A multirresistência aos antibióticos observada em bacilos gram-negativos é um grave problema de saúde pública devido a alta morbidade e mortalidade apresentada, especialmente em instituições assistenciais de saúde. Como consequência do intenso uso de antibióticos, a multirresistência a esses fármacos é principalmente mediada por enzimas hidrolisantes, onde destaca-se as enzimas ?-lactamases, principal mecanismo de resistência aos ?-lactâmicos verificado em bacilos gram-negativos. Os esgotos de origem hospitalar e de estações de tratamento de esgoto (ETE) são considerados como reservatórios de bactérias multirresistentes pela presença de antibióticos que as selecionam e por favorecem a transmissão de determinantes de resistência. Nesse sentido, o presente estudo objetivou avaliar a multirresistência a antibióticos e a produção de enzimas ?-lactamases em bacilos gram-negativos isolados de efluente hospitalar e da estação de tratamento de esgoto, na cidade de Ribeirão Preto, SP. No hospital terciário, amostras de esgotos foram coletadas dos ambulatórios, das enfermarias e da junção do esgoto hospitalar. Na ETE, amostras foram coletadas na caixa de entrada do esgoto bruto e após ao tratamento. Dez microlitros foram semeados em ágar MacConkey, SalmonellaShigella, Cetrimide e TCBS e a identificação dos bacilos gram-negativos foi realizada pelo kit Bactray®. O teste de susceptibilidade aos antibióticos foi realizado pelo método de discodifusão em ágar. A detecção fenotípica de bacilos produtores de ESBL foi realizada pelos testes de sinergia de disco-duplo e disco combinado com ácido clavulânico, e para detecção de isolados produtores de carbapenemases foi utilizado os testes de disco combinado com ácido fenilborônico e EDTA e o teste Blue Carba. A PCR foi utilizada para amplificação dos genes codificadores de ESBL e carbapenemases. No total, 45 bacilos gram-negativos foram isolados, sendo as espécies Klebsiella pneumoniae e Pseudomonas aeruginosa as de maiores prevalências. Ampla resistência foi verificada aos antibióticos ?-lactâmicos, sendo a resistência ao aztreonam, a cefepime e a cefotaxima mais expressiva nos isolados do esgoto hospitalar, com diferenças estatisticamente significante (p<0,05). O fenótipo multidroga resistente foi atribuído a 33,3%, nos isolados exclusivamente do esgoto hospitalar, com diferença estatisticamente significante (p = 0,0025) em relação aos isolados do esgoto da ETE. Genes de ?-lactamases foram encontrados em 35,6% das bactérias, sendo o blaKPC e blaTEM os de maiores ocorrências, ambos em 17,8% dos isolados, e os genes blaSHV e blaCTX-M em 13,3% e 8,9%. Somente em um isolado de Enterobacter cloacae no esgoto tratado da ETE foi identificado o gene blaSHV, os demais isolados portadores dos genes de ?-lactamases foram encontrados no esgoto hospitalar. Os dados obtidos neste estudo são importantes levando em consideração que no Brasil o esgoto hospitalar pode ser lançado in natura na rede coletora municipal, no entanto, acredita-se que tal permissão favorece a disseminação da multirresistência bacteriana, posto que, os resultados demonstram alta frequência de bactérias portadoras de genes de resistência a antibióticos no esgoto hospitalar estudado. Assim, a implementação do tratamento de efluentes hospitalares, especialmente os de hospitais terciários, e adicionalmente ao tratamento da ETE evitaria a propagação dessas bactérias no ambiente e de impactar negativamente os recursos hídricos / Antibiotic multi-resistance observed in Gram-negative bacilli is a serious public health problem due to high morbidity and mortality, especially in health care institutions. As a consequence of the intense use of antibiotics, multi-resistance to these drugs is mainly mediated by hydrolyzing enzymes, in which ?-lactamases, the main ?-lactam resistance mechanism observed in Gramnegative bacilli, are prominent. Hospital sewage and wastewater treatment plants (WWTP) are considered reservoirs of multiresistant bacteria by the presence of antibiotics that select these bacteria and favor the transmission of resistance determinants. In this sense, the present study aimed to evaluate the antibiotics multi-resistance and the production of ?