Rozprawy doktorskie na temat „Bacterial antibiotics”

Bacterial biofilms are a leading cause of life-threatening and device-related infections worldwide. Biofilm related infections are notoriously difficult to treat as they are highly tolerant to conventional antibiotics. This project has designed and synthesised a new class of antibiotics to circumvent biofilm tolerance and shown that the prepared compounds could eradicate several medically important pathogens (P. aeruginosa, E. coli, and S. aureus). Importantly, as these compounds are hybrids of drugs that are already used clinically as stand-alone therapies, they demonstrate great potential to be translated into therapies in the near future.

O presente relatório de estágio pretende espelhar o trabalho desenvolvido, as competências adquiridas e as aprendizagens consolidadas durante a realização do estágio curricular na Clínica Veterinária - Clilegre, entre outubro de 2018 e janeiro de 2019. O documento é composto por duas partes: na primeira descrevem-se as atividades desenvolvidas e a casuística acompanhada; na segunda inclui-se uma monografia sobre a problemática das resistências aos antimicrobianos, com apresentação e discussão dos casos clínicos em que foi realizada colheita bacteriológica e teste de sensibilidade aos antibióticos em animais de companhia. A resistência bacteriana é um fenómeno natural que, aliado ao uso incorreto e excessivo de antimicrobianos, tem favorecido a seleção de estirpes resistentes, tornandose num grave problema de saúde pública mundial. Considerando a crescente proximidade entre animais de companhia e o Homem, torna-se fundamental estudar o problema da resistência aos antimicrobianos, sensibilizando profissionais de saúde para o uso adequado destes fármacos; Abstract: Study of (multi)resistant bacteria in companion animals clinics This internship report intends to mirror the work developed, the skills acquired and knowledge consolidation during the curricular internship at the Clilegre Veterinary Clinic, from october 2018 to january 2019. The document consists of two parts: the first part describes the activities developed and the casuistic followed; the second includes a monography about antimicrobial resistance, with the presentation and discussion of clinical cases in which bacteriological isolation and antibiotic susceptibility testing were performed in companion animals. Bacterial resistance is a natural phenomenon that, combined with the incorrect and excessive use of antibiotics, has favoured the selection of resistant strains, becoming a serious public health problem worldwide. Considering the growing proximity between companion animals and humans, it is essential to study the problem of antibiotic resistance and raising awareness to health professionals to the proper use of these drugs.

Polyphosphate biosynthesis and the stringent response play an important role in the virulence of pathogenic bacteria. Our objective is to validate these pathways as an antimicrobial target and to identify inhibitors of the key enzymes polyphosphate kinase (PPK), (p)ppGpp synthetase I (RelA) and (p)ppGpp synthetase/hydrolase II (SpoT). The role of polyphosphate and (p)ppGpp metabolism in Francisella virulence has been explored with deletion mutants. These exhibited defects for intracellular growth in macrophages and were attenuated in mice, indicating a key role for the FtPPK, FtRelA and FtSpoT in the virulence of Francisella. The development of three in vitro activity assays will enable the discovery of PPK inhibitors. Ion-pairing HPLC analysis has been used to measure substrate kinetics, providing evidence that FtPPK belongs to the PPK2 superfamily with little preference between substrates (KM: ADP - 369 μM; GDP - 624 μM) as displayed by other PPK2 enzymes. 31P NMR spectroscopy has been used to monitor the overall time course of the PPK reaction. To facilitate high-throughput screening, a coupled luminescence based activity assay has been developed in a 96-well plate format. These assays can also be applied to the discovery of inhibitors for FtRelA and FtSpoT. Understanding the structural basis of inhibitor action requires a crystal structure of the target enzyme. For FtPPK, a crystallisation screen has identified conditions for obtaining suitable crystals and data has been collected to 2.1 Å resolution. Future studies will use the high throughput assay to identify PPK inhibitors; NMR and HPLC assays to characterise the mode of action and crystal structures of PPK:inhibitor complexes will identify the precise molecular interactions.

Exposure to antibiotics is an important factor influencing the development of bacterial resistance.  In an era where very few new antibiotics are being developed, a strategy for the development of optimal dosing regimen and combination treatment that reduces the rate of resistance development and overcome existing resistance is of utmost importance. In addition, the optimal dosing in subpopulations is often not fully elucidated. The aim of this thesis was to develop pharmacokinetic (PK) and pharmacokinetic-pharmacodynamic (PKPD) models that characterize the interaction of antibiotics with bacterial growth, killing and resistance over time, and can be applied to guide optimization of dosing regimens that enhance the efficacy of mono- and combination antibiotic therapy. A mechanism-based PKPD model that incorporates the growth, killing kinetics and adaptive resistance development in Escherichia coli against gentamicin was developed based on  in vitro time-kill curve data. After some adaptations, the model was successfully applied for similar data on colistin and meropenem alone, and in combination, on one wild type and one meropenem-resistant strain of Pseudomonas aeruginosa. The developed population PK model for colistin and its prodrug colistin methanesulfonate (CMS) in combination with the PKPD model showed the benefits for applying a loading dose for this drug. Simulations predicted the variability in bacteria kill to be larger between dosing occasions than between patients. A flat-fixed loading dose followed by an 8 or 12 hourly maintenance dose with infusion duration of up to 2 hours was shown to result in satisfactory bacterial kill under these conditions. Pharmacometric models that characterize the time-course of drug concentrations, bacterial growth, antibacterial killing and resistance development were successfully developed. Predictions illustrated how PKPD models based on in vitro data can be utilized to guide development of antibiotic dosing, with examples advocating regimens that (i) promote bacterial killing and reduce risk for toxicity in preterm and term newborn infants receiving gentamicin, (ii) achieve a fast initial bacterial killing and reduced resistance development of colistin in critically ill patients by application of a loading dose, and (iii) overcome existing meropenem resistance by combining colistin and meropenem

Macrolide antibiotics are known as anti-bacterial agents. Erythromycin A, 14-membered macrolide antibiotic is known to exist in two forms-ketone (active) and hemiacetal form (inactive). It shows mild flexibility in silico. Its derivative, clarithromycin, 6-O-methyl erythromycin A, shows rigidity and activity against Gram-positive bacteria. The semisynthetic derivative of erythromycin A, azithromycin, 15-membered macrolide antibiotic, shows flexibility in silico and activity against Gram-negative bacteria. The combination of molecular modelling (molecular mechanics and/or molecular dynamics) with TRNOESY NMR data give us the active conformation of flexible molecules. Constraining the strong intramolecular hydrogen bonds can be helpful in the determination of the active conformation of the drug. We have developed modelling strategy for the construction of new 14- and 15-membered macrolideantibiotics with desired activity.Tylosin A and tylosin B, 16-membered macrolide antibiotics, show rigidity in silico. However, tylosin A is very unstable in aqueous solutions, so precise determination of hydrogen and carbon chemical shifts is extremely difficult. Nobody else before us tried to publish the full assignments of this compound in D2O. Accurate determination of hydrogen and carbon chemical shifts is necessary in order to further explore the properties of this compound.Anti-bacterial activity investigation of tylosin A and its derivative, tylosin B, shows lower activity both against Gram-positives and Gram-negatives compared to clarithromycin and azithromycin. Superposition of two molecules of azithromycin with one molecule of tylosin A reveals that two molecules of azithromycin actually occupy the space of one tylosin A molecule, which can explain found anti-malarial activity of tylosin A (both azithromycin and tylosin A show similar contacts to bacterial ribosomes).Clinical trials show that azithromycin has an anti-malarial activity. In order to investigate the potential anti-malarial activity of macrolide antibiotics, we had to construct the exit tunnel of the apicoplast ribosome from Plasmodium falciparum. Because of the unavailability of the crystal structure of P. falciparum ribosome (it is impossible to separate mitochondria and apicoplast, and both of them contain ribosomes), we used different computational methods and softwares in order to construct it. We used both homology modelling and ab initio modelling server for the construction of L4 (apicoplast-encoded P. falciparum ribosomal protein) and L22 (nuclear genome-encoded P. falciparum ribosomal protein) and RNA_2D3D software to construct the 23S rRNA from apicoplast ribosome of Plasmodium falciparum. Using Pymol software and MOE we have constructed the exit tunnel of apicoplast ribosome from P. falciparum. The model shows that it can bind one azithromycin molecule. It is the first model of the exit tunnel of the apicoplast ribosome from Plasmodium falciparum. Further work can be extended to the docking of other molecules than azithromycin into the modelled exit tunnel of Plasmodium falciparum.

