Browsing by Subject "Antibiotic resistance"

Although antibiotics have proven to be one of the great achievements of modern medicine, their efficacy has dramatically decreased over the past several decades. This is due, in part, to the rapid pace of natural bacterial evolution, but also to the overuse and misuse of antibiotics in general. This often selects for drug-resistant pathogens, and allows them to flourish in the face of antibiotic treatment. In addition to the emergence of genetic resistance, bacteria often utilize a number of population-level behaviors to survive antibiotic treatment. This is referred to as collective antibiotic tolerance (CAT). Taken together, antibiotic resistance and tolerance have led to the re-emergence of infectious diseases throughout the world. In general, there are two strategies to combat this risk: develop novel antibiotics, and/or use existing drugs more effectively, so as to minimize the chance of resistance emergence. Novel drug development is a time- and resource-intensive process, and pharmaceutical companies are not financially incentivized to develop these types of drugs. Therefore, it is of increasing importance to understand the population dynamics underlying various bacterial survival mechanisms, and exploit this knowledge to design better antibiotic treatment protocols.

My dissertation research focuses on a prevalent phenomenon called the postantibiotic effect (PAE), which refers to the transient suppression of bacterial growth following antibiotic treatment. Although PAE has been empirically observed in a wide variety of antibiotics and microbial species, heretofore there has not been a definitive mechanistic explanation for this pervasive observation.

In this work, I use a combination of high-throughput microfluidic experiments and computational modeling to examine the relationship between dosing parameters and the degree of bacterial inhibition, quantified by population recovery time. I found that recovery time is a function of total antibiotic, regardless of how the dose profile. Moreover, a minimal model of transport and binding kinetics was sufficient to recapture this trend, suggesting a unifying explanation for historical observations of PAE in a variety of contexts. I validated this modeling using both in silico and in vitro perturbation studies.

Moreover, I showed that efflux inhibition, a common strategy in antibiotic treatment, is effective in certain dynamic-dependent situations. This work puts forth a possible mechanism for PAE, which could serve as a clinical aid in selecting effective antibiotic/adjuvant combinations, as well as in designing periodic antibiotic treatments.

The composition and function of mammalian gut microbiomes are shaped by complex endogenous and exogenous factors that present on evolutionary and proximate timescales. In the Anthropocene era, host-associated microbiota are inevitably, yet differentially, influenced by natural and anthropogenic factors that vary across individuals and populations. In this dissertation, I used descriptive and experimental approaches, largely within a single species, the ring-tailed lemur (Lemur catta), to probe the roles of host physiology, environmental conditions, anthropogenic perturbation, and microbial environment in shaping primate microbiota across scales. First, I conducted a broad investigation of ring-tailed lemur gut microbiota and soil microbiota across 13 lemur populations (n = 209 individuals) spanning this species’ natural range in Madagascar, as well as multiple captivity settings in Madagascar and the U.S. By analyzing the lemur and soil microbiota, I showed that lemur gut microbiota vary widely within and between wild and captive populations, and that lemur and soil microbiota covary, suggesting a role for environmental acquisition in shaping interpopulation variation. Second, I analyzed vaginal, labial, and axillary microbiota of female ring-tailed lemurs and Coquerel’s sifakas (Propithecus coquereli) at the Duke Lemur Center (DLC) to demonstrate the influences of stable traits (e.g., species identity and mating system) and transient traits (e.g., ovarian hormones and forest access). We found that the effects of transient traits build on underlying differences mediated by stable traits. Third, and further focusing on DLC lemurs, but with a concentration on anthropogenic influence, I worked with a team of researchers to perform an experimental manipulation in ring-tailed lemurs to determine the influence of antibiotic treatment, with or without subsequent fecal transfaunation, on lemur gut microbiomes. I applied ecological frameworks to show that different facets of lemur microbial communities, such as bacterial diversity and composition, followed different recovery trajectories following antibiotic treatment. Fourth, I expanded my focus back to multiple ring-tailed lemur populations in natural and captivity settings to investigate the links between anthropogenic disturbance and antibiotic resistance genes (ARGs). I analyzed ARGs in wild and captive lemurs and soil from their habitats to show that lemur ARGs were correlated with anthropogenic disturbance and covaried with soil ARGs; lemur resistomes reflects multiple routes of ARG enrichment, including via antibiotic treatment or environmental acquisition. Integrating across these four data chapters, my results reveal that (a) the foundations of lemur-associated microbiomes are structured according to broad environmental conditions (e.g., wild vs. captive populations), but that between and within these broad categories, lemur microbiota are sensitive to more nuanced environmental variation, (b) lemur microbiota and resistomes co-vary with environmental microbiota, demonstrating the potential role of environmental acquisition in shaping host-associated communities across varying environments, and (c) integrating host microbial data across scales (e.g., at the individual and population level) with data on multiple facets of microbial communities (e.g., diversity, composition, membership, and resistomes), was key to providing a holistic perspective on host-associated and environmental microbe interactions across different microbial landscapes.

