On the verge of entering the post-antibiotic era, numerous efforts are in place to regain the losing potential of antibiotics which are proving ineffective against common bacterial infections. Engineered nanomaterials, especially gold nanoparticles (GNPs) capped with antibacterial agents are proving to be an effective and novel strategy against multi-drug resistant (MDR) bacteria. In this study, we report a one-step synthesis of kanamycin-capped GNPs (20 ± 5 nm) utilizing the combined reducing and capping ability of the aminoglycoside antibiotic, kanamycin. Antibacterial assays showed dosedependent broad spectrum activity of Kan-GNPs against Gram-positive (Staphylococcus epidermidis and Enterococcus durans), Gram-negative (Escherichia coli and Enterobacter aerogenes) and Kan-resistant and MDR bacterial strains. A significant reduction in the minimum inhibitory concentration (MIC) of Kan-GNPs was observed as compared to free kanamycin against all the sensitive and resistant bacterial strains tested. Mechanistic studies using TEM and fluorescence microscopy showed that Kan- GNPs exerted their bactericidal action through disrupting the cellular membrane resulting in leakage of cytoplasmic content and death of bacterial cells. Results of this study provide a novel method in the development of antibiotic capped GNPs as potent next-generation antibacterial agents.

Secondary plant metabolites have recently been gaining interest in livestock production systems following the ban of in-feed antibiotics within the European Union. The rise in antimicrobial resistance found in pathogenic and non-pathogenic bacteria has lead to increased interest in the research community regarding the use of phythochemicals as an alternative to antibiotics. The purpose of this research was to evaluate the impact of including phytochemicals in a livestock production system. Specifically, a high tannin-containing forage, sainfoin (Onobrychis viciifolia), was evaluated in vitro for its antimicrobial effect on Escherichia coli. We determined that phytochemicals alone are not as inhibitory as synthetic antibiotics. Thus, the use of combination therapy to deter the development of antimicrobial resistance was evaluated. A myriad of plant compounds were screened for their synergistic interactions with ciprofloxacin. Geraniol, an essential oil, was identified to possess good antimicrobial activity and synergistic interactions with ciprofloxacin. Therefore the effect of long term exposure to both ciprofloxacin and geraniol were examined. Results demonstrated that once an antimicrobial concentration threshold was reached, resistance to ciprofloxacin increased markedly in the presence of both geraniol and ciprofloxacin. Finally, an in vivo trial was conducted in which forty steers were fed sainfoin or alfalfa over a 9-week period to evaluate its ability to reduce E. coli shedding and its impact on gut microbiota in the context of popular theoretical ecology concepts. Results from the in vivo study indicate that sainfoin was able to promote a slight decrease in generic E. coli shedding which could be maintained throughout the trial. Using high-throughput sequencing, the effect of sainfoin on the microbial ecosystem of the ruminant gut was evaluated. Sainfoin induced a significant shift in the microbial community structure of the rumen and to a lesser extent in the hindgut. Using ecology theories, a hypothesis was formulated regarding the mechanisms that mediate the development of tolerance and the fundamental ecological processes controlling microbial population shifts. Understanding how the gut ecosystem functions and predicting its behaviour in the presence of various fluctuating environmental conditions will enable more efficient manipulation of the rumen and promote best management practices in livestock production.