Chemistry
Ph.D.
Bacterial resistance is a formidable 21st-century global public health threat. If left unaddressed, we risk moving toward a “post-antibiotic era.” While resistance is a natural consequence of antibiotic use, the rate at which pathogenic bacteria have evaded multiple classes of drugs has markedly outpaced the introduction of new ones. New antibiotics are desperately needed to fill this void. Macrolides are one of the safest and most effective drug classes in medicine; however, resistance has compromised efficacy. To date, three generations have been developed with only the lattermost targeting bacterial resistance. Single next-generation macrolides will not keep pace with the inevitable onset of resistance; thus, there is a critical need to greatly accelerate the procurement of multiple future-generation antibiotics to tackle both current and future resistance mechanisms. My research is to meet this need by designing, synthesizing, and evaluating a novel, future-generation macrolide antibiotics that will serve as an armamentarium to be individually deployed on demand. In the previous research in Andrade group, we synthesized and evaluated various desmethyl ketolide analogs. The fact that 4-desmethyl telithromycin was fourfold less potent than telithromycin against A2058G mutants indicated replacing the 4-Me with hydrogen (i.e., desmethylation) to avoid a steric clash with the 2-amino group of G2058 was insufficient in rescuing bioactivity. Guided by MD simulation, we concluded a logical, superior alternative strategy was the replacement of the 4-Me group with one possessing a smaller vdW radius and capable of establishing favorable interactions with both wild-type and A2058G mutant ribosomes. Specifically, we reasoned that 4-fluoro solithromycin would be ideal candidate. The hypothesis was that the 4-fluoro moiety would engage in dipole-dipole interactions (C-F---H) with the exocyclic 2-amino group of guanine, which is based on accumulated evidence that strategic placement of organofluorine can strongly impact potency, selectivity, and physicochemical properties. In addition, the axially disposed of 4-fluorine would provide conformational stabilization from a gauche effect with the vicinal O5 group. The novel synthetic routes to unexplored desosamine analogs at the C3’-amino substituent to the macrolide antibiotic would play a role in bioactivity and resistance. Hofmann reaction was employed to execute the same 2,3-epoxide ring opening method without removing desosamine and re-glycosylating. This markedly reduces the steps, time, and cost involved in preparing novel desosamine-modified analogs. Significantly, this route enables the first synthesis of N,N’-disubstituted desosamine analogs from an epoxide, which was utilized to prepare novel analogs of clarithromycin. The application of in situ click chemistry toward the discovery of novel macrolide antibiotics first required the synthesis of suitable azide and aryl alkyne reactants. Alkyne partners were procured by commercial vendors or chemical synthesis. We targeted two logical, validated positions to tether the side chains, specifically N11 on the macrolactone and N3’ of desosamine. The first (N11) has been the most utilized. Moreover, extensive structure-activity relationships have revealed a four-carbon tether is ideal. Based on the solithromycin−E.coli X-ray structure, I designed, synthesized, and evaluated dehydro solithromycin, which possesses an (E)-alkene in the side-chain. The use of an unsaturated side chain would conformationally preorganize the bi-aryl side chain in order to pay the entropic penalty and thus favorably contribute to the overall binding. An insightful observation made from MD simulationed ribosomes bound with to solithromycin revealed that the interaction of the side-chain includes H-binding as well as π-stacking. The hypothesis was that employing tethered side-chains bearing motifs that maximize H-bonding and π-stacking would be superior antibiotics for treating resistant bacterial strains bearing erm¬-mediated N6 methyl and dimethylated ribosomes. To test this hypothesis, we developed various analogs with different alkynes by introducing different functional groups at the 3 and 5 positions on the aromatic ring. Another desosamine sugar modification is bis-azide. To date, the use of a two side chain strategy has not been reported. To access the requisite bis-azides, we employed a tactic the oxidative demethylation and alkylation of desosamine to afford bis-click solithromycin analogs.
Temple University--Theses

Bacteriological survey of the fish pathogens in Greek mariculture between 1994- 1997 was followed by analysis of prevalence in sea bass, sea bream, sharpsnout bream and common Dentex and discussion of the impact of various fish pathogens. In addition antibiotic resistance profiles and frequencies were studied using quantitative antibiogram and MIC analysis for the two most commonly used antibiotics Oxolinic acid and Oxytetracycline and clinically relevant MIC breakpoints were extrapolated for different fish species and main fish pathogens. The kinetics of the above antimicrobials were analysed in eight experiments where two fish species namely sea bass and sea bream as well as two water temperatures were employed. Muscle, liver, serum, skin samples were analysed by two HPLC methods and two bioassay methods were developed. The relative importance and significance of these findings was evaluated in the general context of pharmacokinetic studies in fish. Kinetic data were compared to clinical data and practical implications were evaluated. Issues like antibiotic resistance and its implications, the implications of residues and resistance in human health and the environment were analysed in order to put this study in context. Conclusions tackled important aspects of antimicrobial chemotherapy and future work was suggested.

In the last decades, there has been an escalating consumption of antibiotics with the number of antibiotic prescriptions increasing worldwide. Overuse or inappropriate use of antibiotics has resulted in a major increase in the development of multi-drug resistant pathogens. Antimicrobial resistance is one of the world’s most serious public health problems with great implication in terms of morbidity, mortality, and costs. To date, there has been no formal antibiotic use study conducted in the West Wollega zone of Ethiopia to assess antibiotic utilization. The objective of this study was to determine the pattern of antibiotic use in two hospitals in the West Wollega zone of Ethiopia, namely Gimbie Adventist Hospital and Nedjo Hospitals, using drug utilization metrics and the costs associated. In addition it was to assess the correlation between diagnosed infectious diseases and antibiotic prescriptions. This study was a cross-sectional, retrospective, descriptive review of antibiotic usage in the two hospitals in the year 2007. Prescriptions dispensed in the first month of each quarter of 2007 were reviewed. The number of prescriptions screened, antibiotic courses started, antibiotic days by specific agent and overall, the cost of individual and all antibiotics, the number and type of infectious diseases diagnosed were collected from which core drug use indicators were calculated. The correlation between infectious disease diagnosed and the antibiotic days prescribed were analyzed. A total of 18568 antibiotic and non-antibiotic prescriptions were reviewed retrospectively in the four months of the study period, 47 percent of which contained at least one antibiotic. The average number of antibiotics per prescription was 1.33 and 1.09 whilst the percentage of injectable antibiotics prescribed was 83.2 percent and 3.76 percent to outpatients and inpatients respectively. Antibiotics prescribed from the Essential Drug List (EDL) and List of Drugs for District Hospital (LDDH) were 63.0 percent, 74.8 percent, and 90.8 percent and 76.1 percent for outpatients and inpatients respectively. 98.6 percent of outpatient and 97.0 percent inpatient prescribed antibiotics were actually dispensed. Penicillins and quinolones were the most prescribed antibiotics in both inpatient and outpatient departments constituting 43.46 percent and 24.08 percent respectively. The antibiotic utilization ratio, incidence of outpatient antibiotic use, incidence of inpatient antibiotic use, the number of Defined Daily Doses (DDD)/1000inhabitants/year and DDD/100 Occupied Bed Days (OBD) for the zone was 0.16, 17.25, 23.56, 158.61, and 70 respectively. Antibiotic cost constituted 33.7 percent of all expenditure on drug, cost of antibiotic per patient care day and cost per antibiotic day was 3.84 Ethiopian Birr (ETB) ($0.40) and 6.29 ETB ($0.66) respectively. The correlation between infectious diseases diagnosed and antibiotic prescription shows significant variation. At outpatient departments alone an average number of antibiotic courses started was 2.7 at Gimbie Adventist Hospital and 7.6 for Nedjo Hospital. When overall antibiotic days prescribed and required was compared in both hospitals, there were 2.4 and 5 times more antibiotic days prescribed than were required for Gimbie and Nedjo Hospitals respectively. This suggests that the overuse of antibiotic is worse in the government hospital (Nedjo Hospital) than in the mission hospital (Gimbie Adventist Hospital). This study suggested that there was overuse of antibiotics in the West Wollega hospitals although further investigation is needed to identify its underlying causes and nature. It is recommended that the health personnel, the hospital management, the zonal and regional Health Bureau, the regulatory bodies and Non-Governmental Organizations (NGOs) work hand-in-hand to promote the rational use of antibiotics in this region so that the consequences and financial cost of antimicrobial resistance can be prevented.

