Antibiotic resistance is a growing threat to human health and is defined by the World Health Organization as a crisis that must be managed with the utmost urgency. Antibiotic resistant bacteria increase both mortality and morbidity and have a great impact on the global economy. Resistance is not confined to human health care, but is present also among animals and in our environment at large. Indeed, resistant bacterial strains have now been found in virtually all parts of the world, even in locations without direct human contact. The human gastrointestinal tract is populated by a complex, dynamic, diverse and highly interactive collection of microorganisms, including bacteria, archaea, fungi, yeasts and viruses, which constitutes our gastrointestinal microbiota. This microbiota is an important reservoir of resistance genes (our gastrointestinal resistome) and a “melting pot” for transfer of resistance genes between microbes, including potential pathogens. In this thesis I investigated the prevalences of two clinically important kinds of antibiotic resistance: extended-spectrum β-lactamases (ESBL) and vancomycin-resistant enterococci (VRE), as well as asymptomatic carriage of potential enteropathogens among healthy preschool children in Uppsala. Fecal samples from unidentified, individual diapers were collected in 2010 (125+313 samples) and in 2016 (334 samples). In addition, 204 environmental samples from the children’s preschools were collected in autumn 2016. A prevalence of 2.9% ESBL-producing Enterobactericeae was demonstrated in the first samples from 2010. No VRE were found and the occurrence of enteropathogens were reassuringly low. Results on ESBL prevalence in 2016 and transmission of resistance between children will be presented when the manuscript is published and at the dissertation.

The purpose of this study was to see if any soil bacteria were able to use pentachlorophenol or sodium pentachlorophenate either aerobically or anaerobically as a sole carbon source, to see if any soil bacteria could survive in high concentrations of sodium pentachlorophenate, to determine the maximum concentration of sodium pentachlorophenate which permitted the growth of some soil bacteria, to see the effects of varying concentrations of sodium pentachlorophenate on the growth curves of soil bacteria capable of growing in its presence, and to see if any soil bacteria could degrade sodium pentachlorophenate.

ABSTRACT Current guidelines published by the Centers for Disease Control and Prevention (CDC) and American Academy of Pediatrics (AAP) recommend empiric antibiotics for all neonates born to mothers with a diagnosis of chorioamnionitis due to the risk of early onset sepsis (EOS). EOS is difficult to diagnose due to nonspecific symptoms and a lack of reliable tests, can progress quickly, and is potentially fatal or have neurodevelopmental consequences for survivors. Antibiotics are frequently prescribed in the hospital and are lifesaving in the setting of a serious infection. Conversely, overuse of antibiotics has potential negative effects to individuals and the population as a whole. Antibiotic resistant infections are a consequence of antibiotic misuse, are costly and difficult to treat, and pose a risk to patients hospitalized. To examine this problem at The University of Vermont Medical Center (UVMMC) a retrospective chart review was preformed. Data on the maternal risk factors associated with EOS were collected in addition to clinical characteristics of their neonates and entered into a neonatal early onset sepsis (NEOS) calculator to determine the specific risk of infection to each infant. Treatment of the infant was compared to the NEOS calculator and CDC recommendations. Using posterior probability to determine a more specific risk profile better targets antibiotic therapy to ensure all infants that need treatment receive it, while reducing the number of infants treated empirically. UVMMC currently treats 78% of infants according to CDC guidelines. Use of the NEOS calculator would reduce antibiotic treatment to 18% of term neonates born to mothers with a diagnosis of chorioamnionitis. Using a new tool to determine risk of EOS may safely reduce the number of infants receiving antibiotic treatment.

