Master of Public Health - MPH / Background: The irrational use of medicines is a global public health challenge, particularly in developing countries like Nigeria. One of the consequences of irrational medicine use is rising antimicrobial resistance, which continues to contribute to the increase in morbidity, mortality, and high cost of care, despite breakthroughs in medicine and new treatment options. Community pharmacists have been identified as contributors to antimicrobial resistance through their antibiotic dispensing practices. However, there is little research on community pharmacists who provide private healthcare in Nigeria. Aim: This study described the antibiotic dispensing practices of community pharmacists and factors associated with such dispensing practices in Jos, Nigeria. Methodology: The study used a cross-sectional descriptive design. Simple random sampling was used to select a sample of 84 community pharmacies out of a total of approximately 107 community pharmacies in Jos, and one community pharmacist from each community pharmacy. A research assistant was trained to administer the questionnaire along with the researcher and collect information on community pharmacists’ demographics, antibiotic dispensing practices, and the factors associated with those dispensing practices. The socio-demographic data was analysed via descriptive analytical tools such as simple percentages and crosstabulations. These tools were used to generate a descriptive picture of the data, patterns and associations using SPSS version 25. Quantitative content analysis was done on responses to scenario-based questions, and recommendations made as to how the dispensing of antibiotics could be improved. Ethical clearance was obtained from the University of the Western Cape Biomedical Research Ethics Committee and Jos University Teaching Hospital, while informed consent was obtained from all community pharmacists before the commencement of the study. Results: The majority of the community pharmacists (87%) indicated that patients could purchase antibiotics without prescription from their pharmacies, and most pharmacists (98%) asked for reasons why antibiotics were demanded for without prescriptions. While 58% indicated that patients could purchase partial quantities of prescribed antibiotics at their pharmacies, 96% investigated the reasons for partial requests, and 94% counselled on the right dosage and frequency of the prescribed antibiotics. Sixty-seven percent of the pharmacists indicated that one of the major reasons for dispensing antibiotics without prescription was self-medication by patients, while most (87%) indicated that financial constraint was a major reason why patients requested for partial quantities of prescribed antibiotics. Conclusion: The dispensing of non-prescribed and part-prescribed antibiotics is a common practice amongst community pharmacists in Jos, Nigeria. Enhancing the financial status of Nigerians, as well as ensuring stricter regulatory measures on antibiotic use, would help promote rational use of antibiotics and reduce rising antimicrobial resistance rates.

Philosophiae Doctor - PhD / The emergence of virulent and drug-resistant bacterial strains such as methicillin-resistant Staphylococcus aureus (MRSA) is a global public health burden. The World Health Organization (WHO) has placed MRSA and vancomycin-intermediate-sensitive S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) on a high global priority pathogens list of antibiotic-resistant bacteria to promote the research and development of novel and effective antibiotic therapeutic rationales. Uncomplicated S. aureus bacteraemia (e.g., mild skin infections) may be treatable with the conventional regimens of antibiotics, but resistance strains of the bacteria (e.g., invasive infections), often persist as a high load of bacterial DNA in blood, and has been linked to increased mortality in world populations, irrespective of country or location. Several lines of evidence imply that combinations of vancomycin (a glycopeptide antibiotic that targets cell wall synthesis) and ß-lactam antibiotics that target the penicillin-binding proteins (PBPs) improve clearance of MRSA bloodstream infections (BSIs).