The Marine sponge Cinachyrella sp used in this study are commonly found in offshore South Florida and Caribbean waters and appeared to be resilient in closed system aquaculture. Marine sponges host diverse bacterial symbionts that are distinct compared to bacteria found in ambient seawater, however the roles of a large fraction of the bacterial community in marine sponges are unknown. Comparison of symbiotic to aposymbiotic (bacteria-free) sponges could provide information about interactions (metabolic and physiologic) between the bacteria and sponge. In this study, a single Cinachyrella kuekenthali individual was subsectioned into explants (N=240) in order to provide identical bacterial communities to perform comparative studies. Presence of photosymbionts was also analyzed by characterizing bacterial communities from varying light and dark conditions. Tools for characterization included transmission electron microscopy (TEM) and 16S rRNA sequence analysis obtained from Illumina Miseq. High throughput DNA sequencing revealed bacterial taxa belonging to phyla Thaumarchaeota, Chloroflexi, Nitrospira, Acidobacteria and Verrucomicrobia persist in the explants. This study also demonstrated that antibiotics (Ampicillin, Tetracycline, Penicillin-Streptomycin and combination of all) can alter the bacterial community in the marine sponge C. kuekenthali explants in vitro. Bacterial communities of explants treated with different antibiotics were statistically (Unifrac and Bray-Curtis analysis) different from controls (p-value < 0.001, R2=41%). Penicillin-streptomycin and cocktail of antibiotics treatment contributed to the highest difference in the bacterial communities. Also, bacterial communities of explants at difference time points treated with corresponding antibiotics were also statistically significant (p-value<0.05, R2=15%). TEM observations of denatured nucleic acid and osmotic lysis of bacteria, due to the effect of antibiotics were observed, creating a LMA mesohyl. However light versus dark conditions did not produce any statistically significant difference in beta diversity between bacterial communities. These interdisciplinary results indicate that while individual bacterial symbiont taxa may persist after community disruption, significant changes in the overall composition of the bacterial symbiont population can be created

Dissertação de Mestrado Integrado em Medicina Veterinária
Bacterial infections are one of the biggest health issues globally. Antimicrobial therapy experienced `the golden era of antibiotics´ from the 1950s to the 1970s. However, the inadequate and irrational use of antimicrobial agents over the years, has given microorganisms conditions to acquire resistance, compromising the control of infectious diseases. Drug combinations and interactions involve a variety of possible outcomes that are currently explored to create more efficient clinical therapies. The combination of non-antibiotic compounds with antibiotics to achieve synergy, exploring a diversity of substances that could act as antimicrobials, is an exciting approach. Equine clinical practice uses frequently local anaesthetics to examine lameness, which is a common issue in horses. Antibiotics can be used prophylactically during these procedures. Besides, they can be applied during regional limb perfusions and are used in combination with local anaesthetics to treat septic arthritis. Various studies over the years have reported the antimicrobial effects of local anaesthetics against a variety of pathogens, although knowledge regarding interactions with different compounds is scarce. The aim of the present study was to investigate possible interactions between three frequently used local anaesthetics — lidocaine, mepivacaine, and bupivacaine — with three frequent antibiotics — amikacin, gentamicin, and ceftiofur — in equine practice, against sensitive and resistant strains of Escherichia coli and Staphylococcus aureus, collected from infection sites from horses. Disc diffusion tests were performed to verify susceptibility of isolates to the antibiotics, and minimum inhibitory concentrations (MIC) were determined for all compounds by broth microdilution. Interactions between the compounds were analysed by a checkerboard method, quantifying the fractional inhibitory concentration index (FICI). A modified disc diffusion test (MDDT) was implemented to confirm some synergy results, and also to evaluate the correlation between methods. The results obtained confirm the antimicrobial effects of local anaesthetics against equine pathogens. Reproducibility of the checkerboard method is not perfect, nonetheless it allows an initial detection of interactions between different compounds. Most interactions demonstrated additivity/indifference, revealing the use of these compounds combined, to be safe in equine practice. However, some antagonistic interactions were discovered, although most exhibited maximum FICI values close to 4, showing a weak antagonism. A few cases of synergy were detected in methicillin-resistant Staphylococcus aureus strains. Checkerboard and MDDT methods displayed a good correlation.
RESUMO - Avaliação da interação entre antibióticos e anestésicos tópicos em bactérias patogénicas de equinos - As infeções bacterianas são um dos maiores problemas de saúde reconhecidos mundialmente. O recurso a antibióticos experienciou a `época de ouro´ entre os anos 50 e os anos 70. Contudo, o uso irracional de agentes antimicrobianos, permitiu o desenvolvimento de microrganismos multirresistentes, dificultando o controlo de infeções. Combinações entre diferentes compostos podem originar diversos efeitos, os quais são atualmente investigados para produzir tratamentos mais eficazes. Particularmente, a combinação de antibióticos com diferentes compostos, explora diversas combinações com o potencial de aumentar a eficácia de tratamentos. Tais combinações poderão melhorar a interação entre um antibiótico e o seu alvo, desenvolvendo uma interação sinérgica. Na prática clínica de equinos é frequente recorrer a anestésicos locais para examinar casos de claudicação. Durante estes procedimentos é possível utilizar antibióticos profilaticamente. Além disso, os antibióticos podem ser usados em administrações regionais dos membros e é comum a sua co-administração com anestésicos em tratamento de artrites. Ao longo dos anos, diversos estudos reportaram os efeitos antimicrobianos dos anestésicos contra uma variedade de agentes patogénicos. Contudo, o conhecimento relativo das interações com diferentes compostos é escasso. O objetivo do presente estudo foi investigar a eficácia das interações entre os três anestésicos, lidocaína, mepivacaína, e bupivacaína, e os três antibióticos, amicacina, gentamicina, e ceftiofur, contra estirpes sensíveis e resistentes de Escherichia coli e Staphylococcus aureus, colhidas em focos de infeção de equinos. Realizaram-se testes de suscetibilidade aos antibióticos por difusão de disco, e as concentrações inibitórias mínimas foram determinadas por micro-diluição. As interações foram analisadas através do método do checkerboard, calculando o índice de concentração inibitória fracionada. Um teste por discodifusão modificado (MDDT) foi efetuado em alguns casos de sinergia, permitindo avaliar a correlação dos métodos. Os resultados confirmam o poder antimicrobiano dos anestésicos contra as estirpes estudadas. A reprodutibilidade do método checkerboard não é perfeita, porém permite detetar prováveis interações entre compostos. A maioria das interações revelou aditividade/indiferença, demonstrando que o uso destes compostos em combinação é seguro. Algumas interações revelaram antagonismo, apesar de fraco. Poucos casos de sinergia foram detetados em S. aureus resistentes à meticilina. Os métodos checkerboard e MDDT apresentaram uma boa correlação.
N/A

Thesis advisor: Jianmin Gao
Thesis advisor: Eranthie Weerapana
The imminent threat of antibiotic resistant pathogens that have emerged in clinical settings over the past several decades demands novel solutions in the form of both species- and/or strain-specific diagnostic technologies and treatments. Such new developments would aid in the improved management of bacterial infections by accurate diagnosis and targeted bacterial killing, which would mitigate the continued spread of antibiotic resistance as a result of broad-spectrum antibiotic application The cell surface of bacteria presents a unique opportunity towards development of these modalities, as bacterial cell walls possess both universal and unique features that can be targeted by chemical functionalities without the requirement of cell penetration. This work has sought to take advantage of naturally existing and non-natively installed bacterial cell wall chemical functionalities for which we can develop novel chemoselective chemistries and unique peptides that incorporate those chemical functionalities to enable targeted, biocompatible methods of bacterial labeling and targeting. We initially began these endeavors with the goal of improving upon existing readily reversible iminoboronate chemistry with acetylphenyl boronic acid (APBA), which selectively labels bacteria that contain amine-presenting cell wall lipids (e.g. PE and Lys-PG). In our efforts to improve upon the binding potency of this chemical motif, we synthesized a panel of APBA analogues with varying substituents to modulate amine-binding affinity. We additionally characterized these analogues capacity to form thiazolidinoboronates with free and N-terminal cysteine. Furthermore, we applied an APBA dimer presenting phage library towards identification of potent and selective APBA-presenting peptide binders of 1) a cationic antimicrobial peptide (CAMP) implicated in cancer, human beta defensin 3 (hBD3), and 2) colistin-resistant strains of bacteria that attain their resistance through a variety of different mechanisms. This high-throughput technology afforded identification of peptides that are indeed protein or species/strain selective binders, thus enabling targeted labeling of these important biomolecules. In our continued efforts to identify highly potent and selective bacterial targeting chemistries, we also developed an irreversible chemistry that enables the incorporation of chemical motifs, APBA and semicarbazide, into the cell walls of bacteria through cell wall remodeling mechanisms, which then undergo rapid conjugation with fluorescent and turn-on fluorescent reactive partners. While this alternative approach to bacterial detection requires a primary cell-wall incorporation step, the incorporation and subsequent labeling of these chemical motifs are both highly efficient, which enhances the potency of this bacterial labeling approach The chemical approaches to targeted bacterial labeling herein highlight our ability to develop several species- and strain-selective bioorthogonal chemical probes towards the goal of discovering targeted bacteria binding modalities. Beyond identification of such targeted bacterial binding molecules, we hope to translate these findings into effective, narrow-spectrum antibiotics, which is an endeavor currently being pursued in our laboratory
Thesis (PhD) — Boston College, 2019
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry

