Interactions of Pseudomonas aeruginosa and Staphylococcus aureus in biofilm-related infections: insights through network reconstruction and creation of a new online database

Introduction: Despite important advances in biofilm research, these consortia remain a critical concern for many biomedical applications. Their naturally occurring polymicrobial nature is characterised by the development of complex communities, where pathogen interactions can promote disease progression and severity. Intra- and inter-species communication within these consortia is majorly regulated by quorum-sensing, affecting the expression of virulence factors and biofilm formation, making it a promising target for new anti-infective strategies. P. aeruginosa and S. aureus are two major pathogens that co-occur in many biofilm-related infections and whose competitive interaction is highly related to infection resilience. Hypothesis and aims: Information on P. aeruginosa-S. aureus interactions is currently scattered in the ever-growing scientific literature, making it difficult for researchers to grasp critical information. Therefore, this study aimed at systematically collecting and analysing experimental information presented in the biomedical literature on the molecular basis of P. aeruginosa-S. aureus interactions, identifying promising therapeutic targets, and making this data available to the research community. Methodology: Full-text papers were optimally retrieved from PubMed and classified by their relevance. Interaction data was methodically annotated, reconstructed as networks to identify promising therapeutic targets, and integrated with specialized databases to identify promising antimicrobials. A new online database was created to deposit the gathered interaction data in searchable format. Results: Network analysis revealed key entities regulating P. aeruginosa-S. aureus interactions, for instance the PqsABCDE/PqsR quorum-sensing system, which affects S. aureus growth and biofilm formation. By identifying the most reported P. aeruginosa virulence factors affecting S. aureus, e.g. HQNO and siderophores, a list of experimentally validated agents affecting those factors, ranging from synthetic drugs to natural plant extracts, was constructed. Conclusion: The complex experimental data on P. aeruginosa-S. aureus interactions was for the first time thoroughly retrieved, systematized, and made publically available in the new Inter-Species CrossTalk Database (www.ceb.uminho.pt/ISCTD).Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit; European Regional Development Fund under the scope of Norte 2020 - Programa Operacional Regional do Norte for the BioTecNorte operation (NORTE - 01 - 0145 - FEDER - 000004 ) ; COMPETE 2020 and FCT for the project POCI - 01 - 0145 - FEDER - 029841 . The authors also thank FCT for the PhD Grant of Andreia Patricia Magalhães [grant number SFRH/BD/ 132165 / 2017 ] and ESCMID for the Young Scientist Members Attendance Grantinfo:eu-repo/semantics/publishedVersio

The fate of adhering bacteria on antimicrobial surfaces: transcriptomic analysis of resistance associated genes and macrophage-mediated phagocytosis

Book of Abstracts of CEB Annual Meeting 2017info:eu-repo/semantics/publishedVersio

Pseudomonas aeruginosa-Candida albicans polymicrobial biofilms in ventilator-associated infections (VAP): evaluating the post-antimicrobial effect of amphotericin B/polymyxin B combined activity

Introduction: Mixed bacterial-fungal colonization of the endotracheal tubes is now evident, with microbial interplay withstanding common antimicrobial therapy and paying for persistent and severe VAP infections. While alternative therapeutic strategies effectively targeting inter-kingdom biofilms are required, the role of each microorganism need to be appraised to deliver effective treatments. Hypothesis and aims: We earlier reported the combination therapy involving polymyxin B (PMB) and amphotericin B (AMB) as holding an attractive therapeutic option to treat dual-species biofilms. This study aimed to determine the post-antimicrobial phenomenon of PMB/AMB combined action in P. aeruginosa (PA) +Candida albicans (CA) biofilms, and to ascertain the events underlying biofilm growth restoration. Methodology: Post-antimicrobial effect of PMB combined with AMB was assessed in 24-h dual-species biofilms. Cell culturability and viability were evaluated by CFU and Live/Dead staining, respectively. The gene expression profile was assessed by qPCR. Results: Results showed that PA+CA biofilms lost their culturability straightaway being exposed to PMB/AMB combined solution. However, 24h was enough to both species recover their growth onto agar medium, with microbial counts approximating those observed for pre-treated biofilms. Following the subsequent treatment cycle, CFU estimation was only slightly disturbed. L/D results revealed that PA and CA populations displayed a compromised status at the end of the first PMB/AMB treatment cycle. Finishing the 24-h-regrowth cycle, most biofilm-encased species exhibited viability, which endured after the second treatment period. Transcriptional analysis of dual-species biofilms exposed to PMB/AMB combined action showed a high expression level in all PA resistance-encoded genes anrB, galU, mexA and algD and in ERG3 and ALS2 CA genes. Conclusion: Our finsings showed that PA+CA biofilms were able to escape to the combined action of PMB/AMB, and both species had a preeminent role while retaining adaptive resistance mechanisms that likely contributed for their recovery and adaptation on the ensuing treatments.info:eu-repo/semantics/publishedVersio

