Tesi sul tema "Antibiotics"

Thesis (Ph.D.)--Boston University
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Antibiotic-resistant bacterial strains continually arise and their increasing prevalence poses significant clinical and societal challenges. Functional analyses of resistant mutants and the study of general stress responses perturbed by antibiotic treatment have yielded valuable insights into how resistance arises through mutations. However, less is known about the population dynamics and communal interactions that underlie the development of resistance through mutations. In this work, we utilize systems approaches to study the functional dynamics of bacterial populations evolving antibiotic resistance. We follow a continuous culture of Escherichia coli facing increasing levels of antibiotic and show that the vast majority of isolates are less resistant than the population as a whole. We find that the few highly resistant mutants improve the survival of the populations less resistant constituents, in part, by producing indole, a signaling molecule generated by actively growing and unstressed cells. We show, through transcriptional profiling, that indole serves to turn on drug efflux pumps and oxidative stress protective mechanisms. The indole production comes at a fitness cost to the highly resistant isolates, and wholegenome sequencing reveals that this bacterial altruism is enabled by drug-resistance mutations unrelated to indole production. This work establishes a population-based resistance mechanism constituting a form of kin selection whereby a small number of resistant mutants can, at some cost to themselves, provide protection to other more vulnerable cells, enhancing the survival capacity of the overall population in stressful environments. Deeper studies into cooperative strategies bacteria use to evade antibiotics may prove critical for the rational design of more effective antimicrobial interventions.
2031-01-01

There is a tension between the need to use antibiotics to prevent adverse outcomes from infection, and a consequence of their use, which is antibiotic (treatment) resistant infection. Actions taken to control the spread of antibiotic resistant microbes, and constraints on the use of antibiotics both give rise to ethical tensions. I consider the evaluative framework and the principles that might be used to decide a just distribution of burdens and benefits associated with the use of antibiotics. Nussbaum specifies a list of capabilities. A minimum sufficiency of each capability is required for a life of human dignity. Nussbaum’s approach provides a richer framework for the evaluation of the distribution of burdens and benefits associated with the use of antibiotics than prevailing health economic, or prevalence of disease measures. There are contexts in which we cannot assure a sufficiency of capabilities. I consider the potential for Scanlon’s contractualism to provide principles for deciding the distribution of burdens and benefits associated with the use of antibiotics under differing levels of resource constraint. Finally I consider the influence of metaphor and analogy in the context of the human relationship with microbes.

Class of 2011 Abstract
OBJECTIVES: To determine the appropriateness of antimicrobial therapy in patients initiated on empiric meropenem therapy. METHODS: Adult patients prescribed empiric meropenem therapy between January 1, 2010 and March 31, 2010 at a tertiary care, academic medical center were included. Data collected included site of infection, culture and susceptibility data, risk factors for multi-drug resistant organisms, and changes in antimicrobial therapy during the first seven days after meropenem therapy was initiated. Demographic variables included age, sex, weight, and race. RESULTS: RESULTS: A total of 89 patients were included in the study analysis. Initial culture(s) was obtained before administration of antibiotics in only 58% of patients. During the first 24 hours of admission, four or more different antibiotics were prescribed in 26% of patients often with overlapping spectrums of activity. The majority of patients received meropenem for either less than 1 day or greater than 4 days. CONCLUSION: The primary issues identified with appropriate antibiotic prescribing involved the timing of cultures, and multiple changes in antibiotic therapy without culture-driven reasoning.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Este trabalho trata da remoção de fármacos da água por meio do processo de adsorção, utili- zando como adsorvente o carvão ativado obtido da casca de coco da baía. Os fármacos utili- zados foram os antibióticos Amoxicilina, Ampicilina, Cefalexina e Ciprofloxacina. O adsor- vente de carvão ativado foi caracterizado por análise textural, determinação dos grupos funci- onais da superfície pelo método de titulação de Boehm e por FTIR e determinação do pH de ponto de carga zero (pHPCZ). Foi observado que o carvão apresenta uma área superficial de 745,38 m2/g , é constituído principalmente por microporos, é levemente alcalino (pHPCZ: 7,58) e possui tanto grupos funcionais ácidos como básicos em sua superfície. Os resultados mos- traram que a eficiência de remoção não sofre influência significativa do pH na faixa de 2 a 10. Os experimentos de adsorção foram realizados pelo processo de batelada, onde soluções aquosas de cada antibiótico foram colocadas em contato com diferentes dosagens de carvão ativado (0,05 a 2 g) a temperatura ambiente. As concentrações dos fármacos foram determi- nadas por cromatografia líquida de alta eficiência (HPLC), usando como fases móveis água e metanol em sistema de bombeamento gradiente. Os resultados de exatidão (repetibilidade), dos limites de detecção e de quantificação da técnica analítica demonstraram a aplicabilidade do método. A eficiência de remoção foi superior a 90% para todos os antibióticos. O equilí- brio de adsorção dos antibióticos foi alcançado após 4h e foi expresso por meio de isotermas de adsorção de acordo com os modelos de Langmuir, Freundlich, Temkin e D-R. A isoterma de Langmuir foi a que melhor representou os dados experimentais da adsorção dos antibióti- cos em carvão ativado. A cinética de adsorção dos fármacos foi discutida...
This report is about medicine removal from water by using adsorption process, employing activated carbon as absorvent obtained from baía coconut nutshell. The medicines used were the antibiotics Amoxillin, Ampicillin, Cephalexin and Ciprofloxacin. The activated carbon adsorbent was characterized by textural analysis, surface functional group determination by Boehm and FTIR titration method and the pH of point of zero charge (pHpzc) determination. It was observed that carbon presents a 745,38 m2/g superficial area, its essentially composed by micropores, its slightly alkaline (pHpzc: 7,58) and owns either acidy or basic functional groups on its surface. The results showed that removal performance does not suffered signifi- cant influence from pH on a group from 2 to 10. The adsorption experiments were accom- plished by batch process, where watery solutions from each antibiotic were in touch with dif- ferent activated carbon doses (0,05 to 2 g) and environmental temperature. The medicine con- centrations were determined by high performance liquid chromatgraphy (HPLC), employing water as moving phases and gradient methanol pump system. The accuracy results (repeata- bily), detection limits and quantification of analytic technique presented the aplicability of the method. The removal performance was higher thn 90% to all antibiotic. The adsorption bal- ance of antibiotics was reached after 4 hours and it was expressed by adsorption isotherms according to Langmuir, Freundlich, Temkin and D-R models. Langmuir isotherm best repre- sented experimental data of antibiotic adsorption on activated carbon. The adsorption kinec- tics of medicines was discussed employing pseudo first and second order kinetic models, by Elovich and intraparticle difussion. The kinectic model of pseudo second order can better de- scribe medicine... (Complete abstract click electronic access below)