Staphylococci are important opportunistic pathogens often found in the microflora of skin and mucosal surfaces of the upper respiratory tract of man and animals. The coagulase-positive species such as Staphylococcus aureus are capable of causing invasive (eg furuncles and bacteraemia) and non-invasive (food poisoning and toxic shock syndrome) conditions in humans. In animals, S. intermedius and S. aureus have been implicated in a variety of conditions including pyoderma in dogs, mastitis in cows and skin infections in horses with S. intermedius being responsible for more than 95% of staphylococcal infections in dogs. The emergence of antibiotic-resistant bacteria, in humans, animals and the environment, has become a cause for concern following the introduction of antimicrobial agents in clinical practice. Staphylococcal species, in particular, have developed or acquired antibiotic-resistance determinants to almost all the antimicrobial agents in clinical use today. In recent years there has been an increase in reports of the isolation of antibiotic-resistance staphylococci especially methicillin-resistant staphylococci (MRS), from cats and dogs. Cats and dogs are in close contact with humans, especially in advanced countries and therefore the possibility for transfer of antibiotic-resistant staphylococci from these animals to humans or vice versa may exist. The aims of this study were; to determine the species distribution and antibiotic sensitivity of staphylococci obtained from cats and dogs, to investigate the molecular basis of resistance and to examine the genetic relatedness of specific resistant isolates. Many studies have shown that S. intermedius and S. simulans (S. felis) are the predominante species on healthy dogs and cats, respectively, and lesions in these animals are caused by S. aureus and S. intermedius. In this study, a diverse range of coagulase-negative staphylococci was isolated from healthy animals but staphylococci from skin lesions of cats and dogs were identified mainly as S. intermedius as reported in the literature. A limited number of resistant isolates (~20%) were observed in this study and were mostly isolated from dogs. Investigations into the molecular basis of resistance to beta-lactam, macrolide and tetracycline antibiotics were carried out. The resistant isolates were analysed by polymerase chain reaction (PCR) and DNA sequencing techniques. MRS were analysed for the presence of the mecA gene and the staphylococcal cassette chromosome mec (SCCmec). The recombinase genes, cassette chromosome recombinase (ccr) on SCCmec elements were also examined. The SCCmec elements detected were as diverse as those reported in human staphylococcal strains. Comparative analysis of nucleotide and amino acid sequences of mecA and ccr gene complexes revealed that the genes are conserved among MRS of cat and dog orgin. Multilocus sequence typing (MLST) of methicillin-resistant S. aureus (MRSA) and S. epidermidis isolates showed that, the MRSA were of human origin but the S. epidermidis isolates were unique to cats and dogs. In addition, the blaZ gene which codes for ??-lactamases production, the erm genes responsible for erythromycin resistance and the tet genes which encode tetracycline resistances, were found to be identical to those observed in humans and other animal staphylococci and demonstrated similar diversity. The study has provided important information about the molecular basis of resistance in beta-lactamase producing staphylococci as well as the molecular epidemiology of MRS of cat and dog origin and identifies the risk of spread of MRS between humans and pets and vice versa. These findings should form part of a larger surveillance study on staphylococci of cat and dog origin for a better understanding of the epidemiology of antibiotic-resistance genes for improved management and control of resistant staphylococci in the community and in health care settings. / PhD Doctorate

In this project we set out to find when the resistance level against first line antibiotics would reach 20%. This was executed by first defining relevant bacteria and antibiotics for urinary and respiratory tract infections (UTI's, RTI's). The data was collected from the European Center for Disease Control (ECDC) and the Center for Disease Dynamics, Economics & Policy (CDDEP). The data included the level of resistance for specific years for countries in Europe, as well as for the USA. A prediction model was made using the programming language R. A linear model was used to make a five and ten year prediction. The accuracy was tested. The results were then visualized using R and MATLAB. The results show a big variation between different bacteria and antibiotic combinations. For the two E. coli combinations the resistance is already near 20% for many countries and the resistance is increasing. For the three K. pneumoniae combinations the resistance is high in Southern Europe, meaning many countries have reached or are near 20%. For the two P. aeruginosa combinations there is also a higher resistance in Southern Europe but the resistance is decreasing in most countries. The resistance for E. faecalis is also decreasing and is generally very low in all of Europe. For the only RTI relevant combination, S. pneumoniae and penicillins, the resistance is low and many countries except for Sweden show a decrease in resistance. The USA did not have data for the same time span as Europe and was therefore analyzed separately. For many combinations the USA are near the 20% limit. Only for two combinations the USA showed a decrease in resistance level, and for one of those combinations the prediction is too uncertain to make any assumptions about. For the USA there were two more combinations for RTI than for Europe. For the S. pneumoniae and penicillins combination they have, just as most of Europe, a decreasing resistance. The two combinations with Acinetobacter spp. have a high resistance that is increasing. The main challenge during this project was finding relevant data with a long timespan and with high certainty. The data found is based on invasive isolates which means that the disease which the samples are taken from is not known. The timespan and the certainty of the data affected the accuracy of the prediction model and how long period that could be predicted. The prediction model generated 202 predictions that were visualized. An ethical analysis was made concerning both research ethics and general ethics on the topic of antibiotic resistance. This analysis is meant to acknowledge these questions since we believe they are important when discussing antibiotic resistance. The objective of this project turned out to be more difficult to attain than first believed. This was because of the lack of quality data. Even though we cannot give a clear answer when each country will reach a resistance of 20% this report gives a good understanding of how the situation looks for UTI and RTI relevant bacteria.