Electrospinning has the capacity to create fibers of natural or synthetic polymers with dimensions that are similar to analogous fibers in native tissue. Mats consisting of fibers of these sub-micron dimensions have shown promise in provoking little immune response and in offering a habitable environment for cell proliferation. Fibrinogen is a natural protein capable of being electrospun and offers the benefit of existing as part of the natural coagulation cascade. Mats of fibrinogen could be utilized as possible hemostatic dressings or as an early scaffold for cell migration for either wound repair or tissue engineering. The addition of antibiotic into such a dressing/scaffold could prevent infection during healing/incorporation. The goal of this study was to determine any effect that the addition of the antibiotic tetracycline hydrochloride (0%, 2.5%, 5%, 10% by weight) would have on the mechanical properties of electrospun fibrinogen (110 mg/mL, 120 mg/mL, and 130 mg/mL concentrations). Also, the time release of tetracycline from electrospun fibrinogen was investigated. The results show no significant effect of tetracycline loading on the mechanical properties of electrospun fibrinogen under the conditions of this study. The results of the release study demonstrate that initial tetracycline release is dependent upon loading percentage. The release data also demonstrate that the amount of tetracycline released is approximately 20-30% of the tetracycline in the original solution and that the release occurs within approximately 4 hours, with no significant release thereafter. This study demonstrates the feasibility of tetracycline in electrospun fibrinogen for the purposes of short term drug release in fibrinogen-based technologies.

The genus Enterococcus has an exceptional ability to acquire and transmit antibiotic resistance genes and is considered to be a major vector in their dissemination. Enterococci are part of the normal gut microbiota of humans and animals and are frequently encountered in food products including dry fermented sausage. Since fermented sausages are not heat-treated before consumption they might be a vehicle for transmitting resistance and virulence traits of enterococci by conjugation with commensal bacteria present in the human gut and pathogenic bacteria that might be present, such as Listeria species. A PCR-based assay was developed to detect enterococci in dry fermented sausage meat at the generic level by targeting a 16S rRNA sequence and a total of 29 Enterococccus strains (15 E. faecalis, 13 E. faecium, and one E. gallinarum) were identified. The susceptibility of these enterococci to antibiotics was tested and it was found that 27/29 were resistant to more than one antibiotic and possessed antibiotic resistance determinants. All strains were positive for at least one virulence gene. Strong biofilm formation occurred at lower than optimum temperature in all three species of enterococci and probably contributed to their survival in the harsh conditions experienced during dry sausage fermentation and drying. SmaI pulsed-field gel electrophoresis (PFGE) patterns exhibited genomic heterogeneity within and between the two larger groups of isolates. In spite of this heterogeneity, the phenotypic similarities observed suggested that food could still be a vehicle for distribution of antibiotic resistant bacteria among humans. In vitro conjugation experiments demonstrated transfer of the tetracycline resistant determinant, tet(M), from E. faecium S27 isolated from fermented sausage to clinical isolates of both E. faecium and E. faecalis. The streptomycin resistance of E. faecium S27 was also transferred to a clinical strain, E. faecalis 82916, which was confirmed by the presence of the streptomycin resistance gene, aadA, in the donor and transconjugant strains. E. faecium S27 also transferred tet(M) and streptomycin resistance to Listeria monocytogenes GLM-2 by in vitro mating. Evidence suggests that enterococci in fermented meats may contribute to the spread of resistance determinants. / October 2015

MSc (Med), Epidemiology and Biostatistics, Faculty of Health Sciences, University of the Witwatersrand / Introduction Given the increasing prevalence of non-typhoidal salmonella in humans, especially as an opportunistic illness associated with HIV, enhanced surveillance for non-typhoidal salmonella (NTS), including screening for antibiotic resistance, is conducted annually in South Africa. We aimed to determine whether there is an association between trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis and multi-drug resistant NTS infection, to establish whether various factors modify the relationship between TMP-SMX resistance and invasive NTS infection, to examine whether these associations vary by province, and to quantify the resistance rates of NTS to a range of antibiotics. Methods This study was a secondary analysis of enhanced surveillance data on NTS collected between 2003 and 2005. We used descriptive methods to assess the prevalence of NTS by year, province and serotype, and to determine the prevalence