Risco genotóxico de herbicidas em Anguilla anguilla L.

Doutoramento em BiologiaNo contexto dos contaminantes aquáticos, os herbicidas são considerados como um dos grupos mais perigosos. Uma vez aplicados, estes são facilmente transportados para cursos de água, quer devido a uma pulverização pouco cuidada ou devido a fenómenos de escorrência superficial e/ou subterrânea. A presença destes agroquímicos no ambiente tem vindo a ser associada a efeitos nefastos em organismos não-alvo, como é o caso dos peixes. Contudo, existe ainda uma grande lacuna no que diz respeito à informação científica relacionada com o seu impacto genotóxico. Deste modo, a presente tese foi delineada com o intuito de avaliar o risco genotóxico em peixes de duas formulações de herbicidas: o Roundup®, que tem como princípio activo o glifosato, e o Garlon®, que apresenta o triclopir na base da sua constituição, produtos estes largamente utilizados na limpeza de campos agrícolas, assim como em florestas. Foi ainda planeado desenvolver uma base de conhecimento no que diz respeito aos mecanismos de dano do ADN. Como último objectivo, pretendeu-se contribuir para a mitigação dos efeitos dos agroquímicos no biota aquático, nomeadamente em peixes, fornecendo dados científicos no sentido de melhorar as práticas agrícolas e florestais. Este estudo foi realizado adoptando a enguia europeia (Anguilla anguilla L.) como organismo-teste, e submetendo-a a exposições de curta duração (1 e 3 dias) dos produtos comerciais mencionados, em concentrações consideradas ambientalmente realistas. Para a avaliação da genotoxicidade foram aplicadas duas metodologias: o ensaio do cometa e o teste das anomalias nucleares eritrocíticas (ANE). Enquanto o ensaio do cometa detecta quebras na cadeia do ADN, um dano passível de ser reparado, o aparecimento das ANE revela lesões cromossomais, sinalizando um tipo de dano de difícil reparação. O ensaio do cometa foi ainda melhorado com uma nova etapa que incluiu a incubação com enzimas de reparação (FPG e EndoIII), permitindo perceber a ocorrência de dano oxidativo no ADN. No que diz respeito ao Roundup®, o envolvimento do sistema antioxidante como indicador de um estado próoxidante foi também alvo de estudo. Uma vez que as referidas formulações se apresentam sob a forma de misturas, o potencial genotóxico dos seus princípios activos foi também avaliado individualmente. No caso particular do Roundup®, também foram estudados o seu surfactante (amina polietoxilada; POEA) e o principal metabolito ambiental (ácido aminometilfosfórico; AMPA). Os resultados obtidos mostraram a capacidade do Roundup® em induzir tanto dano no ADN (em células de sangue, guelras e fígado) como dano cromossómico (em células de sangue). A investigação sobre o possível envolvimento do stresse oxidativo demonstrou que o tipo de dano no ADN varia com as concentrações testadas e com a duração da exposição. Deste modo, com o aumento do tempo de exposição, os processos relacionados com o envolvimento de espécies reactivas de oxigénio (ERO) ganharam preponderância como mecanismo de dano no ADN, facto que é corroborado pela activação do sistema antioxidante observado nas guelras, assim como pelo aumento dos sítios sensíveis a FPG em hepatócitos. O glifosato e o POEA foram também considerados genotóxicos. O POEA mostrou induzir uma maior extensão de dano no ADN, tanto comparado com o glifosato como com a mistura comercial. Apesar de ambos os componentes contribuirem para a genotoxicidade da formulação, a soma dos seus efeitos individuais nunca foi observada, apontando para um antagonismo entre eles e indicando que o POEA não aumenta o risco associado ao princípio activo. Deste modo, realça-se a necessidade de regulamentar limiares de segurança para todos os componentes da formulação, recomendando, em particular, a revisão da classificação do risco do POEA (actualmente classificado com “inerte”). Uma vez confirmada a capacidade do principal metabolito do glifosato – AMPA – em exercer dano no ADN assim como dano cromossómico, os produtos da degradação ambiental dos princípios activos assumem-se como um problema silencioso, realçando assim a importância de incluir o AMPA na avaliação do risco relacionado com herbicidas com base no glifosato. A formulação Garlon® e o seu princípio activo triclopir mostraram um claro potencial genotóxico. Adicionalmente, o Garlon® mostrou possuir um potencial genotóxico mais elevado do que o seu