-lactamase enzymes in Gram-negative bacilli isolated from hospital effluent and the wastewater treatment plants in Ribeirão Preto city, SP. In the tertiary hospital, sewage samples from the outpatient clinics, rooms patients and the hospital sewage junction were collected. In the WWTP, raw and treated sewage were collected. Ten microliters were seeded on MacConkey, Salmonella-Shigella, Cetrimide and TCBS agar and the identification of Gram-negative bacilli was performed by the Bactray® kit. Antibiotic susceptibility test was performed by agar-diffusion method. Phenotypic detection of ESBL-producing bacilli was performed by double-disc and discsynergy tests combined with clavulanic acid, and for the detection of carbapenemase-producing isolates the combined disk tests with phenylboronic acid and EDTA and Blue Carba test were used. PCR amplification of ESBL and carbapenemases-encoding genes was used. In total, 45 Gram-negative bacilli were isolated, and Klebsiella pneumoniae and Pseudomonas aeruginosa being the most prevalent. Extensive resistance was verified to ?-lactam antibiotics and resistance to aztreonam, cefepime and cefotaxime was more pronounced in hospital sewage isolates, with statistically significant differences (p<0.05). Multidrug-resistant phenotype was attributed to 33.3% in isolates exclusively from hospital sewage, with a statistically significant difference (p = 0.0025) in relation to the sewage isolates from the WWTP. ?-lactamase genes were found in 35.6% of the bacteria, with blaKPC and blaTEM having the highest occurrences, both in 17.8% of the isolates, and the blaSHV and blaCTX-M genes in 13.3% and 8, 9%. Only in an isolate of Enterobacter cloacae in the treated sewage from WWTP was the blaSHV gene identified, the other isolates carrying the ?-lactamases genes were found in hospital sewage. The data obtained in this study are important considering that in Brazil the hospital sewage can be released in nature in municipal collection network, however, it is believed that such permission favors the dissemination of bacterial multi-resistance, since, the results show high frequency of bacteria carrying antibiotic resistance genes in the hospital sewer studied. Thus, the implementation of treatment of hospital effluents, especially those in tertiary hospitals, and in addition to the treatment of WWTP would prevent the spread of these bacteria in the environment and negatively impact water resources

La résistance aux antibiotiques demeure un problème majeur de santé publique particulièrement dans les pays en voie de développement (PVD) où les conditions d’hygiène sont encore précaires et où l’utilisation des antibiotiques est souvent abusive et très peu contrôlée. Cette thèse a pour objectif principal d’étudier la prévalence, la circulation et la caractérisation génétique des bactéries multirésistantes (BMR) dans un PVD, le Burkina Faso. Ce travail permet de mieux comprendre la propagation de ces BMR dans ce pays et de donner des informations indispensables pour les services de santé publique. Dans cette région, c’est la première étude du genre et cette thèse représente donc un travail original et informatif. A partir d’une revue de la littérature réalisée sur la problématique de l’émergence des BMR en Afrique de l’ouest, les objectifs spécifiques sont : i) d’étudier la prévalence et la caractérisation phénotypique et génotypique des bêta-lactamases à spectre élargi (BLSE) chez les entérobactéries isolées en portage et dans les processus infectieux ; ii) d’évaluer la sensibilité des entérobactéries aux carbapénèmes ; iii) de documenter la prévalence du portage nasal de Staphylococcus aureus (SAUR) résistant à la méticilline (SARM) ainsi que d’effectuer la caractérisation moléculaire les isolats de SAUR. Les isolats ont été prélevés par les services de santé publique au sein des 3 CHU du Burkina et ont été remis en culture au laboratoire de bactériologie du CHU de Montpellier [un total de 594 souches, 522 entérobactéries (214 en portage et 308 dans les processus infectieux) et 72 SAUR]. L’identification des espèces bactériennes repose sur la spectrométrie de masse MALDI-TOF. L’évaluation de la sensibilité aux antibiotiques s’appuie sur la méthode de diffusion en milieu gélosé. Les BLSE et les carbapénémases chez les entérobactéries sont identifiées par méthodes moléculaires (PCR et séquençage) et la caractérisation génétique des isolats de SAUR par une technique de puce à ADN. Au Burkina Faso, les points importants sont : des taux très élevés en portage et dans les infections d’entérobactéries sécrétrices de BLSE en hospitalier et en communautaire; les antécédents de consommation d’antibiotiques ou d’hospitalisation sont des facteurs de