La résistance aux antibiotiques est une menace non négligeable pour la santé publique. Ces résistances peuvent être portées par différents supports dont les îlots génomiques. Il a été démontré que les îlots génomiques Salmonella Genomic Island 1 (SGI1) et Proteus Genomic Island 1 (PGI1) sont des acteurs importants de la multirésistance aux antibiotiques. Quelques variants de SGI1 et PGI1 ont déjà été décrits au sein de l’espèce P. mirabilis. Dans ce contexte, ce projet de thèse se proposait d’approfondir notre connaissance de la situation épidémiologique de la diffusion de SGI1 et PGI1 chez P. mirabilis chez l’homme et l’animal en France, en ce qui concerne la diversité des isolats, mais aussi celles des variants de SGI1/PGI1. En parallèle, une autre volonté a été d’identifier d’autres facteurs et acteurs permettant l’acquisition de gènes de résistances d’intérêt au sein des Morganellaceae (β-Lactamases à Spectre Etendu, céphalosporinase AmpC, Plasmid-mediated Quinolone Resistance...). Au final, cette étude a permis en outre de révéler les premiers cas de SGI1 et PGI1 chez P. mirabilis chez l’animal en France. De nouveaux variants de SGI1 ont également été mis en évidence. Et pour la première fois, SGI1 a été décrit chez M. morganii, une autre espèce d’entérobactérie. / The antibiotic resistance is a major treat for public health. These resistances can be hold by different element and genomic islands are one of them. Salmonella Genomic Island 1 (SGI1) and Proteus Genomic Island 1 (PGI1) are important genetic elements for the antibiotic resistance. A few SGI1 and PGI1 variants were already described in P. mirabilis. It is in this context that this thesis project aimed to improve our knowledge about the epidemiological spread of SGI1 and PGI1 in P. mirabilis in humans but also in animals in France (diversity of isolates and SGI1/PGI1 variants). Moreover, another wish was to identify other factors and actors for the acquisition of antibiotic resistance in the Morganellaceae tribe (Extended-Spectrum β-Lactamases, AmpC cephalosporinase, Plasmid-mediated Quinolone Resistance…). Finally, this study revealed the first cases of SGI1 and PGI1 in P. mirabilis in animals in France. New SGI1 variants were also described. And for the very first time, SGI1 was found in M. morganii, another entrobacterial species.