princípio activo. No entanto, a capacidade de infligir dano oxidativo no ADN não foi demonstrada para nenhum dos agentes. No que concerne à avaliação da progressão do dano após a remoção da fonte de contaminação, nem os peixes expostos a Roundup® nem os expostos a Garlon® conseguiram restaurar completamente a integridade do seu ADN ao fim de 14 dias. No que concerne ao Roundup®, o uso de enzimas de reparação de lesões específicas do ADN associado ao teste do cometa permitiu detectar um aparecimento tardio de dano oxidativo, indicando deste modo um decaimento progressivo da protecção antioxidante e ainda uma incapacidade de reparar este tipo de dano. O período de pós-exposição correspondente ao Garlon® revelou uma tendência de diminuição dos níveis de dano, apesar de nunca se observar uma completa recuperação. Ainda assim, foi evidente uma intervenção eficiente das enzimas de reparação do ADN, mais concretamente as direccionadas às purinas oxidadas. A avaliação das metodologias adoptadas tornou evidente que o procedimento base do ensaio do cometa, que detecta apenas o dano nãoespecífico no ADN, possui algumas limitações quando comparado com a metodologia que incluiu a incubação com as enzimas de reparação, uma vez que a última mostrou reduzir a possibilidade de ocorrência de resultados falsos negativos. Os dois parâmetros adoptados (ensaio do cometa e teste das ANE) demonstraram possuir aptidões complementares, sendo assim recomendado a sua utilização conjunta com vista a efectuar uma avaliação mais adequada do risco genotóxico. Globalmente, os resultados obtidos forneceram indicações de grande utilidade para as entidades reguladoras, contribuindo ainda para a (re)formulação de medidas de conservação do ambiente aquático. Neste sentido, os dados obtidos apontam para a importância da avaliação de risco dos herbicidas incluir testes de genotoxicidade. A magnitude de risco detectada para ambas as formulações adverte para a necessidade de adopção de medidas restritivas em relação à sua aplicação na proximidade de cursos de água. Como medidas mitigadoras de impactos ambientais, aponta-se o desenvolvimento de formulações que incorporem adjuvantes selecionados com base na sua baixa toxicidade.Herbicides are considered among the most hazardous contaminants of water bodies, since they easily reach these ecosystems through aerial spray drift, artificial drainage systems and surface or sub-surface runoff. The occurrence of these agrochemicals in the aquatic environment has been associated to deleterious effects in non-target organisms, namely fish. However, a considerable gap is evident regarding the scientific information on their genotoxic impact. Therefore, the present thesis was designed with the intention to evaluate the genotoxic risk to fish of the herbicide formulations Roundup® (glyphosate-based) and Garlon® (triclopyr-based), representing broadly used products worldwide to manage unwanted vegetation in agriculture and forestry. It was also planned to develop of a biologically base knowledge on DNA damage mechanisms. As ultimate goal, it was intended to contribute to mitigate the effects of agrochemicals in aquatic biota, namely fish, providing scientific data able to improve forestry and agriculture managing practices. The study was carried out adopting the European eel (Anguilla anguilla L.) as test organism and performing short-term exposures (1 and 3 days) to environmentally realistic concentrations of the mentioned commercial products. Two different genotoxic endpoints were adopted: comet and erythrocytic nuclear abnormalities (ENA) assays. The comet assay measures DNA stand breaks, a repairable type of damage, whereas the ENA assay identifies chromosomal lesions, signalizing a type of damage hardly repairable. The comet assay was also upgraded with an extra-step involving incubation with repair enzymes (FPG and EndoIII), in order to detect oxidative DNA damage. In what concerns to Roundup®, the involvement of the antioxidant system as indication of a pro-oxidant status was also assessed. Once the aforementioned formulations are presented as mixtures of chemicals, the genotoxic potential of their active ingredients individually was also assessed. In the case of Roundup®, the evaluation of the risk associated to the surfactant (polyethoxylated amine; POEA) and to the major environmental breakdown product of the active principle (aminomethylphosphonic acid; AMPA) was carried