of four MDR patterns. Univariate and multivariate regression models were used to investigate the relationships between TMP-SMX prophylaxis and MDR NTS. Univariate logistic regression was used to assess the relationship between invasive NTS and TMP-SMX resistance. Results TMP-SMX prophylaxis is associated with the ACKSSuT pattern (OR 1.91, 95% CI 1.14 – 3.19, p=0.0080) and the AKSSuT MDR pattern (OR 2.00, 95% CI 1.26 – 3.15, p=0.0015). Being on TMP-SMX prophylaxis is associated with an increased odds of having at least one of the four MDR patterns investigated (OR 1.43, 95% CI 1.00 – 2.04, p=0.0388). We also found high rates of resistance to all antibiotics tested except for ciprofloxacin and imipenem. The highest resistance rate was observed for sulfamethoxazole (>75.85%). S. enterica Isangi isolates showed the highest levels of resistance, with 94.43% having at least one MDR pattern. Other factors significantly associated with MDR NTS were ESBL production, prior treatment with antibiotics, HIV status and resistance to TMP-SMX. Discussion and conclusions Isolates from patients on TMP-SMX prophylaxis were associated with an increased odds of having the ACKSSuT and AKSSuT MDR patterns, not taking into account other explanatory factors. These associations did not remain significant when possible confounders were taken into account. Despite the threat of increased multi-drug resistance, TMP-SMX prophylaxis remains important in certain clinical settings.

Thesis advisor: Steven D. Bruner / The enediyne antitumor antibiotics are produced by complex biosynthetic machinery in acetomycetes. This dissertation will focus on the study of three enzymes involved in key steps in the biosynthesis of two enediynes, neocarzinostatin and C-1027. Neocarzinostatin is biosynthesized by a number of enzymes that synthesize and decorate the enediyne core and the peripheral moieties. NcsB1 is one enzyme involved in functionalizing the naphthoic acid portion of neocarzinostatin, a key group involved in binding to target DNA duplexes. The enzyme has been shown to be a promiscuous (<italic>S</italic>)-adenosylmethionine-dependent <italic>O</italic>-methyltransferase responsible for methylating a variety of hydroxynaphthoic acids. Multiple crystal structures of NcsB1 cocomplexed to substrate and/or cofactor have been solved. These structures revealed a displacement of the C-terminal domain when not bound to substrate, a movement that likely opens up the active site for naphthoate binding. Additionally, the ternary complex structure of 1,4-dihydroxynaphthoic acid, (<italic>S</italic>)-adenosylhomocysteine, and NcsB1 was solved and showed a rotation of this alternate substrate in the binding pocket, allowing for methylation. These results led us to probe NcsB1 activity using active site mutants, demonstrating altered substrate specificity and revealing key residues in substrate binding. The final step of neocarzinostatin biosynthesis involves multiple enzymes that convergently assemble the multiple biosynthetic intermediates to form the chromophore. NcsB2, originally proposed to catalyze the attachment of the naphthoic acid moiety to the enediyne core, has been characterized <italic>in vitro</italic>. Studies into its substrate specificity as an adenylation domain led to a revised biosynthetic pathway of 2-hydroxy-7-methoxy-5-methyl naphthoic acid. Instead of catalyzing the attachment of an enzyme bound naphthoic acid to the enediyne core, NcsB2 was found to act as a CoA-ligase, activating a variety of naphthoic acids and forming a naphthoyl-CoA intermediate. The results of these studies present an outstanding opportunity to produce novel analogs of neocarzinostatin by manipulating its biosynthesis. C-1027 is an architecturally similar enediyne that is also biosynthesized in a convergent route. C-1027 is a member of a class of enediynes that contains a functionalized &beta;-tyrosine derived from L-tyrosine. The first catalytic step towards this beta-tyrosine moiety is achieved by <italic>Sg</italic>TAM, a tyrosine aminomutase that catalyzes a 2,3-amino shift on L-tyrosine to form (<italic>S</italic>)-&beta;-tyrosine. The first X-ray crystal structure of <italic>Sg</italic>TAM was recently solved by our group, revealing structural homology to ammonia lyases. Through site-directed mutagenesis, X-ray crystallography, and biochemical analysis, residues that influence the mechanism by which <italic>Sg</italic>TAM catalyzes this difficult transformation were explored. From these studies, the enzymatic base and other pertinent residues involved in catalysis have been identified. In addition, residues that close the tunnel leading to the active site, thought to play a key role in mutase activity, were probed. Further study of rational mutants of <italic>Sg</italic>TAM will allow us to engineer its activity to alter its substrate specificity and the type of product it produces. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Les bactéries de l'environnement et du sol en particulier sont des producteurs actifs de molécules antibiotiques et les composés antibiotiques utilisés en médecine ont pour la plupart été isolés de bactéries saprophytes du sol qui ont elle mêmes développé une variété de mécanismes pour contrer les effets des antibiotiques conduisant à un arsenal de gènes de résistance à des antibiotiques dans l'environnement (ARGD). Une évaluation de l'abondance et de la diversité en terme de gènes de résistance à des antibiotiques a donc été conduite. Pour cette analyse, nous avons compilé 71 jeux de données de séquences d'ADN métagénomique environnementale variées: océans, et identifié des gènes de résistance pour chacun d'eux. Le sol est confirmé par cette étude comme un environnement extrêmement divers en terme de résistance à des antibiotiques. Cet étude in silico a été complété d'abord par une approche en microcosmes visant à étudier les effets soit de pollution soit par des molécules antibiotiques pures, soit par des effluents de ferme utilisés pour la fertilisation des sols. Les microcosmes de sols ont été incubés pendant 6 mois au laboratoire en conditions contrôlées. L'abondance de gènes de résistance à des antibiotiques a été évaluée au cours du temps par PCR quantitative. Une seconde étude visant à évaluer l'impact de la consommation de molécules antibiotiques par une population humaine sur son environnement immédiat, dont le sol, a été entreprise. Le village de Trois-Sauts est situé sur les berges du haut-Oyapock en Guyane Française. Les prescriptions antibiotiques sont très récentes dans cette région et les molécules distribuées ont été précisément répertoriées. Un transect de sol de 3km a été échantillonné chaque 600m afin de vérifier l'existence d'un gradient d'anthropisation entre le village (0m) et les échantillons de forêt les plus distants (3000m). Tous nos résultats confirment la présence à une forte abondance de gènes de résistance à des antibiotiques dans l'environnement, et en particulier dans le sol. Les facteurs à l'origine de la sélection et de la dissémination des gènes de résistance restent cependant difficiles à appréhender dans des environnements aussi complexes. C'est cependant avec une meilleure compréhension des phénomènes conduisant à l'émergence et à la dissémination des gènes de résistance à des antibiotiques au sein des flores pathogènes, depuis leur réservoir environnemental, que nous pourrons agir en vue de préserver les antibiotiques encore actifs aujourd'hui et ceux encore à développer. / Environmental bacteria and especially soil bacteria are active producers of antibiotic molecules and most drugs used nowadays are isolated from saprophytic soil bacteria and these microorganisms have also evolved numerous resistance pathways leading to an arsenal of Antibiotic Resistance Genes Determinants (ARGD) known as the environmental resistome. A survey of ARGD prevalence is required in order to characterize this natural phenomenon with critical implications in our current infectious diseases management. In order to perform such analysis we compiled a set of 71 metagenomic datasets from various environmental origins: soils, oceans, lakes, human feces, indoor air, etc., and compared their sequences with a database of known antibiotic resistance gene determinants (ARGD). ARGD-annotated reads are found in every environment analyzed confirming their ubiquity. Soil is found to be the richest and shares a large part of ARGD with the human gut microbiome, indicating ARGD transfers between these environments. Experiments using qPCR and metagenomic DNA sequencing on soil samples from two sites with known and distinct antibiotic pollution history were conducted to understand how ARGD abundance and diversity in soil are affected when impacted by antibiotic molecules. The first site is a reference soil from a long-term experiment without history of antibiotic pollution (Rothamsted Park Grass, UK). Soil microcosms are setup with addition of either antibiotic-containg animal manure or pure molecules and incubated for 6 months to monitor changes in ARGD concentration following these perturbations. Our second study-site is a very remote settlement in French Guiana where antibiotics are available since recently and may have impacted the local soil microbial community. Soil samples are taken following a line-transect going from the village (antibiotic source) to 3km deep in the forest in a gradient of human-impact. Our results all confirm prevalence of ARGD in soil at significant abundance but also that ARGD distribution is more correlated to environmental factors such as soil type, microbial taxonomy composition or microcosms incubation conditions than antibiotic molecules exposure in both sites. Pathogens ARGD diversity is far lower than ARGD diversity found in the environment and not all the soil resistome is readily accessible for transfer. In order to characterize the soil mobile gene pool, a strategy is proposed to isolate specifically mobile DNA directly from the environment for sequencing purposes. Better knowledge on the microbial ecology factors limiting ARGD transfers to pathogens may greatly help us reduce the current threat on our limited medical antibiotic molecules resource.