risque de portage ; par les isolats BLSE, Escherichia coli est l’espèce prédominante ; la majorité des BLSE sont des CTX-M-15, suivi de CTX-M-14, 27 et SHV-12 ; l’étude des phylogroupes d’E. coli suggère une expansion des gènes CTX-M-15 d’origine plasmidique et non liée à une épidémie de clone ; des isolats d’E. coli producteurs de carbapénémase OXA-181 avec un support génétique plasmidique de type IncX3 sont identifiés. On observe un taux de portage nasal de SAUR classique avec une très faible prévalence de SARM ; on note dans une forte proportion des gènes de virulence (Leucocidine de Panton Valentin et du facteur EDIN) parmi les SAUR en portage constituant des facteurs de risques de gravité lors des infections ; l’analyse génétique des isolats de SAUR montre une grande diversité de clones. Les études de prévalence et de génétiques apportent des informations fondamentales pour l’épidémiologie des BMR, base indispensable pour appréhender la lutte et le contrôle de ces organismes pathogènes résistants. En conclusion, ces premières études réalisées au Burkina permettent de définir les perspectives de recherche et les stratégies à développer pour le contrôle l’émergence et la diffusion de ces BMR. / Antibiotic resistance is a major public health issue, particularly in developing countries where health conditions are still inadequate and antibiotic use is often unjustified and not properly regulated. The main objective of this thesis was to study the prevalence, spread and genetic features of multi-resistant bacteria (MRB) in Burkina Faso, a developing country. This work allows better understanding MRB spread in this country and gives essential information for the development of public health policies. This is the first study in this region and this thesis is therefore an original and informative work. Starting from a review of the literature data on the issue of MRB emergence in West Africa, the specific objectives are: i) to study the prevalence and to characterize phenotypically and genotypically the extended-spectrum beta lactamases (ESBL) in enterobacterial samples isolated from healthy carriers and during the clinical phase of infection; ii) to assess the sensitivity of such enterobacterial samples to carbapenem drugs; iii) to document the prevalence of nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) and to molecularly characterize all S. aureus isolates. Samples were collected by the public health services of three Burkina Faso University Hospital Centers (UHC) and were then cultured at the Laboratory of Bacteriology of Montpellier UHC [total n= 594 isolates of which 522 were Enterobacteriaceae isolates (214 from healthy carriers and 308 from patients with clinical infection) and 72 S. aureus isolates]. Bacterial species were identified by MALDI-TOF mass spectrometry. Antibiotic sensitivity was tested using the agar diffusion method. ESBL and carbapenemases in enterobacterial isolates were identified using molecular methods (PCR amplification and sequencing). S. aureus isolates were genetically characterized by using DNA arrays. This work shows that in Burkina Faso, ESBL-producing enterobacterial strains are frequent both in healthy carriers and hospitalized patients and that previous antibiotic use or hospitalizations are risk factors for colonization. Among the ESBL-producing enterobacterial strains, Escherichia coli was the predominant species and most ESBL-producing isolates were CTX-M-15, followed by CTX-M-14, 27 and SHV-12. The analysis of the E. coli phylogenetic groups suggests a plasmid-mediated spread of CTX-M-15 genes and not linked to an epidemic clone. Moreover, E. coli strains that produce the carbapenemase OXA-181 and with an IncX3-type plasmid have been identified. Concerning S. aureus, the rate of nasal carriage was classical with a low prevalence of MRSA. Among the S. aureus nasal samples, a high proportion of virulence genes (Panton Valentin leukocidin and EDIN) was detected. This constitutes an important risk of severe disease during clinical infection. The genetic analysis of the S. aureus isolates showed great clone diversity. These data are essential for understanding MRB epidemiology in Burkina Faso and represent a base for fighting and controlling resistant pathogens. In conclusion, these first studies carried out in Burkina Faso allow defining the future research perspectives and strategies to be developed for controlling the emergence and spread of these MRB.