The continuing rise of antibiotic resistance is threatening a return to the world of pre-antibiotic medicine. Multi-drug resistant pathogens are already claiming lives and causing economic losses in developing and developed countries alike. We need, therefore, to understand what allows resistant strains to spread; what makes them evolutionarily competitive in and amongst other strains and species. The majority of laboratory studies of antibiotic resistance focus on simple growth in liquid culture. By contrast, microbes commonly grow as surface-associated communities, in which interactions between neighbouring cells have strong consequences for competition and evolution. My first goal was to understand how growth in such environments affects the success of a resistant strain. By competing an antibiotic resistant and susceptible strain of the pathogenic bacterium Pseudomonas aeruginosa, I found that growth in dense colonies on agar allowed a resistant strain to protect susceptible strains, to the extent that the susceptible strain may even prevail under antibiotic treatment. This effect was specific to a cooperative mechanism of antibiotic resistance, however; a β-lactamase enzyme that digests the antibiotics surrounding a resistant cell. A further, unexpected reason that susceptible cells could prevail was that they elongate under antibiotic treatment, allowing them to push shorter resistant cells aside in the competition for the growing edge of a colony. My work suggests that the rise of cooperative resistance mechanisms should be more easily suppressed than for non-social mechanisms. However, one major strategy to overcome antibiotic resistance is the use of antibiotic-adjuvants, drugs which inhibit a mechanism of antibiotic resistance. It is not clear if these adjuvants will tend to suppress or promote cooperative resistance mechanisms. I performed experiments to test the effects of inhibitory adjuvants on cooperative resistance. These revealed that the effects of adjuvants are varied. In liquid culture, an adjuvant inhibited resistance evolution, while, in colony experiments, it promoted resistance evolution by removing the cross protection of susceptible strains. Given the complexity and importance of antibiotic adjuvants, I developed an eco-evolutionary model to dissect these complexities associated with the combination of interacting microbial and molecular species. As in my experiments, the models identified conditions where an inhibitory adjuvant can increase selection for resistance. However, the theory also identifies scenarios for which adjuvants will delay resistance evolution by shutting down the associated evolutionary pathway. Broadening the modelling framework to include the stochastic effects of rare mutation, I found that early administration of adjuvant inhibitors can be a powerful way to suppress the emergence of antibiotic resistance. Microbial interactions are complex and affected by the growth environment. My thesis underlines that the study of antibiotic resistance will benefit from greater consideration of how bacteria interact and, more broadly, how their ecology and evolution determine the rise, or fall, of resistance.

Secondary multidrug transporters use the energy stored in transmembrane ion gradients to bind and extrude a variety of weakly related chemical structures. These polyspecific antiporters challenge the notions of high-affinity conformation and strict ion-substrate coupling, inherent to the alternating-access model of transport. In order to investigate the mechanism of secondary multidrug transport at a molecular level, we study LmrP, a Major Facilitator Superfamily (MFS) multidrug transporter from Lactococcus lactis, which relies on the proton-motive force to achieve the transport of its diverse substrates. We carried out Double Electron Electron (DEER) distance measurements to elucidate the conformational dynamics underlying the transport cycle. We monitored the conformational response of a library of labeled double cysteine mutants to the presence of ligand(s) and proton(s). We investigated the role of the lipid environment by performing the measurements on mutants reconstituted in nanoscale soluble lipid bilayers (nanodiscs). During this work, we have demonstrated that the transporter oscillates between two main conformations, the outward-open and the inward-open. We have shown that the protonation of conserved acidic residues is the driving force of the conformational transition. The lipid bilayer modulates the equilibrium and allows the transition to occur at higher and more physiological pH values. By using specific lipid compositions, we observe that the lipid headgroup is crucial in the regulation of the conformational equilibrium. Based on our data, we propose a model of secondary multidrug transport wherein substrate binding initiates the transport cycle by catalysing proton entrance from the extracellular side. Subsequent protonation of membrane-embedded acidic residues triggers a cascade of conformational changes that