Thesis (M.Sc. (Microbiology))--Mahidol University, 2006. / LICL has E-Thesis 0014 ; please contact computer services.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-06-22T13:49:01Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE DOUTORADO MARIA CECILIA SANTOS C MELO.pdf: 1417870 bytes, checksum: b618daedc0a0d5458d1a1e355c421b86 (MD5) / Made available in DSpace on 2016-06-22T13:49:01Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE DOUTORADO MARIA CECILIA SANTOS C MELO.pdf: 1417870 bytes, checksum: b618daedc0a0d5458d1a1e355c421b86 (MD5) Previous issue date: 2015-12-23 / CAPEs / Objetivo: Investigar as alterações dos encéfalos e olhos de conceptos recém-nascidos de ratas que se submeteram a peritonite fecal autógena e avaliar as respostas com a intervenção de dois esquemas terapêuticos com moxifloxacino-dexametasona e meropenem realizados durante a prenhez. Métodos: Foram incluídas, de forma aleatória, 30 ratas Wistar para acasalamento. Dessas, 15 ratas tiveram esfregaço vaginal positivo para prenhez. O experimento foi realizado em duas fases: 1. Desenvolvimento do modelo experimental: As ratas foram distribuídas em : AGrupo estudo com cinco ratas após peritonite fecal autógena com suspensão de fezes a 10 %, na dose de três ml/Kg no nono dia de prenhez, B- Grupo estudo com cinco ratas após peritonite fecal autógena com suspensão de fezes a 10 %, na dose de quatro ml/Kg no nono dia de prenhez e C- grupo de cinco ratas prenhes sem peritonite fecal autógena (controle). Após o parto, foi realizada a eutanásia e o inventário das cavidades abdominal e torácica das ratas dos grupos estudo A e B e os dados de seus conceptos. 2.Intervenção com esquemas terapêuticos: Grupo1: Duas ratas prenhes após peritonite fecal autógena com suspensão de fezes a 10 %, na dose de quatro ml/Kg no nono dia de prenhez que receberam moxifloxacino - dexametasona intraperitoneal em 48 e 72 horas e Grupo 2: Duas ratas prenhes após peritonite fecal autógena com suspensão de fezes a 10 %, na dose de quatro ml/Kg no nono dia de prenhez que receberam meropenem intravenoso em 48 e 72 horas. Após o parto, foi realizada a eutanásia e o inventário das cavidades abdominal e torácica destas e a decapitação com inspeção do crânio, da consistência dos cérebros e os olhos de todos os conceptos. O projeto foi aprovado pelo Comitê de Ética da Faculdade de Ciências Médicas de Campina Grande- Paraíba. P ≤ 0.05 foi usado para rejeição da hipótese de nulidade. Resultados: O modelo de peritonite fecal autógena selecionado foi o que utilizou a dose de quatro ml/Kg da suspensão de fezes a 10%. Das 15 ratas com esfregaço vaginal positivo dez estavam prenhas, sendo três no grupo A, quatro no grupo B e três no grupo C. Os cérebros dos conceptos das ratas que receberam quatro ml/kg da suspensão de fezes a 10% se mostraram, significantemente, menores e com consistência menos firme que aqueles do grupo controle, assim como, comparados com os das intervenções terapêuticas. Cataratas congênitas foram observadas em nove de 26 (34,6%) conceptos das ratas que receberam quatro ml/Kg da suspensão de fezes a 10% e não receberam intervenção terapêutica, sendo sete bilateral e dois unilateral. Nenhuma catarata congênita foi observada nos 20 recém-nascidos das ratas que receberam a combinação de moxifloxacino - dexametasona intraperitoneal. Catarata congênita foi observada em três (13.6%) dos 22 recém-nascidos das ratas que receberam meropenem intravenoso. Conclusões: Septicemia gestacional em ratas pode produzir alteração cerebral e catarata congênita nos conceptos. Com a utilização dos esquemas terapêuicos, nas ratas prenhas submetidas à peritonite fecal autógena com a dose de quatro ml/Kg da suspensão de fezes a 10%, houve menor número de casos de conceptos com alterações encefálicas e oculares. / Purpose: To investigate the abnormalities of brains and eyes of newborn fetuses of rats that underwent autologous fecal peritonitis and evaluate the responses with the intervention of two treatment regimens with moxifloxacin and meropenem - dexamethasone performed during pregnancy. Methods: Randomly, 30 Wistar rats for mating. Of these, 15 rats had a positive vaginal smear for pregnancy. The experiment was conducted in two phases: 1. Development the experimental model: The rats were divided into: Group A- study with five rats after fecal peritonitis autogenous with faeces of a 10% suspension at a dose of three ml/kg on the ninth day of pregnancy, group B study with five rats after fecal peritonitis autogenous with faeces of a 10% suspension at a dose four ml/kg on the ninth day of pregnancy and C-group of five pregnant rats without autologous fecal peritonitis (control). After delivery, euthanasia and inventory of abdominal and thoracic cavities of study groups A and B and the data of their fetuses. 2.Intervention with therapeutic regimens : Group 1 : Two pregnant rats after fecal peritonitis autogenous suspension of faeces to 10 % at a dose four ml / kg on the ninth day of pregnancy who received moxifloxacin - intraperitoneal dexamethasone at 48 and 72 hours and Group 2 : Two pregnant rats, autogenous after peritonitis with stool suspension at 10 % in doses four ml / kg on the ninth day of pregnancy who received intravenous meropenem in 48 and 72 hours. After delivery, euthanasia and inventory of abdominal and chest cavities of these and the beheading with skull inspection was carried, the consistency of the brain and the eyes of all fetuses . The project was approved by the Ethics Committee of the Faculty of Medical Sciences of Campina Grande - Paraíba. P ≤ 0.05 was used to reject the null hypothesis. Results: The fecal peritonitis autogenous model selected was that using 10% faeces suspension at a dose four ml/kg. Of the 15 rats with positive vaginal smear ten were pregnant, three in group A, four in group B and three in group C. The brains of fetuse of rats given four ml/kg of 10% faeces suspension is shown, significantly, smaller and less firm consistency than those of the control group, as compared with the therapeutic interventions. Congenital cataract was observed in nine of 26 (34.6%) fetuses of rats given four ml/kg of 10% faeces suspension received no therapeutic intervention, seven bilateral and two unilateral. No congenital cataract was observed in 20 newborns of rats given the combination of intraperitoneal moxifloxacin - dexamethasone. Congenital cataract was observed in three (13.6%) of 22 infants of rats given intravenous meropenem. Conclusions: Gestational septicemia in rats can produce brain abnormalities and congenital cataracts in fetuses. With the use of treatment regimens, in pregnant rats submitted autologous fecal peritonitis with a dose of four ml/kg of 10% faeces suspension, there were few cases of fetuses with brain and eye abnormalities.

No description available.

Yes / The use of methylmalonyl-CoA epimerase (MCEE) to improve stereoselectivity in crotonase-mediated biocatalysis is exemplified by the coupling of MCEE, crotonyl-CoA carboxylase reductase and carboxymethylproline synthase in a three-enzyme one-pot sequential synthesis of functionalised C-5 carboxyalkylprolines starting from crotonyl-CoA and carbon dioxide. / Biotechnology and Biological Sciences Research Council, The Wellcome Trust, and CONACyT and FIDERH (Mexico, RGC) The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from AbbVie, Boehringer Ingelheim, the Canada Foundation for Innovation, the Canadian Institutes for Health Research, Genome Canada, GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, Takeda, and the Wellcome Trust (092809/Z/10/Z).

