Synergistic effects of combinations of antibiotics on coagulase negative staphylococcal (CoNS) biofilms

Background. Previous studies examining synergistic effects of antibiotics against CoNS biofilms often relied on drug concentrations higher than the peak serum (PS) concentration, as determined by MIC assays, therefore having little clinical applicability. Other studies investigated synergistic effects of a wide range of antibiotics, however many are not routinely used in a clinical setting. Dicloxacillin is the major antibiotic used in Portugal to treat staphylococci infections, and we evaluated the synergistic effect of antibiotics with different mechanisms of action combined with dicloxacillin and other cell wall synthesis inhibitors. Methods. We used combinations of 5 antibiotic agents (cefazolin, vancomycin, dicloxacillin, tetracycline and rifampicin) at the PS concentration of each agent, and evaluated the killing rate over a 24 h period using biofilms made by 10 CoNS clinical isolates. Results from all of the assays were compared using ANOVA. Results. Among all of the combinations tested there was an increase in the killing rate of the antibiotics when compared to the killing rates when only one antibiotic was used. However, only the combination of dicloxacillin with rifampicin resulted in a true synergistic effect for most CoNS strains. When tetracycline was used in combination with any other antibiotic, a decrease in the killing rate was obtained. Conclusions. Our results indicate that dicloxacillin could be useful against CoNS biofilms when combined with a small amount of rifampicin (10 μg/ml). Antibiotics highly effective against planktonic cells, like vancomycin, were minimally effective against cells in biofilms, even after adding more antibiotics with distinct mechanisms of action. Also, except for dicloxacillin, adding more antibiotics to the biofilm did not result in an increase in killing efficiency, and there were even antagonistic in some instances. Screening for synergistic effects of different combinations of antibiotics might be a useful tool to determine a therapeutic approach to infections involving CoNS biofilms

A study on the immune response elicited in mice challenged with Staphylococcus epidermidis planktonic - or biofilm - grown cells

In this study a biofilm-forming strain of Staphylococcus epidermidis was used in order to evaluate, in a murine model, the immune response to bacteria grown either as planktonic cells or biofilm. Groups of male BALB/c mice were infected intra-peritoneally (i.p.), with 2×10^8 S. epidermidis cells per mice, obtained either from a mature biofilm (grown for 48h) or from a planktonic cell culture. Mice were sacrificed 6h, 24h and 8 days after the i.p. infection. Flow cytometric and qualitative cytospin analysis of cells recovered from peritoneal exsudates, 24h after infection, indicates that the bacterial cells obtained from the biofilm were less inflammatory than planktonic. Splenomegaly was observed in mice 8 days after the bacterial challenge, more pronounced in mice challenged with planktonic cells. Flow cytometric analysis of spleen cells showed in both groups of bacteria-challenged mice an increase in the number of lymphocyte cells, more marked in the animals challenged with planktonic cells. The isotypic profile of the imunoglobulins present in the sera of each group of mice was analysed by ELISA. Extracellular polymeric substance (EPS) isolated from a S. epidermidis biofilm grown for 48h inhibited in vitro nitrite production by macrophages stimulated with LPS and interferon-gamma. Our results indicate that S. epidermidis biofilm-grown cells were less inflammatory than planktonic cells, and suggest that EPS could diminish the inflammatory response elicited in the host by this bacterium

Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry

Published by Oxford University Press on behalf of the British Society for Antimicrobial ChemotherapyObjectives: To quantitatively compare the antibiotic susceptibility of biofilms formed by the coagulasenegative staphylococci (CoNS) Staphylococcus epidermidis and Staphylococcus haemolyticus with the susceptibility of planktonic cultures. Methods: SeveralCoNSstrains were grown planktonically or as biofilms to determine the effect of themode of growth on the level of susceptibility to antibiotics with different mechanisms of action. The utility of a new, rapid colorimetric method that is based on the reduction of a tetrazolium salt (XTT) to measure cell viability was tested by comparison with standard bacterial enumeration techniques. A 6 h kinetic study was performed using dicloxacillin, cefazolin, vancomycin, tetracycline and rifampicin at the peak serum concentration of each antibiotic. Results: In planktonic cells, inhibitors of cell wall synthesis were highly effective over a 3 h period. Biofilms were much less susceptible than planktonic cultures to all antibiotics tested, particularly inhibitors of cell wall synthesis. The susceptibility to inhibitors of protein and RNA synthesis was affected by the biofilm phenotype to a lesser degree. Standard bacterial enumeration techniques and the XTT method produced equivalent results both in biofilms and planktonic assays. Conclusions: This study provides a more accurate comparison between the antibiotic susceptibilities of planktonic versus biofilm populations, because the cell densities in the two populations were similar and because we measured the concentration required to inhibit bacterial metabolism rather than to eradicate the entire bacterial population. While the biofilm phenotype is highly resistant to antibiotics that target cell wall synthesis, it is fairly susceptible to antibiotics that target RNA and protein synthesis.Fundação para a Ciência e a Tecnologia (FCT

Dormant bacteria within Staphylococcus epidermidis biofilms have low inflammatory properties and maintain tolerance to vancomycin and penicillin after entering planktonic growth

Staphylococcus epidermidis is the most commonly isolated etiological agent of nosocomial infections mainly due to its ability to establish biofilms on indwelling medical devices. Detachment of bacteria from S. epidermidis biofilms and subsequent growth in the planktonic form is a hallmark in the pathogenesis of these infections leading to dissemination. Here we showed that S. epidermidis cells collected from biofilms cultured in conditions that promote cell viability present marked changes in their physiological status upon initiating a planktonic mode of growth. When compared to cells growing in biofilms, they displayed an increased SYBR green I staining intensity, increased transcription of the rpiA gene, decreased transcription of icaA gene as well as higher susceptibility to vancomycin and penicillin antibiotics. When bacteria collected from biofilms with high proportions of dormant cells were subsequently cultured in the planktonic mode, a large proportion of cells maintained a low SYBR staining intensity and increased resistance to vancomycin and penicillin, a profile typical of dormant cells. This phenotype further associated with a decreased ability of these biofilm-derived cells to induce the production of pro-inflammatory cytokines by bone marrow-derived dendritic cells in vitro, as determined by pro-inflammatory cytokine quantification. These results demonstrated that cells detached from the biofilm maintain a dormant cell-like phenotype, having a low pro-inflammatory effect and decreased susceptibility to antibiotics suggesting these cells may contribute for the recalcitrant nature of biofilm infections.This work was funded by Fundacao para a Ciencia e a Tecnologia (FCT) and COMPETE grants PTDC/BIA-MIC/113450/2009 and FCOMP-01-0124-FEDER-014309. Filipe Cerca, Angela Franca and Virginia Carvalhais were funded by FCT fellowships SFRH/BD/27638/2006, SFRH/BD/62359/2009 and SFRH/BD/78235/2011, respectively. Parts of the work were also supported by National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIAID) grants AI46706 and AI057159, a component of award number U54 AI057159. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health. The authors acknowledge the technical assistance of Encarnacao Rebelo

Staphylococcus epidermidis biofilms with higher proportions of dormant bacteria induce a lower activation of murine macrophages

Staphylococcus epidermidis an opportunistic pathogen due to its ability to establish biofilms on indwelling medical devices. The presence of high amounts of dormant bacteria is a hallmark of biofilms, making them more tolerant to antimicrobials and to the host immune response. We observed that S. epidermidis biofilms grown in excess glucose accumulated high amounts of viable but non-culturable (VBNC) bacteria, as assessed by their low ratio of culturable bacteria over the number of viable bacteria. This effect, which was a consequence of the accumulation of acidic compounds due to glucose metabolism, was counteracted by high extracellular levels of calcium and magnesium added to the culture medium allowing modulation of the proportions of VBNC bacteria within S. epidermidis biofilms. Using bacterial inocula obtained from biofilms with high and low proportions of VBNC bacteria, their stimulatory effect on murine macrophages was evaluated in vitro and in vivo. The inoculum enriched in VBNC bacteria induced in vitro a lower production of TNF-α, interleukin-1 and interleukin-6 by bone-marrow-derived murine macrophages and, in vivo, a lower stimulatory effect on peritoneal macrophages, assessed by increased surface expression of Gr1 and MHC class II molecules. Overall, these results show that environmental conditions, such as pH and extracellular levels of calcium and magnesium, can account to induce dormancy in S. epidermidis biofilms. Moreover, they show that bacterial suspensions enriched in dormant cells are less inflammatory suggesting that dormancy can contribute to the immune evasion of biofilms.This work was supported by Fundação para a Ciência e a Tecnologia (FCT) PTDC/BIAMIC/113450/2009 and FEDER FCOMP-01-0124-FEDER-014679. FC, AF and EBA were respectively supported by FCT fellowships SFRH/BD/27638/ 2006, SFRH/BD/62359/2009 and SFRH/BD/38380/200