results in substrate extrusion to the extracellular side and proton release in the cytosol. We suggest the opening and closing of the extracellular site is tightly regulated while the cytoplasmic side is more flexible. To our knowledge, this work provides the first direct structural evidence of the role of the lipids in the regulation of the conformational dynamics of a membrane transporter. / La surexpression de transporteurs capables d’expulser des molécules cytotoxiques est un mécanisme connu de résistance aux antibiotiques de la cellule bactérienne. Certains transporteurs ont développé la capacité de reconnaitre et d’expulser des substrats de structures diverses, donnant lieu à une résistance multidrogue de la part de leur hôte. Ces transporteurs multidrogues sont présents dans une variété de classes de protéines, distribués dans tous les règnes du vivant. Parmi celles - ci, la famille MFS (Major Facilitator Superfamily) comprend la majorité des transporteurs multidrogues activé par une source d’énergie secondaire, et jouent un rôle crucial dans la propagation de maladies nosocomiales d’origine bactérienne. Une meilleure compréhension des mécanismes fondamentaux du transport multidrogue secondaire est le prérequis indispensable à l’élaboration de thérapies adaptées. En particulier, une description détaillée des changements conformationnels impliqués dans le transport, et une identification des mécanismes moléculaires qui permettent de lier la source d’énergie au transport fait actuellement défaut. Afin de pallier ce manque, ce travail vise à étudier LmrP (Lactococcus lactis multidrug resistance Protein) un transporteur MFS qui confère à son hôte Lactococcus lactis la résistance à divers antibiotiques et agents cytotoxiques de structure et de charge variable. Cette extrusion active est alimentée par un cotransport énergétiquement favorable de protons. Nous avons étudié le mécanisme de transport de LmrP à l’échelle moléculaire en utilisant la technique spectroscopique Double Electron Electron Resonance (DEER), qui permet de mesurer des variations de distances à l’échelle nanométrique, idéale pour observer les mouvements intramoléculaires d’un transporteur MFS. Différents aspects moléculaires susceptibles de réguler le cycle de transport sont étudiés de façon indépendante et couplée :le rôle des protons, des différents substrats, et de l’environnement lipidique. Sur base de cette cartographie conformationnelle, un mécanisme de transport couplant tous les acteurs moléculaires est proposé :la liaison du proton à un motif d’acides aminés conservé constitue la base de la transition conformationnelle, les divers substrats ayant pour rôle de permettre aux protons d’accéder à ce motif. La compétition substrat-proton est la base du transport couplé. Notre travail a mis en évidence le rôle fondamental de l’environnement lipidique, qui module l’équilibre conformationnel du transporteur en interagissant avec un ou plusieurs motif(s) conservé(s). Par ailleurs, notre étude questionne le paradigme actuel de transport au sein de la famille MFS car elle démontre que les changements conformationnels globaux passent par des réarrangements locaux et coordonnés. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Class of 2006 Abstract / 1College of Pharmacy, University of Arizona 2El Rio Special Immunology Associates Objectives: To compare antibiotic resistance patterns of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) in HIV patients and the general population. We hypothesized that CA- MRSA strains in HIV patients may show a decreased susceptibility to trimethoprim/sulfamethoxazole (TMP/SMX) because of its widespread use for prophylaxis of Pneumocystis carinii pneumonia (PCP) in this population. Methods: Susceptibility reports for all Staphylococcus aureus isolates collected between November 1, 2004 and November 1, 2005 from Special Immunology Associates (SIA), an HIV clinic, and the neighboring Carondelet St. Mary’s Hospital Emergency Center (SMH) were analyzed. Results: Twenty-five cases in HIV patients at SIA (Mean age 40, Race: 21 Caucasian, 4 Hispanic) and 102 cases at SMH (Mean age 37, Race: 45 Caucasian, 34 Hispanic, 12 Native American, 8 African American) were identified as CA-MRSA. More than 95% of patients presented with skin infections and a seasonal peak was identified between June and October. No cases of TMP/SMX resistance were found at either institution despite the fact that 40% of the SIA patients with CA-MRSA had been exposed to TMP/SMX for PCP prophylaxis in the past. Susceptibility varied between SIA and SMH, with significant differences in susceptibility to tetracycline (57% vs. 86%, p<0.001) and levofloxacin (38% vs. 60%, p < 0.001). Erythromycin resistance in both institutions was greater than 90%. Conclusions: All CA-MRSA isolates from an HIV clinic and a neighboring health care facility were susceptible to TMP/SMX. HIV patients with CA-MRSA did not show a decrease in susceptibility to TMP/SMX, despite its routine use for prophylaxis in this population.