Student Number : 0000127R - MPharm dissertation - School of Pharmacy and Pharmacology - Faculty of Health Sciences / Traditional medicines form a significant part of the lives of many people around the world and in South Africa almost 60 % of people consult traditional healers in addition to the modern medical services available. Plants form a significant part of traditional healing and hence, selected species of a traditionally used plant genus, Hermannia, were chosen for biological and chemical investigation to determine a scientific basis for the traditional use of these plants. A phytochemical investigation was carried out, firstly using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) and then isolation and identification of compounds from various Hermannia species. TLC analysis indicated significant similarities between the various species with only H. saccifera displaying chemical anomalies. This was further corroborated by the HPLC analysis although very conservative profiles were produced. Isolation of compounds from H. saccifera yielded a novel labdane compound, E-17, 19-diacetoxy - 15 - hydroxylabda - 7,13 - diene, as well as two flavones, 5,8- dihydroxy-6,7,4’- trimethoxyflavone and cirsimaritin which have previously been isolated. In addition, two commonly found compounds, lupeol and β- sitosterol were isolated from H. cuneifolia and H. salviifolia respectively. This is the first report on the isolation and identification of all five compounds from Hermannia species. Antimicrobial activity was assessed using two methods i.e. minimum inhibitory concentrations as well as the death kinetics assay. Minimum inhibitory concentrations were determined using four Gram-positive and two Gram-negative bacteria as well as two yeasts. All species investigated indicated antimicrobial activity with H. saccifera showing good activity against S. aureus and B. cereus. E-17, 19-diacetoxy - 15 - hydroxylabda - 7,13 - diene isolated from H. saccifera indicated activity (MIC = 23.6 μg/ml against S.aureus) although the activity was less than that of the crude extract (MIC = 19.5 μg/ml), thus, demonstrating that there are a number of compound contributing to the promising activity of the crude extract. This was further corroborated by the bioautograms developed of the H. saccifera extract. Time-kill studies on H. saccifera against S. aureus indicated that at concentrations of 0.1, 0.25 and 0.5 % bacteriostatic activity was observed while at 0.75% the extract achieved complete bactericidal activity after 240min. Free radical scavenging activity was assessed using the 2,2-diphenyl-1-picrylhydrazy (DPPH) and 2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) assays. Ten of the twelve species indicated good activity with H. cuneifolia demonstrating the most promising activity (IC50 = 10.26 μg/ml for DPPH and 10.32 μg/ml for ABTS). Two of the isolated compound, 5,8- dihydroxy-6,7,4’- trimethoxyflavone and cirsimaritin displayed insignificant activity. The 5-lipoxygenase assay was used to assess the anti-inflammatory activity of Hermannia species. All species exhibited intermediate activity with the exception of H. cuneifolia (IC50 = 15.32 μg/ml). In addition, four isolated compounds, 5,8- dihydroxy-6,7,4’- trimethoxyflavone, cirsimaritin, lupeol and β-sitosterol showed moderate inhibition of the enzyme indicating that while these compounds do contribute to the activity of the extracts they are not individually responsible for any significant activity. Antimalarial activity was assessed using the titrated hypoxanthine incorporation assay while toxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay. Only three species indicated any good antimalarial activity i.e. H. saccifera, H. muricata and mostly H. trifurca (IC50 = 25.30, 28.17 and 18.80 μg/ml respectively). However, the activity of H. saccifera and H. trifurca are probably due to a general cytotoxicity as these species exhibited a low safety index. All other species appear safe for use. Several Hermannia species have indicated in vitro biological activity in a number of assays which is related to their use in traditional medicines to treat a number of disease states. Hence, a scientific basis, albeit in vitro, has been established for the use Hermannia species in traditional healing.

ABSTRACT Twenty essential oil constituents (EOC′s) from seven structural groups were tested for their antimalarial, antimicrobial (both bacterial and fungal), anti-oxidant, anticholinesterase and toxicity properties. To test for their antimalarial property, the tritiated hypoxanthine incorporation assay was used, while the disc diffusion and minimum inhibitory concentration (MIC) microplate assays were employed for the antimicrobial properties. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) method was used to test the anti-oxidant property and their toxicity profile was assessed with the 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cellular viability assay. The anticholinesterase activity was determined using the thin layer chromatography (TLC) bioautographic method. The EOC´s were found to inhibit the growth of Plasmodium falciparum with IC50 values ranging between 0.9 to 1528.8μM with E- and Z-(±)-nerolidol, (-)-pulegone, (+)-α-pinene and linalyl acetate being the most active. In combination p-cymene (the least active) and E- and Z-(±)-nerolidol (the most active) displayed the most synergistic interaction (ΣFIC = 0.09), with their antimalarial activity comparable to that of the interaction between E- and Z-(±)-nerolidol and quinine (ΣFIC = 0.01). Eugenol had the most favourable safety index and was the only EOC with anti-oxidant activity comparable to vitamin C. Combination studies showed that E- and Z-(±)-nerolidol and (-)-pulegone or quinine, p-cymene and γ-terpinene or (-)-pulegone potentiated each other′s toxicity. The EOC´s inhibited the growth of Gram-positive, Gram-negative bacteria and yeast with MIC values ranging from 1.66 to >238.4mM. When combined, synergism was observed between (+)-β-pinene and carvacrol or γ-terpinene; γ-terpinene and geranyl acetate when tested against Staphylococcus aureus, while (+)-β-pinene and (-)-menthone showed antagonism against C. albicans. The combinations of EOC′s and a standard antimicrobial resulted in synergistic interactions between carvacrol and ciprofloxacin against Bacillus cereus, eugenol and ciprofloxacin against Eschericia coli, carvacrol and amphotericin B against C. albicans. The trans-geraniol and E- and Z-(±)-nerolidol combination demonstrated an additive interaction against B. cereus, while for eugenol and E- and Z-(±)-nerolidol an indifferent interaction against E. coli was noted. These results show that the biological activities of EOC′s can vary when used alone and in combination. They do have the potential to be used as templates for novel drugs and as adjuncts to modern medicines in the combat against drug resistance.