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
A degradação foto-Fenton dos antibióticos sulfonamidas sulfadiazina (SDZ) e sulfatiazol (STZ) mediados por Fe(III)-oxalato foi estudada nesse trabalho. A influência da complexação de íons ferro, a concentração de H2O2 e o pH na velocidade inicial de degradação dos antibióticos foi avaliada. A degradação de SDZ e STZ foi melhorada na presença de Fe(III)-oxalato em comparação a Fe(NO3)3, alcançando completa degradação após 8 minutos de irradiação em pH 2,5 na presença de 5 mM H2O2 (equivalente a [H2O2]/[antibiótico] = 50). Também foi possível estender a faixa de pH do processo foto-Fenton na presença de Fe(III)-oxalato alcançando mais de 69% de degradação em pH 6, embora sem significante mineralização. A comparação da cinética de degradação dos antibióticos indicou que a STZ é mais recalcitrante provavelmente devido à baixa densidade eletrônica do seu anel tiazol em relação ao anel pirimidínico na SDZ. Experimentos de fotodegradação das sulfonamidas por irradiação gama da água na presença de N2O foram executados e seus produtos de degradação, identificados por análise LC/MS, foram comparados àqueles formados por processo foto-Fenton. Os intermediários de degradação formados são compostos hidroxilados após entrada de grupos hidroxilas no anel benzênico assim como no anel pirimidínico na molécula da SDZ e no anel tiazol na molécula da STZ, seguido por abertura dos anéis durante processo de descarbonilação
The photo-Fenton degradation of the sulfonamide antibiotics sulfadiazine (SDZ) and sulfathiazole (STZ) mediated by Fe(III)-oxalate was studied in this work. The influence of iron complexation, H2O2 concentration and pH on the initial SDZ and STZ degradation rate was evaluated. Degradation of both antibiotics is improved in the presence of Fe(III)-oxalate in comparison to free iron, achieving complete degradation after 8 min irradiation at pH 2.5 in the presence of 5 mM H2O2 (equivalent to H2O2/antibiotic = 50). It was also possible to extend pH range of the photo-Fenton reaction by the use of Fe(III)-oxalate reaching more the 69% degradation at pH 6, however without significant mineralization. Comparison of the degradation kinetics of both sulfonamides indicated higher recalcitrance of STZ due to the lower electron density of its thiazol ring in relation to pyrimidine ring in SDZ. The sulfonamides photodegradation experiments by water gamma-irradiation in presence of N2O were also executed and their by-products, identified by LC/MS analysis, were compared to that formed from photo-Fenton process. The intermediates of degradation formed are hydroxylated compounds with entrance of hydroxyl group in the benzenic ring as well as in both pyrimidine ring in the SDZ molecule and thiazole group in the STZ ring, followed by rings opening during decarbonylation process

Class of 2008 Abstract
Objectives: To evaluate changes in parental knowledge regarding antibiotic use and antibiotic resistance with an educational intervention given at elementary school parent-teacher association (PTA) meetings. Methods: This was an analytical pre-test/post-test study of an educational intervention given at two elementary schools in the Phoenix metro area. The primary dependent variable was a knowledge measure, calculated as a total score. The changes between the pre- and post-test total score means were compared using a dependent t-test. The a-priori alpha level used was 0.05. Results: The study sample consisted of 25 participants. Study data were collected between September 2007 and December 2007. The mean (SD) pre- and post-test scores were 33.7 (4.4) and 40.7 (2.7), respectively (p < 0.05). Conclusions: The educational intervention presented at elementary school PTA meetings resulted in a significant knowledge increase regarding the appropriate use of antibiotics when pre- and post-test scores were compared.

Antibiotic use in agricultural ecosystems has the potential to increase resistance to antibiotics in soil microbial communities since 40-95% of an antibiotic dose administered to livestock is excreted intact or as metabolites. Exposure to antibiotics is also known to alter microbial community composition, biomass, and physiology, but the potential influences of antibiotic residues on the essential ecosystem processes that microbes regulate, e.g., carbon and nitrogen cycling are not well understood. I investigated the effects of antibiotic residues associated with dairy cattle operations on soil microbial communities and the ecosystem processes they regulate. I examined the effects of antibiotic exposure on the biogeochemical functioning of soil microbial communities by measuring the activity of extracellular enzymes associated with organic matter processing and nutrient mineralization in soils collected from dairy cattle operations across the United States. At each experimental station paired sites were identified by local managers that represented sites with high and low stocking rates of dairy cows who had been treated prophylactically with antibiotics to prevent mastitis. Responses varied among individual enzymes, but I found an overall significant decrease in total hydrolytic enzyme activity under high cattle stocking rates indicating a change in the functioning of the microbial community in soils exposed to antibiotic laden manure. Principle components analysis suggest that while some of the variation in enzyme activities are associated with the abundance of antibiotic resistance genes, soil organic matter (total organic, mineralizable, and particulate organic carbon) was the most significant variable accounting for differences in enzyme activities. This reflects an inherent challenge in studies of antibiotic exposure in agricultural landscapes: the difficulty of distinguishing direct effects of antibiotic residues from the organic matter and nutrient subsidy associated with manure applications. To address this concern I conducted a series of incubation experiments manipulating soils to isolate the influences of antibiotics, manure resource subsidies, and bovine microbiome inoculants into soils. Specifically, I examined soil respiration and antibiotic resistance gene counts using qPCR following treatment with cephapirin, pirilimycin and a positive and negative control. I found that pre-exposure to antibiotics and manure is important in modulating the response of microbial communities (soil respiration, and gene copy numbers of AmpC and TetO) to further antibiotic exposure. I conclude that antibiotics themselves have a direct effect on soil communities and their functioning that is additive to the effect of manure (i.e., as a resource subsidy). This effect is mediated by the history of previous exposure to antibiotics, i.e., cattle stocking density. These results suggest that antibiotic residues from dairy cattle operation may have significant effects on microbial communities and the biogeochemical cycling they regulate in agricultural ecosystems.
Master of Science

Thesis advisor: Roche P. John
The growing threat of antibiotic resistance makes it extremely important that citizens be informed about the risks posed by antibiotic-resistant bacteria, and measures with which they can reduce these risks. The print media are major sources of such information for members of the public. In the present study, articles from major newspapers in the United States and Canada appearing between 1998 and 2002 were surveyed to determine the extent to which mention was made of antibiotic resistance and the risks associated with antibiotic resistance, the contextual precision with which this information was communicated, and the extent to which information was presented about causes, and risk-reduction measures, associated with antibiotic resistance. The majority of articles surveyed mentioned antibiotic resistance, but most failed to mention associated risks (i.e., the risk of illness and/or the risk of mortality). Articles that did report risks, did so only at a low level of contextual precision. A relatively low percentage of articles mentioned causes of antibiotic resistance, and even fewer mentioned risk reduction measures. These findings suggest that the print media could improve the efficacy with which they inform the public about issues associated with antibiotic resistance
Thesis (BS) — Boston College, 2003
Submitted to: Boston College. College of Arts and Sciences
Discipline: Biology
Discipline: College Honors Program

Antibiotics are today one of the most important cornerstones in modern healthcare when it comes to treating bacterial infections. It is an asset human kind have been leaning on for the last century, but excessive and widespread misuse of antibiotics have left deep scars in the form of multi resistant pathogenic strains of bacteria that we soon will not be able to treat. A lot of research have been invested in understanding the mechanisms and spread of resistance within bacteria living in planktonic form, overlooking the fact that there are more lifestyles that causes problems. In this study, focus has been put on antibiotic resistance within bacteria living as biofilms, a lifestyle that causes problems in chronic infections and prosthetics/medical implants. By constructing resistant mutants derived from a biofilm forming strain of Escherichia coli, the minimal selection concentration has been investigated in both planktonic and biofilm assays for Streptomycin and Ciprofloxacin. By comparing the results, it is possible to evaluate if and how the antibiotic resistance properties differ between the two lifestyles. Focus has been put on concentrations of antibiotics below the minimal inhibitory concentration with the objective to see how selection of antibiotic resistant mutants take place with the susceptible strain still growing, although with reduced growth rate. The hope is that the results gained in this study will provide a foundation for future research regarding antibiotic resistance in biofilms, and be part of the solution to the excessive resistance problem before it is too late.