out as well. The results obtained showed the Roundup® ability to induce both DNA (in blood, gills and liver cells) and chromosomal damage (in blood cells). The investigation on the causative involvement of oxidative stress demonstrated that the type of DNA damage varies with tested concentrations and exposure duration. Thus, ROS-dependent processes gained preponderance as a mechanism of DNA damage with the increase of exposure length, which was corroborated by the antioxidant activation observed in gills as well as the net FPG-sensitive sites elevation detected in liver. Glyphosate and the surfactant POEA were also found to be genotoxic. Moreover, POEA induced the highest extent of DNA damage, when compared to glyphosate and the commercial mixture. Though both components showed to contribute to the overall genotoxicity of the herbicide formulation, the sum of their individual effects was never observed, pointing out an antagonistic interaction between them, indicating that POEA does not increase the risk associated to the active ingredient. These findings also emphasized the need to define regulatory thresholds for all the formulation components, recommending, in particular, the revision of the hazard classification of POEA (classified as “inert” until date). Since the ability of the main environmental metabolite of glyphosate - AMPA - in exert DNA and chromosomal damage was also confirmed, it was pointed out the silent problem that the products of environmental degradation of the active ingredients can constitute. In addition, the importance to include AMPA in risk assessment studies concerning the glyphosate-based herbicides was highlighted. The formulation Garlon® and its active ingredient triclopyr also showed a clear genotoxic potential. In addition, it was demonstrated the higher genotoxicity of the formulation, in comparison to the active ingredient. However, their ability in exert oxidative DNA damage could not be demonstrated. In what concerns to the evolution of the damage progression after removal of the contamination source, neither fish exposed to Roundup® nor Garlon® achieved a complete restoration of DNA integrity in 14 days. In relation to Roundup®, the use of the DNA lesion-specific repair enzymes allowed understanding the occurrence of a late oxidative DNA damage, indicating a progressive decay of cell antioxidant protection as well as the incapacity to repair this particular type of damage. The Garlon® post-exposure period revealed a tendency to decrease damage levels, although not enough to be regarded as an effective recovery. However, an efficient intervention of DNA repair enzymes specifically directed to oxidized purines became evident. Evaluating the performance of the adopted genotoxic endpoints, it was evident that the standard comet procedure, detecting only non-specific DNA damage, displayed some limitations when compared to the methodology that includes the incubation with the repair enzymes, since the latter reduced the possibility of false negative results. The two adopted endpoints (comet and ENA assays) demonstrated complementary aptitudes, being recommended their jointly application since it allows a more effective genotoxic risk assessment. Overall, the results obtained provided useful recommendations for policymaking, contributing to (re)formulate regulatory procedures for protecting the health of aquatic environment. In this direction, the data gathered in this work point to the importance of performing a genotoxic evaluation in order to actually determine the hazard posed by herbicides and their by-products. The magnitude of risk detected for both formulations strongly advise the adoption of restrictive measures in relation to their application in the proximity of watercourses. As mitigation measures, the development of formulations incorporating adjuvants selected on the basis of their lower toxicity emerged as a recommended path

Indicadores químicos, bioquímicos e citogenéticos em Liza aurata após exposição ambiental a mercúrio