Bakgrund: Multiresistenta bakterier är ett globalt problem och ett hot mot folkhälsan. Smittförebyggande åtgärder som basala hygienrutiner är en central del i det preventiva arbetet mot multiresistenta bakterier. Patienter som är bärare av en multiresistent bakterie kan uppleva känslor av otrygghet och skam, därför är det av stor vikt att all hälso- och sjukvårdspersonal tillämpar en personcentrerad vård. Syfte: Att undersöka hälso- och sjukvårdspersonalens upplevelser av att vårda patienter med multiresistenta bakterier. Metod: Studien har genomförts som en litteraturöversikt baserad på 15 stycken artiklar. Artiklarna var av både kvalitativ, kvantitativ samt kombinerad kvalitativ och kvantitativ metod. Databaser som använts i sökningen är PubMed och CINAHL. Analysen resulterade i tre teman. Resultat: Resultatet presenteras under tre olika teman: Upplevelser av rädsla och osäkerhet, upplevd okunskap och behov av utbildning samt upplevelser av hinder för utförandet av infektions-preventiva åtgärder. Slutsats: Rädsla och osäkerhet hos vårdpersonalen framkom tydligt och ledde till brister i utförandet av omvårdnaden. Resultatet visade även en upplevd okunskap om multiresistenta bakterier och ett behov av utbildning efterfrågades. Kunskap genom utbildning skulle resultera i en större förståelse om multiresistenta bakterier och minska rädslan hos vårdpersonalen. / Background: Multiresistant bacteria is a global problem and a threat to public health. Infection prevention such as basic hygiene routines are a key part of preventive work against multiresistant bacteria. Patients who is a carrier of a multiresistant bacteria can experience feelings of insecurity and shame, therefore it is important that all healthcare professionals apply a person-centered care. Aim: To study the healthcare professionals' experience of caring for patients with multiresistant bacteria. Method: The study has been conducted as a literature review based on 15 articles. The articles were of both qualitative and quantitative method as well as mixed method with combined qualitative and quantitative method. Databases used in the search are PubMed and CINAHL. The analysis resulted in three themes. Result: The results are presented in three different themes: Experiences of fear and insecurity, experienced lack of knowledge and need for education and experiences of obstacles to the performance of infection prevention measures. Conclusion: Fear and insecurity in healthcare staff clearly emerged and led to shortcomings in the performance of nursing care. The result also showed an experienced ignorance of multiresistant bacteria and a need for education was requested. Knowledge through education would result in a greater understanding of multiresistant bacteria and reduce the fear among healthcare professionals ’.

Les bactéries multirésistantes (BMR) représentent un véritable problème de santé publique. Les antibiotiques conventionnels commencent à devenir inefficaces et des bactéries qu’à une époque étaient contrôlées, commencent à devenir de plus en plus pathogènes. L’utilisation des actifs dérivant des plantes comme certains composants issus des huiles essentielles ont déjà démontré une action bactérienne contre plusieurs bactéries dites résistantes. Pour l’administration de ces actifs, à nature lipophile, nous les avons encapsulés à l’intérieur des nanocapsules au cœur lipidique (NCL) grâce à une modification de la méthode d’inversion de phase développé dans le laboratoire Inserm U1066 au début des années 2000. Le but de cette thèse est de trouver une association avec des antibiotiques qui puisse s’avérer synergique contre des BMR. Nous avons obtenu une synergie sur plusieurs souches bactériennes avec un mélange avec trois actifs différents : l’eugénol, le carvacrol et le cinnamaldéhyde et la doxycycline (antibiotique bactériostatique de la famille des tetracyclines). Cette synergie a été également testée in vivo sur un modèle pneumopatique de souris avec des résultats qui semblent en concordance avec les résultats in vitro. Nous nous sommes servis des méthodes physico-chimiques comme l’angle de contact ou la mobilité électrophorétique, dans le but d’étudier les interactions entre les NCL et les bactéries. Les résultats obtenus semblent indiquer que les NCL recouvrent la surface de la bactérie et libèrent leurs actifs, néanmoins nous n’écartons pas l’hypothèse qu’une certaine quantité des NCL puisse aussi pénétrer la bactérie. / Multiresistant bacteria (MRB) present today a public health problem. Some antibiotics are ineffective and bacteria formerly controlled, begin to become more and more pathogenic. The use of drugs derived from plants as some components from essential oils have demonstrated a bactericidal effect against resistant bacteria.For the administration of these lipophilic drugs, they have been encapsulated in the core of lipidnanocapsules (LNC), formulated via the phase inversion method. This method was developed in the laboratory Inserm U1066 in the early 2000s.The aim of this thesis is to find a combination with an antibiotic that prove to be synergistic against MRB. We have found a synergistic effect with the combination of doxycycline (bacteriostatic antibiotic of the tetracycline class) and the LNC of a mixture of three aromatic components of essential oils (eugenol,carvacrol and cinnamaldehyde). This synergy was also tested in vivo in a pneumopathicmice model with interesting results which appear consistent with in vitro results.The interactions between LNC and bacteria were studied using physicochemical method as electrophoretic mobility and contact angle measurement. Obtained results suggest that LNC cover the surface of the bacteria and release the aromatic components. These results are also observed by confocal microscopy. Nevertheless, we also accept the hypothesis that, a certain amount of LNC can cross the bacteria.