The University Hospital of Liège has 955 beds in 8 intensive care units, 15 medical wards, 10 surgical wards and 1 paediatric ward. Approximately 36,000 patients are admitted each year, giving a total of 265,000 patient-days hospitalization. Extended-spectrum beta-lactamase-producing Enterobacteriaceae (E-ESBL) constitute, along with methicillin-resistant Staphylococcus aureus (MRSA), the main multi-resistant bacteria recovered in our hospital. The aims of the present study were to: - evaluate the epidemiology of E-ESBL - evaluate the impact of an infection control programme to reduce the spread of E-ESBL in the University Hospital of Liège. In order to do this, several studies were carried out between 2001 and 2007: 1. Determination of the high risk units in the CHU (2001) The high risk units were determined by comparing the incidence rates of each type of unit. Two types of high risk unit were identified in this way: the Intensive Care Units (ICUs) and the Onco-Haematology Unit. 2. Epidemiology of E-ESBL in the Onco-Haematology unit (2002-2003 and 2005-2006) Digestive tract colonization by E-ESBL was found to be relatively high (7.3%) and this explains the high incidence of E-ESBL in Onco-Haematology in comparison with the rest of the hospital. However, the clinical gravity associated with exposure to the risk factor (digestive tract colonization by E-ESBL) was found to be relatively weak. 3. Importance of digestive tract colonization by E-ESBL in General ICUs (2002-2003) Digestive tract colonization by E-ESBL was found to be relatively common (8.8%) and faecal carriage of E-ESBL was found to be a good marker for infection with E-ESBL at another body site. Even though the number of infected patients was found to be low, the risk of infection due to E-ESBL was multiplied by 14.7 in a group of digestive carriers of E-ESBL with regard to a group of non carriers. Our data also showed that Enterobacter aerogenes is the most frequent species producing extended-spectrum beta-lactamase (ESBL) and that TEM-24 is the most prevalent ESBL produced by E-ESBL species in our ICUs. No CTX-M-type genes were identified. With regard to antibiotic susceptibility, meropenem and cefepime appeared to be the most active agents against the majority of isolates. 4. Impact of an infection control programme to reduce the spread of E-ESBL (2006-2007) A surveillance programme was carried out to evaluate the implementation of infection control procedures including surveillance of ESBL-producing strains, utilization of computer alerts for E-ESBL positive patients and the application of contact precautions for colonized or infected patients. Infection control compliance observations were performed by trained referring nurses. During the 2 years of application, one or more E-ESBL were identified in 500 patients. A total of 2268 internal messages regarding the identification of E-ESBL were sent within the hospital, among which 91.84 % were received (at least 1 for every patient). An alert was associated with 406 patients, who were always hospitalized as the identification of the E-ESBL by the laboratory was obtained. A total of 257 registration forms were filled in by the referring nurses, resulting in a survey compliance of 63%. This survey showed that door signs identifying positive patients, hydro-alcoholic solution and gloves were present in 90% of the cases, but that gowns were only present in 59%. The overall incidence of nosocomial acquisition of E-ESBL between 2006 and 2007 was 0.92/1000 patient-days, more or less the same as in 2002. In relation to this research, several questions remain: - Even though the rates of digestive tract colonization with E-ESBL in the 2 types of high risk unit were found to be more or less the same (7.3 and 8.8%), the impact on infections due to E-ESBL was very different. - Are the infections due either to E-ESBL endogenous infections (owing notably to the use of broad spectrum antibiotics) or to secondary infections (resulting from cross-transmission) or to both? The implementation of an infection control programme to limit the spread of E-ESBL has been based on the limitation of the cross-transmission of these micro-organisms. An enhanced barrier precautions policy has been in place in our institution for 2 years, and we have seen no erosion in compliance. We should not however lose sight of the fact that, whatever the institutional policy for the management of multi-resistant bacteria, the correct application of standard precautions for all patients is the first measure to limit the cross-transmission of all micro-organisms.

ESBL-producing Enterobacteriaceae are emerging worldwide and they are frequently multi-drug resistant, thus limiting treatment options for infections caused by these pathogens. The overall aim of the thesis was to investigate ESBL-producing Enterobacteriaceae in a Swedish county. First, we developed a molecular method, a multiplex PCR assay for identification of SHV, TEM and CTX-M genes in clinical isolates of Enterobacteriaceae with an ESBL phenotype. From 2002 until the end of 2007 all isolates of ESBL-producing Enterobacteriaceae in Östergötland, Sweden were further investigated. The prevalence of ESBL-producing Enterobacteriaceae was low, <1%, but increasing,while the antibiotic consumption remained unchanged. CTX-M enzymes, particularly CTX-M group 1, dominate in our region as well as in the rest of Europe. Furthermore, we have investigated antimicrobial susceptibility by performing MIC-testing in a large, well-characterized population of CTX-M-producing E. coli. Only three oral antimicrobial agents (fosfomycin, nitrofurantoin and mecillinam) demonstrated susceptibility above 90%. High susceptibility, >90%, was also demonstrated for carbapenems, colistin, tigecycline and amikacin. Sixty-eight per cent of ESBL-producing E. coli was multi-resistant, and the most common multi-resistance pattern was the ESBL phenotype with decreased susceptibility to trimethoprim, trimethoprim-sulfamethoxazole, ciprofloxacin, gentamicin and tobramycin. Isolates belonging to CTX-M group 9 are generally more susceptible to antibiotics than the CTX-M group 1-producing E. coli. Finally, a prospective multicentre case-control study examined the prevalence of ESBL-producing Enterobacteriaceae in faecal samples before and after travel abroad and the risk factors of acquisition. Sixty-eight of 226 travellers (30%) had ESBL-producing Enterobacteriaceae in the faecal flora. The geographical area visited had the highest impact on acquisition, with highest the risk for travellers visiting the Indian subcontinent, followed by Asia and Africa north of the equator. Also, acquisition of ESBL-producing Enterobacteriaceae during travel is associated with abdominal symptoms such as diarrhoea. Age also seemed to affect the risk of acquiring ESBL-producing Enterobacteriaceae, the highest risks were found among travellers ≥ 65 years. This thesis has contributed to increased understanding of the epidemiology of ESBL-producing Enterobacteriaceae and their susceptibility to both beta-lactam and non-beta-lactam agents.