Mestrado em ToxicologiaA presença de mercúrio no ambiente aquático é, actualmente, consideradacomo um problema à escala global, com repercussão generalizada em todo obiota. A conhecida contaminação por mercúrio numa área particular da Ria de Aveiro (Portugal) - Largo do Laranjo -serviu como motivação para o presente estudo. Foram definidos como objectivos centrais (i) a avaliação do estado dapopulação de Liza aurata (Tainha-garrento) nessa área e (ii) contribuir paraum melhor conhecimento da toxicidade do mercúrio e mecanismos subjacentes empeixes, clarificando a relação entre a contaminação no ambiente e efeitosquímicos, bioquímicos e citogenéticos. Deste modo, o presente trabalho encontra-se divido em duas componentes, sendo a primeira baseada na amostragem sazonal de espécimesselvagens de L. aurata. Em paralelo, foi levada a cabo uma segunda componente pondo em prática uma experiência in situ com animais da mesma espécie engaiolados durante 3 dias no Largo do Laranjo, em três locais diferentes (LAR1, LAR2 e LAR3, por ordem crescente de proximidade àfonte de contaminação). Para avaliar a influência da distância ao sedimento, em cadalocal foi colocada uma gaiola próxima do fundo e outra a alguns centímetros dasuperfície. Os resultados obtidos no Largo do Laranjo foram comparados, emambas as componentes, com resultados correspondentes relativos a uma áreapouco contaminada - S. Jacinto. O mercúrio total (Hgt) foi quantificado na coluna de água e no sedimento, assim como em três diferentes tecidos alvo –sangue, fígado e músculo. Ao nível bioquímico/metabólico, foram estudadas respostas de stresse oxidativo nofígado com a análise de enzimas antioxidantes como a catalase (CAT) e aglutationa-S-transferase (GST), do conteúdo total de glutationa (GSHt) e ainda da peroxidação lipídica (LPO). A interferência do mercúrio com o metabolismohepático de biotransformação associado ao citocromo P450 (P450) foi tambémanalisada através da actividade da etoxiresorufina-O-desetilase (EROD), assim como da determinação do conteúdo em P450 e da sua degradação (conteúdoem P420). Ao nível citogenético, foi avaliada a acção genotóxica do mercúriopor intermédio do teste das anomalias nucleares eritrocíticas (ANE). Tendo em conta os objectivos gerais anteriormente referidos, foi investigada a correlação entre o nível de Hgt no fígado e as respectivasrespostas metabólicas, assim como o Hgt no sangue e a frequência de ANE. Os peixes selvagens capturados no Laranjo não apresentaram alterações significativas nos parâmetros antioxidantes (excluindo um aumento esporádicodo conteúdo GSHt), nem dos níveis de LPO. Os parâmetros associados ao citocromo P450 também não evidenciaramdiferenças significativas. Em paralelo, foi observado um claro aumento dos níveis hepáticos de Hgtnos peixes desta área contaminada, o que pode serentendido como uma evidência de que o metal se encontra acumulado numaforma inócua. Os níveis de Hgtencontrados no músculo dos peixes provenientes do Laranjo foramsignificativamente elevados em todas as estações do ano, sem, contudo, exceder os limites regulamentados pela UE.Todavia, o potencial risco para a saúde humana não pode ser excluído dada aforte dependência da taxa de consumo de peixe de cada população. Este aspecto assume particular importância para o caso português, uma vez quePortugal é um dos maiores consumidores de peixe da Europa. Na avaliação da genotoxicidade, os peixes selvagens oriundos do Laranjo,pescados no Verão e Outono, demonstraram uma elevada frequência de ANE, evidenciando também uma correlação com os altos níveis de Hgtencontrados no sangue. Surpreendentemente, etendo em conta os níveis determinados no sangue, no Inverno não se observou indução de ANE. Numa tentativa deexplicar estefacto, foi avaliada a dinâmica hematológica pela determinação dafrequência de eritrócitos imaturos (EI), que sinalizou alterações (redução daeritropoiese e/ou o aumento da remoção de eritrócitos) capazes de mascarar aexpressão de genotoxicidade. No que respeita à experiência in situ, os peixes engaiolados no Laranjo mostraram um aumento generalizado no Hgthepático, sendo acompanhado pelos aumentos da actividade da CAT e de GSHt, assim como pela inibição daactividade da EROD. As gaiolas de fundo em LAR2 e LAR3 mostraram um aumento dos níveis hepáticos de Hgt e do conteúdo de GSHt, respectivamente, quando comparadas com as gaiolas de superfície, sublinhando a importânciada proximidade ao sedimento. Foi ainda demonstrada a relevância da absorçãodo mercúrio a partir da água, uma vez que a absorção via alimento foirestringida nesta componente. Analisando o efeito genotóxico do mercúrio,observou-se uma induçãode ANE no local mais próximo da fonte de contaminação (LAR3). Deste modo, foi possível observar uma correlação entre o Hgt acumulado no sangue e a genotoxicidade expressa pela frequência