Staphylococcus aureus is a gram positive coccoid pathogen that causes intractable infections in hospitals and communities around the world, and tens of thousands of people die of these infections every year. In order to combat these antibiotic-resistant infections, we need to better understand the genes involved in resistance to the cell stress caused by antibiotic treatment, which will enable the discovery of new antimicrobials and the development of novel therapeutic strategies. We chose to use an approach to this problem that utilizes a new phage-based high frequency of transposition system. In this work, we adapted this system so that transposon mutant libraries can be made and sequenced using next-generation sequencing (NGS) in any strain of S. aureus. We validated our new platform by performing a temperature screen and identifying mutants that are significantly resistant or sensitive to temperature-stress. Next, we created transposon libraries in two MRSA strains to show that this system can be broadly applied to other S. aureus strains, and we used one of these libraries to identify a new interaction between two genes involved in the secretion of sortase-anchored surface proteins. To better understand antibiotic-resistance, we performed Tn-Seq on transposon libraries treated with a small panel of six different antibiotics to identify intrinsic resistance factors to these antibiotics. We identified two new intrinsic resistance factors, SAOUHSC_01025 and SAOUHSC_01050, that sensitize to many cell envelope targeting antibiotics and may be involved in hemolysin regulation. Finally, we expanded this approach to sequence transposon libraries treated with 25 different antibiotics. Based on these data, we were able to develop methods for predicting the mechanism of action of new antibiotics. These methods involve identifying genes upregulated by transposon insertion and applying machine learning algorithms to identify similarities to a curated panel of well-studied antibiotics with known mechanisms of action. This work will enable many new functional genomics studies in S. aureus, and it will allow us to gain a better understanding of antibiotic resistance in this dangerous pathogen.
Chemical Biology

The aims of this thesis were to investigate the antibiotic susceptibility patterns of C. difficile with relation to the S-type of the isolates over a period of 18 months. Detailed growth curves were performed on strains NCTC 11223, the sequenced strain 630 and an endemic isolate 338a. Toxin A was shown to be produced upon entry to stationary phase in agreement with other studies. OD₆₀₀ was found to be a good predictor of growth phase and allowed this measurement to be used for subsequent experiments. MICs were performed on 186 random isolates of C. difficile collected during an 18-month epidemiological study to investigate the patterns of sensitivity to six different antibiotics. No evidence of resistance was seen to the two treatment antibiotics and all strains were resistant to cefoxitin (MICs 64-256mg/ml), the antibiotic used in most selective media. Most strains (98.9%) had intermediate resistance to ceftriaxone. The MIC₅₀ and MIC₉₀ of the strains to amoxicillin and clindamycin were very close (8 and 16 for amoxicillin and 16 for clindamycin) but the range of MICs was great. Clindamycin resistance was common with 67% of strains resistant (MICs of > 8mg/ml), 25% with intermediate resistance (MIC > 4mg/ml) and only 8% sensitive (MICs of < 2mg/ml). Twelve isolates from six different patients had very high resistance to clindamycin with MICs > 128mg/ml. Multiple isolates from the same patient, taken at different times, showed changes in susceptibility patterns over time. The only major change in susceptibility over the time period was in clindamycin resistance; some strains appearing to become more resistant while others became less resistant. No differences were apparent in the MIC₅₀ and MIC₉₀ of the different S-types of C. difficile identified, although some S-types were present in very small numbers. No links between antibiotics prescribed and susceptibility patterns were found. Three strains (NCTC 11223, strain 630 and endemic isolate 338a) were cultured in sub-lethal concentrations of the six antibiotics (1/2,1/4 and1/8 of the MIC) over 104 hours and growth and toxin A measured three times a day. The effects varied between strain and antibiotic. The most common effect on the growth of the strains was to increase the initial lag period by ca. 4h compared to the antibiotic-free controls through the clindamycin resistant strain NCTC 11223, (MIC >512mg/ml) showed not lag whatsoever in comparison to the controls when grown in this antibiotic. The most common effect on toxin A production was in the onset of toxin elaboration. Normally toxin began to appear in low levels in early stationary phase before accumulating to high levels by the start of decline.

The emergence of multidrug resistant Gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii has been recognized worldwide and some clinical isolates of these bacteria are now resistant to most of the antibiotics currently available. Unfortunately, there are at present not enough new antimicrobial drugs being produced by the pharmaceutical industry to keep pace with the continuing development of antibiotic resistance. Colistin is an old antibiotic and in the form of colistin methanesulphonate sodium (CMS) has re-emerged as a major treatment for burn patients colonised with Acinetobacter baumannii. However, this antibiotic can lead to profound toxicity and for this reason the blood levels of CMS should be carefully monitored. Unfortunately the methods available for measuring it in serum are not robust and are not able to differentiate between the two forms of colistin i.e. colistin sulphate (CLS) and CMS. In this study the chemical (Thin Layer Chromatography and High Performance Liquid Chromatography) and microbiological methods for CLS and CMS analysis were investigated to develop a methodology for reproducible quantification of CLS and CMS in water or serum. Since CMS in aqueous solution has the potential to hydrolyze to produce a complex mixture of colistin sulphomethylated derivatives as well as colistin base, the optimized chemical and microbiological methods were used to determine the degradation of CLS or CMS in aqueous solution and serum. The bacteriostatic and bactericidal activity of CLS and CMS were probed by calculations of minimum inhibitory concentrations and time survivor studies. Depending on their concentrations, both antibiotics were found to exhibit bacteriostatic and bactericidal properties against a range of Gram-negative bacteria. Membrane damage caused by both forms of colistin was investigated using Acinetobacter lwoffii R46383 anddetennining intracellular potassium leakage and 260nm absorbing materials leakage. CLS was shown to cause substantial membrane damage, indicated by rapid, gross potassium leakage, while the effect of CMS on the membrane appeared to be more subtle, with cells exhibiting a more concentration dependent loss of potassium. Adsorption isotherms ofCLS gave results that were indicative of high affmity isotherm (H- shape), while adsorption isotherms of CMS were indicative of co-operative sorption (S- shape). Investigations into CLS and CMS cytotoxicity were performed using normal rat kidney (NRK-52E) cell line; the investigations revealed that neither forms of colistin has a major adverse effect on the rat renal cells even at concentrations higher than the therapeutic doses.

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).