The worldwide rise of antibiotic resistance stimulates the search of new strategies for killing bacteria, whose mechanisms of action are different from those of antibiotics. Two promising strategies in this respect are the use of cationic antimicrobial peptides (CAMPs) and photodynamic therapy (PDT). Both approaches have been considered and some aspects elucidated during my PhD. Therefore, studies along two different research lines, having the same final aim and merging in the last part of the project, have been carried out. The first research line focused on PDT, in particular I investigated how several experimental factors affect the phototoxic activity against bacteria of cationic porphyrins, and I identified some of their molecular targets in Staphylococcus aureus. The second research line focused on the mechanism of action of a CAMP, apidaecin 1b, that was then conjugated to a photoactive porphyrin to obtain a new and broad-spectrum antimicrobial agent. PDT utilizes a visible light absorbing molecule, called photosensitizer (PS), that in the presence of oxygen generates cytotoxic reactive species that kill bacterial cells previously loaded with the PS. I studied the effects of some experimental factors that could affect the binding of the PS to the bacterial cell, and, as a consequence, the efficiency of bacteria photoinactivation. In particular, the effects of the cell washing after incubation with the PS, and of the presence of cations in the incubation medium, were considered. For these studies a dicationic porphyrin was used, and its ability to photoinactivate S. aureus and E. coli was measured after irradiation of the bacteria with increasing doses of blue light. Illumination was carried out with the unbound PS left in the suspension, as well as after one or four washings of the cells. The washings produced a completely different effect in the two microorganisms: slightly decreased the photokilling of S. aureus and strongly increased the photokilling of E. coli. The increased photokilling of E. coli was explained with a re-localisation, occurring during the time needed for the washing procedure, of the porphyrin molecules in inner cellular sites critical for survival. This is very likely to occur, considering the short (5 min.) incubation time of the cells with the PS. The presence of a monovalent cation (Na+) in the cell medium during incubation and illumination did not affect negatively the photoinactivation of S. aureus, that was slightly increased by the presence of Ca2+ and Mg2+. On the contrary, the phoinactivation of E. coli was slightly decreased in the presence of both mono- and divalent cations, and the effect was more evident in cells illuminated without washings. In all cases, the bacteria photoinactivation with this dicationic porphyrin was little affected by the presence of cations, in comparison to other cationic porphyrins. A classical proteomic approach, that included two-dimensional gel electrophoresis (2-DE) and mass spectrometry analysis, was used to identify some proteins that are potential primary molecular targets of PDT. Towards this aim, the change of the proteomic profile of S. aureus after PDT treatments was investigated using two different cationic porphyrins. For each of these two porphyrins, that strongly differ in their photoinactivation activity, I have selected two PDT treatments on the base of the different survival of treated bacteria: a sublethal one, which allows a bacterial survival of 60 to 80%, and a stronger one, which allows only a bacterial survival of about 1%. 2-DE maps from PDT-treated and untreated samples were compared using the Proteomweaver software for 2-DE maps analysis. All proteins showing a significant difference in their intensity levels between untreated and PDT treated 2-DE maps were identified by mass spectrometry (MALDI-TOF/TOF). All identified proteins were then divided into functional classes on the basis of their cellular function, in order to identify the pathways that were more damaged by PDT. Among the 265 proteins identified by the Proteomweaver analysis, 70 changed their intensity levels after PDT treatments; most of these are involved in the response to oxidative stress, in the energy metabolism and in the uptake of sugar. The comparison of the effects of PDT treatments between the two porphyrins revealed that several proteins were modified by both of them, and generally showing the same trend. Interestingly, the modifications of the proteomic patterns induced by PDT treatments are not consistent with a response of S. aureus cells to oxidative stress induced by other oxidizing agents (hydrogen peroxide, superoxide or diamide), therefore suggesting a selective targeting of specific proteins. These findings provide new insight in the understanding of the mechanism of action of PDT, and may be useful to design new photosensitizing agents with improved antibacterial activity. In the second research line I studied the antibacterial activity, and the ability to translocate into the bacterial cell, of the insect antimicrobial peptide apidaecin 1b. The improved understanding of the translocation mechanism of apidaecin was then used to investigate the possibility to use apidaecin itself as a cargo delivering molecule, by conjugating it with a porphyrin, in an attempt to obtain a new broad-spectrum antimicrobial agent. Apidaecin 1b is a small (18 residues) peptide that is extensively studied because of its unique properties, which include the ability to inhibit some Gram (-) bacteria growth and a high capability to translocate into cells with a non pore-forming mechanism. Moreover, no toxicity against eukaryotic cells has been reported even at concentrations that totally inhibit bacterial growth. In order to study its mechanism of action, each of the three arginine residues (or all of them together) were replaced with the corresponding peptoid residue, N-(3-guanidinopropyl) glycine (NArg), and the minimal inhibitory concentrations (MICs) of all of these peptide-peptoid hybrids were evaluated against E. coli, P. aeruginosa and S. aureus. No antimicrobial activity was detected for apidaecin and its peptide-peptoid hybrids against S. aureus and P. aeruginosa. On the contrary, against E. coli apidaecin showed a MIC value of 8-16 µM, while its peptide-peptoid hybrids substituted in arginine 4 and 12 showed a little decrease of antimicrobial activity and those substituted in arginine 17 or in all three arginines completely lost their activity. These results suggested that the arginine in position 17 is particularly important in the translocation mechanism into the bacterial cell. Therefore, apidaecin and its peptide-peptoid hybrids were labelled with fluorescein, in order to monitor by means of fluorescence microscopy and flow cytometry their ability to tightly bind to bacterial cells. The fluorescein-labelling completely abolished the antimicrobial activity of all our compounds and inhibited to a great extent their binding and translocation into bacterial cells. This effect was mainly due to the additional mass brought by fluorescein to apidaecin and its hybrids. Anyway, it was observed that the peptide-peptoid hybrid substituted in arginine 17 did not show any binding to bacterial cells. This finding confirmed the importance of this residue for antimicrobial activity, and, in general, the importance of the C-terminal of apidaecin in the binding and translocation into the bacterial cells. The conjugate (called T-api), resulting from the coupling of an anionic porphyrin (monocarboxy-tetraphenylporfine, cTPP) with the N-terminal of apidaecin 1b, did not show any antimicrobial activity in the dark. On the contrary, following activation with blue light, T-api caused a significant reduction of bacteria survival with an efficacy strongly dependent on the bacteria species. The phototreatment was extremely successful against E. coli and S. aureus, while the photokilling efficiency against P. aeruginosa was much lower but sufficient to induce a significant reduction of survival. Irradiation experiments were also performed by treating bacteria with cTPP alone, apidaecin 1b alone or the two agents together but not conjugated. None of these treatments affected the survival of both E. coli and P. aeruginosa, while S. aureus survival was decreased by cTPP, but to a lesser extent in comparison to T-api. Thus, it was demonstrated that the conjugation of an antimicrobial peptide with a photoactive molecule can lead to the formation of antimicrobial agents very effective and displaying a broader activity in comparison to the single components.
cIl forte aumento, a livello mondiale, del fenomeno della resistenza agli antibiotici richiede lo sviluppo di nuove strategie antimicrobiche, basate su meccanismi d’azione diversi da quelli degli antibiotici, per combattere le infezioni batteriche. Due delle strategie più promettenti in quest’ottica sono la terapia fotodinamica (PDT) e l’utilizzo di peptidi cationici antimicrobici (CAMPs). Durante il mio dottorato di ricerca ho studiato alcuni aspetti peculiari di entrambe queste strategie, in due linee di studio che alla fine sono state riunite. Nella prima linea di ricerca, focalizzata sulla PDT con porfirine cationiche, ho studiato quale impatto abbiano alcune condizioni sperimentali sull’efficienza di fotosensibilizzazione di batteri, ed ho identificato alcuni dei bersagli molecolari della loro azione su Staphylococcus aureus. Nella seconda linea di ricerca ho studiato il meccanismo d’azione di un particolare CAMP, l’apidaecina 1b, che ho successivamente coniugato con un fotosensibilizzatore allo scopo di creare un nuovo, più efficiente, agente antimicrobico. La PDT utilizza molecole capaci di assorbire la luce visibile, dette fotosensibilizzatori (PS), che, quando illuminate in presenza di ossigeno molecolare, generano specie reattive dell’ossigeno, che hanno un forte effetto citotossico su cellule batteriche precedentemente incubate col PS stesso. Nella prima fase di studio ho valutato l’effetto di alcune condizioni sperimentali sulla capacità del PS di legarsi alla cellula batterica e, di conseguenza, sull’efficienza di fotoinattivazione di batteri. In particolare sono stati valutati gli effetti di lavaggi effettuati dopo l’incubazione dei batteri con il PS, nonché della presenza di diversi cationi nel mezzo di incubazione. In questo studio è stata utilizzata una porfirina dicationica, la cui efficienza nella fotoinattivazione di Staphylococcus aureus ed Escherichia coli è stata misurata mediante irradiamento con dose crescenti di luce blu. L’irradiamento è stato effettuato sia lasciando nella sospensione batterica il PS non legato alle cellule che rimuovendolo mediante uno o quattro lavaggi. Questi lavaggi hanno prodotto effetti completamente opposti nei due microrganismi oggetto di studio: da un lato si è riscontrato un forte aumento dell’efficienza di fotosensibilizzazione di E. coli, dall’altro un decremento di quella di S. aureus. L’aumento dell’efficienza di fotosensibilizzazione in E. coli è probabilmente dovuto al fatto che, nel tempo necessario per effettuare i lavaggi, la frazione di porfirina legata alle cellule batteriche riesce a raggiungere siti cellulari più sensibili alla PDT. Il fatto che si sia utilizzato un tempo di incubazione molto breve (5 minuti) rende molto plausibile questa ipotesi. L’aggiunta di un catione monovalente (Na+) nel mezzo di irradiamento non ha causato alcuna variazione dell’efficienza di fotosensibilizzazione di S. aureus, che invece è stata fortemente incrementata da quella di cationi bivalenti (Ca2+ e Mg2+). Al contrario, la fotosensibilizzazione di E. coli è stata sensibilmente diminuita in presenza di cationi (sia mono che bivalenti), con un effetto più marcato in assenza di lavaggi. In ogni caso, utilizzando questa porfirina dicationica gli effetti prodotti sia dai lavaggi che dalla presenza di cationi sono stati minori di quelli riscontrati in precedenza con altri fotosensibilizzatori. Per l’identificazione di alcune delle proteine che sono bersaglio della PDT è stato scelto un approccio di tipo proteomico, comprendente la separazione con elettroforesi bidimensionale dei lisati batterici e l’identificazione di proteine con tecniche di spettrometria di massa. Al fine di ottenere un’analisi il più possibile dettagliata, sono stati valutati i cambiamenti nel profilo proteomico di S. aureus causati dalla PDT con due diverse porfirine cationiche. Per ciascuna di queste porfirine, che differiscono notevolmente nell’attività fotosensibilizzante, sono stati selezionati, sulla base della differente mortalità indotta in sospensioni di S. aureus, due trattamenti fotodinamici: uno subletale, che consente una sopravvivenza dal 60 all’80% dei batteri, e l’altro più forte, che consente la sopravvivenza di circa l’1% dei batteri. Le mappe bidimensionali ottenute da lisati proteici di batteri non sottoposti a PDT sono quindi state confrontate, mediante l’utilizzo dell’apposito software Proteomweaver, con quelle ottenute da lisati di batteri sottoposti ai diversi trattamenti fotodinamici. Tutte le proteine delle mappe bidimensionali che, a seguito dell’analisi, hanno mostrato di essere state significativamente modificate dai trattamenti fotodinamici, sono quindi state identificate tramite spettrometria di massa (MALDI-TOF/TOF). Sulla base delle loro funzioni nella cellula, le proteine identificate sono quindi state assegnate a diverse classi funzionali, al fine di scoprire quali funzioni cellulari venissero maggiormente colpite dalla PDT. Tra le 265 proteine globalmente identificate dall’analisi con Proteomweaver, 70 hanno mostrato significative variazioni di intensità dovute ai trattamenti fotodinamici; tra queste, la maggioranza era composta da proteine implicate nella risposta allo stress ossidativo, nel metabolismo energetico e nella captazione di zuccheri. Comparando gli effetti della PDT tra le due porfirine, si è scoperto che i livelli di intensità di molte proteine sono stati modificati da entrambe, ed in genere nella stessa direzione. Particolarmente interessante è stata la scoperta che le tipologie di modifica del profilo proteomico di S. aureus, causate dai trattamenti fotodinamici, non sono compatibili con le risposte ad agenti ossidanti (come per esempio perossidi o superossidi) da parte della cellula batterica; questo suggerisce che la PDT ha come bersagli specifiche proteine. I risultati ottenuti sono di particolare importanza perché, approfondendo la conoscenza del meccanismo d’azione della PDT, potrebbero aiutare nel disegno di nuovi fotosensibilizzatori più efficienti di quelli attualmente in uso. Nella seconda linea di ricerca ho studiato alcune proprietà legate all’attività antimicrobica ed alla capacità di ingresso nella cellula batterica di un peptide cationico antimicrobico, l’apidaecina 1b. I risultati ottenuti da questo studio hanno quindi permesso di utilizzare l’apidaecina stessa come vettore di altre molecole, coniugandola con una porfirina al fine di ottenere un nuovo agente antimicrobico, con un maggior spettro d’azione rispetto ai suoi singoli costituenti. L’apidecina 1b è un piccolo (soli 18 amminoacidi) peptide che viene molto studiato per via di alcune sue particolari capacità, tra cui una buona inibizione della crescita di batteri Gram (-) e, soprattutto, un’eccezionale abilità nell’entrare nelle cellule mediante un meccanismo che non comporta la formazione di pori nelle membrane. Inoltre, è stato dimostrato che l’apidaecina non presenta tossicità per cellule eucariotiche a concentrazioni che sono invece letali per i batteri. Allo scopo di studiare il meccanismo d’azione dell’apidaecina sono stati sintetizzati degli ibridi peptide-peptoide dell’apidaecina stessa, nei quali ognuno dei tre residui di arginina è stato sostituito con residui di N-(3-guanidinopropyl)-glicina, e si sono quindi valutate le MIC (minima concentrazione inibente) di ognuno degli analoghi nei confronti di diversi batteri. Né l’apidaecina né i suoi ibridi peptide-peptoide hanno mostrato attività antimicrobica nei confronti di Staphylococcus aureus e Pseudomonas aeruginosa. Invece, in Escherichia coli si è potuta osservare una lieve diminuzione del valore della MIC, rispetto al peptide naturale, con gli ibridi sostituiti nelle posizioni 4 e 12, mentre l’attività antimicrobica veniva completamente persa nell’ibrido sostituito nella posizione 17. Questi risultati suggeriscono che l’arginina in posizione 17 possa giocare un ruolo particolarmente importante nel meccanismo di traslocazione dell’apidaecina all’interno della cellula. Quindi, sia l’apidaecina che i suoi ibridi peptide-peptoide sono stati marcati mediante legame con una molecola di fluoresceina, allo scopo di monitorarne la capacità di legame con la cellula batterica mediante tecniche di microscopia di fluorescenza e citometria di flusso. Sfortunatamente, la marcatura con la fluoresceina ha causato la perdita dell’attività antimicrobica e di gran parte della capacità di legarsi ed entrare nella cellula batterica sia dell’apidaecina che dei suoi ibridi. Questo effetto è dovuto principalmente alla massa aggiuntiva portata dalla fluoresceina ai peptidi. Tuttavia, si è osservato che, a differenza degli altri, l’ibrido peptide-peptoide con la sostituzione dell’arginina 17 non presentava alcuna capacità di legarsi alle cellule batteriche. Questo risultato ha confermato sia l’importanza di questa arginina per la capacità antimicrobica dell’apidaecina che, in generale, l’importanza del dominio C-terminale sulla capacità di legame e di ingresso nella cellula batterica. Il coniugato (che è stato chiamato T-api), ottenuto dall’unione di una porfirina anionica (monocarbossi-tetrafenil porfirina, cTPP) con il dominio N-terminale dell’apidaecina 1b, non ha mostrato alcuna attività antimicrobica al buio. Tuttavia, in seguito all’irradiamento con luce blu, T-api si è dimostrato un efficiente fotosensibilizzatore, con un’efficienza fortemente dipendente dalle diverse tipologie di batteri considerate. In particolare, il trattamento fotodinamico con T-api è stato estremamente efficace con E. coli e S. aureus, e leggermente meno efficace, ma comunque sufficiente per indurre un’apprezzabile mortalità, in P. aeruginosa. Esperimenti analoghi, eseguiti trattando i batteri con la porfirina oppure l’apidaecina da sole, oppure con le due insieme ma non coniugate, non hanno causato alcuna mortalità in E. coli e P. aeruginosa. In S. aureus, invece, si è riscontrata una marcata mortalità in seguito al trattamento con cTPP e luce, ma comunque minore di quella ottenuta con T-api. Quindi, ho dimostrato che la coniugazione di un peptide antimicrobico con una molecola fotosensibilizzante può portare alla sintesi di agenti antimicrobici estremamente efficaci, e con uno spettro d’azione superiore rispetto a quelli dei suoi singoli componenti.