deANE. No geral, quer os peixes selvagens, quer os engaiolados, revelaram maioracumulação de Hgtno fígado, quando comparado com os níveis encontradosno músculo e no sangue. Assim, o fígado revelou-se como o órgão com maiorcapacidade de bioacumulação, provavelmente devido ao seu activo papel nadesintoxicação de xenobióticos. A ausência de dano peroxidativo neste órgãopoderá ser atribuída a esta capacidade de desintoxicação e/ou a uma eficaz defesa antioxidante. Globalmente, o mercúrio mostrou ser um agente genotóxico, tendoevidenciado a capacidade de inibir algumas respostas enzimáticas, não sepodendo, com base nos dados obtidos, considerar um indutor de stresse oxidativo. A estratégia adoptada, baseada na análise integrada de diferentesindicadores aplicados ao mugilídeo L. aurata, demonstrou a sua aplicabilidade na monitorização ambiental de contaminação por metais em águas costeiras.Mercury contamination of aquatic ecosystems became a worldwideenvironmental problem, representing a worrying threat to biota. In this context, the intense mercury contamination historically observed at Laranjo basin, in Ria de Aveiro (Portugal), was the motivation for the present study.Hence, the main goals of this research were (i) to evaluate the health status ofthe local Liza aurata (gold grey mullet) population, and (ii) to improve the knowledge about mercury toxicity and its subjacent mechanisms, clarifying therelation between the environmental contamination and chemical, biochemicaland cytogenetic effects in fish. The present investigation is divided in two components, being the first based on a wild L. aurataseasonal sampling at the contaminated area (Laranjo basin = LAR), and the second, an in situ experiment where L. aurataspecimens were caged, during 3 days, at three different sites (LAR1, LAR2 and LAR3, increasing proximity to the mercury contamination source). To assessthe influence of the sediment distance on fish responses, at each site, one cagewas placed closed to the bottom and other in the surface. For bothcomponents, the results obtained at Laranjo basin were compared with the corresponding results in a low contaminated area – S. Jacinto. Total mercury (Hgt) concentration was quantified in the water column andsediment, as well as in three different target tissues – blood, liver and muscle. Biochemical/metabolic effects were assessed by measuring the followinghepatic oxidative stress indicators: catalase (CAT) and glutathione-S-transferase (GST) activities, total glutathione content (GSHt) and lipid peroxidation (LPO). In addition, the mercury interference on the hepatic cytochrome P450 (P450) dependent biotransformation was evaluated throughethoxyresorufin-O-deethylase (EROD) activity, total P450 content and itsdegradation (P420 content). In a cytogenetic level, the mercury genotoxicity was determined, using the erythrocytic nuclear abnormalities (ENA) assay.Taking into account the previous objectives, the correlation between Hgtlevels in liver and blood and respective responses was also investigated. Wild fish from Laranjo showed noalterations in antioxidant parameters (excluding a sporadic GSHtincrease), LPO or P450 parameters. Considering the concomitant increment on hepatic Hgtlevels, these results were regarded as an indication that metal was accumulated in a detoxified form. Muscle Hgt concentration was significantly increased in L. auratacaptured at Laranjo for all sampling seasons, although the EU regulatory limit was notexceeded. However, the potential risk to human health can not be excluded,taking into account the high fish consumption rate observed in Portugal. Wild L. auratafrom Laranjo displayed elevated ENA frequency in summerand autumn in concomitance with increased blood Hgt levels. Surprisingly, no ENA induction was observed in winter, where the highest blood Hgtlevel was measured. This particular result may be explained by an altered haematologicaldynamics related to high blood Hgtlevels, as supported by a decreased immature erythrocytes frequency, affecting the ENA generation. Considering the in situ experiment, fish caged at Laranjo showed a general hepatic Hgt increase, followed by an elevation of CAT activity and GSHt content, as well as an EROD activity inhibition. Bottom cages from LAR2 andLAR3 revealed higher hepatic Hgt levels and GSHt content, respectively, when compared with surface cages, underlining the importance of sedimentproximity. This