Background: Gram-negative resistance is a growing problem worldwide. It is generally believed that rates of resistant bacteria within a hospital are a function of antibiotic use, resistant organisms brought into the hospital, infection control efforts, and underlying severity of patient illness. The relative contribution of each to a particular resistance phenotype is unclear. P. aeruginosa is responsible for many hospital acquired infections and it may become resistant to carbapenems. In addition, newer threats to the future utility of the carbapenems are carbapenemase-producing K. pneumoniae Purpose: To determine if there is an association between the volume and composition of antibiotic use, geography, severity of illness and rates of carbapenem-resistant P. aeruginosa and K. pneumoniae. Methods: This is a retrospective ecological longitudinal investigation within the University HealthSystem Consortium affiliated academic medical centers. Antibiotic use data between January 1, 2006 and December 31, 2009 were obtained from billing records and reported as days of therapy per 1000 patient days (DOT/1000 PD), in addition to hospital characteristics (e.g. geographical location, bed size, case mix index). “Whole house” antibiograms were obtained to determine rates and proportions of carbapenem-resistant P. aeruginosa (CR-PA) and carbapenem resistant K. pneumoniae (CR-KP). Also, CR-KP isolation was generated as a binary outcome. Generalized estimating equations (GEE) were used to model CR-KP and CR-PA. Results: CR-KP rates (1000PDs) increased from 0.07 in 2006 to 0.15 in 2009 (P= 0.0118) and CR-KP proportions increased from 1.3% in 2006 to 3.1% in 2009 (0.0003) within 40 hospitals over 2006-2009. However, CR-PA rates and proportions were stable over the same period. Geographical location, carbapenems use, and antipseudomonal penicillins use were significantly associated with CR-KP isolation. Thus, for every ten DOT/1000 PDs increase in carbapenem use, the odds of CR-KP isolation increased by 42% (P=0.0149). In contrast, for every ten DOT/1000 PDs increase in antipseudomonal penicillin use, the odds of CR-KP isolation decreased by 14%. However, there was no significant model to explain CR-PA rates and proportions. Conclusion: Carbapenems, antipseudomonal penicillins, and geographical location were identified as risk factors associated with CR-KP isolation. These findings emphasize the challenges associated with the treatment of multidrug- gram-negative bacteria.

Introdução: O Acinetobacter spp. é um cocobacilo gram-negativo de grande importância nas infecções hospitalares, especialmente em pacientes internados em unidades de terapia intensiva (UTI); podendo levar a um aumento na morbidade e mortalidade desses pacientes. Há evidências sustentando associação entre infecção por Acinetobacter baumannii e aumento das taxas de mortalidade bruta e atribuível. Contudo, o papel desse agente como causa direta de mortalidade ainda não está suficientemente caracterizado. Dentre os fatores relacionados com o aumento da mortalidade estão: gravidade do paciente, infecção relacionada à A. baumannii multirresistente, tratamento com antimicrobiano inadequado, tempo de hospitalização com alta permanência, choque séptico e imunossupressão. Objetivos: Estimar a mortalidade bruta e atribuível às infecções hospitalares causadas pelo Acinetobacter baumannii resistente a antimicrobianos carbapenêmicos (CRAB) por meio de revisão sistemática e metanálise de estudos observacionais. Métodos: Foi desenvolvida uma revisão sistemática e metanálise de estudos observacionais publicados nas bases de dados: MEDLINE/Pubmed, CENTRAL/Cochrane Library, EMBASE/Elsevier, SCOPUS/Elsevier, Web of Science/Thomson Reuters e LILACS/BVS, para estimar a mortalidade bruta e atribuível à infecção hospitalar causada pela bactéria A. baumannii resistente a antimicrobianos carbapenêmicos em pacientes adultos e pediátricos internados em unidades de tratamento intensivo e nãointensivo. Os estudos incluídos caracterizaram fatores preditores de mortalidade associada à infecção por CRAB, comparando com pacientes infectados por A. baumannii sensível a carbapenêmicos (CSAB). Primeiramente, foi estimado um modelo de efeitos aleatórios para a medida agregada de mortalidade atribuível não ajustado a fim de avaliar a contribuição direta das infecções na morte. Na sequência, foram avaliados descritivamente os principais aspectos metodológicos necessários aos estudos observacionais, para a análise dos fatores de risco relacionados a mortalidade atribuível em pacientes infectados por CRAB, por meio de instrumento