate that donor cells also control response to pheromone by changing the number of copies of pCF10 plasmid inside the cell. Cells with higher copies show increased robustness of the bistable switch and lower sensitivity to pheromone. Once bistable genetic-switch is ON, expression of genes encoding various proteins involved in the transfer of the plasmid are induced, however, this also causes production of an inhibitor of conjugation, thus giving rise to negative feedback loop which causes the donor to return to OFF state. Modeling and experimental analysis of dynamic response to induction indicate that this negative feed-back loop causes a brief surge of expression of the entire operon. We show that the inhibitor signaling peptide for pCF10 based system, acts as quorum-sensing signal with the role of turning-OFF conjugation at a population-wide scale. An interplay of positive and negative feedback loops allows the donor cell to quickly transition between ON and OFF states and is critical both for the transfer of plasmid and survival of the cell. Studying both the turning-ON and turning-OFF mechanisms of the switch allows identification of potential drug targets for blocking transmission of antibiotic resistance for use in future therapy.
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    Determining Mechanism Used By Bacteria To Evade Bactericidal Conditions, By The Way Of Resistance And Persistence
    (2020-02) Ostrer, Lev
    The three main chapters of this thesis (Ch2,Ch3,Ch4) address the topics of antibiotic resistance, persistence and thymineless death respectively. The antibiotic resistance chapter (Ch2) focused on determining mutation hierarchy and predicting mutational paths different species of bacteria take to reach clinical levels of fluoroquinolone resistance. Based on the findings described in the chapter, many bacterial species appear to have a distinct mutational pattern, that can be used to predict future development of fluoroquinolone resistance.The persistence-focused chapter (Ch3) links the clinically relevant antibiotic resistance mutations to the high-persistence phenotype, while identifying some of the underlying molecular mechanisms used by bacteria to induce persister state. Finally, the thymineless death chapter (Ch4) focuses on identifying the mechanism of killing bacteria via thymine limitation. In this chapter we show that premature deposition of ftsZ rings triggered by prolonged thymine starvation plays a crucial role in determining bacterial survival. Altogether, this thesis work attempts to gain insights into strategies bacteria have at their disposal to evade antibiotic-mediated death, while at the same time attempting to offer real world solutions. It is my hope that this body of work, in combination with others’ research, will someday usher changes that ultimately will help to reign in the rampant spread of antibiotic resistance.
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    Population-level antibiotic treatment policies in the setting of antibiotic resistance: A mathematical model of mass treatment of Helicobacter pylori in Mexico
    (SAGE, 2017-10-23) Alarid-Escudero, Fernando
    Purpose: Helicobacter pylori (H. pylori) is the strongest known risk factor for gastric cancer and peptic ulcer disease. Programs under consideration in high risk countries to prevent H. pylori- related diseases via broad population treatment could be complicated by increasing levels of antibiotic resistance (ABR). We evaluate the impact of different mass-treatment policies on H. pylori infection and ABR in Mexico using a mathematical model. Methods: We developed an age-structured, susceptible-infected-susceptible (SIS) transmission model of H. pylori infection in Mexico that included both treatment-sensitive and treatment- resistant strains. Antibiotic treatment was assumed to either clear sensitive strains or induce acquired resistance. In addition, the model included the effects of both background antibiotic use and antibiotic treatment specifically intended to treat H. pylori infection. Model parameters were derived from the published literature and estimated from primary data. Using the model, we projected H. pylori infection and resistance levels over 20 years without treatment and for three hypothetical population-wide treatment policies assumed to be implemented in 2018: (1) treat children only (2-6 year-olds); (2) treat older adults only (>40 years old); (3) treat everyone regardless of age. Clarithromycin -introduced in Mexico in 1991- was the antibiotic considered for the treatment policies. In sensitivity analyses, we considered different mixing patterns and trends of background antibiotic use. We validated the model against historical values of prevalence of infection and ABR of H. pylori. Results: In the absence of a mass-treatment policy, our model predicts infection begins to rise in 2021, mostly caused by treatment-induced resistant strains as a product of background use of antibiotics. The impact of the policies is immediate on decreasing infection but also increasing ABR (see Figure). For example, policy 3 decreases infection by 11% but increases ABR by 23% after the first year of implementation. The relative size of the decrease in infection is 50% the increase in ABR for policies 2 and 3, and 20% for policy 1. These results agree across all scenarios considered in sensitivity analysis. Conclusions: Conclusions: Mass-treatment policies have a higher effect on increasing ABR letting resistant strains take over infection. Given the high proportion of ABR at the time of the policy implementation, mass treatment strategies are not recommended for Mexico.