caging experiment also demonstrated the relevance of aqueousmercury uptake, since food intake was restricted due to caging. Analyzing themercury genotoxicity, an ENA frequency induction was observed in LAR3, theclosest site to the contamination source. Moreover, a correlation between bloodHgt concentration and ENA frequency was found. Globally, both wild and caged fish revealed that liver accumulateshigher mercury concentrations, when compared with blood and muscle. Thus, liverwas found to be the organ with the most bioaccumulation capacity, probablydue to its role in xenobiotics detoxification. The absence of mercury-induced hepatic peroxidative damage can be attributed to an effective detoxificationand antioxidant defense. Mercury showed to be a genotoxic agent, being alsoresponsible by enzyme inhibition. However, based on the obtained results, thismetal was not confirmed as an oxidative stress inducer. The adopted strategy based on the integrated evaluation of differentindicators, using the mugilide L. aurataas biosensor, demonstrated its usefulness on the assessment of metal pollution in coastal ecosystems

Evaluation of DNA Damage Induced by Environmental Exposure to Mercury in Liza aurata Using the Comet Assay

Mercury (Hg) is one of the major aquatic contaminants even though emissions have been reduced over the years. Despite the relative abundance of investigations carried out on Hg toxicity, there is a scarcity of studies on its DNA damaging effects in fish under realistic exposure conditions. This study assessed the Hg genotoxicity in Golden grey mullets (Liza aurata) at Laranjo basin, a particularly contaminated area of Ria de Aveiro (Portugal) well known for its Hg contamination gradient. (1) Fish were seasonally caught at Laranjo basin and at a reference site (S. Jacinto), and (2) animals from the reference site were transplanted and caged (at bottom and surface), for 3 days, in two different locations within Laranjo basin. Using the comet assay, blood was analyzed for genetic damage and apoptotic cell frequency. The seasonal survey showed greater DNA damage in the Hg-contaminated area for all sampling seasons excluding winter. The temporal variation pattern of DNA lesions was: summer ≈ autumn > winter > spring. Fish caged at Laranjo also exhibited greater DNA damage than those caged at the reference site, highlighting the importance of gill uptake on the toxicity of this metal. No increased susceptibility to apoptosis was detected in either wild or caged fish, indicating that mercury damages DNA of blood cells by a nonapoptotic mechanism. Both L. aurata and the comet assay proved to be sensitive and suitable for genotoxicity biomonitoring in mercury-contaminated coastal systems

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved

Epidemiology of intra-abdominal infection and sepsis in critically ill patients: "AbSeS", a multinational observational cohort study and ESICM Trials Group Project

PURPOSE: To describe the epidemiology of intra-abdominal infection in an international cohort of ICU patients according to a new system that classifies cases according to setting of infection acquisition (community-acquired, early onset hospital-acquired, and late-onset hospital-acquired), anatomical disruption (absent or present with localized or diffuse peritonitis), and severity of disease expression (infection, sepsis, and septic shock). METHODS: We performed a multicenter (n = 309), observational, epidemiological study including adult ICU patients diagnosed with intra-abdominal infection. Risk factors for mortality were assessed by logistic regression analysis. RESULTS: The cohort included 2621 patients. Setting of infection acquisition was community-acquired in 31.6%, early onset hospital-acquired in 25%, and late-onset hospital-acquired in 43.4% of patients. Overall prevalence of antimicrobial resistance was 26.3% and difficult-to-treat resistant Gram-negative bacteria 4.3%, with great variation according to geographic region. No difference in prevalence of antimicrobial resistance was observed according to setting of infection acquisition. Overall mortality was 29.1%. Independent risk factors for mortality included late-onset hospital-acquired infection, diffuse peritonitis, sepsis, septic shock, older age, malnutrition, liver failure, congestive heart failure, antimicrobial resistance (either methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, extended-spectrum beta-lactamase-producing Gram-negative bacteria, or carbapenem-resistant Gram-negative bacteria) and source control failure evidenced by either the need for surgical revision or persistent inflammation. CONCLUSION: This multinational, heterogeneous cohort of ICU patients with intra-abdominal infection revealed that setting of infection acquisition, anatomical disruption, and severity of disease expression are disease-specific phenotypic characteristics associated with outcome, irrespective of the type of infection. Antimicrobial resistance is equally common in community-acquired as in hospital-acquired infection.status: publishe