Pathogens on surfaces in child care centers can contribute to illness among attendees and may thereby contribute to medical visits as well. This intervention study was conducted to assess the effect of using specific sanitizing products and cleaning protocols in child care centers on the incidences of lower respiratory infections, diarrheal illness, antibiotic use, and medical visits among children attending the centers and on the levels and antibiotic resistance of indicator bacteria in those centers. During the ten-week study period, children from twelve centers were observed. Six of the centers were randomly assigned to the intervention. The other six were controls. Intervention centers were given cleaning protocols and sanitizing products. Control centers were asked to retain their original procedures and products.Acute illness was determined from records kept by the center directors and telephone calls made to parents of ill children. A call was also made to one randomly selected healthy child's parents for every two ill children recorded. Parents were given a questionnaire requesting information including bedroom sharing status, environmental tobacco smoke exposure, and chronic illnesses.After controlling for within-center clustering and zero-inflation, statistically non-significant trends of reduction were seen in the weeks of lower respiratory infections, diarrheal illness, and medical visits. Multivariable zero-inflated Poisson regression revealed that the number of weeks intervention center children were using antibiotics was 32% lower than among the control center children. This was a statistically significant reduction (95% CI = 0.54-0.86; p = 0.001).Bacterial samples were collected from ten sites within each center at the beginning and the end of the study period to determine the effect of the intervention on the microbial population. The study determined the heterotrophic plate count bacteria numbers and the rates of resistance to ampicillin and cephalothin. Neither heterotrophic bacterial concentrations nor antibiotic resistance rates significantly changed over the course of the study.

[ES] La presencia de residuos de antibióticos en la leche y los productos derivados representa un riesgo para los consumidores. Límites Máximos de Residuos (LMRs) no se han fijado para los productos lácteos y la transferencia de antibióticos de la leche a la cuajada y al lactosuero durante el proceso de elaboración del queso apenas ha sido estudiada. El objetivo de esta tesis fue evaluar la transferencia de antibióticos de la leche al queso y lactosuero, así como la validación de las características de varios métodos de cribado para la detección de antibióticos en muestras de suero de leche. En el primer estudio, el objetivo fue validar un método UHPLC-HRMS multi-residuo utilizando el analizador Orbitrap ExactiveTM, para el cribado cuantitativo de antibióticos en muestras de leche, queso fresco y lactosuero de acuerdo con los criterios especificados en la Decisión 657/2002/CE de la Comisión. El estudio de distribución mediante el método UHPLC-HRMS indicó que la mayor parte de antibióticos se transfirieron principalmente de la leche a la fracción lactosuero (hasta el 85,9%) durante la elaboración de queso. Por tanto, los porcentajes de retención de antibióticos en la cuajada fueron inferiores al 50%, excepto en el caso del ceftiofur (59,7%) y la dicloxacilina (52,8%), y muy variables entre los distintos antimicrobianos. En la mayor parte de los casos, la distribución de medicamentos no se vio afectada por la concentración de antibióticos presente en la leche, y estuvo escasamente relacionada con la lipofilicidad de los antibióticos. En el segundo estudio, se evaluaron las características de diferentes métodos de detección de antibióticos en muestras de lactosuero de acuerdo con la Decisión 657/2002/CE de la Comisión. En primer lugar, la especificidad (porcentaje de falsos positivos) y la capacidad de detección (CCß) del método de detección de inhibidores Eclipse Farm provisto del dispositivo e-Reader y de los métodos de unión a receptores 3Aminosensor, Quinosensor, Twinsensor y Tylosensor se evaluaron en muestras de lactosuero de leche de cabras, obteniendo en general, resultados similares a los obtenidos cuando se aplican para el análisis de la leche. También se evaluaron tres bioensayos en placa microtiter y respuesta dicotómica, que contenían Bacillus subtilis, Geobacillus thermocatenulatus y Geobacillus thermoleovorans, respectivamente, para ser aplicados simultáneamente con los métodos comerciales basados en la utilización de Geobacillus. stearothermophilus var. calidolactis. Elevados valores de especificidad (98-100%) se obtuvieron cuando las muestras de lactosuero fueron tratadas térmicamente (85ºC, 10 min). Bacillus subtilis, con menores valores de CCß para quinolonas y macrólidos, fue la opción más interesante para mejorar el perfil de detección del Eclipse 100. En cuanto al sistema multiplaca Screening Test for Antibiotic Residues (STAR) que utiliza cinco microorganismos diferentes (Geobacillus stearothermophilus para betalactámicos y sulfonamidas, Bacillus subtilis para aminoglucósidos, Kocuria varians para macrólidos, Escherichia coli para quinolonas y Bacillus cereus para tetraciclinas) aplicado al lactosuero, presentó una elevada especificidad (>=98%) en la mayor parte de casos. Los valores de CCß obtenidos con el protocolo STAR en muestras de lactosuero superan el LMR establecido en la leche, aunque podría representar una adecuada herramienta en la etapa de post-cribado y reducir el número de muestras destinadas al análisis cuantitativo por LC-MS/MS. La producción de queso a partir de leche con antibióticos genera residuos en el lactosuero. Por tanto, adecuados métodos de cribado para la detección de residuos de antibióticos en este importante subproducto de la elaboración del queso permitirían el establecimiento de una apropiada estrategia de control para prevenir la presencia de antibióticos en el lactosuero y, así, evitar los posibles efectos
[CA] La presència de residus d'antibiòtics en la llet i els productes derivats representa un risc per a la salut del consumidor. Límits Màxims de Residus (LMRs) no s'han fixat per als productes lactis i la transferència d'antibiòtics de la llet al formatge i al sèrum durant el procés d'elaboració del formatge quasi no s'ha estudiat. L'objectiu d'aquesta tesi va ser avaluar la transferència d'antibiòtics de la llet a les fraccions formatge i sèrum, així com la validació de la resposta de diversos mètodes per a la detecció d'antibiòtics en mostres de sèrum de llet. En el primer estudi, l'objectiu va ser validar un mètode UHPLC-HRMS multi-residu utilitzant l'analitzador Orbitrap ExactiveTM, per al garbellat quantitatiu d'antibiòtics en mostres de llet, formatge fresc i sèrum, d'acord amb els criteris especificats en la Decisió 657/2002/CE de la Comissió. L'estudi de partició utilitzant UHPLC-HRMS mètode va indicar que la major part d'antibiòtics es van transferir principalment de la llet a la fracció sèrum de llet (fins al 85,9%) durant l'elaboració de formatge. Per tant, els percentatges de retenció d'antibiòtics en la quallada van ser inferiors al 50%, excepte en el cas del ceftiofur (59,7%) i la dicloxacilina (52,8%), i molt variables entre els diferents fàrmacs. En la majoria dels casos, la distribució de medicaments no es va veure afectada per la concentració d'antibiòtics present en la llet per a la producció de formatge, i va estar escassament relacionada amb la lipofilicitat dels antibiòtics. En el segon estudi, es van avaluar les característiques de diferents mètodes de detecció d'antibiòtics en mostres de sèrum d'acord amb la Decisió 657/2002/CE de la Comissió. L'especificitat (percentatge de falsos positius) i la capacitat de detecció (CCß) d'una prova d'inhibició microbiana (Eclipse Farm acoblat al dispositiu e-Reader) i d'assajos d'unió a receptors (3Aminosensor, Quinosensor, Twinsensor i Tylosensor) es van avaluar en mostres de sèrum de llet de cabra, obtenint en general, resultats similars als obtinguts quan s'apliquen per a l'anàlisi de la llet. Es van avaluar tres bioassatjos en placa microtiter i resposta dicotòmica, que contenien Bacillus subtilis, Geobacillus thermocatenulatus i Geobacillus thermoleovorans, respectivament, per a ser aplicats simultàniament amb els mètodes comercials basats en la utilització de Geobacillus stearothermophilus var. calidolactis. Elevats valors d'especificitat (98-100%) es van obtindre quan les mostres de sèrum van ser tractades tèrmicament (85°C, 10 min). Bacillus subtilis, amb menors valors de CCß per a quinolones i macròlids, va ser l'opció més interessant per a millorar el perfil de detecció del mètode Eclipse 100. Respecte al sistema multiplaca Screening Test for Antibiotic Residues (STAR) que utilitza cinc microorganismes diferents (Geobacillus stearothermophilus per a betalactàmics i sulfonamides, Bacillus subtilis per a aminoglucòsids, Kocuria varians per a macròlids, Escherichia coli per a quinolones i Bacillus cereus per a tetraciclines), va presentar una elevada especificitat (>=98%) en la major part de casos. Els valors de CCß obtinguts amb el protocol STAR en mostres de sèrum superen el LMR establit en llet. No obstant això, aquest mètode podria convertir-se en una eina adequada en el post-garbellament per a la identificació preliminar dels residus d'antibiòtics presents en el sèrum de llet i reduir el nombre de mostres destinades a l'anàlisi quantitativa per LC-MS/MS, que és un mètode més complex i car. La producció de formatge a partir de llet amb antibiòtics genera residus en el sèrum. L'adequada prestació dels mètodes de garbellat per a la detecció de residus d'antibiòtics en aquest subproducte d'elaboració del formatge permetria l'establiment d'una estratègia de control per a evitar el risc derivat de la presència d'aquestes substàncies en el sèrum, amb efectes negatius sobre la
[EN] The presence of antibiotic residues in milk and dairy products poses a risk for consumer health, mainly the development of antimicrobial resistance. Maximum Residue Limits (MRLs) for veterinary drugs have not been established for dairy products. Furthermore, the transfer of antibiotics from milk to cheese and whey fractions during cheese-making has been scarcely studied and, therefore, the impact of the use of whey containing antibiotics for the manufacture of foodstuffs for human and animal consumption is unknown. The aim of this thesis was to evaluate the transfer of antibiotics from milk to cheese and whey fractions, as well as the validation of the performance of several methods to screen antibiotics in whey samples. In the first study, a multiresidue UHPLC-HRMS method using the Orbitrap ExactiveTM analyser for the quantitative screening of antibiotics in milk, fresh cheese, and whey samples was validated according to Commission Decision 2002/657/EC, using samples from three different dairy matrices (milk, fresh cheese and whey) from cows, sheep and goats. The partitioning study by UHPLC-HRMS method indicated that most antibiotics were mainly transferred from milk to whey fraction (up to 85.9%) during cheese-making. Thus, retention rates in the rennet curd fraction were lower than 50%, except for ceftiofur (59.7%) and dicloxacillin (52.8%), and very variable between drugs. In most cases, drug distribution was unaffected by the antibiotic concentration present in milk for cheese production and was poorly related to the drug lipophilicity. In the second study, the performance of different methods for screening antibiotics in whey samples was evaluated in accordance with Commission Decision 2002/657/EC, by conducting three experiments focused on commercially available screening tests, microtiter plate bioassays, and a semi-quantitative multi-plate system, respectively. Specificity (false-positive rate) and Detection Capability (CCß) of a microbial inhibitor test (Eclipse Farm coupled to e-Reader device) and receptor-binding assays (3Aminosensor, Quinosensor, Twinsensor, and Tylosensor) were evaluated in whey samples from goats, having in general, similar results than those obtained when they are applied for milk analysis. Three microtiter plate bioassays with dichotomous response containing Bacillus subtilis, Geobacillus thermocatenulatus and Geobacillus thermoleovorans, respectively, were evaluated simultaneously with commercially available tests using Geobacillus stearothermophilus var calidolactis. High specificity values (>=98%) were obtained when whey samples were heat treated (85ºC, 10 min) prior to analysis. Bacillus subtilis, having lower CCß values for quinolones and macrolides, was the most interesting option to improve the detection profile of the Eclipse 100. Regarding the multiplate system Screening Test for Antibiotic Residues (STAR) using Geobacillus stearothermophilus for ß-lactams and sulfonamides, Bacillus subtilis for aminoglycosides, Kocuria varians for macrolides, Escherichia coli for quinolones and Bacillus cereus for tetracyclines, high specificity values (>=98%) were obtained in most cases. The CCß values obtained using the STAR protocol in whey samples exceed the MRL established in milk for most of the substances considered. However, this method could become an adequate tool in post-screening and reduce the number of samples destined for the quantitative analysis by LC-MS/MS, which is a more complex and expensive method. The production of cheese using milk containing antibiotics generates drug residues in whey. Thus, the suitable performance of screening methods for the detection of veterinary drug residues in this cheese-making by-product will allow the establishment of an adequate control strategy to prevent the presence of antibiotic residues in whey and to avoid the hazards associated to human and animal health and environment.
This research forms part of the Project AGL-2013-45147-R financed by the Ministerio de Ciencia e Innovación
Giraldo Gómez, J. (2020). Transfer of antibiotics from goat's milk to cheese and whey [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/155899
TESIS