Thesis: Ph. D., Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Department of Biology; and the Woods Hole Oceanographic Institution), 2019
Cataloged from PDF version of thesis.
Includes bibliographical references.
Antibiotics are naturally occurring chemicals in bacteria that were recently discovered and utilized by humans. Despite a relatively short time of use, anthropogenic use of antibiotics has increased natural levels of antibiotic resistance, which has caused a looming antibiotic resistance crisis, where antibiotics may not work. Understanding resistance patterns is critical to allow for continued therapeutic use of antibiotics. While resistance is often thought of in hospitals, antibiotics and antibiotic resistance genes from human activity are disposed of into nature where they are able to interact with naturally occurring antibiotics and resistance. In this dissertation, I examine the ocean as an understudied region of the environment for antibiotic resistance. The ocean represents an area of human activity with recreation and food consumption and it is an enormous region of the planet that is affected by both land and sea activities.
In Chapter 2, I explore the policies that have contributed to the antibiotic resistance crisis. I offer explanations of market and political failures that contributed to the situation, areas for growth in terms of assessing scientific knowledge, and finally, recommendations for mitigating antibiotic resistance. In Chapters 3 and 4, I collected individual bacterial cultures from Cape Cod, MA beaches to assess the phenotypic response to antibiotic resistance. I show that 73% of Vibrio-like bacteria and 95% of heterotrophic bacteria (both groups operationally defined) are resistant to at least one antibiotic. These results indicate that antibiotic resistance is prevalent and persistent on beaches over both spatial and temporal scales. In Chapter 5, I used metagenomics to assess the abundance and types of resistance genes at coastal impacted Massachusetts sites. I found that, even in sites that seem distinct in terms of anthropogenic impact, prevalence of resistance remained the same.
Finally, in Appendix A, I examined part of the TARA Ocean dataset for prevalence of antibiotic resistance genes across the world's ocean. Here, I found that there are distinctions between different ocean biomes based upon antibiotic, metal, and mobile genetic elements. This dissertation has increased the understanding of temporal and spatial dynamics of antibiotic resistance in the coastal and open ocean.
"This work has be funded by the National Science Foundation Graduate Research Fellowship under Grant No. 1122374 and a Martin Fellows for Sustainability Fellowship (both to MKM). Grants from Woods Hole Oceanographic Institution from the Coastal Ocean Institute, Grassle Family Foundation, Hill Family Foundation, and Biology Department also supported this work"--Page 6
by Megan Katherine May.
Ph. D.
Ph.D. Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Department of Biology; and the Woods Hole Oceanographic Institution)