elaborado conforme recomendações internacionais - ORION, TREND, STROBE e CONSORT. Resultados: Com base nos 29 estudos incluídos na metanálise, observou-se um risco atribuível aumentado na mortalidade bruta em pacientes com infecção por CRAB comparativamente aos pacientes com infecção por CSAB (RA = 0,19 (IC95% = 0,14-0,24) com elevada heterogeneidade (I2 = 66,4%, p-valor < 0,001). Como fontes de heterogeneidade investigou-se o tempo de internação, sítio de infecção, gravidade da doença e uso de terapia inapropriada. Entre os estudos que avaliaram exclusivamente pacientes com bacteremia, o risco de mortalidade atribuível foi maior (RA = 0,27; IC95% = 0,19-0,34). Utilizando-se metarregressão foi observada relação linear positiva entre o risco atribuível de mortalidade e a diferença da média padronizada do escore de APACHE II. Para a investigação da presença de risco de viés e confundimento avaliou-se descritivamente os principais aspectos metodológicos necessários aos estudos observacionais que identificam os fatores de risco associados com a mortalidade atribuível em pacientes com infecções por CRAB. Observou-se nesta revisão que os estudos estão sujeitos a confundimento, incluindo a forma inadequada do ajuste para fatores de confusão de variáveis importantes (ex.: seleção de grupo controle, exposição prévia aos antimicrobianos, mensuração do tempo em risco e a gravidade), além da grande heterogeneidade entre os estudos, devido aos desenhos, unidades de análise e abordagens na medida de exposição e desfecho, tornando difícil a comparação e a sumarização das informações. Conclusões: Os dados dessa revisão sistemática fornecem evidências que a mortalidade atribuível relacionada à presença de infecção por CRAB é maior que em pacientes com infecção por CSAB. Contudo, a investigação da mortalidade atribuível apresenta muitas limitações e ainda não é conclusiva em razão da adequação do desenho do estudo aos seus objetivos; definições de medidas de exposição e desfecho; métricas utilizadas na aferição dos resultados; seleção de grupo controle e fatores de confusão. A consciência de todos esses elementos para a interpretação epidemiológica é vital na análise da mortalidade bruta e atribuível. / Introduction: Acinetobacter spp. is a gram-negative coccobacillus of great importance in hospital infections, especially in patients in intensive care units (ICUs); may lead to an increase in morbidity and mortality of these patients. There is evidence supporting association between infection by Acinetobacter baumannii and the increase in crude and attributable mortality rates. However, the role of this agent as a direct cause of death is not sufficiently characterized yet. Among the factors related to the increase of mortality are: severity of the patient, infection related to A. baumannii multidrug-resistant, inappropriate antimicrobial treatment, hospital stay with high permanence, septic shock and immunosuppression. Objectives: To estimate the crude and attributable mortality to hospital-acquired infections caused by carbapenem-resistant Acinetobacter baumannii through systematic review and meta-analysis of observational studies. Methods: A systematic review and metaanalysis of observational studies published in the databases has been developed: MEDLINE/PubMed, CENTRAL/Cochrane Library, EMBASE/Elsevier, SCOPUS/Elsevier, Web of Science/Thomson Reuters and LILACS/BVS to estimate the crude and attributable mortality to hospital infection caused by the bacterium carbapenem-resistant A. baumannii (CRAB) in adult and pediatric patients in intensive and non-intensive care units. The studies included characterized predictors of mortality associated to infection with CRAB, compared to patients infected with carbapenem-susceptible A. baumannii (CSAB). First, a random effects model was estimated for the aggregate measure of non-adjusted attributable mortality in order to assess the direct contribution of infections in death. Following were descriptively assessed the main methodological aspects necessary to observational studies for the evaluation of risk factors related to attributable mortality in patients infected with carbapenem-resistant A. baumannii through instrument designed according to international recommendations - ORION, TREND, STROBE and CONSORT. Results: Through the 29 studies included in the meta-analysis, there was an increased attributable risk in the crude mortality in patients with infections by CRAB compared to patients with infections by CSAB (RA = 0.19 (95% CI = 0.14 to 0.24) with high heterogeneity (I2 = 66.4%, p <0.001). As sources of heterogeneity, it