This research aimed to delineate the final stages of polyether biosynthesis, using the monensin pathway as a model system. The metabolites produced by mutants in which the gene encoding the putative epoxidase MonCI (monCI) along with the proposed isomerase genes monBII and monBII (BHΔCIBIBII) were disrupted were found to be (stereo)chemically identical to those produced by a strain deficient in monCI only (ABΔCI), suggesting that MonBI and MonBII do not play a role in double bond isomerisation. In agreement with this, site-directed mutagenesis of a residue in MonBI previously implicated in authentic double bond isomerase did not abolish the activity of MonBI. The type II thiesterases (TEs) MonAIX and MonAX were investigated as candidates for the enzyme that releases the polyketide chain. However, neither of the genes monAIX or monAX is essential for monensin biosynthesis and these TEs are proposed to act as conventional PKS-editing enzymes, and it is proposed here that this offloading step is catalysed by MonCII. In vitro assays of MonCII, previously assigned as the putative episode hydrolase, showed that it has thioesterase activity, in particular against a thioester derivative of monensin A, supporting previous proposals that oxidative cyclisation might occur while the substrate is enzyme bound. In collaboration with Dr. S.F. Haydock, the biosynthetic gene cluster for nigericn biosynthesis was sequenced from Streptomyces violaceoniger sp. DSM4137 and found to be similar to those of monensin and nanchangmycin.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2011.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 64-66).
The purpose of the research was to identify the clinical and commercial benefits of switching from intravenous (IV) to buccal delivery of antibiotics. then, the research continued to select 3-5 antibiotics that best met the buccal delivery and market requirements. Methods: The research began with the hypothesis that some injectable antibiotics are good candidates for buccal delivery even with the limitations imposed by the buccal tissue. The thesis captures a two-year research period encompassing three critical fronts - the clinical viability of switching from IV to buccal delivery for antibiotics, the market's desire and readiness to switch, and the antibiotic brands available for commercialization. Then the research moved to drug identification and selection in order to assess the antibiotics that would best function in the buccal delivery model. Results: Intravenous (IV) antibiotics are usually reserved for severe infections that require faster treatment. Less aggressive bacterial growths are treated with oral antibiotics, which has fewer side effects and complications. In the past two decades, the understanding of drug transport across different tissues has increased resulting in improved patient adherence to the therapeutic regimen and pharmacologic response. The administration of drugs by transdermal or transmucosal routes are relatively painless, offers patients more choices, and reduces the need to establish intravenous access, which is a particular benefit for children and elderly. These alternative methods also provide clinical care providers with more choices to better manage their patient's course of treatment. In the past, clinicians administered sedatives, narcotics, and a variety of other medications by transdermal, sublingual, nasal, rectal, and even tracheal-mucosal routes. These delivery options have provided flexible practice settings and this paper intends to show that antibiotics could be the next set of drugs to be administered in variety of ways to provide patients and clinicians the best array of choices. Conclusion: A few years ago, the buccal delivery method was fairly unknown. However, advances in nano encapsulation, physiology, toxicity, and the availability of certain drugs make the timing ideal for introducing antibiotics that have undergone a highly selective process for delivering through the buccal tissue.
by Manijeh Nazari Goldberg.
S.M.