Coliform bacteria have long been used to indicate fecal contamination of food, water, and solid surfaces, and thus the presence of a health hazard. In this study, the in-mill water and external effluent treatment systems of seven typical Canadian pulp and paper mills were shown to support the growth of numerous coliforms, especially Klebsiella spp, Escherichia coli, Enterobacter spp., and Citrobacter spp . Mill coliforms were shown to be not just simple transients from feedwater or furnish (wood), but to be continuously growing, especially in the primary clarifiers. Therefore, coliforms and fecal coliforms cannot be used as fecal contamination indicators in pulp and paper mill water and effluent treatment systems. / N2-fixing coliform populations were detected in mill water systems and were analyzed using N2-fixation assays and nitrogenase gene (nifH) probing. Both active in situ populations and cultured microbial isolates were tested. Active N2-fixation was demonstrated in six primary clarifiers. Measurement of the numbers and composition of the total culturable bacterial community in a primary clarifier revealed that approximately 50% of all aerobic cells contained nifH , of which >90% were Klebsiella. Coliforms growing on MacConkey agar plates from the primary clarifier were all identified as Klebsiella and 100% of these Klebsiella contained the nifH gene. Preliminary estimates indicate that the amount of N2 fixed per day is substantial in some clarifiers.

A presença de Salmonella é uma das mais importantes barreiras sanitárias à exportação de alimentos. A ração contaminada representa potencial fonte de introdução de Salmonella nos rebanhos suínos, além do risco indireto de infecção ao consumidor. Um estudo transversal conduzido em quatro fábricas de ração teve como objetivos avaliar a frequência de isolamento de Salmonella e coliformes totais nestas unidades, verificar a presença desses agentes nas diversas etapas do processo de produção, correlacionar grupos clonais de Salmonella sp. obtidos pela análise de macrorestrição associada à eletroforese em campo pulsado (PFGE), além de avaliar a concordância entre o escore obtido na aplicação de roteiro de inspeção da Instrução Normativa (IN) 4 do Ministério da Agricultura e Abastecimento e os níveis de coliformes totais encontrados. De 1.269 amostras analisadas, sessenta e três (4,96%) apresentaram Salmonella sp. e 38,53% (n=489) apresentaram presença de enterobactérias. As freqüências de contaminação por Salmonella nas quatro fábricas avaliadas (A, B, C e D) foram, respectivamente, 3,5% (n=11/317), 1,7% (n=5/289), 7,1% (n=23/308) e 7% (n=25/355). Já para coliformes totais foram, respectivamente, 40,7% (129/317), 30,1% (87/289), 52,6% (162/308) e 31,3% (111/355). Nas fábricas A e D, Salmonella foi detectada em amostras de produto final. Estirpes apresentando genótipos semelhantes foram identificadas nos sorovares Orion, Montevideo, Worthington e Agona. Foi possível verificar que o sorovar Montevideo obteve o maior número de grupos clonais, apresentando pulsotipos distribuídos entre ingredientes, poeira, equipamentos e ração. O isolamento de Salmonella foi significativamente mais frequente (p =0,002) em amostras com a presença (36/489; 7,36%) do que com ausência de coliformes totais (27/780; 3,46%). As contagens logarítmicas médias encontradas de coliformes totais, considerando todas as amostras analisadas por fábrica, foram: A: 0,97 (IC 95%: 0,81-1,13); B: 0,78 (IC 95%: 0,61-0,94); C: 1,32 (IC 95%: 1,16-1,49) e D: 0,91 (IC 95%: 0,75-1,06). Constatou-se elevada variabilidade no número de coliformes totais em todos os pontos amostrados nas quatro fábricas, e não houve diferenças significativas entre os pontos amostrados e entre as fábricas (p=0,174). A regressão logística tendo como variável resposta a presença de coliformes totais apontou maiores razão de chance (OR) de isolamento no transportador (OR=3,67; IC 95%: 2,28-5,89), dosagem (OR=9,51, IC 95%: 4,43-20,41), moagem (OR=7,1; IC 95%: 3,27-15,40), mistura (OR=4,08; IC 95%: 2,04-8,17), poeira (OR=3,50; IC 95%:2,10-5,84), resíduos (OR=6,22; IC 95%: 3,88-9,95) e fábrica C (OR=2,43; IC 95%: 1,68-3,53). Da mesma forma, o transportador (OR= 4,43; IC95%: 2,43-8,09) foi o local com maior probabilidade de isolamento de Salmonella, seguidos da poeira coletada nas dependências da fábrica (OR=2,88; IC95%: 1,41-5,88). Comparadas à fábrica B, as unidades C e D apresentaram, respectivamente, 2,74 e 2,83 mais chances de isolamento de Salmonella. Ao analisar os 128 itens necessários no Roteiro de Inspeção da IN4, a fábrica B obteve o menor número de não conformidades. Nas demais fábricas, as maiores inconformidades foram encontradas quanto à estrutura da área interna e externa das fábricas. As fábricas A e D foram as que apresentaram o maior número de itens não conformes relacionados à limpeza e higienização de equipamentos. Entre os 29 itens imprescindíveis na Avaliação de Estabelecimento e Procedimentos Operacionais Padrão, oito não foram cumpridos em pelo menos uma das fábricas. A fábrica B apresentou