Acinetobacter baumannii is an opportunistic pathogen prevalent in nosocomial infections, most commonly infecting humans with compromised immune systems during their hospital stays. The organism's success in such circumstances has to do with its ability to survive on dry, abiotic surfaces (e.g. catheters, bed railings, and other medical equipment) and its increasingly apparent antibiotic resistance. These factors make A. baumannii a serious problem for healthcare professionals and in public health generally. A. baumannii is paradigmatic and representative of the issues confronting healthcare in the ongoing antibiotic crisis, and many strains are showing multidrug resistant (MDR) phenotypes. Given that the patients infected by A. baumannii tend to be very vulnerable and traditional antibiotic treatment seems to be getting less and less effective, it is imperative to explore alternative treatment options that may lead to better outcomes, especially if their mechanisms are not the same as the traditional antibiotics that exert the selective pressures that have led to the current antibiotic crisis. A small molecule called M64 is known to inhibit a LysR-type transcription regulator (LTTR) important for virulence, but not cell growth or viability, in another opportunistic pathogen, Pseudomonas aeruginosa. The experiments presented here show that M64 was able to rescue mice infected with A. baumannii and to downregulate the expression of important metabolic genes downstream from A. baumannii LTTRs BenM and CatM in vitro while having no effect on bacterial growth. BenM and CatM regulate genes involved in the metabolism of benzoate and catechol respectively, both of which are parts of tryptophan metabolism and are eventually broken down to form acetyl-CoA and succinyl-CoA for energy production in the citric acid cycle. Such a pharmacodynamic profile offers a starting point in the design of alternative treatments of MDR bacterial infections, as successful outcomes are observed without the direct killing of cells in vitro seen in traditional antibiotics. In this case, as catechol metabolism is important for siderophore biosynthesis and thus bacterial virulence, inhibition of the transcription of genes involved in catechol metabolism may be playing a role in the observed rescue of infected mice. Further studies are required to ascertain the nature of the inhibitor's effect, however.

Work was undertaken towards the synthesis of the promising antibiotic lactonamycin (iii). Following the work of Parsons et al. it was proposed that cyclisation of the ene-diyne (i) would give access to advanced pentacyclic intermediate (ii) and that from this a total synthesis of lactonamycin would be achieved (scheme I). Scheme I : Proposed Parsons, Board, Waters cyclisation to form the pentacycle (iii)(For image refer to pdf). A synthesis towards the cyclisation precursor (i) was carried out and a route to the key tetrasubstituted phthalide (v) established. Further chemistry was proposed to complete the synthesis of lactonamycin (scheme II). Scheme II : Formation of a fully substituted benzolactone.(For image refer to pdf). During attempts to introduce the β-bromoallyl group of key intermediate (v) using a high temperature Claisen rearrangement it was established that the benzodioxin (vii) underwent thermolysis to generate the reactive quinone methide intermediate (viii) and that in the presence of a nucleophilic solvent the adduct (ix) was formed (scheme III). Model studies showed the reaction to be both general and high-yielding. Scheme III : Novel quinone methide methodology. (For image refer to pdf).