The history of the discovery of bacteriophages began almost a century ago. In spite of the appearance of antibiotics, bacteriophages are still extremely promising. Bacterial infections are an urgent public health problem, due to the increasing of antibiotic resistance and negative health consequences. Thus, phages are currently considered as an alternative treatment, which are going to replace antibiotics.

In this study, we investigated the potential applications of carbohydrates in the development of new antibiotics. To tackle the problem of multidrug-resistant variants of M. tuberculosis (MDR-TB), we investigated the biosynthesis pathways of trehalose, which has contributed to significant drug resistance. Some new methods were developed for the synthesis of potential inhibitors (6-azido-trehalose and 6,6'-diazido-trehalose) that have been designed to imitate the intermediate molecule (trehalose 6-phosphate, TPP) of OtsA-OtsB pathway. At the same time, some new antibacterial agents based on trehalose have been synthesized. Members of the 1,2,3-triazole family have shown interesting biological properties. Steroid derivatives have been developed in antibiotics area. Therefore click reaction was utilized to build a 1,2,3-triazole ring and combine cholesterol with different carbohydrate moieties. A series of new compounds has been synthesized and their bioactivities have been tested.

Staphylococcus aureus est un pathogène opportuniste responsable d’infections difficiles à traiter dues à l’inefficacité des antibiotiques actuels. En 2019, cette bactérie a causé le taux de mortalité mondial le plus élevé. La nécessité de recherche de nouveaux antibiotiques a permis de développer de nouvelles molécules inhibant la synthèse des acides gras de type II (FASII). Cependant, ces molécules anti-FASII présentent une faiblesse : S. aureus et les pathogènes Firmicutes sont capables de contourner l’inhibition de FASII par incorporation d’acides gras environnementaux présents dans l’hôte (eFA). L’objectif de ma thèse est d’élucider et comprendre comment le contournement des anti-FASII permettent un regain de développement bactérien. Ceci a permis d’identifier les points faibles de l’adaptation de S. aureus aux anti-FASII afin de développer des traitements en bi-thérapie associant d’autres molécules. Deux principales pistes sont développées. 1) Une approche protéomique couplée aux études physiologiques ont permis de mettre en évidence que l’anti-FASII provoque une résistance accrue au stress oxydant associée à une baisse de la virulence via une reprogrammation des fonctions bactériennes. Les données in vitro sont confirmées in vivo dans un modèle d’insecte montrant que la mortalité des bactéries adaptées à l’anti-FASII est plus lente que les bactéries non traitées. Un pré-traitement par H₂O₂ avant l’ajout des anti-FASII accélèrerait l’adaptation. Par ailleurs, il a été montré qu’un tel traitement active l’efflux des antibiotiques. Ce prétraitement stimulerait l’incorporation des eFA permettant une adaptation rapide à l’anti-FASII. En réponse à l’infection les cellules hôte se défendent en produisant du H₂O₂ suggérant que les bactéries adaptées à l’anti-FASII pourrait présenter un avantage sélectif dans de tels biotopes engendrant un état de persistance des bactéries. 2) Les analyses des données protéomiques ont révélé que UgtP, enzyme impliquée dans la synthèse de l’acide lipotéichoique (LTA), n’était plus produite dans les bactéries adaptées à l’anti-FASII. Après son adaptation à l’anti-FASII, le LTA n’est plus détectable par immunoprécipitation. Ce phénomène est indépendant de la nature des acides gras présents dans les phospholipides membranaires. Les fonctions du LTA dans la division et la croissance sont décrites comme essentielles, comment la bactérie prolifère en son absence ? Contrairement au LTA, l’acide teichoique pariétal (WTA) et la cardiolipine sont surproduits. L’une ou l’autre molécule peut compenser la perte d’expression du LTA. L’absence simultanée du LTA et du WTA est létale pour la bactérie. Ainsi la synergie d’un anti-FASII couplé à un inhibiteur de la synthèse du WTA (le targocil) a été étudiée. Tous ces résultats montrent que les bactéries adaptées à un anti-FASII sont moins virulentes et plus résistantes au stress oxydatif. Un lien entre le stress oxydatif et l’incorporation des acides gras est pour la première fois démontré. Un élément du métabolisme des acides gras (FASII) impacte la synthèse du LTA. Ce travail souligne l’importance des lipides membranaires dans la pathogénie de S. aureus et ouvre de nouvelles perspectives de traitements en bi-thérapie
Staphylococcus aureus is a common opportunist pathogen causing infections that are notoriously difficult to treat due to antibiotic failure. In 2019, S. aureus caused the highest mortality among pathogens. The need for new treatments spurred the development of a novel pipeline class of antibiotics targeting the fatty acid synthesis (FASII) pathway. However, the use of FASII antibiotics has an inherent flaw: S. aureus, and several Firmicute pathogens, are able to compensate FASII inhibition by incorporating environmental fatty acids (eFAs) as present in the host. The aim of my thesis project is to elucidate the factors contributing to this FASII antibiotic adaptation and to analyze the consequences on bacterial fitness. This information will be used to identify the weak points of anti-FASII-adapted S. aureus, and to develop a bi-therapy treatment coupled with anti-FASII. Two main findings are reported. 1) Using combined omics and physiological approaches, we show that anti-FASII adaptation reprograms S. aureus to be more oxidative stress resistant, but less virulent. These in vitro results were in accordance with studies in an insect model, showing that anti-FASII-adapted S. aureus killed insects slowly compared to non-treated S. aureus. Priming S. aureus with H₂O₂ accelerated FASII-antibiotic adaptation. Previously, H₂O₂ priming was reported to stimulate antibiotic efflux. In contrast, H2O2 priming stimulates fatty acid (FA) incorporation, thereby accelerating anti-FASII adaptation via a unique mechanism. As the host produces H₂O₂ as part of its antibacterial defense mechanism, FASII-antibiotic-adapted S. aureus may have a fitness advantage in some host biotopes. Collectively, reduced virulence and greater oxidative stress resistance may provide S. aureus a means to escape detection and persist in the host. Noted, this is the first report showing the link between oxidative stress and FA incorporation. 2) Our proteomics analyses revealed that a lipoteichoic acid (LTA) synthesis enzyme, “UgtP”, was turned off in anti-FASII-treated bacteria. Remarkably, after S. aureus adapts to anti-FASII, LTA was nearly non-detectable. This loss was observed regardless of the phospholipid FA composition. In view of LTA reported essentiality for S. aureus cell division and growth, we then questioned how S. aureus compensates LTA depletion. In contrast to LTA, wall teichoic acid (WTA) and cardiolipin (CL) pools increased in anti-FASII-adapted bacteria. This suggests that WTA and/ or CL might compensate LTA depletion and enable S. aureus to grow. Simultaneous removal of both TAs is reportedly lethal. Consequently, the LTA depletion observed in anti-FASII-adapted bacteria enabled us to demonstrate synergistic inhibition between anti-FASII and the WTA inhibitor, targocil, in S. aureus. Our work reveals that anti-FASII-adapted bacteria produce less virulence factors and are more oxidative stress-resistant. These findings reveal a link between oxidative stress and FA incorporation. They further suggest that an element of the FASII pathway exerts control on LTA synthesis. Altogether this work underlines the importance of membrane lipids in S. aureus pathogenesis and opens perspectives for designing bi-therapy treatments