RNA-based qPCR as a tool to quantify and to characterize dual-species biofilms

Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-50094-3.While considerable research has focused on studying individual-species, we now face the challenge of determining how interspecies interactions alter bacterial behaviours and pathogenesis. Pseudomonas aeruginosa and Staphylococcus aureus are often found to co-infect cystic-fibrosis patients. Curiously, their interaction is reported as competitive under laboratory conditions. Selecting appropriate methodologies is therefore critical to analyse multi-species communities. Herein, we demonstrated the major biases associated with qPCR quantification of bacterial populations and optimized a RNA-based qPCR able not only to quantify but also to characterize microbial interactions within dual-species biofilms composed by P. aeruginosa and S. aureus, as assessed by gene expression quantification. qPCR quantification was compared with flow-cytometry and culture-based quantification. Discrepancies between culture independent and culture dependent methods could be the result of the presence of viable but not-cultivable bacteria within the biofilm. Fluorescence microscopy confirmed this. A higher sensitivity to detect viable cells further highlights the potentialities of qPCR approach to quantify biofilm communities. By using bacterial RNA and an exogenous mRNA control, it was also possible to characterize bacterial transcriptomic profile, being this a major advantage of this method.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Grant of APM (SFRH/BD/132165/2017).info:eu-repo/semantics/publishedVersio

Unveiling co-infection in cystic fibrosis airways: transcriptomic analysis of Pseudomonas aeruginosa and Staphylococcus aureus dual-species biofilms

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene.2022.883199/ full#supplementary-materialCystic fibrosis (CF) is a common heritable genetic disorder caused by a defect in the cystic fibrosis conductance regulator gene, resulting in several complications in the human body (Kreda et al., 2012). So far, the pathological changes in the lungs are best studied due to the high mortality rates linked to poor lung function and the recurrent development of severe biofilm-related infections (Flume et al., 2009; Ciofu et al., 2015). Staphylococcus aureus and Pseudomonas aeruginosa are the most prevalent pathogens that colonize structurally abnormal airways such as those diagnosed with CF and other chronic obstructive lung diseases (Lyczak et al., 2002; Hubert et al., 2013). Although these bacteria seem to succeed with one another, CF patients acquire coinciding P. aeruginosa and S. aureus pulmonary infections, being co-infection usually associated with decreased lung function and increased frequency of pulmonary exacerbations (Limoli et al., 2016). Furthermore, P. aeruginosa and S. aureus pathogens adopt a biofilm mode of growth, which contributes to high tolerance to antibiotic treatment (Schobert and Jahn, 2010) and the recalcitrant nature of these chronic co-infections (Burmølle et al., 2006; Lopes et al., 2012), leading to significant patient morbidity and mortality (Cox et al., 2010). Interactions between P. aeruginosa and S. aureus have been widely studied, and it is commonly admitted that P. aeruginosa outcompetes S. aureus, perhaps outcompeting S. aureus for limited nutrients (Mashburn et al., 2005) or producing antistaphylococcal compounds (DeLeon et al., 2014; Fugère et al., 2014), having S. aureus a minimal contribution to the overall course of the CF-associated biofilm infections (Bragonzi et al., 2012; Filkins et al., 2015). However, P. aeruginosa and S. aureus have been identified in the same lobe of CF lungs (Hogan et al., 2016; Wakeman et al., 2016) and are frequently diagnosed (Limoli et al., 2016; Zolin et al., 2019) as co-infecting species in CF patients. Moreover, P. aeruginosa strains isolated from early infection outcompete S. aureus, while strains isolated from chronic infection are less aggressive and can be co-cultivated with S. aureus (Frydenlund Michelsen et al., 2016; Limoli et al., 2017), suggesting that these pathogens can interact in vivo. In a previous study, we showed that S. aureus can grow and coexist with P. aeruginosa under dualspecies biofilm conditions (Magalhães et al., 2021). Following up on these findings, and acknowledging that the molecular mechanisms behind these interactions are largely unknown, the purpose of the present study was, therefore, to identify the major transcriptomic features of P. aeruginosaS. aureus dual-species biofilms, using high-throughput RNA-sequencing (RNA-seq). Herein, we described the full transcriptome of P. aeruginosa and S. aureus single- and dual-species biofilms and used a data analysis approach based on direct and functional gene interactions, namely gene set enrichment. These results will be invaluable for future functional studies involving P. aeruginosaS. aureus interactions.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte. The authors also acknowledge the support, through the Programa Operacional Competitividade e Internacionalização (COMPETE 2020) and by national funds, through FCT, of the PhD Grant of APM (SFRH/BD/132165/2017).info:eu-repo/semantics/publishedVersio