A Periodontite é uma doença de etiologia multifatorial que acomete os tecidos de suporte dentário resultando em perda progressiva de inserção e perda óssea. Este estudo clínico controlado, aleatorizado e duplo-cego avaliou a eficácia da Terapia Fotodinâmica antimicrobiana (TFDa), em aplicações múltiplas, como terapia adjuvante ao tratamento periodontal cirúrgico em pacientes com Periodontite Crônica Avançada (PCA). Dezesseis voluntários foram submetidos ao modelo de estudo do tipo boca dividida, recebendo tratamento de acesso cirúrgico associado a raspagem e alisamento radicular (RAR) adjunto à TFDa em protocolo de aplicação nos períodos de 0, 2, 7 e 14 dias pós-operatórios (Grupo Teste - GT), ou apenas tratamento de acesso cirúrgico associado a RAR (Grupo Controle - GC). Todos os pacientes receberam orientação de higiene oral e acompanhamento por 90 dias pós-cirúrgicos. Os seguintes parâmetros clínicos e microbiológicos foram avaliados: Nível clínico de inserção (NCI), Profundidade de sondagem (PS), Recessão gengival (RG), Sangramento à sondagem (SS), Índice de placa (IP) e contagem de 40 espécies microbianas subgengivais (checkerboard DNA-DNA hybridization). Os dados foram coletados nos períodos baseline (pré-terapia básica), 60 dias (30 dias após terapia não cirúrgica) e 150 dias (90 dias pós-cirurgia). Uma redução significativa na PS foi observada aos 150 dias para o GT, quando comparado ao GC no mesmo período (p < 0,05). O ganho do NCI foi significativamente maior no GT entre os tempos de 60 e 150 dias (p < 0,05). As mudanças da microbiota subgengival foram similares entre os grupos (p > 0,05), mas o Grupo Teste apresentou quantidades mais elevadas de bactérias compatíveis com saúde periodontal no período final do experimento em relação ao GC (p < 0,05). Concluiu-se que a utilização da TFDa em aplicações múltiplas, como terapia adjuvante ao tratamento cirúrgico periodontal, produziu melhoras significativas nos parâmetros clínicos no período de 90 dias de avaliação / Chronic Periodontitis is a multifactorial disease which results in tooth supporting tissues loss. This double-blind randomized controlled clinical trial assessed the efficacy of multiple sessions of antimicrobial photodynamic therapy (aPDT) as an adjunct to surgical periodontal treatment in patients with severe chronic periodontitis (SCP). Sixteen volunteers were selected into this Split-mouth study. They were subjected to scaling and root planning in open flaps (SRP) combined with aPDT at 0, 2, 7, and 14 postoperative days (Test Group - TG), or only SRP (Control Group CG). All patients were instructed about oral hygiene and were followed up for 90 days after surgery. The following clinical and microbiological parameters were assessed: clinical assessment level (CAL), probing depth (PD), gingival recession (GR), bleeding on probing (BOP), plaque index (PI). Levels of 40 subgingival species were measured by checkerboard DNA-DNA hybridization at baseline, 60 (30 days after non-surgical therapy) and 150 days (90 days post-surgery). Data were collected at baseline (pre-intervention), at 60 days (30 days after the end of nonsurgical therapy), and at 150 days (90 days after surgery). A significant reduction in PD was observed at 150 days for the TG, when compared to the CG (p < 0.05). CAL gain was significantly higher in the TG at 60 and 150 days (p < 0.05). Changes in the subgingival microbiota were similar between the groups (p > 0.05), but the TG revealed a larger number of bacteria associated with periodontal disease at the end of the experiment compared to the CG (p < 0.05). In conclusion, multiple sessions of aPDT as an adjunct to surgical periodontal treatment significantly improved clinical parameters at 90 postoperative days

A presença de resíduos de antimicrobianos no leite representa um problema de saúde pública, podendo causar reações alérgicas e a seleção de bactérias resistentes aos antimicrobianos. É também um problema econômico interferindo nas culturas lácteas utilizadas na fabricação de derivados. A mastite é a causa mais freqüente para o tratamento de bovinos leiteiros com antimicrobianos. Foram objetivos deste trabalho estudar a influência do processo inflamatório no tempo de eliminação de diferentes antimicrobianos, administrados por via sistêmica ou intramamária em vacas em lactação e avaliar a presença de resíduos detectáveis em tanques resfriadores de propriedades leiteiras, correlacionando com os níveis de mastite nos rebanhos. Foi utilizado teste microbiológico comercial (Delvotest) para a avaliação da corrência de