was investigated the length of stay, the site of infection, disease severity and use of inappropriate therapy. Among the studies that evaluated only patients with bacteremia, the risk of attributable mortality was higher (RA = 0.27; 95% CI = 0.19 to 0.34). Using meta-regression was observed a positive linear relationship between the attributable mortality risk and the standardized mean difference of APACHE II score. For investigating the presence of bias and confounding risk was evaluated descriptively the main methodological aspects necessary to observational studies evaluating the risk factors associated with attributable mortality in patients with infections caused by carbapenem-resistant A. baumannii. It was observed in this review that these studies are subject to pitfalls, including the inappropriate mode for adjustment for confounding factors of important variables (eg.: control group selection, previous exposure to antimicrobials, measurement of time in risk and gravity); besides the great heterogeneity between studies due the drawings, units of analysis and approaches to the extent of exposure and outcome, making it difficult comparison and summarization of information. Conclusions: The data of this systematic review provide evidence that attributable mortality related to the presence of infection by CRAB is higher than in patients with infection by CSAB. However, the investigation of attributable mortality has many limitations and is not conclusive yet because of the design adequacy of the study to their goals, definitions of exposure and outcome measures, metrics used in measuring results, control group selection and confounding factors. The awareness of all these elements is vital in analyzing the crude and attributable mortality.

A.baumannii é um importante patógeno em infecções nosocomiais principalmente por sua capacidade de se tornar resistente aos antimicrobianos. Surtos de A.baumannii resistente aos carbapenêmicos (ABRC) têm sido descritos em todo mundo. Devido à emergência de resistência aos antimicrobianos e ausência de novas opções de tratamento, as polimixinas reemergiram como opção de terapia contra infecções causadas por A.baumannii. O uso de polimixina é associado a maior mortalidade e menor eficácia comparada a outros antimicrobianos. Alguns estudos in vitro têm avaliado a combinação de polimixina com outros antimicrobianos a fim de aumentar a eficácia dos tratamentos. O objetivo deste estudo foi avaliar o sinergismo entre a polimixina B com outros antimicrobianos em isolados de ABRC, pelo método de Curvas Tempo-Morte bacteriana (Time- Kill Curves). Os isolados foram provenientes de banco de amostras e foram avaliadas as combinações de polimixina B com carbapenêmicos (imipenem e meropenem), tigeciclina, rifampicina, amicacina e ceftazidima. As combinações foram testadas nos tempo 0, 30’, 1,4,12 e 24 h. Sinergismo entre polimixina B foi demonstrado contra todos antimicrobianos para ambos isolados, exceto para ceftazidima e imipenem no isolado 1. Nosso estudo mostrou que tigeciclina, amicacina e rifampicina são agentes mais ativos combinados com polimixina B, sendo assim estes agentes podem apresentar efeito benéfico em combinação com a polimixina no tratamento de ABRC. / A.baumannii is an important pathogen in nosocomial infections primarily for its ability to become resistant to antimicrobials. Outbreaks carbapenem- resistant A.baumannii (CRAB) has been described worldwide. Due to the emergence of antimicrobial resistance and the absence of new treatment options, the polymyxins reemerged as an option therapy against infections caused by A.baumannii. The use of polymyxin is associated with higher mortality and lower effectiveness compared to other antimicrobials. In vitro studies have evaluated the combination of polymyxin with other antimicrobial agents to enhance the effectiveness of the treatments. This study was to evaluate the synergy between polymyxin B with other antimicrobials in isolates from ABRC, by Time-Kill Curves. The isolates were from stool samples and were evaluated combinations of polymyxin B with carbapenems (imipenem and meropenem), tigecycline, rifampin, amikacin and ceftazidime. The combinations were tested at time 0, 30 ', 1,4,12 and 24 h. Synergism between polymyxin B was demonstrated against all antimicrobials for both isolates, except for ceftazidime and imipenem in isolated 2. Our study showed that tigecycline, amikacin and rifampicin more active agents are combined with polymyxin B, and thus these agents may have a beneficial effect in combination with a polymyxin in treating CRAB.