In India, there is no regulation of antibiotics and allegedly the use has doubled since 2006. Indiscriminate use of antibiotics gives rise to development of resistant bacteria. The media has, according to the theories used in this study, a responsibility to educate and empower the people to make personal judgments about health risks. This study focuses on the extent to which two of the largest English-language newspapers in India, the Hindu and Times of India, report on antibiotic resistance; and also, how the journalists and editors on these newspapers look upon their profession and responsibilities when it comes to reporting on health issues. In addition to the quantitative content analysis, which comprises 162 articles about antibiotic resistance published between 2006 and 2012, six in-depth interviews were conducted. The results show that the amount of coverage on antibiotic resistance increased 2010 when the Lancet published a report on new findings of multi-resistant bacteria in India. This indicates that an event was needed to qualify antibiotic resistance for the news pages. Our study also shows that preventive measures which can be taken to reduce the emerge of resistant bacteria are often included in the articles and that they are addressed to doctors as well as to the general public. On the other hand, information on the magnitude of the problem is rarely presented. Scientists are often quoted or referred to, and the journalists of the investigated newspapers state that they have a great confidence in them. Furthermore, the respondents express that they have a responsibility to report on health issues. They believe that their newspapers have a major influence on its readership, and that their reporting can make a difference in the health situation in India. Some of them mention, however, that their overall impact is limited since their newspapers only reach the literate middle-class.

The hypotheses of this study are that reduction and rational usage of antibiotics reduces development of antibiotic resistance. In Sweden, the trends do not follow this pattern. Despite a decrease in prescriptions of antibiotics, there is an increase in the number of patients infected with Methicillin-resistant Staphylococcus Aureus (MRSA), Extended Spectrum Beta-Lactamases (ESBL) and ESBL selecting for carbapenem-resistance (ESBLCARBA). This study aims to study factors affecting antibiotic resistance management. An additional aim is to use a multidisciplinary approach for a subject that has mostly been studied with quantitative methods. First, linear regressions investigated any possible significant changes of prescription rates in outpatient care, hospital usage of antibiotic groups and antibiotic resistance. After this, nine interviews were conducted with physicians in outpatient care, hospital care and with representatives from the Swedish Strategic Programme for the Rational Use of Antimicrobial Agents and Surveillance of Resistance (Strama), a network working for Swedish prevention against antibiotics resistance. There was a significant decrease in the number of prescriptions of antibiotics in outpatient care among all Swedish counties and a small, but significant increase of antibiotics used in hospitals. The number of patients infected with multidrug resistant bacteria also show a significant increase. The interviews revealed that health care workers in all counties follow the same guidelines and try to be as specific as possible in choosing antibiotics to hit specific bacteria. The respondents suggested migration and extended travelling as explanations to the growing number of cases of multidrug resistant bacteria. Further, two major factors emerged as important for an efficient antibiotic resistance management; Education/knowledge and Discussion. The results indicate a need for further research on rational usage of antibiotics and the use of broad-spectrum antibiotics in hospital care, rather than the reduction through prescriptions. The results indicate that rational usage has a bigger impact than reduction. Using a multidisciplinary approach gave a broader perspective on the issue and future studies should see the possibilities of mixing quantitative and qualitative studies.

In a world where the human population is increasing, new innovations to produce enough food are required. Aquaculture’s part of the global animal protein production has increased in recent years and could be a possible solution. However, if aquaculture is poorly managed, it can result in negative consequences and one such consequence is the development of antibiotic resistance. In this review, I examine how aquaculture affect antibiotic resistance by studying what the literature says on accumulation of antibiotics in different organisms and sediment, if antibiotics can be transferred to humans through consumption of antibiotic treated products, and if human pathogens in aquaculture farms may acquire antibiotic resistance. Furthermore, I examine what factors are contributing to irresponsible antibiotic use and how such use is managed. The result of this review indicate that antibiotics are able to accumulate in organisms and sediment. It is not clear however how consumption of these affect human microbiomes. In contrast, it is clear that antibiotic resistance can be transferred from antibiotic-resistant bacteria to human pathogens. Regarding antibiotic use, irresponsible use foremost exists in low-income countries and the main drivers behind such use are socioeconomic ones, such as lack of knowledge, poverty and food security. Finally, I propose possible solutions that might improve future management.
I en värld där den mänskliga befolkningen ökar krävs nya innovationer för att producera tillräckligt med mat. Vattenbrukets andel av den globala animaliska proteinproduktionen har ökat de senaste åren och kan ses som en potentiell lösning. Om vattenbruk dock hanteras ansvarslöst kan det uppstå negativa konsekvenser. En sådan konsekvens är utveckling av antibiotikaresistens hos skadliga bakterier. I denna litteraturstudie undersöker jag vattenbrukets påverkan på antibiotikaresistens genom att studera vad litteraturen säger om ackumulation av antibiotika i olika organismer och sediment, om antibiotika kan överföras till människor genom konsumtion av antibiotikabehandlade produkter, och om mänskliga patogener i vattenbruksodlingar kan förvärva antibiotikaresistens. Jag undersöker också vilka faktorer som bidrar till ansvarslös antibiotikaanvändning och hur den hanteras ur ett hållbarhetsperspektiv. Resultaten i denna studie tyder på att antibiotika kan ackumuleras i organismer och sediment men att det råder oklarheter huruvida konsumtion av antibiotikabehandlad mat påverkar mänskliga bakteriekulturer. Antibiotikaresistens kan dock överföras från antibiotikaresistenta bakterier till mänskliga patogener. Ansvarslös antibiotikaanvändning sker huvudsakligen i fattigare länder och det är förmodligen i stor utsträckning till följd av socioekonomiska faktorer som okunskap, fattigdom och livsmedelstrygghet. Slutligen föreslår jag lösningar som möjligen kan bidra till bättre hantering av framtida antibiotikaanvändning.