resíduos de antimicrobianos em 60 amostras de leite de tanques resfriadores de propriedades leiteiras e individualmente de 135 vacas em lactação com e sem processo inflamatório na glândula mamária. Foi verificada correlação entre a ocorrência de resíduo de antimicrobiano no tanque resfriador e o aumento da freqüência de mastite clínica (r= 1,0; teste de Spearman).O aumento do período de carência, no leite de glândulas com processo inflamatório, foi observado tanto nos animais tratados por via sistêmica quanto naqueles tratados por via intramamária, ultrapassando o período estipulado pelo fabricante do medicamento. Tal fato foi observado em relação a todos os grupos tratados com diferentes antimicrobianos (betalactâmicos, aminoglicosídeos, tetraciclinas e sulfa) variando de 11 a 60 % das amostras analisadas. Após 72 horas da aplicação de medicamento por via sitêmica, houve diferença significante (p < 0,05, Fisher) entre as amostras de animais com mastite e as de animais com ausência de processo inflamatório, indicando uma maior persistência de resíduo nas amostras de animais com mastite. Na avaliação da influência de inibidores naturais observou-se que 33% das amostras de quartos com mastite clínica e 18% das amostras de quartos com mastite subclínica apresentaram resultados positivos, sendo que na ausência de processo inflamatório todas as amostras apresentaram resultados negativos. A diferença de positividade foi estatisticamente significante, quando foram comparadas as amostras de quartos mamários com mastite clínica com as amostras de quartos mamários com ausência de processo inflamatório (p < 0,05, Fisher). Em síntese concluiu-se que houve uma correlação entre o nível de ocorrência de mastite clínica e a presença de resíduo no leite dos tanques resfriadores de propriedades leiteiras e que glândulas mamárias com processo inflamatório apresentaram maior período de eliminação de resíduos de antimicrobiano pelo leite, em relação às glândulas sem processo inflamatório, muitas vezes ultrapassando o período estipulado pelos fabricantes de medicamentos. Observou-se também que as amostras de leite de animais não tratados, analisadas sem aquecimento prévio, apresentaram positividade demonstrando a influência dos inibidores naturais, produzidos em decorrência do processo inflamatório, nos resultados dos testes microbiológicos, indicando a necessidade de aquecimento das amostras antes da realização dos testes microbiológicos para a detecção de resíduos, para evitar a ocorrência de resultados falso positivos. Como conclusão geral, a análise dos resultados obtidos no presente estudo demonstrou que a mastite interferiu na ocorrência de antimicrobianos no leite. / The presence of antimicrobial residues im milk is a public health concern, because they may cause allergic reactions and may lead to the development of resistance to antimicrobial agents by bacterial strains. It is also an economic problem because it interferes with starter cultures used in dairy products. Mastitis, the inflammatory process of the mammary gla nd, is the main reason to treat dairy cows with antimicrobial agents. The purpose of this study was to verify the influence of mastitis on the withdrawal period of several intramammary and parenteral antimicrobial agents used in lactating cows, as well as to evaluate the presence of detectable antimicrobial residues in bulk tank milk, in relation to the mastitis rates among the studied dairy farms. A total of 60 bulk tank samples of dairy farms and 135 individual samples of healthy and mastitic dairy cows were screened to verify the occurrence of antimicrobial residues with a microbiologic test (Delvotest®). A correlation was detected between antimicrobial residues in bulk tank and high clinical mastitis rates (r= 1.0; Spearman\'s test). A higher withdrawal period, exceeding that determined by the manufacturer, was observed in the milk of mammary glands presenting inflammatory processes, both in animals treated by the parenteral route, and those treated by the intramammary route. This observation ranged from 11 to 60% of the analyzed samples and was verified in all groups treated with different antimicrobial agents (betalactam, aminoglycosides, tetracycline and sulfas). Statistically significant difference was detected (p<0.05, Fisher\'s test) at seventy two hours after the parenteral treatment in samples from animals presenting inflammatory processes, in relation to samples from animals that did not present mastitis. That is, there was a higher withdrawal period in the samples from animals presenting