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    Single cell analysis of bacterial communication and gene transfer by Enterococcus faecalis
    (2019-02) Erickson, Rebecca
    Enterococcus faecalis is a commensal member of the gastrointestinal tract of animals including humans but is also an opportunistic pathogen and a major cause of healthcare-associated infections. Its pathogenicity is thought to arise in immunocompromised people and after infection, treatment is difficult due to antibiotic resistance. E. faecalis is particularly good at transferring antibiotic resistance by mechanisms like conjugation and conjugative transfer of plasmids can occur at a high frequency without antibiotic selection. Conjugative plasmid pCF10 encodes tetracycline resistance and transfer between E. faecalis cells is facilitated by cell-to-cell communication. This signaling triggers expression of genes from pCF10 that encode for transfer machinery. The response to signaling is robust and has been extensively studied at the population level. However, it has recently become apparent that there is response variation. Understanding the mechanisms that underlie variation in response initiation is important to preventing transfer. Studies presented in this dissertation adapt fluorescence in situ Hybridization Chain Reaction (HCR) for single cell analysis of transcripts and explore questions about the pCF10 conjugation system that would not have otherwise been possible. In chapter 3, variation in the signaling response was assessed and the response was shown to be very heterogenous. When the level of signal is low, (like what might occur naturally), a minority of cells respond. Although stochasticity in the system may give rise to such heterogeneity, work in chapter 4 investigates the response impact of a few specific mechanistic players (PrgX, C, and I). Changing the levels of these components was shown to change the outcome. Lastly, single cell analysis was used in chapter 5 to assess the expression of genes required for conjugative transfer. These results show that the few responding cells commit to expression of all the genes encoding for production of the conjugation machinery. Overall, these results suggest that the pCF10 system is evolutionarily tuned for specific levels of each component and poised to have response variation for a population of cells. Thus, a small percent of cells can respond and since the majority of responding cells are able to conjugate, plasmid transfer is highly efficient. These results also exemplify how small differences in two cells can precipitate different responses in otherwise identical cells exposed to very similar conditions. Information about variation in the initiation of the signaling response required for pCF10 transfer is important to understanding the general biology of gene transfer among bacteria. In the future, this information will be important for successful design of effective interventions to the transfer of genes conferring antibiotic resistance.
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    Statistical and mathematical modeling to evaluate the cost-effectiveness of Helicobacter pylori screening and treating strategies in Mexico in the setting of antibiotic resistance
    (2017-08) Alarid Escudero, Fernando
    Helicobacter pylori (H. pylori), a bacterium that is present in the stomach of half of the world’s population with disproportionate burden in developing countries, is the strongest known biological risk factor for gastric cancer. Gastric cancer is the fourth most common type of cancer and the second cause of cancer death in the world. In particular, in Mexico gastric cancer is the third highest cause of cancer death in adults, with some regions having cancer mortality rates that are twice the national average (8.0 vs. 3.9 per 100,000, respectively). H. pylori can be treated with antibiotics, but widespread treatment may lead to significant levels of antibiotic resistance (ABR). ABR is one of the main causes of H. pylori treatment failure and represents one of the greatest emerging global health threats. In this thesis, we use statistical and mathematical modeling to investigate the health benefits, harms, costs and cost-effectiveness of screen-and-treat strategies for identifying and treating persons with H. pylori to inform public health practice in three steps. First, we estimated the age-specific force of infection of H. pylori --defined as the instantaneous per capita rate at which susceptibles acquire infection-- using a novel hierarchical nonlinear Bayesian catalytic epidemic model with data from a national H. pylori seroepidemiology survey in Mexico. Second, we developed an age-structured, susceptible-infected-susceptible (SIS) transmission model of H. pylori infection in Mexico that included both treatment-sensitive and treatment-resistant strains. Model parameters were derived from the published literature and estimated from primary data. Using the model, we projected H. pylori infection and resistance levels over 20 years without treatment and for three hypothetical population-wide treatment policies assumed to be implemented in 2018. In sensitivity analyses, we considered different mixing patterns and trends of background antibiotic use. We validated the model against historical values of prevalence of infection and ABR of H. pylori. Third, we expanded the SIS model to incorporate the natural history of gastric carcinogenesis including gastritis, intestinal metaplasia, dysplasia and ultimately non-cardia gastric cancer. We then estimated the cost-effectiveness of various screen-and-treat strategies for H. pylori infection and ABR in the Mexican population from the health sector perspective.