Antibiotic resistance rates have increased in both clinical and environmental bacteria over the past several decades. While the causes underlying these trends have been investigated in a clinical setting, little work has been done to estimate the contribution of antibiotic resistance genes (ARGs) derived from non-traditional sources like transgenic (GM) crops. The cultivation and consumption of transgenic crops continues to be a widely debated topic, as the potential ecological impacts are not fully understood. In particular, because ARGs have historically been used as selectable markers in the genetic engineering of transgenic crops, it is important to determine if the genetic constructs found in decomposing or consumed transgenic crops persist long enough in the environment and if they can be transferred horizontally to indigenous microorganisms.

The first Objective of this dissertation addresses the question of persistence. Others have also estimated the DNA adsorption capacity of various clays, but have done so by manipulating the surface charge and size of particles tested which may overestimate sorption and underestimate the DNA available for horizontal transfer. In the present study, isotherms were generated using model Calf Thymus DNA and transgenic maize DNA without surface modification. Montmorillonite, kaolinite, and 3 soil mixtures with varying clay content were used in this study. The adsorption capacity of pure montmorillonite and kaolinite minerals was found to be one to two orders of magnitude less than previously estimated likely due to the distribution of clay particle sizes and heteroionic particle surface charge. However, it appears that a substantial amount of DNA is still able to adsorb onto these matrices (up to 200 mg DNA per gram of clay) suggesting the potential availability of free transgenic DNA in the environment may still be significant.

In addition to the soil environments described above, the persistence of these genes should be investigated in agricultural soils. Two important tools exist for understanding the potential contribution of transgenic crop-derived ARGs to environmental antibiotic resistance: (A) the microbiomes associated with transgenic maize and (B) decomposition behavior of transgenic crop biomass. The purpose of Objective Two is to characterize GM maize microbiomes and biomass decomposition to better how transgenic maize may be affecting antibiotic resistance rates among soil bacteria. To investigate this, bulk soil, rhizosphere soil, and internal endophyte prokaryotic microbiomes associated with conventional and transgenic maize were compared using lllumina MiSeq 16S amplicon sequencing. Internal endophytes were significantly influenced by time and location within the maize, but not by maize type. Nitrogen-cycling bacteria in the rhizosphere of transgenic pest-resistant (BT) maize experienced a decrease in diversity at day 56 of maize cultivation that was moderately correlated with the level of Cry1Ab protein exudates in soils surrounding transgenic BT maize. Secondly, bla ARG expression was tracked across 40 weeks of maize biomass decomposition in soils associated with conventional and transgenic BT maize. Bla expression significantly increased in soils associated with BT maize relative to conventional maize soils after 32 weeks or decomposition.

In addition to agricultural environments, wastewater treatment plants (WWTPs) may also contain genes derived from transgenic crops. Consumption of transgenic crops and foods containing them is common in the United States. Once ingested, DNA within transgenic crops can conceivably behave in three ways: 1) DNA will be degraded by acids or DNase I and II enzymes found within the digestive system, 2) DNA will be taken up by cells in the gut (host or bacterial), or 3) DNA will pass through the digestive system and excreted partially or wholly intact. In addition to degradation or uptake, transgenic DNA may be excreted as solid waste by the host. As free DNA contained within food matrices is protected from both enzymatic digestion and acid hydrolysis, the genes contained within foods containing GMOs may be allowed to pass through the host’s digestive tract intact and enter into WWTP environments. Objective 3 assessed this possibility by screening activated sludge and digester sludge obtained from France and the United States for the following four possible transgenes: p35 promoter gene, nos terminator gene, nptII ARG, and bla ARG. All wastewater samples were first screened using end-point PCR and all positive results were further analyzed with qRT-PCR to determine the relative concentration of each gene in wastewater. All domestic activated and digester sludge samples were found to contain at least one transgene, with the majority of samples containing all four. However, wastewater from France, where transgenic crops are neither cultivated nor consumed, contained significantly fewer copies of p35, nos, and nptII genes. Based on these findings, it is possible that some of these genes are derived from partially digested transgenic crop material present in human waste. This is supported in part by the significant differences in p35, nos, and nptII concentrations in French WWTP systems relative to domestic WWTP systems, as well as additional screening for Cauliflower Mosaic Virus (CaMV) ORF VII genes and larger fragments of possible transformation plasmids. Overall it is possible for genes contained within transgenic crops and processed foods containing their byproducts to survive the human digestive process and enter into WWTP environments, though the source of the detected genes could not be definitely confirmed.