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In micropropagation, plant tissue fragments called explants are isolated aseptically, disinfected and cultured in culture medium. However, oxidation and contamination are the main problems in the initiation of the tissue culture plants. In order to initiate the establishment of in vitro methodologies for selected sugarcane varieties a series of decontaminating treatments were performed in shoot tips with different exposure times and concentrations of sodium hypochlorite, casugamicin, ethanol and mercuric chloride. There were no problems with oxidation in any treatment. The use of reagent concentrations and times tested showed not be phytotoxic. Treatment using longer times of exposure to casugamicin and sodium hypochlorite obtained the lowest levels of contamination by fungi and bacteria. However, bacterial contamination rates were high which indicates the necessity of using new procedures for the decontamination process. It is known that the sugarcane is associated with different endophytic bacteria. In order to better study these endophytic bacteria and their relationship with the host plant, promoted the isolation of bacteria in explants of five different varieties of sugarcane: RB98710, RB34068, RB034130, RB034120 and RB034041 to determinate of morphological and physiological characteristics. The thirteen isolates have diversity for morphological and biochemical staining and are characterized as gram-positive. All of them had a production capacity of plant growth factors, and after antimicrobial susceptibility testing, two antibiotics: Ciprofloxacin and Ceftazidime showed the greatest potential for controlling the growth of the isolates. With microbial contamination controlled, studies were conducted to evaluate the effect of growth regulators on in vitro multiplication of shoot tip culture and its roots in three sugarcane varieties: RB98710, RB034041 and RB034130. We used liquid MS medium containing BAP, KIN, NAA and GA3 in different combinations and concentrations. The results showed that, for the three varieties tested, MS medium with 0.2 mg.L-1 BAP and 0.1 mg L-1 KIN are most suitable for higher production of shoots and that the best rooting results were obtained in semi-solid ½ MS medium with 6% sucrose, 5.0 mg L-1 NAA and 0.2% activated charcoal. The plantlets were acclimatized and survivability under ex vitro conditions was 90%, three weeks after the transfer to the greenhouse.
Na micropropagação, fragmentos de tecidos vegetal chamados explantes são isolados, desinfetados e cultivados assepticamente em meio de cultura. Porém, a oxidação e a contaminação são os principais problemas no início do processo de cultura de tecidos de plantas. Com o objetivo de iniciar metodologias de estabelecimento in vitro de variedades selecionadas de cana-de-açúcar foram realizados tratamentos de descontaminação de ápices caulinares com diferentes tempos de exposição e concentrações de hipoclorito de sódio, casugamicina, etanol e bicloreto de mercúrio. Não houve problemas de oxidação em nenhum dos tratamentos. O uso dos reagentes nas concentrações e tempos testados não mostrou ser fitotóxico, no tratamento utilizando os maiores tempos de exposição à casugamicina e ao hipoclorito de sódio obteve os menores índices de contaminação por fungos e bactérias. Porém, os índices de contaminação bacteriana foram elevados, o que indica a necessidade de uso de novos procedimentos para o processo de descontaminação. Sabe-se que a cana-de-açúcar está associada a diferentes bactérias endofíticas. Com o objetivo de melhor estudar estas bactérias endofíticas e sua relação com a planta hospedeira, promoveu-se o isolamento de bactérias contaminantes de explantes de cinco diferentes variedades de cana-de-açúcar: RB98710, RB034068, RB034130, RB034120 e RB034041 visando à determinação de características morfológicas e fisiológicas. Os treze isolados obtidos possuem diversidade para características morfo-tintoriais e bioquímicas sendo caracterizados como bastonetes gram-positivos. Apresentaram capacidade de produção de fatores de crescimento vegetal e, após teste de sensibilidade a antimicrobianos, dois antibióticos: Ciprofloxacino e Ceftazidima apresentaram maior potencial de controle do crescimento dos isolados. Com as contaminações microbianas controladas, estudos foram realizados para avaliar o efeito de reguladores de crescimento na multiplicação in vitro de culturas de ápices caulinares e seu enraizamento em três variedades de cana: RB98710, RB034041 e RB034130. Utilizou-se meio MS líquido contendo BAP, KIN, ANA e GA3 em diferentes combinações e concentrações. Os resultados mostraram que, para as três variedades testadas, o meio MS com 0,2 mg.L-1 de BAP e 0,1 mg.L-1 de KIN são os mais apropriados para a maior produção de brotos e que os melhores resultados de enraizamento foram obtidos em meio ½ MS semissólido com 6% de sacarose, 5,0 mg.L-1 de ANA e 0,2% de carvão ativado. As plântulas foram aclimatizadas e a capacidade de sobrevivência sob condições ex vitro foi de 90%, três semanas após a transferência para a casa de vegetação.