todos os itens em conformidade, ao passo que as fábricas A e D não cumpriram os oito itens. A fábrica C apresentou três itens não conformes, relacionados à limpeza de piso e parede, recepção e armazenamento de matérias-primas. Não houve concordância entre os escores obtidos no roteiro de avaliação e a média de coliformes totais encontrada nas fábricas, demonstrando que apenas a adequação à legislação pode não garantir a inocuidade do alimento produzido. A partir disso, conclui-se que é necessário implantar programas de monitoramento e controle microbiológico ao longo da linha de produção, independente da situação da fábrica em relação à legislação vigente. A elaboração e produção de equipamentos de fácil limpeza e com mínimo acúmulo de poeira e resíduos deve ser incluída nos programas de controle de Salmonella concomitante às demais medidas de controle preconizadas. / The presence of Salmonella is one of the most important sanitary barriers to food exports. The contaminated feed is a potential source of introduction of Salmonella in pig herds, and may also represent a risk to the consumer. A cross-sectional study conducted in four feed mills was carried out to evaluate the frequency of isolation of enterobacteria and Salmonella at various stages of feed production, to identify clonal groups of Salmonella sp. Obtained by macrorestriction analysis associated with pulsed field gel electrophoresis (PFGE), and to verify the association between the score obtained by feed mills after the application of the checklist of the Normative 4 (IN4) of the Ministry of Agriculture and Food Supply and the mean of total coliforms found in the unit. Among 1,269 samples analysed, sixty-three (4.96%) showed the presence of Salmonella. The four feeds mills evaluated (A, B, C and D) presented, respectively, 3,5% (n=11/317), 1.7% (n=5/289), 7,1% (n=23/308), and 7% (n=25/355) of positive samples. Total coliforms were isolated from 40.7% (129/317), 30.1% (87/289), 52.6% (162/308) e 31.3% (111/355) samples taken at unit A, B, C and D, respectively. In the units A and D, Salmonella was detected in samples of feed. Strains showing similar genotypes have been identified as Serovar Orion, Montevideo, Worthington and Agona. Serovar Montevideo presented the highest number of clonal groups, with common pulsotypes distributed among ingredients, dust, equipment and feed. The isolation of Salmonella was significantly higher (p=0,002) in samples with the presence of total coliforms (36/489; 7.36%) than in the negative ones (27/780; 3.46%). The mean logarithmic counts of total coliforms considering all samples were determined for each plant: A: 0.97 (95% CI: 0.81 to 1.13), B: 0.78 (95% CI: 0.61 to 0.94), C: 1.32 (95% CI: 1.16 to 1.49) and D: 0.91 (95% CI: 0.75 to 1.06). A high variability on the number of total coliforms in all the sampled points was found in all feed mills, and there was no statistical diference between the sampling sites and units (p=0.174). The logistic regression, with isolation of total coliforms as the response variable, showed a high odds ratio (OR) of isolation from conveyors (OR = 3.67, 95% CI: 2.28 to 5.89), dosing (OR = 9.51, 95% CI: 4.43 to 20.41), grinding (OR = 7.10, 95% CI = 3.27 to 15.40), mixing (OR = 4.08, 95 % CI: 2.04 to 8.17), dust (OR = 3.50, 95% CI: 2.10 to 5.84), crusts (OR = 6.22, 95% CI: 3.88 to 9.95) and unit C (OR = 2.43, 95% CI: 1.68 to 3.53). The conveyors (OR = 4.43, 95% CI: 2.43-8.09) were the most likely sites of isolation of Salmonella, followed by the dust collected on the premises of the plant (OR = 2.88, 95% CI: 1.41-5.88). Feed mills C and D showed, respectively, 2.74 and 2.83 higher odds for Salmonella isolation than feed mill B. The verification of the 128 items addressed on the Guidelines for Inspection of Normative 4 (IN 4) of the Brazilian Ministry of Agriculture and Food Supply pointed out that factory B had the lowest number of nonconformities. The other three feed mills, presented most faults related to the physical structure of the facilities. Feed mills A and D presented the largest number of nonconformities related to cleaning and sanitizing of equipment. Among the 29 mandatory items in IN4, eight were not met in at least one of the factories. Feed mill B presented all items accordingly, while plants A and D did not meet any of the eight items. Feed mill C had three non-compliant items related to the floor and wall cleaning, reception and storage of raw materials. There was no association between the score obtained on the inspection checklist and the level of total coliforms found in the sampled units, demonstrating that only the compliance to the legislation cannot guarantee the safety of feed produced. It was concluded that programs for monitoring and control microbial contamination throughout the production line are needed, regardless of the status of the plant in the relation to the current legislation.The design of equipment that allow easy cleaning and with minimal accumulation of dust and debris should be added to other measures in Salmonella control programs.