Background: Infections caused by carbapenem-resistant Enterobacteriaceae (CRE) such as Escherichia coli and Klebsiella pneumoniae are among the most urgent threats of the infectious disease realm. The incidence of these infections has been increasing over the years and due to very limited treatment options, mortality is estimated at about 50%. By 2050, mortality from antimicrobial resistant infections is expected to surpass cancer at 10 million deaths annually. Methods: We evaluated 18 contemporary antimicrobials against 122 carbapenem-resistant Enterobacteriaceae using a variety of antimicrobial susceptibility testing methods according to Clinical Laboratory Standards Institute guidelines. Time-kill studies were performed on clinical isolates with variable resistance to meropenem, amikacin, and polymyxin B. Phenotypic expression assays were performed on all isolates and whole genome sequencing was performed on 8 isolates to characterize molecular resistance mechanisms. Pharmacodynamic modeling of meropenem and polymyxin B was also conducted. Results: CRE were primarily K. pneumoniae, and Enterobacter spp. 60% expressed Klebsiella pneumoniae carbapenemase (KPC) only, 16% expressed Verona Integron-encoded Metallo-beta-lactamase (VIM) only, 5% expressed KPC and VIM, and 20% expressed other mechanisms of resistance. Antimicrobial susceptibility testing indicated the most active antimicrobials against CRE were ceftazidime/avibactam, imipenem/relebactam, amikacin, tigecycline, and the polymyxins. Etest® strips did not reliably measure polymyxin B resistance. The automated testing system, BD Phoenix™, consistently reported lower MICs than the gold standard broth microdilution. Time-kill studies showed regrowth at clinically achievable concentrations of meropenem alone (4, 16, and 64 mg/L), polymyxin B alone (0.25 and 1 mg/L), or amikacin alone (8 and 16 mg/L), but combinations of meropenem with either polymyxin B or amikacin were bactericidal and synergistic. Meropenem administered simultaneously or prior to polymyxin B exhibited superior activity to polymyxin B administered first. Conclusions: Novel carbapenemase-inhibitor combinations (ceftazidime/avibactam and imipenem/relebactam) exhibit the best activity against KPC-producing CRE. The polymyxins, amikacin, and tigecycline exhibit the best activity against VIM-producing CRE. Meropenem in combination with polymyxin B is bactericidal and synergistic when the meropenem MIC is ≤32 mg/L, and meropenem should never be administered after polymyxin B. Meropenem and amikacin is bactericidal and synergistic when the amikacin MIC is ≤16 mg/L. Etest® strips should not be used for characterizing polymyxin B or colistin activity. Clinicians should be aware that automated testing systems may produce biased susceptibility results relative to the gold standard method, broth microdilution, which may influence interpretation of in vitro results.