Antimicrobial Lessons From a Large Observational Cohort on Intra-abdominal Infections in Intensive Care Units

evere intra-abdominal infection commonly requires intensive care. Mortality is high and is mainly determined by disease-specific characteristics, i.e. setting of infection onset, anatomical barrier disruption, and severity of disease expression. Recent observations revealed that antimicrobial resistance appears equally common in community-acquired and late-onset hospital-acquired infection. This challenges basic principles in anti-infective therapy guidelines, including the paradigm that pathogens involved in community-acquired infection are covered by standard empiric antimicrobial regimens, and second, the concept of nosocomial acquisition as the main driver for resistance involvement. In this study, we report on resistance profiles of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis and Enterococcus faecium in distinct European geographic regions based on an observational cohort study on intra-abdominal infections in intensive care unit (ICU) patients. Resistance against aminopenicillins, fluoroquinolones, and third-generation cephalosporins in E. coli, K. pneumoniae and P. aeruginosa is problematic, as is carbapenem-resistance in the latter pathogen. For E. coli and K. pneumoniae, resistance is mainly an issue in Central Europe, Eastern and South-East Europe, and Southern Europe, while resistance in P. aeruginosa is additionally problematic in Western Europe. Vancomycin-resistance in E. faecalis is of lesser concern but requires vigilance in E. faecium in Central and Eastern and South-East Europe. In the subcohort of patients with secondary peritonitis presenting with either sepsis or septic shock, the appropriateness of empiric antimicrobial therapy was not associated with mortality. In contrast, failure of source control was strongly associated with mortality. The relevance of these new insights for future recommendations regarding empiric antimicrobial therapy in intra-abdominal infections is discussed.Severe intra-abdominal infection commonly requires intensive care. Mortality is high and is mainly determined by diseasespecific characteristics, i.e. setting of infection onset, anatomical barrier disruption, and severity of disease expression. Recent observations revealed that antimicrobial resistance appears equally common in community-acquired and late-onset hospital-acquired infection. This challenges basic principles in anti-infective therapy guidelines, including the paradigm that pathogens involved in community-acquired infection are covered by standard empiric antimicrobial regimens, and second, the concept of nosocomial acquisition as the main driver for resistance involvement. In this study, we report on resistance profiles of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis and Enterococcus faecium in distinct European geographic regions based on an observational cohort study on intra-abdominal infections in intensive care unit (ICU) patients. Resistance against aminopenicillins, fluoroquinolones, and third-generation cephalosporins in E. coli, K. pneumoniae and P. aeruginosa is problematic, as is carbapenem-resistance in the latter pathogen. For E. coli and K. pneumoniae, resistance is mainly an issue in Central Europe, Eastern and South-East Europe, and Southern Europe, while resistance in P. aeruginosa is additionally problematic in Western Europe. Vancomycin-resistance in E. faecalis is of lesser concern but requires vigilance in E. faecium in Central and Eastern and South-East Europe. In the subcohort of patients with secondary peritonitis presenting with either sepsis or septic shock, the appropriateness of empiric antimicrobial therapy was not associated with mortality. In contrast, failure of source control was strongly associated with mortality. The relevance of these new insights for future recommendations regarding empiric antimicrobial therapy in intra-abdominal infections is discussed