É necessário debater a política de emprego científico em Portugal

Artigo de opinião publicado no jornal "Público", Domingo, 4 de Junho de 2023.[Excerto] O Estatuto de Carreira de Investigação Científica foi publicado há 24 anos, com o intuito de promover investigação de qualidade e reforçar as instituições com pessoal altamente qualificado e integrado. Parece-nos, no entanto, óbvio que o sistema científico tem falhado numa componente essencial: condições estáveis para se fazer ciência em Portugal. Faltam soluções efetivas para combater a precariedade, permitindo aos investigadores que se possam focar no essencial: fazer investigação científica de excelência

Extracellular matrix in Staphylococcus epidermidis biofilms: a consequence of bacterial production or cell wall degradation?

Staphylococcus epidermidis is a leading pathogen accounting for nosocomial infections. The ability to form biofilms is considered the major virulence factor of this bacterium. The hallmark of this type of infection is the presence of an extracellular polymeric matrix that, in the case of S. epidermidis biofilms, is mainly constituted by an N-acetylglucosamine polymer. We have identified a subpopulation of bacteria that we believe to be the responsible for the extracellular matrix accumulation in S. epidermidis biofilms as they have comparative significant higher amount of surface N-acetylglucosamine. Flow cytometric evaluation of cell wall permeability and transmission electronic microscopy are highly suggestive of primary wall degradation in these bacteria. In overall, these results suggest that the extracellular matrix in S. epidermidis biofilms is a consequence of the degradation of the bacteria cell wall and that propide iodium should be used with care when used as a marker for bacteria dead in biofilms

Questionário endereçado à comunidade académica sobre perspetivas de carreiras para doutorados