L’anthropisation médicamenteuse des eaux usées favorise l’émergence et la diffusion dans l’environnement de microorganismes résistants aux antibiotiques. Les effluents hospitaliers pourraient être doublement impliqués en véhiculant antibiotiques et bactéries multirésistantes. L’objectif de ce travail est de caractériser les effluents hospitaliers d’un Centre Hospitalo-Universitaire en évaluant simultanément les concentrations d’antibiotiques (fluoroquinolones et imipénème) et la diversité des bactéries résistantes à ces antibiotiques au sein de biofilms constitués in situ. Les concentrations en antibiotiques mesurées par chromatographie en phase liquide - spectrométrie de masse après collecte via un échantillonnage passif pendant 15 jours sont égales à 2,08±0,88μg/L (ciprofloxacine), 101,06±18.47 μg/L (ofloxacine), 6,43±0.56 μg/L (norfloxacine) et indétectable pour l’imipénème. Comparées aux données de consommation à l’hôpital pendant cette même période, les concentrations estimées sont 5,84±1,78μg/L (ciprofloxacine), 11.22±1.09μg/L (ofloxacine), 7.68±3,7μg/L (norfloxacine) et 3,61±0,24ug/L (imipénème). La mesure du risque potentiel écotoxicologique s’est avérée positive pour la ciprofloxacine et la norfloxacine (hazard quotient >1). En parallèle, des bactéries résistantes aux fluoroquinolones (n=115) ou aux carbapénèmes (n=38) ont été isolées de biofilms formés dans les effluents hospitaliers. 60 % des isolats, constitués majoritairement de bacilles à Gram négatif, notamment Aeromonas spp et Klebsiella spp, sont résistants à plusieurs familles d’antibiotiques dont certains sont exclusivement utilisés à l’hôpital. La majorité des souches hébergent des éléments génétiques mobiles dont des plasmides conjugatifs porteurs de la résistance à l’imipénème ou aux fluoroquinolones. La présence combinée de bactéries résistantes aux antibiotiques hébergeant des éléments génétiques mobiles en lien avec ces résistances et de faibles concentrations en antibiotiques permet de qualifier l’interface hôpital-environnement comme un lieu propice au transfert des résistances. / The presence of pharmaceutical compounds in waste water favors the emergence and the spreading of antibiotic resistant microorganisms. The hospital effluents could be involved gathering antibiotics and multiresistant bacteria. The aim of this work is to characterize the hospital effluents of a teaching hospital measuring simultaneously the concentrations of antibiotics (fluoroquinolones and imipenem) and the diversity of the bacteria resistant to these antibiotics within hospital effluent biofilms.The antibiotics concentrations were measured by liquid-phase chromatography - mass spectrometry via a passive sampling during 15 days. The measured environmental concentrations were 2.08 ± 0.88μg/L (ciprofloxacin), 101.06 ± 18.47 μg/L (ofloxacine), 6.43 ± 0.56 μg/L (norfloxacine). Imipenem was not detected. Compared with the data of hospital consumption during the same period, the predicted estimated concentrations are 5.84±1.78µg/L(ciprofloxacin), 11.22 ± 1.09µg/L (ofloxacin), 7.68 ± 3.7µg/L, 7.68 ± 3.7μg/L (norfloxacin) and 3.61 ± 0.24ug/L (imipenem). The ecotoxicological risk was confirmed for the ciprofloxacin and the ofloxacin (hazard quotient > 1).In parallel, fluoroquinolones (n=115) and carbapenem (n=38) resistant bacteria were isolated from hospital effluent biofilm. Sixty % of isolates, mainly composed by Gram negative bacilli in particular Aeromona spp and Klebsiella spp, are resistant to several antibiotics among which some are exclusively used at the hospital. The majority of these strains have mobile genetic elements such as conjugative plasmids harboring imipenem or fluoroquinolones resistances.The presences of both antibiotics resistant bacteria harboring mobile genetic elements in connection with these resistances and low antibiotics concentrations make the hospital effluent a convenient place for the transfer of resistance between the hospital and the environment.