Poor timing and failure of source control are risk factors for mortality in critically ill patients with secondary peritonitis

Purpose: To describe data on epidemiology, microbiology, clinical characteristics and outcome of adult patients admitted in the intensive care unit (ICU) with secondary peritonitis, with special emphasis on antimicrobial therapy and source control. Methods: Post hoc analysis of a multicenter observational study (Abdominal Sepsis Study, AbSeS) including 2621 adult ICU patients with intra-abdominal infection in 306 ICUs from 42 countries. Time-till-source control intervention was calculated as from time of diagnosis and classified into 'emergency' (< 2 h), 'urgent' (2-6 h), and 'delayed' (> 6 h). Relationships were assessed by logistic regression analysis and reported as odds ratios (OR) and 95% confidence interval (CI). Results: The cohort included 1077 cases of microbiologically confirmed secondary peritonitis. Mortality was 29.7%. The rate of appropriate empiric therapy showed no difference between survivors and non-survivors (66.4% vs. 61.3%, p = 0.1). A stepwise increase in mortality was observed with increasing Sequential Organ Failure Assessment (SOFA) scores (19.6% for a value ≤ 4-55.4% for a value > 12, p < 0.001). The highest odds of death were associated with septic shock (OR 3.08 [1.42-7.00]), late-onset hospital-acquired peritonitis (OR 1.71 [1.16-2.52]) and failed source control evidenced by persistent inflammation at day 7 (OR 5.71 [3.99-8.18]). Compared with 'emergency' source control intervention (< 2 h of diagnosis), 'urgent' source control was the only modifiable covariate associated with lower odds of mortality (OR 0.50 [0.34-0.73]). Conclusion: 'Urgent' and successful source control was associated with improved odds of survival. Appropriateness of empirical antimicrobial treatment did not significantly affect survival suggesting that source control is more determinative for outcome

Epidemiology of intra-abdominal infection and sepsis in critically ill patients: "AbSeS", a multinational observational cohort study and ESICM Trials Group Project

Purpose To describe the epidemiology of intra-abdominal infection in an international cohort of ICU patients according to a new system that classifies cases according to setting of infection acquisition (community-acquired, early onset hospital-acquired, and late-onset hospital-acquired), anatomical disruption (absent or present with localized or diffuse peritonitis), and severity of disease expression (infection, sepsis, and septic shock). Methods We performed a multicenter (n = 309), observational, epidemiological study including adult ICU patients diagnosed with intra-abdominal infection. Risk factors for mortality were assessed by logistic regression analysis. Results The cohort included 2621 patients. Setting of infection acquisition was community-acquired in 31.6%, early onset hospital-acquired in 25%, and late-onset hospital-acquired in 43.4% of patients. Overall prevalence of antimicrobial resistance was 26.3% and difficult-to-treat resistant Gram-negative bacteria 4.3%, with great variation according to geographic region. No difference in prevalence of antimicrobial resistance was observed according to setting of infection acquisition. Overall mortality was 29.1%. Independent risk factors for mortality included late-onset hospital-acquired infection, diffuse peritonitis, sepsis, septic shock, older age, malnutrition, liver failure, congestive heart failure, antimicrobial resistance (either methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, extended-spectrum beta-lactamase-producing Gram-negative bacteria, or carbapenem-resistant Gram-negative bacteria) and source control failure evidenced by either the need for surgical revision or persistent inflammation. Conclusion This multinational, heterogeneous cohort of ICU patients with intra-abdominal infection revealed that setting of infection acquisition, anatomical disruption, and severity of disease expression are disease-specific phenotypic characteristics associated with outcome, irrespective of the type of infection. Antimicrobial resistance is equally common in community-acquired as in hospital-acquired infection