La découverte récente du premier gène de résistance plasmidique à la colistine mcr-1 a mis en évidence le besoin urgent d'améliorer la détection des isolats résistant à la colistine dans les laboratoires de microbiologie clinique afin de pouvoir rapidement isoler les patients porteurs. L’objectif de ma thèse a ainsi été de répondre à cette problématique par le développement et l’évaluation d’outils spécifiques. Une revue a été rédigée visant à résumer les nouveaux outils développés pour détecter la résistance aux polymyxines, y compris la présentation des méthodes actuelles phénotypiques, antibiogrammes et génotypiques. Le milieu de culture LBJMR a été développé pour détecter toutes les bactéries à Gram négatif résistantes à la colistine et également les souches d’entérocoques résistants à la vancomycine qui représentent un autre problème majeur de santé publique. LBJMR est polyvalent et a permis de dépister 3 bactéries d'intérêt clinique à partir d’un même échantillon: E. coli et K. pneumoniae résistantes à la colistine, et E. faecium résistant à la vancomycine. Un brevet a été déposé. Le kit UMIC Colistine est un dispositif commercial de détermination de la CMI basé sur la méthode de référence. Évalué conformément à la norme ISO 20776, sa sensibilité était excellente pour la détection des souches porteuses du gène mcr-1. Le kit UMIC Colistine constitue ainsi une méthode rapide et fiable pour évaluer la CMI des isolats cliniques dans les laboratoires de microbiologie clinique. Enfin, mes travaux de thèse ont compris l’analyse d’une souche de K. pneumoniae hétérorésistante à la colistine et la description d’une nouvelle espèce bactérienne : Pseudomonas massiliensis. / The recent discovery of mcr-1, the first plasmid-mediated colistin resistance gene, highlighted the urgent need to implement the detection of colistin-resistant isolates in clinical microbiology laboratories, in order to isolate carrier patients. My thesis aimed to address this issue by developing and evaluating specific tools. A review was redacted to summarize the new tools developed to detect polymyxin resistance including the current phenotyping, susceptibility testing and genotyping methods. The LBJMR culture medium has been developed to detect all colistin-resistant Gram-negative bacteria and also the vancomycin-resistant enterococci which represent another major problem of public health. The LBJMR medium allowed the detection of different bacteria of interest from the same clinical sample: colistin-resistant E. coli and K. pneumoniae and also a vancomycin-resistant E. faecium. This project led to a patent filing. The UMIC Colistine kit is a ready-to-use device based on the reference method to determine the polymyxin MIC. Evaluated in accordance with ISO 20776 standard, its sensitivity was excellent for the detection of colistin-resistant strains harboring the mcr-1 gene. The UMIC Colistin system is a rapid and reliable method to determine colistin MIC of clinical isolates in clinical microbiology laboratories. My thesis project also included the analysis of a colistin-heteroresistant Klebsiella pneumoniae strain, and the description of a new bacteril species, Pseudomonas massiliensis. These studies highlight the need to improve the detection of colistin-resistant strains by using reliable methods, suitable for diagnosis in clinical microbiology laboratories.

A presença de Salmonella é uma das mais importantes barreiras sanitárias à exportação de alimentos. A ração contaminada representa potencial fonte de introdução de Salmonella nos rebanhos suínos, além do risco indireto de infecção ao consumidor. Um estudo transversal conduzido em quatro fábricas de ração teve como objetivos avaliar a frequência de isolamento de Salmonella e coliformes totais nestas unidades, verificar a presença desses agentes nas diversas etapas do processo de produção, correlacionar grupos clonais de Salmonella sp. obtidos pela análise de macrorestrição associada à eletroforese em campo pulsado (PFGE), além de avaliar a concordância entre o escore obtido na aplicação de roteiro de inspeção da Instrução Normativa (IN) 4 do Ministério da Agricultura e Abastecimento e os níveis de coliformes totais encontrados. De 1.269 amostras analisadas, sessenta e três (4,96%) apresentaram Salmonella sp. e 38,53% (n=489) apresentaram presença de enterobactérias. As freqüências de contaminação por Salmonella nas quatro fábricas avaliadas (A, B, C e D) foram, respectivamente, 3,5% (n=11/317), 1,7% (n=5/289), 7,1% (n=23/308) e 7% (n=25/355). Já para coliformes totais foram, respectivamente, 40,7% (129/317), 30,1% (87/289), 52,6% (162/308) e 31,3% (111/355). Nas fábricas A e D, Salmonella foi detectada em amostras de produto final. Estirpes apresentando genótipos semelhantes foram identificadas nos sorovares Orion, Montevideo, Worthington e Agona. Foi possível verificar que o sorovar Montevideo obteve o maior número de grupos clonais, apresentando pulsotipos distribuídos entre ingredientes, poeira, equipamentos e ração. O isolamento de Salmonella foi significativamente mais frequente (p =0,002) em amostras com a presença (36/489; 7,36%) do que com ausência de coliformes totais (27/780; 3,46%). As contagens logarítmicas médias encontradas de coliformes totais, considerando todas as amostras analisadas por fábrica, foram: A: 0,97 (IC 95%: 0,81-1,13); B: 0,78 (IC 95%: 0,61-0,94); C: 1,32 (IC 95%: 1,16-1,49) e D: 0,91 (IC 95%: 0,75-1,06). Constatou-se elevada variabilidade no número de coliformes totais em todos os pontos amostrados nas quatro fábricas, e não houve diferenças significativas entre os pontos amostrados e entre as fábricas (p=0,174). A regressão logística tendo como variável resposta a presença de coliformes totais apontou maiores razão de chance (OR) de isolamento no transportador (OR=3,67; IC 95%: 2,28-5,89), dosagem (OR=9,51, IC 95%: 4,43-20,41), moagem (OR=7,1; IC 95%: 3,27-15,40), mistura (OR=4,08; IC 95%: 2,04-8,17), poeira (OR=3,50; IC 95%:2,10-5,84), resíduos (OR=6,22; IC 95%: 3,88-9,95) e