mastitis. In the evaluation of the influence of inflammatory reaction natural inhibitors on Delvotest®, it was verified that 33% of the samples from clinical mastitis quarters and 18% from subclinical mastitis quarters were positive, and that quarter samples without inflammatory processes were negative. These differences were statistically significant (p<0.05, Fisher\'s test). Among the main onclusions, it should be pointed out that there was a correlation between high clinical mastitis rates in the dairy herd and antimicrobial residues in bulk tank milk. Another important result was that mammary glands presenting inflammatory processes exhibited higher withdrawal periods when compared with mammary glands that did not present inflammatory processes, and that sometimes this withdrawal period exceeded the period recommended by the manufacturer. It was also observed that milk samples from untreated animals, when tested without preliminary heating at 80ºC, were positive, due to the occurrence of natural inhibitors produced by the inflammatory process, showing their interference on microbiologic tests. Therefore, it was demonstrated that previous heating is helpful to avoid false positive results when microbiologic tests are used to detect antimicrobial agents residues in milk samples. As an overall conclusion, it was well demonstrated that mastitis interferes in antimicrobial residues in milk.

O uso indiscriminado de antimicrobianos na suinocultura nacional e mundial tem sido uma prática comum, visando minimizar possíveis falhas no manejo e no ambiente em que vivem os animais, no entanto, este uso é descrito como potencial fator de risco na seleção de estirpes resistentes à antimicrobianos, entre elas as estirpes de Staphylococcus aureus resistentes à meticilina (MRSA). O presente estudo revelou o uso médio de 358,0 mg diferentes antimicrobianos/ kg de suíno produzido nos 25 sistemas de produção pesquisados, sendo este valor considerado elevado, quando comparado a tendência global de 172,0 mg, bem como período médio de exposição de 66,3% da vida dos animais e exposição à 7 diferentes princípios ativos em média, variando de 2 a 11. A pesquisa de animais carreadores de estirpes MRSA revelou 80,0% dos sistemas de produção positivos, sendo 68,0% positivos para LA-MRSA-ST398, e 60,0% positivos para a presença do gene czrC, codificador de resistência ao óxido de zinco e cádmio. Não foi evidenciada correlação significativa entre o uso de antimicrobianos, nível de biossegurança, produtividade, status dos rebanhos para Mycoplasma hyopneumoniae, tipo de criação em sítio único (ciclo completo) ou dois sítios e positividade para MRSA. Apesar da ausência das correlações significativas ficou evidente no presente estudo a possibilidade de grandes melhorias nos programas de biossegurança, manejo, assim como nos programas de utilização de antimicrobianos. A alta frequência de sistemas de produção positivos para as estirpes MRSA são um importante alerta para o risco de disseminação deste agente para os seres humanos. / The indiscriminate use of antimicrobials in Brazilian and global pig farms has been a common practice in order to minimize possible failures in the management and environment in which animals live, however, this use is described as a potential risk factor in the selection of strains resistant to antimicrobials, including strains of methicillin resistant Staphylococcus aureus (MRSA). The present study revealed the average use of 358.0 mg of different antimicrobials / kg of pig produced in the 25 production systems surveyed, which is considered high when compared to the overall trend of 172.0 mg, as well as the mean exposure period of 66.3% of the animals life and exposure to 7 different active principles on average ranging from 2 to 11. Research on MRSA strains showed 80.0% of the positive production systems, and 68.0% were positive for MRSA strains. LA-MRSA-ST398, and 60.0% positive for the presence of the czrC gene, encoding resistance to zinc oxide and cadmium. There was no significant correlation between antimicrobial use, biosafety level, productivity, herd status for Mycoplasma hyopneumoniae, single site (complete cycle) or two sites and MRSA positivity. Despite the absence of significant correlations, it was evident in the present study the possibility of great improvements in biosafety programs, management, as well as in programs for the use of antimicrobials. The high frequency of positive production systems for MRSA strains is an important warning for the risk of dissemination of this agent to humans.