Relatório elaborado pela Comissão Especializada de Educação, Investigação e Interação com a Sociedade (CEEIIS)[Sumário executivo] Mais de vinte anos passados sobre a publicação do Estatuto da Carreira de Investigação Científica é natural que a comunidade Académica se interrogue sobre as suas limitações e eventual necessidade de adaptação à realidade atual. Também na Universidade do Minho o muito significativo aumento do número de Investigadores tornou esta questão mais premente. É neste contexto que o Conselho Geral da Universidade do Minho decidiu auscultar a opinião da comunidade sobre a Carreira de Investigação, e possíveis evoluções da mesma, e eventuais outras Carreiras para Doutorados, quer a nível nacional, quer a nível interno. Aproveitou-se também esta oportunidade para fazer o levantamento das condições de trabalho dos investigadores e de algumas questões conexas. Para tal foram disponibilizados questionários online anónimos destinados aos Investigadores/as, Docentes e Diretores/as dos Centros de Investigação da Universidade do Minho. Foram recebidas 1779 respostas a este inquérito, distribuídas da seguinte forma: 1236 respostas de Investigadores/as, 517 respostas de Docentes e 26 respostas das Direções dos Centros de Investigação. Tal corresponde a taxas de resposta de 84,3% para os/as Investigadores/as doutorados (e 46,5% incluindo os investigadores/as não doutorados), 38,1% para os/as Docentes e 54,2% para as Direções de Centro inquiridas. No que diz respeito ao caminho a seguir relativamente à estrutura da carreira de investigação na UMinho, 95% dos/as respondentes Investigadores/as concordam que a carreira de investigação necessita de uma revisão legislativa. A maioria (63%) considera que a carreira de investigação deve ser mantida separada da Carreira Docente, sendo que 53% dos/as respondentes não concorda com a possibilidade da fusão das duas carreiras. Em concordância com os dados dos investigadores/as, a ampla maioria (91%) dos/as docentes concorda com a necessidade de uma revisão legislativa da Carreira de Investigação. No entanto 55% dos respondentes Docentes consideram que as duas carreiras devem manter-se separadas. Verifica-se nas respostas recebidas um consenso alargado em que o financiamento público é insuficiente para suportar de forma sustentada as posições necessárias no âmbito da Carreira de Investigação. É de assinalar que 91% dos/as respondentes Investigadores/as concordam que para os/as doutorados/as que não pretendam assumir funções de liderança científica deve ser criada uma nova carreira técnica de apoio à ciência. Adicionalmente 81% dos/as respondentes concordam com a integração da posição de Investigador Júnior na Carreira de Investigação, sendo que apenas 22% dos/as respondentes consideram que a posição de Investigador Júnior deve existir apenas para contratos temporários. A população docente diverge da opinião da população investigadora em relação a este último ponto, sendo que 53% dos/as respondentes consideram que a posição de Investigador Júnior deverá apenas ser incluída como posição temporária e não de carreira. A totalidade das direções de centros respondentes considera importante ou muito importante a criação de uma posição de técnico/a doutorado/a de gestão de ciência. 92% considera importante ou muito importante a criação da posição de técnico/a doutorado/a de comunicação em ciência, e 74% considera importante ou muito importante a criação da posição de técnico/a doutorado/a de laboratório. Foram também incluídas perguntas sobre a avaliação de desempenho, com respostas que apontam para a pertinência da revisão das regras e processo associados, bem como sobre as condições de trabalho científico na Universidade do Minho. Os indicadores melhor valorados pelos/as Investigadores/as foram as bibliotecas (72% consideraram-nas adequadas ou muito adequadas), os laboratórios de investigação (65%) e as salas de reuniões (64%). Já os espaços de socialização (27% consideraram-nos desadequados ou muito desadequados), o acesso a equipamentos científicos (26%) e o apoio administrativo (20%) foram os indicadores menos positivos. Verificou-se maior diversidade de opiniões entre Docentes e Diretores/as de Centros de Investigação. A comunidade de Investigadores/as da Universidade do Minho foi também auscultada relativamente a diversos aspetos da sua participação em processos eleitorais destinados à eleição de órgãos da Unidade Orgânica que integram. 74% dos/as respondentes indicou nunca ter participado (em qualquer capacidade) numa iniciativa deste tipo. Quanto aos motivos para essa não participação, pouco menos de metade dos/as respondentes indicou não ser elegível, com cerca de um quarto a indicar falta de oportunidade. Não obstante o reduzido envolvimento reportado, uma larga maioria dos/as Investigadores/as considera importante a representação deste corpo nos vários órgãos de governo da UMinho, matéria que poderá merecer reflexão por parte da Universidade. Mais do que apontar conclusões, o presente relatório pretende documentar as respostas recebidas por parte dos/as Investigadores/as, Docentes e Direções de Centros de Investigação, na convicção de que estas podem fornecer um importante contributo para a reflexão em curso, no País, bem como na Universidade do Minho, sobre a possível evolução da Carreira de Investigação e a criação de novas carreiras de apoio à atividade científica, no quadro das políticas de emprego científico. O inquérito e o presente relatório foram elaborados por um grupo de trabalho constituído por Investigadores e Docentes da Universidade do Minho, que foi apoiado por um grupo consultivo constituído por Ana Maria Marote, Anabela Alves, Anabela Gonçalves, António Salgado, Artur Ribeiro, Bruna Silva, Cláudia Botelho, Cristiana Abay, Diana Ferreira da Conceição, Diego Martínez, Elizabete Mercês, Filipe Teixeira Gil, Giuseppe Ballacci, Hugo Oliveira, Isabel Macedo, Jorge Ribeiro, Nídia Sousa, Pedro Domingues, Renato Gonçalves, Ricardo Vilaça, Richard Breia, Silvana Martins, Sílvia Monteiro, Sílvio Santos, Vítor Correlo Silva e Vítor Correia