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    Systems analysis of pheromone signaling and antibiotic resistance transfer in Enterococcus faecalis
    (2018-01) Bandyopadhyay, Arpan Anup
    Antibiotics have been an extremely important weapon in the fight against bacterial infections for over half a century. However, excessive use of antibiotics has led to increased frequencies of resistance among bacteria. Antibiotic resistance is an inevitable outcome of natural selection as organisms undergo random mutations to escape lethal selective pressure. Many of these resistant bacteria can also transfer their genetic material to other bacteria through direct cell-cell contact via conjugation, further facilitating the spread of resistance. The human gastrointestinal tract, replete with a high density of bacteria and often exposed to antibiotics, provides an ideal environment for antibiotic resistance genes to arise and propagate through bacterial populations. Enterococcus faecalis, a commensal bacterium of the human intestinal tract, has emerged as a major cause of healthcare-associated infections. Treatment of these infections has become increasingly difficult with the emergence of E. faecalis strains that are resistant to multiple major classes of antibiotics. The organism’s ability to acquire and transfer resistance genes and virulence determinants through conjugative plasmids poses a serious clinical concern. Here we present our study on conjugation of a tetracycline-resistance plasmid pCF10 which is regulated by intercellular communication using two antagonistic signaling peptides. An inducer peptide produced by the plasmid-free recipient cells functions as a “mate-sensing” signal and triggers the conjugative plasmid transfer in donors. The donors encode an inhibitor peptide on the plasmid which represses conjugation and functions as a "self-sensing" signal, reducing the response to the inducer in a density-dependent fashion. This form of dual signaling-controlled conjugation was also found to be prevalent across other pheromone-responsive plasmids, including pAD1 and pAM373. Though the donors calibrate their conjugation response in accordance with the relative abundance of donors and recipients, plasmid transfer can occur under otherwise unfavorable conditions, such as low inducing pheromone and high inhibitor concentrations. To better understand this apparent inconsistency, we formulated a stochastic mathematical model that integrates intracellular molecular regulation of conjugation and interactions between donors and recipients through the signaling peptides. Kinetic parameters for the model were estimated from literature and augmented by experimental RNA-Seq data and binding constant measurements. Simulations of the stochastic model and single-cell analysis using transcript quantification by HCR-FISH and GFP reporter fusions revealed distinct subpopulations of rapid responders under unfavorable conditions for plasmid transfer. We developed a series of fluorescent reporters to track the uninduced/induced donors, recipients, and uninduced/induced transconjugants in real-time using confocal microscopy and flow cytometry. We are further developing a microfluidic gut model which allow for co-culturing of human and bacteria cells in an in vivo-simulated microenvironment. This system will be used to model the in vivo biology of conjugation and gain a better mechanistic understanding of the community balance between the microbial inhabitants of the GI tract. A better understanding of the bacterial signaling mechanisms in vivo and the downstream effects on microbiome community balance may help us identify alternate strategies to prevent the spread of antibiotic resistance.