The public, as the users of antibiotics, can contribute to the control of bacterial resistance. National and international campaigns have recommended public education to promote the judicious and safe use of antibiotics, and in particular, reducing antibiotic use and misuse in upper respiratory tract infections. Campaigns, however, have not been informed by detailed understanding of public attitudes to the problem. Although previous studies have explored lay perceptions of common infections and antibiotics, public attitudes to bacterial resistance, beliefs about antibiotic use in relation to bacterial resistance and the attitudes towards respiratory tract infection influencing antibiotic use are under researched. This thesis addresses this gap using a grounded theory approach. Semi-structured interviews with members of community groups were conducted across South East Wales. By analysing patterns and connections between various beliefs this thesis shows that historical antecedents and beliefs about dirt and germs act as prototypes for current beliefs about resistant infection. Most respondents did not feel that they have a personal role in either the cause or control of bacterial resistance. Lay beliefs about aetiology resided in both traditional and biomedical models. There was a reliance on medicines, and specifically antibiotic attachment, which contributed to self-medication and expectations for antibiotics during upper respiratory tract infection (URTI). Promoting public engagement in the control of bacterial resistance requires a number of approaches to behavioural change. In relation to antibiotic use efforts to promote adherence to antibiotic regimes need to address beliefs about antibiotics, forgetfulness and practical barriers to adherence but also to reduce public expectations for antibiotics for upper respiratory tract infections by enhancing understanding about the microbial causes of URTI. Efforts to reduce antibiotic use, however, need also to address the wider meaning and the reliance on antibiotics. Public engagement in the control of infection through hand washing should be promoted as an effective way to reduce the risk community acquired resistant infection.

The gut microbiome is a community of closely interacting microbes living in the gastrointestinal tract. Its structure has direct relevance to health. Disturbances to the microbiome, such as due to antibiotic use, have been implicated in various diseases. The goal of this study was to determine how the gut microbiome reacts to and recovers from disturbance caused by antibiotics. Because diet also influences the microbiome, this study included the interaction between diet and antibiotics. Half of the mice in each diet treatment were given antibiotics to disturb their microbiomes. After cessation of antibiotics, mice were paired in combinations within diets to determine whether the microbiomes of control mice influenced the disturbed microbiomes of formerly antibiotic mice. Chapter 1. Diet significantly altered the structure of the gut microbiome but its effect was significantly smaller than the effect of antibiotics. There was a significant interaction between diet and antibiotics; the antibiotic effect was larger in the cornstarch diet than in the glucose diet. Dysbiotic microbiomes resulting from antibiotics were characterized by an increase in Bacteroidetes and Proteobacteria, and a decrease in Firmicutes. Antibiotic administration also resulted in an initial increase OTU diversity, mainly because it reduced the abundance of dominant OTUs, resulting in greater evenness. Chapter 2. Seven weeks after the cessation of antibiotics (experiment termination), the effect of the antibiotics on the microbiome was still evident. The structure of the dysbiotic microbiome had not returned to that of control mice. Antibiotics significantly increased the relative abundance of some taxa and significant decreased the relative abundance of others. It was unexpected that the taxonomic hierarchy within the microbiome did not recover after 7 weeks following cessation of antibiotics. It would appear, therefore, that antibiotics established a new, semi-stable hierarchy. Chapter 3. When paired together, the assumption was that dysbiotic microbiomes of antibiotic mice would be positively influenced by microbiomes of control mice, based on the assumption that the control mouse would act as a probiotic for the antibiotic mouse, either via coprophagy or consumption of food contaminated by feces. Contrary to that hypothesis, the microbiomes of control mice became more similar to that of antibiotic mice. One can offer at least two hypotheses to explain this result, but neither was tested. First, compared to the control microbiome, the dysbiotic microbiome may have been more stable and thus more resistant to change due to invasion by OTUs from the control microbiome. Other research has shown that dysbiotic microbiomes have a high degree of stability. If this were true, the use of probiotics is questionable. Second, one or more of the antibiotics could still have been active at the initial phase of pairing, and coprophagy caused the microbiome of the control mice to rapidly become dysbiotic. If this is true, the experiment should have been conducted with a waiting period between the cessation of antibiotic administration and pairing.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
S. aureus é responsabilizado por diversos problemas clínicos em suinocultura e em humanos. Estudos epidemiológicos comprovam o potencial deste microrganismo em adquirir resistência a antibióticos. Atualmente estirpes resistentes a meticilina (MRSA), responsabilizados por casos de infecções nosocomiais, são as mais estudadas uma vez que o MRSA encontra-se disseminado em ambientes extra-hospitalares e frenquentemente tem sido isolado de vários animais domésticos inclusive suínos. O objetivo desde trabalho foi determinar a presença de S. aureus em granjas de suínos, identificar a ocorrência dos genes mecA, icaA e icaD e o perfil de resistência a antimicrobianos. Ao todo foram colhidas 458 amostras de cinco granjas e dois frigoríficos. As amostras foram semeadas em ágar Braid - Parker e ágar sangue seguido de provas bioquímicas. As amostras sugestivas, foram submetidas a PCR para confirmação de espécie, detecção do gene coa, mecA para avaliar a resistência a meticilina além dos genes de virulência icaA e icaD que expressam capacidade para formação de biofilmes. Na sequência, realizou-se o antibiograma para a avaliação de 11 antimicrobianos. Ao todo foram identificados 81 (79%) S. aureus isolados de todas as granjas e frigoríficos incluindo, três amostras isoladas de funcionários das granjas. Nenhuma amostra foi positiva para o gene mecA. Em relação aos genes icaA e icaD, observou predomínio do gene icaD e que 41% das amostras foram positivas para os dois genes. O antibiograma demonstrou grande resistência às penicilinas e tetraciclinas, além de grande quantidade de S aureus multirresistentes
Staphylococcus aureus are involved in a wide range of clinical problems to swine industry as son in humans. Epidemiological researchs prove his potential to acquire resistantence to antibiotics. Nowadays, methicillin-resistant S. aureus (MRSA) are responsabilized for nosocomial infections and many studies are done because MRSA are spread to extra hospitalar enrivonment and frequentely isolated from domestic animals including pigs. The aim of this study was to determine the presence o S. aureus at swine farms and identify the mecA, icaA and icaD genes and the resistant proflife to antibiotics. Overal, 458 swabs were taked from five pigeris and two slautherhouses. All the samples were placed on Braid – Parker and blood agar follow by biochemical analyses. The suspect colonies were submitted to PCR to confirm the S. aureus species, by the detection of the coa gene, mecA to avaible meticillin-resistant as son to the virulence gens icaA and icaD that can determine slime production. Antibiogram were done to evaluate the response to 11 antibiotics. All pigeris and slautherhouse were positive and 81 (79%) samples were S. aureus positive including three isolates from pigs employeers. The mecA gene was not detected. The icaD gene was most frequent and 41% were positive to both genes. The antibiogram show a lot of samples penicillin and tetraciclin resistant. Most of the samples were multirestant

In this historical period known as the antibiotic crisis era, the ever faster rise of bacterial strains resistant to the clinically used antibiotics along with the scientific research silent gap in the antibacterial field is treating seriously to the worldwide public health. Hence, we urgently need to develop new antibacterials agents with an innovative mode of action, able to trick the mechanisms of the pathogen resistance. In this alarming frame, aware of the antibacterial properties of guanidine moieties, Prof. M. Botta and his research group have evaluated the biological activity of a linear polyalkylguanidino series, synthesized for different medicinal purpose, toward a panel of bacterial microorganisms. Only one compound (1) emerged to have an interesting broad-spectrum antibacterial activity. Later, the serendipitous discovery that the test batch of compound 1 was actually a mixture of oligomers led us to identify the chemical structure of the main component, dimer 2, which was the responsible for the activity. From its scaffold, we designed and synthesized a small library of analogs to make some preliminary consideration on the pharmacophores with the aim of improving the selectivity index and studying the mode(s) of action.