Bacterial resistance to antibiotics is an emerging epidemic throughout the world and there is a desperate need for new antibiotics and new strategies to maintain the effectiveness of current agents. ??-Lactams, such as the penicillins and cephalosporins, have been the most important class of antibiotic for several decades and represent half of the global antibacterial market, but the continued use of ??-lactams is threatened by ??-lactamases, enzymes that efficiently inactivate ??-lactams through hydrolysis. Class A, C, and D ??-lactamases use an active-site serine residue for hydrolysis and achieve turnover through an acylenzyme intermediate while the class B metallo-??-lactamases (MBLs) use a zinc-bound hydroxide as the active-site nucleophile. Two successful approaches to combat ??-lactamase-mediated resistance have involved the development of ??-lactam antibiotics which bind poorly to ??-lactamases and the combination of ??-lactams with ??-lactamase inhibitors. These strategies have been effective for overcoming resistance due to class A ??-lactamases, but the ever-increasing prevalence of extended-spectrum ??-lactamases (ESBLs), metallo-??-lactamases, and carbapenemases compromises the effectiveness of current penicillins, cephalosporins, carbapenems, and mechanism-based ??-lactamase inhibitors. Cyclobutanone analogues of ??-lactam antibiotics were explored in the early 1980s as potential inhibitors of ??-lactamases and D-Ala-D-Ala transpeptidases, but simple analogues showed only weak inhibitory activity and this approach was subsequently abandoned. The increasing threat of multidrug-resistant ??-lactamase-producing organisms in recent years, however, has inspired a re-evaluation of these inhibitors since cyclobutanones have the potential to exhibit broad-spectrum inhibition of both serine- and metallo-??-lactamases through the formation of enzyme-bound hemiketals or hydrates. 7,7-Dichloro-2-thia-bicyclo[3.2.0]heptan-6-one-4-carboxylic acid (65), a dichlorocyclobutanone that had shown modest inhibition of the class B and D ??-lactamases IMP-1 and OXA-10 in earlier work in this laboratory, was prepared in an efficient seven-step sequence from triethyl phosphonoacetate (103) with an overall yield of 28%. Initial efforts to improve upon the potency of the cyclobutanones involved functionalization at C3 and a highly stereoselective chlorination with sulfuryl chloride provided the 3??-chloro derivative 117?? in nearly quantitative yield. Elimination of HCl from 117?? was achieved under a variety of conditions and 3-alkoxy derivatives were prepared from 117?? through diastereoselective substitution reactions with alcohols. Cyclobutanones with 3??-OR substituents were found to favour an endo envelope conformation while the 3??-OR derivatives adopt the exo envelope conformation. Evidence from X-ray crystal structures and ab initio molecular orbital calculations suggests that an anomeric effect contributes to the large conformational preference of the tetrahydrothiophene ring that favours the 3-alkoxy substituent in an axial orientation. In addition, the conformation of the bicyclic system was found to have a dramatic effect on the tendency of the cyclobutanone to undergo hemiketal formation. Cyclobutanone analogues of penicillins, including 3-alkoxy derivatives, and cyclobutanone analogues of penems were evaluated against class A, B, C, and D ??-lactamases and found to be moderate inhibitors of KPC-2, IMP-1, GC1, and OXA-10. The cyclobutanones found to be most potent were those which are hydrated to a larger extent in aqueous solution. Dichlorocyclobutanones were found to be better inhibitors than dechlorinated cyclobutanones and a 3??-methoxy derivative 152??, which favours the exo envelope conformation in which the C4 carboxylate is equatorial, was found to be a better inhibitor than cyclobutanones that favour the endo envelope conformation. A 3,4-unsaturated penem analogue, 153, showed comparable potency to that of 152?? and molecular models of enzyme-inhibitor complexes indicate that an equatorial carboxylate is required for binding to ??-lactamases. An X-ray crystal structure of 152?? bound to the class D ??-lactamase OXA-10 confirms that a serine hemiketal is formed in the active site and that the inhibitor adopts the exo envelope. The biochemical data described above demonstrate that cyclobutanones can indeed act as inhibitors of serine- and metallo-??-lactamases and these cyclobutanones represent the first class of reversible inhibitors to show moderate inhibition of all four classes of ??-lactamase. Although the inhibitory potency of these compounds is modest (low micromolar IC50 values), penem analogue 153 was able to enhance the potency of meropenem against carbapenem-resistant MBL-producing clinical isolates of Chryseobacterium meningosepticum and Stenotrophomonas maltophilia and the synergy demonstrated in these antimicrobial assays is encouraging. Synthetic studies toward other C3-alkyl and C3-thioalkyl-substituted inhibitors are described and the design and synthesis of C7-monochloro- and 7??-hydroxymethyl-7??-chloro cyclobutanone derivatives is presented.