fábrica C (OR=2,43; IC 95%: 1,68-3,53). Da mesma forma, o transportador (OR= 4,43; IC95%: 2,43-8,09) foi o local com maior probabilidade de isolamento de Salmonella, seguidos da poeira coletada nas dependências da fábrica (OR=2,88; IC95%: 1,41-5,88). Comparadas à fábrica B, as unidades C e D apresentaram, respectivamente, 2,74 e 2,83 mais chances de isolamento de Salmonella. Ao analisar os 128 itens necessários no Roteiro de Inspeção da IN4, a fábrica B obteve o menor número de não conformidades. Nas demais fábricas, as maiores inconformidades foram encontradas quanto à estrutura da área interna e externa das fábricas. As fábricas A e D foram as que apresentaram o maior número de itens não conformes relacionados à limpeza e higienização de equipamentos. Entre os 29 itens imprescindíveis na Avaliação de Estabelecimento e Procedimentos Operacionais Padrão, oito não foram cumpridos em pelo menos uma das fábricas. A fábrica B apresentou todos os itens em conformidade, ao passo que as fábricas A e D não cumpriram os oito itens. A fábrica C apresentou três itens não conformes, relacionados à limpeza de piso e parede, recepção e armazenamento de matérias-primas. Não houve concordância entre os escores obtidos no roteiro de avaliação e a média de coliformes totais encontrada nas fábricas, demonstrando que apenas a adequação à legislação pode não garantir a inocuidade do alimento produzido. A partir disso, conclui-se que é necessário implantar programas de monitoramento e controle microbiológico ao longo da linha de produção, independente da situação da fábrica em relação à legislação vigente. A elaboração e produção de equipamentos de fácil limpeza e com mínimo acúmulo de poeira e resíduos deve ser incluída nos programas de controle de Salmonella concomitante às demais medidas de controle preconizadas. / The presence of Salmonella is one of the most important sanitary barriers to food exports. The contaminated feed is a potential source of introduction of Salmonella in pig herds, and may also represent a risk to the consumer. A cross-sectional study conducted in four feed mills was carried out to evaluate the frequency of isolation of enterobacteria and Salmonella at various stages of feed production, to identify clonal groups of Salmonella sp. Obtained by macrorestriction analysis associated with pulsed field gel electrophoresis (PFGE), and to verify the association between the score obtained by feed mills after the application of the checklist of the Normative 4 (IN4) of the Ministry of Agriculture and Food Supply and the mean of total coliforms found in the unit. Among 1,269 samples analysed, sixty-three (4.96%) showed the presence of Salmonella. The four feeds mills evaluated (A, B, C and D) presented, respectively, 3,5% (n=11/317), 1.7% (n=5/289), 7,1% (n=23/308), and 7% (n=25/355) of positive samples. Total coliforms were isolated from 40.7% (129/317), 30.1% (87/289), 52.6% (162/308) e 31.3% (111/355) samples taken at unit A, B, C and D, respectively. In the units A and D, Salmonella was detected in samples of feed. Strains showing similar genotypes have been identified as Serovar Orion, Montevideo, Worthington and Agona. Serovar Montevideo presented the highest number of clonal groups, with common pulsotypes distributed among ingredients, dust, equipment and feed. The isolation of Salmonella was significantly higher (p=0,002) in samples with the presence of total coliforms (36/489; 7.36%) than in the negative ones (27/780; 3.46%). The mean logarithmic counts of total coliforms considering all samples were determined for each plant: A: 0.97 (95% CI: 0.81 to 1.13), B: 0.78 (95% CI: 0.61 to 0.94), C: 1.32 (95% CI: 1.16 to 1.49) and D: 0.91 (95% CI: 0.75 to 1.06). A high variability on the number of total coliforms in all the sampled points was found in all feed mills, and there was no statistical diference between the sampling sites and units (p=0.174). The logistic regression, with isolation of total coliforms as the response variable, showed a high odds ratio (OR) of isolation from conveyors (OR = 3.67, 95% CI: 2.28 to 5.89), dosing (OR = 9.51, 95% CI: 4.43 to 20.41), grinding (OR = 7.10, 95% CI = 3.27 to 15.40), mixing (OR = 4.08, 95 % CI: 2.04 to 8.17), dust (OR = 3.50, 95% CI: 2.10 to 5.84), crusts (OR = 6.22, 95% CI: 3.88 to 9.95) and unit C (OR = 2.43, 95% CI: 1.68 to 3.53). The conveyors (OR = 4.43, 95% CI: 2.43-8.09) were the most likely sites of isolation of Salmonella, followed by the dust collected on the premises of the plant (OR = 2.88, 95% CI: 1.41-5.88). Feed mills C and D showed, respectively, 2.74 and 2.83 higher odds for Salmonella isolation than feed mill B. The verification of the 128 items addressed on the Guidelines for Inspection of Normative 4 (IN 4) of the Brazilian Ministry of Agriculture and Food Supply pointed out that factory B had the lowest number of nonconformities. The other three feed mills, presented most faults related to the physical structure of the facilities. Feed mills A and D presented the largest number of nonconformities related to cleaning and sanitizing of equipment. Among the 29 mandatory items in IN4, eight were not met in at least one of the factories. Feed mill B presented all items accordingly, while plants A and D did not meet any of the eight items. Feed mill C had three non-compliant items related to the floor and wall cleaning, reception and storage of raw materials. There was no association between the score obtained on the inspection checklist and the level of total coliforms found in the sampled units, demonstrating that only the compliance to the legislation cannot guarantee the safety of feed produced. It was concluded that programs for monitoring and control microbial contamination throughout the production line are needed, regardless of the status of the plant in the relation to the current legislation.The design of equipment that allow easy cleaning and with minimal accumulation of dust and debris should be added to other measures in Salmonella control programs.