The investigations detailed herein have been conducted to address various aspects of the biopharmaceutics and pharmacokinetics of josamycin which to-date, have received little or no attention in the literature. Areas of investigation have included the selective determination of josamycin in serum and urine samples, the stability of josamycin in stored biological samples, intrinsic dissolution rates, solubility, acid and alkali stability and bioavailability and pharmacokinetics after dosing with a solution, powder and tablets. High performance liquid chromatography (HPLC) was used as the main analytical tool throughout these studies and proved to be highly versatile for the determination of josamycin in a number of different media. HPLC analysis afforded simple yet accurate determination of josamycin in samples from dissolution, solubility, tablet content and stability studies. Furthermore, the specificity afforded by HPLC was particularly useful for the separation of josamycin from degradation products formed in acid and alkali media. Since metabolites of josamycin are microbiologically active, microbiological assays do not determine the concentration solely of josamycin. An analytical method capable of the selective determination of josamycin in serum and urine samples is therefore required for the procurement of reliable bioavailability and pharmacokinetic data. HPLC affords this selectivity and a method for the selective determination of josamycin in serum and urine was successfully developed. The assay was simple yet precise, accurate and sensitive. Furthermore, it was well suited to the determination of josamycin in a large number of biological samples. Its success was largely due to the use of a solid phase extraction step using C₁₈ extraction columns, with a highly specific wash sequence followed by a phase separation step after elution from the extraction column. Chromatography was performed on a C₁₈ reversed-phase analytical column with UV detection of josamycin and internal standard at 231 nm and at 204 nm respectively using a programmable multi-wavelength detector. Only slight modification of the assay described should enable the selective determination of the metabolites of josamycin. This assay, therefore, lays the groundwork for future investigations into the pharmacokinetics of these metabolites. The re-usability of extraction columns was assessed in an attempt to reduce the cost of sample analysis. It was found that extraction columns could be used twice for the extraction of serum samples and up to four times for the extraction of urine samples. The difference between the re-usability of extraction columns for serum and urine samples was ascribed to various differences in the composition of the sample matrix. The stability of josamycin in stored serum and urine samples was also assessed.

The stereoselective synthesis of urdamycinone B (17) was achieved in a 21% overall yield from C-glycosyl-naphthoquinone 197. The key reaction was the Diels-Alder cycloaddition reaction of 197 and siloxydiene (�)-117 promoted by a chiral Lewis acid derived from (S)-3,3�-diphenyl-1,1�-binaphthalene-2,2�-diol (291), BH₃.THF and acetic acid. An effective kinetic resolution of (�)-117 occurred. Four cycloadducts 199a-d were formed in a ratio between 84:8:2:6 and 70:9:2:19. Aromatisation of the mixture by treatment with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) gave 200a and 200b in 4:1 ratio. A sequence of reactions involving deacetylation, conversion of a phenyldimethylsilyl group into a hydroxyl group and photooxidation gave a 4:1 mixture of urdamycinone B (17) and its C-3 epimer (154). Separation of these products was achieved by high performance liquid chromatography (HPLC). The C-glycosyl donor, 1,3,4-tri-O-acetyl-2,6-dideoxy-D-glucopyranose (204), was synthesised from readily accessible tri-O-acetyl-D-glucal (237) using two approaches. The first involved a sequence of deacetylation, tosylation, lithium aluminium hydride (LiAlH₄) reduction and acetylation to give di-O-acetyl-6-deoxy-D-glucal (242). The triphenylphosphine hydrogen bromide (TPPHBr) catalysed addition of acetic acid to 242 gave 204 in overall yields ranging from 0 to 32%. The step involving the reduction of the tosylate intermediate was the cause of the variable yields. The alternative synthesis started with the TPPHBr catalysed addition of benzyl alcohol to 237. Subsequent deacetylation, tosylation and reduction with LiAlH₄ gave benzyl 2,6-dideoxy-D-glucopyranoside (250). Acetylation and hydrogenolytic debenzylation gave 3,4-di-O-acetyl-2,6-dideoxy-D-glucopyranose (247). Acetylation gave 204 in 40% overall yield. A third approach to 204 involved selective tosylation of methyl α-D-mannopyranoside (258) and subsequent treatment with 2,2-dimethoxypropane under acidic conditions to give acetonide 255. LiAlH₄ reduction of the tosylate gave methyl 6-deoxy-2,3-O-isopropylidene-α-D-mannopyranoside (256). Acidic hydrolysis of 256 and subsequent acetylation afforded 1,2,3,4-tetra-O-acetyl-6-deoxy-α-D-mannopyranoside (260). Treatment of 260 with hydrogen bromide in acetic acid and subsequent reductive elimination with a zinc-copper couple gave 242. The addition of acetic acid catalysed by TPPHBr afforded 204 in 18% overall yield. The final synthesis of 204 started with thiophenyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (269). A sequence of deacetylation, tosylation and LiAlH₄ reduction gave thiophenyl 2,3-O-isopropylidene-6-deoxy-α-D-mannopyranoside (274). The structure of 274 was confirmed from a single crystal X-ray diffraction study. Hydrolysis of the isopropylidene group of 274 and subsequent acetylation afforded thiophenyl 6-deoxy-2,3,4-tri-O-acetyl-α-D-mannopyrannoside (282). Treatment of 282 with iodine monobromide and subsequent reductive elimination with zinc-copper couple gave 242. The TPPHBr catalysed addition of acetic acid to 242 afforded 204 in 19% overall yield. Differentially protected C-glycosyl donor, 1,3-di-O-acetyl-4-O-benzyl-2,6-dideoxy-D-mannopyranose (265), was synthesised from 274. The benzylation of 274 gave thiophenyl 6-deoxy-2,3-O-isopropylidene-4-O-benzyl-α-D-mannopyranoside (276). Acidic hydrolysis followed by acetylation afforded thiophenyl 6-deoxy-1,2-di-O-acetyl-4-O-benzyl-α-D-mannopyranoside (278) which, upon bromination by iodine monobromide, gave thiophenyl 6-deoxy-1,2-di-O-acetyl-4-O-benzyl-α-D-mannopyranosyl bromide (279). The reductive elimination of 279 with zinc-copper couple gave 3-O-acetyl-4-O-benzyl-6-deoxy-D-glycal (264). The TPPHBr catalysed addition of acetic acid to 264 afforded 1,3-di-O-acetyl-4-O-benzyl-2,6-dideoxy-D-mannopyranose (265) in 16% overall yield from 274. The instabillity of bromide 279 affected the yield of 265. A C-glycosylation study of 2-naphthol 227 and 1,4-dimethoxy-5-hydroxynaphthalene (205) with 2-deoxy-glycosyl acetates was undertaken. Boron trifluoride diethyl etherate (BF₃.Et₂O) and scandium triflate [Sc(OTf)₃] proved effective promoters. For example, the glycosylation reaction of donor 265 and 227, promoted by 0.5 equivalents of Sc(OTf)₃, afforded C-glycoside 2-hydroxy-1-[3�-O-acetyl-4�-O-benzyl-2,6-dideoxy-β-D-manno-hexopyranosyl]-naphthalene (289) in 85% yield.

Thesis (Ph. D.)--University of California, San Diego, 2007.
Title from first page of PDF file (viewed January 14, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.

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.