The fate and behavior of these for genes in WWTPs remains largely uncharacterized. Further investigation of these trends in WWTP anaerobic digesters is of paramount importance, especially in the case of the bla and nptII ARGs, as a large fraction of WWTP-generated biosolids is used for land application. In addition, because of the relatively high background levels of antibiotic contaminants in wastewaters there is the further possibility that free ARGs may be horizontally transferred to WWTP bacteria, thus increasing antibiotic resistance in the environment. The ability of WWTP bacteria to take up these transgenes in this environment is a factor of (1) the observed rates of horizontal gene transfer (HGT) in anaerobic environments, (2) the abundance of genes of interest within WWTP anaerobic digesters, and (3) the presence of a selective pressure. Like most other topics related to HGT, observing and quantifying the rates of these event in anaerobic environments has proved to be a difficult task. To investigate the behavior of free ARG DNA in WWTP environments, anaerobic digesters were constructed using anaerobic feed sludge from the North Durham (NC) WWTP. Digesters belonged to one of the following treatments and were operated at 47oC in quadruplicate for a period of 60 days: 102 added bla and nptII transgene copies/mL, 104 added bla and nptII transgene copies/mL, or 106 added bla and nptII transgene copies/mL. Control digesters were also constructed. Across 60 days of operation, persistence of free DNA in anaerobic digester wastewater was significantly influenced by DNA fragment length. LUG genes significantly decreased after day 7, but nptII and bla ARGs persisted past 30 days of operation. Bla ARGs were only detected in iDNA within 106 transgene copies/mL treatments at day 60 of incubation. It is possible that this increase is due to bacterial uptake of free bla in eDNA as a result of an antibiotic-driven selective pressure, inducing the integration of bla into bacterial cells. NptII genes were below detection in iDNA at no time points after digester construction. Finally, the naturally occurring concentrations of bla and nptII ARGs in anaerobic digesters are consistent with the findings presented in Chapter 5. However, the existence of these ARGs as primarily eDNA is novel and presently unreported in the literature.

Biomolecules such as proteins are highly dynamic, and undergo a wide variety of motions at different timescales. Movements as small as a bond vibration or as large as a domain rearrangement can be critical for the function of a protein, making consideration and investigation of protein dynamics necessary for understanding biological systems and developing therapeutics. In this work, we describe the development and implementation of novel techniques to study dynamics in proteins and protein-bound ligands, and discuss our investigation of the crucial role of dynamics in two disease-relevant systems.

First, we have expanded the utility of Chemical Exchange Saturation Transfer (CEST) NMR techniques to aid in the characterization of dynamics for nitrogen- and carbon-attached protons, as well as fluorine nuclei. Protons and fluorine nuclei can be exceptionally sensitive to their chemical environment, allowing detection and measurement of protein motions which may not be readily identified by conventional heteronuclear experiments. Additionally, we discovered the motion of a protein-bound ligand and utilized such information to improve the potency of an antibiotic molecule.

Next, we undertook the investigation and optimization of an inhibitor targeting translesion synthesis, a process that cancer cells can employ to resist the killing action of chemotherapeutics. Early work on this project demonstrated that inhibition of Rev1, an important scaffold in the translesion synthesis process, by the compound JH-RE-06 sensitizes cancer cells to cisplatin chemotherapy and prevents drug resistance. Surprisingly, we found that this inhibition occurs through inhibitor-induced dimerization of Rev1, which masks the protein-protein interface required for assembly of the translesion machinery. We further investigated a transient conformational change in the C-terminal tail of Rev1 and validated dimerization in solution using NMR. Our structure- activity relationship investigation of JH-RE-06 yielded a number of insights into how to develop more potent inhibitors. Most significantly, we found that small changes in the chemical structure of the inhibitor resulted in improved inhibitory activity and also led to a novel dimer arrangement. Our combination of Rev1 crystal structures and dynamics studies has led to a deeper understanding of the inhibitory mechanism of JH-RE-06 and will guide the optimization of this potential chemotherapy adjuvant.

Finally, we have investigated a mechanism of resistance to beta-lactam antibiotics in Neisseria gonorrhoeae, which relies on modulation of conformational dynamics. Neisseria gonorrhoeae is a major growing health concern due to the rapid spread of multi-drug resistance. We have discovered conformational exchange in PBP2, the target of beta-lactam antibiotics in Neisseria gonorrhoeae, between a low-affinity state and a high- affinity state. A histidine residue was found to be the key mediator of interconversion between these states via a network of molecular interactions, and we found that drug resistance-conferring mutations shift the equilibrium toward the low-affinity state by modulating these interactions. This work describes a novel mechanism of drug resistance in bacteria in which conformational dynamics are restricted.