A presença de Salmonella é uma das mais importantes barreiras sanitárias à exportação de alimentos. A ração contaminada representa potencial fonte de introdução de Salmonella nos rebanhos suínos, além do risco indireto de infecção ao consumidor. Um estudo transversal conduzido em quatro fábricas de ração teve como objetivos avaliar a frequência de isolamento de Salmonella e coliformes totais nestas unidades, verificar a presença desses agentes nas diversas etapas do processo de produção, correlacionar grupos clonais de Salmonella sp. obtidos pela análise de macrorestrição associada à eletroforese em campo pulsado (PFGE), além de avaliar a concordância entre o escore obtido na aplicação de roteiro de inspeção da Instrução Normativa (IN) 4 do Ministério da Agricultura e Abastecimento e os níveis de coliformes totais encontrados. De 1.269 amostras analisadas, sessenta e três (4,96%) apresentaram Salmonella sp. e 38,53% (n=489) apresentaram presença de enterobactérias. As freqüências de contaminação por Salmonella nas quatro fábricas avaliadas (A, B, C e D) foram, respectivamente, 3,5% (n=11/317), 1,7% (n=5/289), 7,1% (n=23/308) e 7% (n=25/355). Já para coliformes totais foram, respectivamente, 40,7% (129/317), 30,1% (87/289), 52,6% (162/308) e 31,3% (111/355). Nas fábricas A e D, Salmonella foi detectada em amostras de produto final. Estirpes apresentando genótipos semelhantes foram identificadas nos sorovares Orion, Montevideo, Worthington e Agona. Foi possível verificar que o sorovar Montevideo obteve o maior número de grupos clonais, apresentando pulsotipos distribuídos entre ingredientes, poeira, equipamentos e ração. O isolamento de Salmonella foi significativamente mais frequente (p =0,002) em amostras com a presença (36/489; 7,36%) do que com ausência de coliformes totais (27/780; 3,46%). As contagens logarítmicas médias encontradas de coliformes totais, considerando todas as amostras analisadas por fábrica, foram: A: 0,97 (IC 95%: 0,81-1,13); B: 0,78 (IC 95%: 0,61-0,94); C: 1,32 (IC 95%: 1,16-1,49) e D: 0,91 (IC 95%: 0,75-1,06). Constatou-se elevada variabilidade no número de coliformes totais em todos os pontos amostrados nas quatro fábricas, e não houve diferenças significativas entre os pontos amostrados e entre as fábricas (p=0,174). A regressão logística tendo como variável resposta a presença de coliformes totais apontou maiores razão de chance (OR) de isolamento no transportador (OR=3,67; IC 95%: 2,28-5,89), dosagem (OR=9,51, IC 95%: 4,43-20,41), moagem (OR=7,1; IC 95%: 3,27-15,40), mistura (OR=4,08; IC 95%: 2,04-8,17), poeira (OR=3,50; IC 95%:2,10-5,84), resíduos (OR=6,22; IC 95%: 3,88-9,95) e fábrica C (OR=2,43; IC 95%: 1,68-3,53). Da mesma forma, o transportador (OR= 4,43; IC95%: 2,43-8,09) foi o local com maior probabilidade de isolamento de Salmonella, seguidos da poeira coletada nas dependências da fábrica (OR=2,88; IC95%: 1,41-5,88). Comparadas à fábrica B, as unidades C e D apresentaram, respectivamente, 2,74 e 2,83 mais chances de isolamento de Salmonella. Ao analisar os 128 itens necessários no Roteiro de Inspeção da IN4, a fábrica B obteve o menor número de não conformidades. Nas demais fábricas, as maiores inconformidades foram encontradas quanto à estrutura da área interna e externa das fábricas. As fábricas A e D foram as que apresentaram o maior número de itens não conformes relacionados à limpeza e higienização de equipamentos. Entre os 29 itens imprescindíveis na Avaliação de Estabelecimento e Procedimentos Operacionais Padrão, oito não foram cumpridos em pelo menos uma das fábricas. A fábrica B apresentou todos os itens em conformidade, ao passo que as fábricas A e D não cumpriram os oito itens. A fábrica C apresentou três itens não conformes, relacionados à limpeza de piso e parede, recepção e armazenamento de matérias-primas. Não houve concordância entre os escores obtidos no roteiro de avaliação e a média de coliformes totais encontrada nas fábricas, demonstrando que apenas a adequação à legislação pode não garantir a inocuidade do alimento produzido. A partir disso, conclui-se que é necessário implantar programas de monitoramento e controle microbiológico ao longo da linha de produção, independente da situação da fábrica em relação à legislação vigente. A elaboração e produção de equipamentos de fácil limpeza e com mínimo acúmulo de poeira e resíduos deve ser incluída nos programas de controle de Salmonella concomitante às demais medidas de controle preconizadas. / The presence of Salmonella is one of the most important sanitary barriers to food exports. The contaminated feed is a potential source of introduction of Salmonella in pig herds, and may also represent a risk to the consumer. A cross-sectional study conducted in four feed mills was carried out to evaluate the frequency of isolation of enterobacteria and Salmonella at various stages of feed production, to identify clonal groups of Salmonella sp. Obtained by macrorestriction analysis associated with pulsed field gel electrophoresis (PFGE), and to verify the association between the score obtained by feed mills after the application of the checklist of the Normative 4 (IN4) of the Ministry of Agriculture and Food Supply and the mean of total coliforms found in the unit. Among 1,269 samples analysed, sixty-three (4.96%) showed the presence of Salmonella. The four feeds mills evaluated (A, B, C and D) presented, respectively, 3,5% (n=11/317), 1.7% (n=5/289), 7,1% (n=23/308), and 7% (n=25/355) of positive samples. Total coliforms were isolated from 40.7% (129/317), 30.1% (87/289), 52.6% (162/308) e 31.3% (111/355) samples taken at unit A, B, C and D, respectively. In the units A and D, Salmonella was detected in samples of feed. Strains showing similar genotypes have been identified as Serovar Orion, Montevideo, Worthington and Agona. Serovar Montevideo presented the highest number of clonal groups, with common pulsotypes distributed among ingredients, dust, equipment and feed. The isolation of Salmonella was significantly higher (p=0,002) in samples with the presence of total coliforms (36/489; 