Nós avaliamos a atividade antimicrobiana do mel coletado de ninhos de 12 espécies de abelhas nativas sem ferrão comumente encontrados no Brasil e 25 amostras de mel de Apis mellifera não pasteurizados que foram identificadas como sendo unifloral e uma amostra de mel multifloral. A atividade antimicrobiana de cada amostra de mel foi testada contra cinco espécies de bactérias patogênicas, uma espécie de fungo patogênico e uma espécie de levedura patogênica, comparando esta atividade com o mel terapêutico de manuka produzido por abelhas Apis mellifera na Nova Zelândia a partir do néctar de Leptospermum scoparium (Myrtaceae). Cinco das treze amostras de mel das abelhas sem ferrão foram bactericidas e oito foram fungicidas contra o fungo patogênico Trichophyton rubrum. Somente a levedura Candida albicans foi resistente a todas as amostras de mel. As amostras de mel de Apis mellifera que apresentaram atividade bactericida foram caju, romã e cana, nenhuma das amostras de mel de A. mellifera afetou o fungo T. rubrum. Os meis de Nannotrigona testaceicornis, Plebeia remota, Tetragona clavipes e Scaptotrigona depilis todos com alto nível de atividade antimicrobiana, foram significativamente mais eficiente em termos de atividade antimicrobiana que o mel de manuka, especialmente contra Escherichia coli, Klebsiella pneumoniae e T. rubrum. O mel artificial feito com os principais açúcares encontrados no mel e com teor de água semelhante, não teve atividade antimicrobiana em nossas análises. Nós também fizemos análises de espectrometria de massas das amostras de mel e encontramos alguns \"fingerprint\" característicos que foram associadas com a atividade antimicrobiana. / We evaluated the antimicrobial activity of honey collected from nests of 12 species of native stingless bees commonly found in Brazil and 25 unpasteurized honey samples of Apis mellifera that were identified as being unifloral and one multifloral honey sample. The antimicrobial activity of each honey sample was tested against five species of pathogenic bacteria, a pathogenic fungal species and a pathogenic yeast species, comparing this activity with therapeutic manuka honey produced by Apis mellifera bees in New Zealand from the nectar of Leptospermum scoparium (Myrtaceae). Five of thirteen samples of honey from the stingless bees were bactericidal and eight were fungicidal against the pathogenic fungus Trichophyton rubrum. Only the yeast Candida albicans was resistant to all honey samples. The Apis mellifera honey samples that showed bactericidal activity were caju, romã e cana, none of the A. mellifera honey samples affected the fungus T. rubrum. The honeys from Nannotrigona testaceicornis, Plebeia remote, Tetragona clavipes and Scaptotrigona depilis all had a high degree of antimicrobial activity, they were significantly more efficient in terms of antimicrobial activity than manuka honey, especially against Escherichia coli, Klebsiella pneumoniae and T. rubrum. Artificial honey, made with the main sugars found in honey and with similar water content, had no antimicrobial activity in our analyses. We also made mass spectrometry analyses of the honey samples and found some fingerprint characteristics that were associated with antimicrobial activity.