Using three complementary approaches, this work sought to tackle the widespread problem of antibiotic resistance. To circumvent the resistance mechanisms developed by bacteria, it is necessary to establish drug candidates that act on novel therapeutic targets, such as the ion channels used by bacteria to modulate homeostasis. Examples include the potassium efflux channel, Kef, and the mechanosensitive channel of small conductance, MscS, which are not found in humans. How these targets function must be well understood before drug candidates can be developed, as such, their identification and investigation is often accompanied by the evolution of the analytical techniques used to study them. Membrane protein mass spectrometry is one technique showing potential in the study of ion channels. However, spectra can be clouded by the detergents used to solubilise ion channels from their native membranes. Undertaken herein was the synthesis of some fluorescent glycolipid detergents, which it was hypothesised could be encouraged to dissociate from ion channels via laser-induced excitation within the gas phase of a mass spectrometer, thereby improving the clarity with which spectra can be obtained. For Kef, an unconfirmed mechanism of action had previously been proposed. To explore the suggestion that sterically-demanding central residues are important for channel activation, solid phase peptide synthesis was used to isolate three tripeptide analogues of N-ethylsuccinimido glutathione, a known activator with a high affinity for Kef. A competition fluorescence assay suggested these tripeptides bound to Kef with an affinity lower than predicted, allowing the conclusion that a more detailed assessment of the steric bulk required for activation was necessary before a mechanism of action could be confirmed. Lysophosphatidylcholine has been shown to activate MscS, although it is not known how. Affinity chromatography between MscS and lysophosphatidylcholine was proposed as a means by which specific binding interactions could be investigated. For this technique an amino-derivative of lysophosphatidylcholine was necessary and its challenging synthesis is also detailed herein.

Thesis advisor: Michelle Meyer
The primary bacterial targets for most antibiotics are well known. To survive the stress of an antibiotic a bacterium must decrease the antibiotic to target binding ratio to escape from harmful effects. This can occur through a number of different functions including down-regulation of the target, mutation of the binding site on the target, and decreasing the intake or increasing the efflux of the antibiotic. However, it is becoming more evident that an antibiotic stress response influences more than just the primary target, and that a wave of secondary responses can be triggered throughout the bacterium. As a result resistance mutations may arise in genes that are indirectly affected by the initial interaction between the antibiotic and target. These indirect responses have been found to be associated with metabolism, regulation, cell division, oxidative stress, and other critical pathways. One technique recently developed in our lab, called transposon insertion sequencing (Tn-seq), can be used to further understand the complexity of these indirect responses by profiling growth rates (fitness) of mutants at a genome-wide level. However, Tn-seq is normally performed with large libraries of pooled mutants and thus it remains unclear how this may influence fitness of some independent mutants that may be compensated by others in the population. Additionally, since the original method has only utilized planktonic culture, it is also not clear how higher order bacterial structures, such as biofilms or microcolonies, influence bacterial fitness. To better understand the dynamics of pooled versus individual mutant culture, as well as the effect of community structure in microcolony development on the influence of fitness, we adapted a droplet microfluidics-based technique to encapsulate and culture single mutants. We were able to successfully encapsulate at least 7 different species of bacterial pathogens, including Streptococcus pneumoniae, and culture them planktonically, or as microcolonies, in either monodisperse liquid or agarose droplets. These experiments, however, raised an important challenge: the DNA yield from one encapsulation experiment is insufficient to generate samples for sequencing by means of the traditional Tn-seq method. This led us to develop a novel Tn-seq DNA library preparation method, which is able to generate functional Tn-seq library molecules from picogram amounts of DNA. This method is not ideal yet because fitness data generated through the new method currently does not correlate well with data from traditional Tn-seq library preparation. However, we have identified one major culprit that should be easily solvable. We expect by modifying the binding site of the primer used for linear amplification of transposon ends that the new preparation method will be able recapitulate results from the traditional Illumina preparation method for Tn-seq. This will enable us to prepare robust Tn-seq samples from very small amounts of DNA in order to probe stress responses in single mutants as well as in microcolonies in a high-throughput manner
Thesis (MS) — Boston College, 2016
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

Mestrado em Microbiologia
Mobile phones are daily used and in a frequent manner. There is no awareness in the general public of their potential to be a reservoir of specific bacteria. The use of touch screen mobile phones is exponentially growing and the hospital outbreaks with touch screens as contamination source is more frequently being registered. Touch screens are not perceived as a method of transmission of potentially pathogenic and antibiotic resistant bacteria, thus posing as a health risk due to being overlooked in terms of disinfection standards in healthcare settings. Bacteria are acquiring resistance to various antibiotics, possibly becoming multiresistant such as HA-MRSA. This poses a public health risk when faced with the possibility that these bacteria can adhere and remain on mobile phones over a great length of time. These devices may serve as vector of transmitting bacteria to their owners and third parties. This is even more preoccupying when individuals are healthcare professionals. This study aimed to identify and quantify the bacteria present on mobile phones and the hands of their users. The bacteria were submitted to antibiotic screening and MRSA were selected and genotypically characterized, and the SCCmec element typified. Bacillus spp. was detected in 7.5% of the individuals and in 28% of the mobile phones, hemolytic bacteria were detected in 82% of the individuals, Staphylococcus spp. in 96.5%, S. aureus in 82%, Enterobacteriaceae in 1% and MRSA in 6%. Erythromycin resistance in staphylococci was verified to be 44.7% in general. Oxacillin and Clindamycin resistance was 12.5% and 9.8%, respectively. 0.8% of the screened bacteria were multiresistant, and 3.3% of the individuals presented multiresistant bacteria on their hands. Four samples were identified as being MRSA, all multiresistant and from those, two samples were presumptively identified as SCCmec type II and SCCmec type III, both HA-MRSA. Male individuals have "dirtier" hands than female students in terms of potential pathogenic bacteria. Various factors such as, keyboard type, hand and mobile phone hygiene, nail length, manicure type and presence, taking device to the bathroom, owning pets and hand washing have influence on the bacterial count of the hands. Mobile phones can serve as reservoirs of specific bacterial that may be pathogenic and multiresistant to antibiotics, and should be publically perceived as a possible contamination source.
Atualmente os telemóveis são utilizados diariamente e de modo frequente. Não se observa uma consciencialização por parte da comunidade geral para o seu potencial como reservatório para bactérias específicas. O uso de telemóveis com ecrãs tácteis encontra-se em crescimento exponencial, e os surtos hospitalares em que se verifica que o ecrã táctil é uma fonte de contaminação estão a ser registados com maior frequência. Os ecrãs tácteis não são encarados como um meio de transmissão de bactérias potencialmente patogénicas e bactérias resistentes a antibióticos, podendo ser assim um risco para a saúde pública devido à negligência em termos de desinfeção apropriada em ambientes hospitalares. As bactérias estão a adquirir várias resistências a antibióticos, tornando-se multirresistentes tal como o HÁ-MRSA. Isto representa um risco para a saúde pública quando confrontados com a possibilidade destas bactérias aderirem e permanecerem nos telemóveis durante um longo período de tempo. Estes dispositivos podem servir como vetor na transmissão de bactérias presentes para o seu utilizador e a terceiros. Sendo ainda mais preocupante quando os indivíduos são profissionais de saúde. Este estudo teve como objetivo identificar e quantificar as bactérias presentes nos telemóveis e nas mãos dos seus utilizadores. As bactérias foram analisadas em termos de resistência a antibióticos e MRSA foram selecionados e geneticamente caracterizados, e o elemento SCCmec tipificado. Bacillus spp. foi detetado em 7.5% dos indivíduos e em 28% dos telemóveis, bactérias hemolíticas foram detetadas em 82% dos indivíduos, Staphylococcus spp. em 96.5%, S. aureus em 82%, Enterobacteriaceae em 1% e MRSA em 6%. A resistência à Eritromicina por staphylococci foi 44.7% em geral. A resistência à Oxacilina e Clindamicina foi de 12.5% e 9.8%, respetivamente. 0.8% das bactérias submetidas a antibiograma apresentaram resistência a múltiplas classes de antibióticos, e 3.3% dos participantes apresentaram bactérias multirresistentes nas mãos. Quatro amostras foram identificadas como sendo MRSA, todas multirresistentes, e destas, duas foram presuntivamente identificadas como sendo SCCmec tipo II e SCCmec tipo III, ambas HÁ-MRSA. Indivíduos do sexo masculino têm as mãos mais "sujas" do que as estudantes do sexo feminino, isto em termos de bactérias potencialmente patogénicas. Vários fatores como, tipo de teclado, higiene das mãos e telemóvel, tamanho das unhas, tipo e presença de manicure, levar o telemóvel para a casa de banho, ter animais de estimação e lavar as mãos influenciam o número de CFU nas mãos. Os telemóveis podem servir de reservatório para bactérias específicas que podem ser patogénicas e multirresistentes a antibióticos, por isso devem ser reconhecidos publicamente como uma possível fonte de contaminação.