7.36%) than in the negative ones (27/780; 3.46%). The mean logarithmic counts of total coliforms considering all samples were determined for each plant: A: 0.97 (95% CI: 0.81 to 1.13), B: 0.78 (95% CI: 0.61 to 0.94), C: 1.32 (95% CI: 1.16 to 1.49) and D: 0.91 (95% CI: 0.75 to 1.06). A high variability on the number of total coliforms in all the sampled points was found in all feed mills, and there was no statistical diference between the sampling sites and units (p=0.174). The logistic regression, with isolation of total coliforms as the response variable, showed a high odds ratio (OR) of isolation from conveyors (OR = 3.67, 95% CI: 2.28 to 5.89), dosing (OR = 9.51, 95% CI: 4.43 to 20.41), grinding (OR = 7.10, 95% CI = 3.27 to 15.40), mixing (OR = 4.08, 95 % CI: 2.04 to 8.17), dust (OR = 3.50, 95% CI: 2.10 to 5.84), crusts (OR = 6.22, 95% CI: 3.88 to 9.95) and unit C (OR = 2.43, 95% CI: 1.68 to 3.53). The conveyors (OR = 4.43, 95% CI: 2.43-8.09) were the most likely sites of isolation of Salmonella, followed by the dust collected on the premises of the plant (OR = 2.88, 95% CI: 1.41-5.88). Feed mills C and D showed, respectively, 2.74 and 2.83 higher odds for Salmonella isolation than feed mill B. The verification of the 128 items addressed on the Guidelines for Inspection of Normative 4 (IN 4) of the Brazilian Ministry of Agriculture and Food Supply pointed out that factory B had the lowest number of nonconformities. The other three feed mills, presented most faults related to the physical structure of the facilities. Feed mills A and D presented the largest number of nonconformities related to cleaning and sanitizing of equipment. Among the 29 mandatory items in IN4, eight were not met in at least one of the factories. Feed mill B presented all items accordingly, while plants A and D did not meet any of the eight items. Feed mill C had three non-compliant items related to the floor and wall cleaning, reception and storage of raw materials. There was no association between the score obtained on the inspection checklist and the level of total coliforms found in the sampled units, demonstrating that only the compliance to the legislation cannot guarantee the safety of feed produced. It was concluded that programs for monitoring and control microbial contamination throughout the production line are needed, regardless of the status of the plant in the relation to the current legislation.The design of equipment that allow easy cleaning and with minimal accumulation of dust and debris should be added to other measures in Salmonella control programs.

La résistance aux antibiotiques est devenue un problème majeur de santé publique à travers le monde pouvant conduire à l’impasse thérapeutique. L’utilisation abusive et inappropriée des antibiotiques en médecine humaine mais également en médecine vétérinaire est en grande partie responsable de la multiplication et de la propagation des bactéries multirésistantes (BMR). Les entérobactéries, hôtes naturels du tube digestif de l’homme et des animaux, ont particulièrement subi ces pressions de sélection antibiotiques et ont pu, grâce à leur capacité à échanger du matériel génétique, acquérir de plus en plus de mécanismes de résistance aux antibiotiques. L’environnement joue un rôle de diffuseur par l’intermédiaire des déchets humains et animaux qu’il reçoit mais est également un pourvoyeur de gènes de résistance grâce aux bactéries naturellement résistantes qu’il héberge. En Guadeloupe, à l’exception des données de surveillance hospitalière des BMR, aucune étude portant sur la résistance aux antibiotiques n’avait été réalisée. Les objectifs de ce travail de thèse étaient donc (i) d’évaluer l’importance de la résistance en milieu communautaire en étudiant la résistance aux antibiotiques des entérobactéries isolées des infections urinaires communautaires et (ii) d’étudier la diffusion environnementale des entérobactéries résistantes aux antibiotiques (ERAs) dans les rivières et les eaux de mer recevant des effluents de stations d’épuration (STEPs) mais également au sein de la faune sauvage terrestre de Guadeloupe.Nous avons donc pu grâce à ce travail mettre en évidence une diffusion environnementale de souches d’ERAs en lien avec les activités humaines. Les rejets de STEPs ont été identifiés comme une des sources d’ERAs et en particulier d’EBLSEs dans l’environnement. Néanmoins, nos résultats montrent que d’autres activités humaines, qui feront l’objet de futures investigations, peuvent être des sources potentielles d’ERAs. La prévention passe donc par une amélioration globale de la gestion des déchets avec notamment une remise à niveau des STEPs et le rejet au large des eaux usées. / Antibiotic resistance has become a major public health concern worldwide that could lead to therapeutic impasse. The overuse and misuse of antibiotics in human medicine but also in veterinary medicine is largely responsible for the proliferation and spread of multi-drug resistant (MDR) bacteria. Enterobacteriaceae are subject to this selective pressure, as the digestive tract is their main reservoir. Moreover, thanks to their ability to exchange genetic material, they can acquire new antibiotic resistance determinants. Through human and animal waste, antimicrobial resistant bacteria can spread in the environment. However, the environment is also a supplier for antibiotic resistance since environmental bacteria naturally harbor antibiotic resistance determinants.In Guadeloupe, except for data from MDR bacteria surveillance in the hospital, no studies concerning antibiotic resistance had been carried out. The objectives of this thesis were (i) to evaluate the antimicrobial resistance in the community by studying antimicrobial susceptibility of Enterobacteriaceae strains isolated from community-acquired urinary tract infection and (ii) to study the environmental spread of antimicrobial resistant Enterobacteriaceae (AREs) in river and sea waters receiving effluents from wastewater treatment plants but also in terrestrial wildlife.Our work highlighted the environmental spread of AREs linked to human activities. WWTPs discharge has been identified as a source of AREs, especially ESBLEs, in the environment. Nevertheless, other human activities may release ARB in the environment, and some will be explored in further studies. Thus, prevention requires an overall improvement in waste management, and wastewater discharge should occur in open sea as often as possible