With the increase of antibiotic resistant bacteria strains, the need to determine the mechanisms of antimicrobial resistance is similarly rising. <i>Serratia marcescens</i>, a ubiquitous, Gram-negative opportunistic pathogen is known to have strong, natural resistance to diverse antimicrobial agents including antibiotics and antimicrobial peptides. Recently, we identified <i>S. marcescens</i> as one of the few bacteria resistant to antimicrobial compounds produced by <i>Stemphylium vesicarium</i>, an isolated fungal spinach endophyte. To identify the mechanism of antimicrobial resistance to the unknown <i>Stemphylium</i> antimicrobial compounds, we designed a transposon mutant screen identifying mutants sensitive to antimicrobial inhibition of bacterial growth. A transposon mutant library was constructed using the Tn5 EZ-Transposome (Epicentre) system and contains 1,824 individual mutants with 127 being identified as having a decreased resistance to the <i>Stemphylium</i> antimicrobial compounds. The transposon growth inhibition screen initially evaluates the mutants for reduced growth in the presence of 25% fungal metabolite over 24 hours. The growth phenotype is then confirmed in triplicate in a 12-hour time course growth experiment. Identification of the genomic insertion site of the Tn5 transposon utilized a multi-step modified nested-PCR protocol, termed TAIL-PCR. Following PCR purification, nanodrop spectroscopy and gel electrophoresis were performed to ensure the amplification purity of the extracted DNA and was subsequently sequenced via WideSeq analysis. BLAST identified insertions in genes necessary for membrane biogenesis, drug transport, pili formation, and iron metabolism. Future work is aimed at confirming these results and understanding the role of iron sequestration. Not only will this research contribute to our understanding of <i>S. marcescens</i> antimicrobial resistance mechanisms, but it aids in our understanding of the mechanisms of antimicrobial resistance development in other human pathogens.

La multiplication des résistances aux antibiotiques constitue une grave menace qui nécessite de nouvelles stratégies antimicrobiennes. Le but de ce travail est d'étudier le potentiel des nanocapsules lipidiques (NCLs) pour l'administration de peptides antimicrobiens (PAMs). Les premiers travaux ont porté sur le développement et l'optimisation de NCLs chargées en PAMs. Différentes stratégies d’association ont été testé (adsorption à la surface ou encapsulation dans le coeur de NCLs). Les résultats ont démontré une efficacité d'association comprise entre 20 et 40%, lorsque les peptides sont adsorbés à la surface et plus de 80%,lorsqu’ils sont encapsulés. La deuxième partie s’est concentrée sur l'évaluation de l’activité des complexes PAMs et NCLs ainsi que leur stabilité vis-à-vis des protéases. Les résultats ont montré que l'adsorption entraîne une potentialisation de l'activité antimicrobienne des PAMs, associée à une protection partielle contre la dégradation enzymatique. Inversement, l’encapsulation des PAMs montre une meilleure stabilité aux enzymes, corrélée à une efficacité d'encapsulation supérieure sans amélioration de l'activité antimicrobienne in vitro. Dans une troisième partie, les mécanismes impliqués dans les interactions LNC/PAM ainsi que l'interaction du complexe avec un modèle de membrane bactérienne ont été étudiés. Il a été montré que la structure et la flexibilité des PAMs aux interfaces solide-liquide gouverneraient l'adsorption des peptides à la surface des NCLs, entrainant un changement de leur comportement avec les membranes bactériennes. L’ensemble de ces résultats démontre le potentiel prometteur des NCLs en tant que vecteur de PAMs. / The rapid increase in drug-resistant infections presents an acute problem in the healthcare sector, generating interest in novel antimicrobial strategies. The aim of this work is to explore the potential of lipid nanocapsules (LNCs) for Antimicrobial peptides (AMPs) delivery. Firstly, the experiments were focused on the development and optimization of AMP-loaded LNCs. Different strategies were investigated to deliver AA230,LL37 and DPK060 using LNCs (peptides adsorption atthe surface or encapsulated in the core of modified LNCs). The results demonstrated an association efficiency of 20 to 40%, when peptide is adsorbed, and over 80% encapsulation efficiency, when peptides are encapsulated. The second part concerned the study ofthe influence of peptides loading on their activity and stability against proteases. The results showed that peptides adsorption induced a potentiation of the antimicrobial activity of the native peptides, with a partial protection against proteolytic degradation. Conversely, peptides encapsulation allowed better peptide stability, correlated with higher encapsulation efficiencies and a preservation of the in vitro antimicrobial activity. In a third part, the mechanisms involved in LNC/AMP interactions and the complex interaction with model bacterial membrane have been evaluated. It has shown that structure and flexibility at solid-liquid interfaces govern peptide adsorption on the surface of the LNCs, which in turn is expected to change LNCs properties and interaction with bacterial membranes. Taken together, these results demonstrate the potential of LNCto deliver AMPs as an alternative anti-infective therapy.