Antibiotic resistance is a major threat to public health. Understanding how it evolves, and the genes that underlie resistance, is the main goal of my Ph.D. research. After a resistance mutation arises, it’s fate within a pathogen population will be etermined in part by its fitness: mutations that suffer little or no fitness cost are more likely to persist in the absence of antibiotic treatment. My research centers on understanding this process better by gaining knowledge about the spectrum of fitness effects associated with antibiotic resistance mutations. Using a meta-analysis framework I find that, across a range of antibiotics and pathogens, on average single resistance mutations exhibit fitness costs in the absence of drug, however, there are instances of cost-free mutations. To evaluate the conditions leading to the persistence of resistance in the absence of antibiotic, I use experimental evolution of the opportunistic pathogen Pseudomonas aeruginosa and the antibiotic ciprofloxacin to investigate the phenotypic and genetic differences associated with constant and fluctuating drug treatment. I find that fluctuating drug treatment leads to the evolution of cost-free resistance. At the genetic level, cost-free resistance is the result of second-site mutations that compensate for the fitness cost associated with ciprofloxacin-resistance mutations. Further examination of the resistance mutations shows a lack of epistatic interactions between co-occurring mutations that confer resistance within a single isolate. To investigate the repeatability of the genetic causes of resistance, I execute a second evolution experiment using multiple clinical strains of P. aeruginosa adapting to a constant ciprofloxacin selective pressure. I find a remarkable lack of parallel evolution at the genomic level both within and between different P. aeruginosa strains. I have shown that antibiotic resistance is costly, and that these costs can be ameliorated by second-site mutations that readily arise over short time scales. Additionally, different strains of the same bacteria can gain resistance through a diverse set of genetic mutations. On an applied level these results are not positive; combating resistance evolution will be difficult because pathogens can easily compensate fitness costs of resistance, and resistance itself can be gained via a large number of genetic targets.

The cell envelope of mycobacteria is critical for the survival and virulence of pathogenic species during infection, and its biosynthesis has been a proven drug target. Therefore, finding new targets in the biosynthetic pathway of cell envelope components is of great interest. Mycobacterium smegmatis is a model organism for the study of the devastating pathogen Mycobacterium tuberculosis. Previously, lipomannan elongation factor A (LmeA) has been identified as a cell envelope protein that is critical for the control of mannan chain length of lipomannan (LM) and lipoarabinomannan (LAM), lipoglycan components of the cell envelope. The deletion mutant, ∆lmeA, accumulates abnormal LM/LAM with fewer mannan residues. To understand the importance of this protein, the antibiotic sensitivity of ∆lmeA was tested using a resazurin-based viability assay. We found that the lmeA deletion leads to increased sensitivities to antibiotics such as vancomycin and erythromycin, and lmeA overexpression leads to increased antibiotic resistance. To directly test if the increased antibiotic sensitivity is due to the defective permeability barrier, we used an ethidium bromide uptake assay and found that ∆lmeA is more efficient in taking up ethidium bromide in the cell. We have also found that LmeA is important for protein stabilization under stress conditions. MptA is an α1,6-mannosyltransferase involved in elongation of LM and LAM mannan chain. During stress conditions in the ΔlmeA mutant, levels of MptA decrease significantly relative to wild-type. This also results in delayed doubling time after stress, a phenotype not seen in this mutant under normal growth conditions. In addition, the ΔlmeA mutant has differential protein expression during stress conditions relative to ΔlmeA in log phase, or to wild-type in either condition. To help elucidate the role of LmeA at the molecular level, binding behavior of this protein to membrane fractions was determined. In a subcellular fractionation analysis, LmeA localizes to fractions containing plasma membrane, which is tightly bound to cell wall layers. To test the binding of LmeA to membrane further, LmeA was heterologously expressed in Escherichia coli, purified, and mixed M. smegmatis cell lysate. LmeA localized to intracellular domain fractions (IMD), indicating that LmeA is capable of localizing to fractions containing only plasma membrane. Consistent with this finding, LmeA is capable of binding to spheroplasts in both an ELISA setting as well as in a sucrose gradient fractionation setting. It has also been determined that ΔlmeA has a defective capsular layer with a unique phenotype relative to other strains. We have concluded that LmeA is important for antibiotic resistance, cell envelope permeability, capsule formation, stress response, and have also determined its binding properties.