Parmi les plus grandes menaces de la santé publique dans le monde entier, la résistance aux antibiotiques est à la pointe. Ceci en partie est dû à l'augmentation des infections causées par des bactéries pathogènes résistantes aux antibiotiques ainsi que la diminution du nombre actuel de nouveaux antibiotiques. Dans le souci de remédier à cette situation malheureuse, il y a eu récemment la ré-surfaçage des antibiotiques anciens et abandonnés comme les polymyxines. Colistine, un membre des antibiotiques de polymyxine, est maintenant considéré comme un antibiotique de «dernier recours» pour le traitement des infections bactériennes à Gram-négatives graves en raison de son action puissante contre ces agents pathogènes. Cependant, la résistance à la colistine parmi ces agents pathogènes a émergé dans plusieurs pays et est actuellement en augmentation. En raison de la nouvelle réintroduction relative de cet antibiotique, il ya un manque d'information complètes sur ses propriétés pharmacologiques ainsi que des mécanismes par lequel les bactéries développent une résistance contre celle-ci.Afin de combler ce manque d'information en ce qui concerne le mécanisme de résistance, nous avons donc entrepris ce projet. Tout d'abord, pour procéder à une surveillance épidémiologique des bactéries résistantes à la colistine chez les humains et les animaux domestiques et d'autre part, de décrypter les mécanismes moléculaires de résistance à la colistine parmi les bactéries résistantes isolées. / Among one of the greatest threats facing public health worldwide, antibiotic resistance is at the forefront. This is partly due to increase in infections caused by antibiotic-resistant pathogenic bacterial as well as the current dwindling number of new antibiotics. In a way to address this unfortunate situation, there have been recent resuscitation of old and abandoned antibiotics such as polymyxins. Colistin, a member of polymyxin antibiotics, is now regarded as a 'last-resort' antibiotic for the treatment of severe Gram-negative bacterial infections owing to its potent action against these pathogens. However, resistance to colistin among these pathogens has emerged in several countries and is currently on increase. Due to the relatively new reintroduction of this antibiotic, there is a lack of comprehensive information on its pharmacological properties as well as mechanisms by which bacteria develop resistance against it.In order to bridge this information gap in relation to the mechanism of resistance, we therefore undertook this project. First, to carry out an epidemiological surveillance of colistin-resistant bacteria in humans and domesticated animals and secondly, to decipher the molecular mechanisms mediating colistin resistance among the isolated resistant bacteria.

La diffusion des entérobactéries multirésistantes aux antibiotiques (MDR) à l’échelle mondiale constitue une menace de santé publique majeure. Résistantes à au moins trois classes d’antibiotiques, les entérobactéries MDR entrainent des infections échappant aux traitements de première intention. La première partie de ce travail s’intéresse à l’épidémiologie moléculaire des souches d’entérobactéries MDR isolées dans les infections et les colonisations des patients hospitalisés en Languedoc-Roussillon, en France, et dans un pays où cette épidémiologie est encore peu connue, l’Algérie. Nous avons montré, dans notre région et au niveau national, que la résistance aux carbapénèmes était essentiellement liée à des modifications de la perméabilité membranaire (87,4% des entérobactéries résistantes, au niveau national). Dans la deuxième partie de ce travail, nous avons étudié les modulations de la perméabilité membranaire et de l’efflux chez Escherichia coli ST131, l’exemple-type d’un clone MDR. Nous avons montré que ce clone mondial présentait une remarquable adaptabilité à la pression antibiotique. Cette adaptabilité avait un impact significatif sur la virulence et le fitness de E. coli. Les capacités de formation de biofilm et la virulence chez Caenorhabditis elegans étaient augmentées chez les souches de phénotypes « efflux ». Inversement, les souches de phénotypes « imperméabilité » présentaient un faible potentiel de virulence, associé à une diminution significative de la formation de biofilm et de la mobilité par swimming. / The spread of multidrug-resistant (MDR) Enterobacteriaceae is a major public health threat worldwide. Resistant to at least three classes of antibiotics, MDR Enterobacteriaceae cause infections for which first-line treatments are inefficient. The first part of this work focused on the molecular epidemiology of MDR Enterobacteriaceae strains isolated in infections and colonizations of patients hospitalized in Languedoc-Roussillon, in France and in Algeria, a country where few data are currently available. We showed in our region and nationally, that resistance to carbapenems was mainly due to changes in membrane permeability (87.4% of resistant Enterobacteriaceae, nationally).In the second part of this work, we studied the modulation of membrane efflux and permeability in the quintessential example of an international MDR high-risk clone, Escherichia coli ST131. We showed that this global clone had a remarkable adaptability to antibiotic pressure. This adaptability had a significant impact on the virulence and the fitness of E. coli. The biofilm formation and virulence capacities in Caenorhabditis elegans model were increased in strains overexpressing an efflux system. Conversely, the strains with altered porins expression had a low potential virulence, associated with a significant reduction in biofilm formation and swimming mobility.