This document illustrates a small sample of the important roles molecular motions have in biology, and the power of dynamics studies in understanding protein function, developing drugs, and elucidating resistance mechanisms.

Antibiotic discovery and widespread usage has revolutionized the treatment of infectious diseases. However, this golden age of modern-day medicine is threatened by the increasing prevalence of antibiotic-resistant pathogens. As the antibiotic development pipeline increasingly slows, we find ourselves falling behind in the race between innovation and evolution.

Among the various means of bacterial evolution, horizontal gene transfer (HGT) – or the non-genealogical transmission of DNA between organisms – is the dominant mode responsible for the acquisition of antibiotic resistance genes. Combined with antibiotic overuse and misuse, HGT primarily via conjugation, has compromised the efficacy of nearly every single antimicrobial available. Tight coupling between HGT and antibiotic-mediated selection, along with a lack of quantitative experiments, has led to the general belief that antibiotics themselves promote gene transfer; however, antibiotic action could modulate the rate of gene transfer (as is assumed), the resulting population dynamics, or both. Therefore, it is critical to decouple these two processes to definitively determine the influence of antibiotics on conjugation.

In my dissertation, I quantified the extent to which antibiotics influence conjugation in the presence and absence of antibiotic-mediated selection dynamics. To do so, I implemented a synthetically engineered conjugation system, which facilitates precise quantification of conjugation dynamics. Using this platform, I quantified the rate of gene transfer, or the conjugation efficiency, in the absence of confounding selection dynamics. I discovered that, in contrast to conventional wisdom, antibiotics did not significantly increase the conjugation efficiency. This finding was general to 10 antibiotics, as well as nine native and clinically relevant plasmids. Instead, antibiotic selection dynamics alone could account for conjugation dynamics.

I next investigated the potential strategies to minimize, or ideally reverse, plasmid-mediated resistance. Traditionally, reducing overall antibiotic use has been the primary approach to reversing resistance; minimizing selection takes advantage of costly resistance genes to competitively displace resistant bacteria with their sensitive counterparts. However, despite widespread antibiotic stewardship initiatives, even costly resistance persists for long periods of time. One potential explanation is that sufficiently fast conjugation enables plasmid persistence in the absence of selection. Similar challenges in quantifying conjugation have prevented general conclusions, and overall, the extent to which conjugation enables persistence is unknown.

Using the same platform, I showed that conjugation enables the persistence of costly plasmids, even in the absence of selection. Conjugation-assisted persistence was true for nine common conjugal plasmids, and in microbial populations consisting of varying degrees of complexity. Finally, I showed that by reducing the conjugation efficiency and promoting plasmid loss, it is possible to reverse resistance. Together, these findings contribute to basic understanding of the propagation and persistence plasmids, and elucidate a novel therapeutic strategy by taking advantage of ecological/evolutionary dynamics to to reduce or even reverse the spread of resistance.

Plasmids are key vehicles of horizontal gene transfer (HGT), mobilizing antibiotic resistance, virulence, and other traits among bacterial populations. Their fitness is directed by two orthogonal processes—vertical transfer through cell division and horizontal transfer through conjugation. When considered individually, improvements in either mode of transfer can promote how well a plasmid spreads and persists. Together, however, the metabolic burden of conjugation could create a tradeoff between the two and constrain plasmid evolution. Furthermore, the environmental and genetic forces that drive plasmid transfer are poorly understood, due to the lack of definitive quantification coupled with genomic analysis. Knowing how conjugation influences plasmid fitness and, subsequently, how to influence conjugation opens the door for evolutionary therapies targeting antibiotic resistance and bacteria in general.

Here, we integrate conjugative phenotype with plasmid genotype to provide quantitative analysis of HGT in clinical Escherichia coli pathogens. We find a substantial proportion of these pathogens (>25%) able to readily spread resistance to the most common classes of antibiotics. Antibiotics of varied modes of action had less than a 5-fold effect on conjugation efficiency in general, with one exception displaying 31-fold promotion upon exposure to macrolides and chloramphenicol. In contrast, genome sequencing reveals plasmid incompatibility group strongly correlates with transfer efficiency. These findings offer new insights into the determinants of plasmid mobility and have implications for the development of treatments that target HGT.

We also present evidence for the presence, consequences, and molecular basis of a conjugation-burden tradeoff among 40 plasmids derived from clinical E. coli pathogens. We discover that most plasmids avoid a fitness tradeoff by operating below a conjugation efficiency threshold for burden, indicating strong selection for vertical transfer. In this region, E. coli demonstrates a remarkable growth tolerance to over four orders of magnitude change in conjugation efficiency. This tolerance fades as nutrients become scarce and horizontal transfer attracts a larger share of host resources. These results provide insight into evolutionary constraints directing plasmid fitness and strategies to combat the spread of antibiotic resistance.