A internet das coisas como uma ferramenta auxiliar no ensino superior

A Internet das Coisas (IdC) é essencialmente constituída por uma rede de objetos e dispositivos físicos que, como têm capacidade computacional e de comunicação via WEB, são capazes de recolher e transmitir dados de forma autónoma. Esta possibilidade tem sido utilizada, por exemplo, em sistemas remotos de telemedicina e monitoramento de pacientes. De forma a verificar se a IdC tem potencial para promover uma aprendizagem ativa de natureza interdisciplinar que promova o trabalho em equipa, o desenvolvimento de projetos e competências de comunicação científica no ensino superior, surgiu esta prova de conceito, de cariz misto, resultado do trabalho conjunto entre a Faculdade de Educação e Psicologia e a Escola Superior de Biotecnologia, ambas da Universidade Católica Portuguesa – Centro Regional do Porto. Para tal, um grupo de alunos do curso de Bioengenharia, através da integração dos conhecimentos e competências adquiridas, desenvolveu um dispositivo eletrónico, controlado por um microcontrolador do tipo arduino, para monitoramento de sinais vitais, frequência cardíaca e oxímetro, com a plataforma SOLL, Smart Objects Linked to Learnings, servindo como interface WEB. A partir dos dados recolhidos, provenientes de técnicas de observação e inquérito por questionário, verifica-se que a maioria dos alunos não conhecia a ferramenta e os que conheciam poucos já tinham utilizado. No entanto, os estudantes foram unânimes em considerar a ferramenta versátil, inovadora, intuitiva, com potencial para a aplicação prática e útil dos conteúdos ministrados em sala de aula.info:eu-repo/semantics/publishedVersio

Antimicrobial resistance three ways: healthcare crisis, major concepts, and the relevance of biofilms

Worldwide, infections are resuming their role as highly effective killing diseases, as current treatments are failing to respond to the growing problem that is antimicrobial resistance (AMR). The social and economical burden of AMR seems ever rising, with health- and research-related organizations rushing to collaborate on a worldwide scale to find effective solutions. Resistant bacteria are spreading even in first-world nations, being found not only in healthcare-related settings, but also in food and in the environment. In this mini-review, the impact of AMR in healthcare systems and the major bacteria behind it are highlighted. Ecological aspects of AMR evolution and the complexity of its molecular mechanisms are explained. Major concepts, such as intrinsic, acquired, and adaptive resistance, as well as tolerance and heteroresistance, are also clarified. More importantly, the problematic of biofilms and their role in AMR, namely its main resistance and tolerance mechanisms, is elucidated. Finally, some of the most promising anti-biofilm strategies being investigated are reviewed. Much is still to be done regarding the study of AMR and the discovery of new anti-biofilm strategies. Gladly, considerable research on this topic is generated every day and increasingly concerted actions are being engaged globally to try and tackle this problem.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors also acknowledge COMPETE2020 and FCT for the project POCI-01-0145-FEDER-029841, and FCT for the PhD Grants of Andreia Magalhães [grant number SFRH/BD/132165/2017] and Tânia Grainha [grant number SFRH/BD/136544/2018].info:eu-repo/semantics/publishedVersio

Viable but non-culturable state: a strategy for Staphylococcus aureus survivable in dual-species biofilms with Pseudomonas aeruginosa

While considerable research has focused on studying individual-species, we now face the challenge of determining how interspecies interactions alter bacterial behaviours and pathogenesis. Of particular interest is the ecological behaviour between P. aeruginosa-S. aureus, since microbial communities containing both pathogens can display enhanced virulence. Curiously, their interaction is often reported as competitive under laboratory conditions. Here we investigate the interaction between P. aeruginosa-S. aureus in a biofilm co-culture model. The growth of 24- and 48h-old dual-species biofilms was monitored by CFU and flow-cytometry counting, and the gene expression profile inspected by qPCR. Plating experiments showed that S. aureus growth significantly decreased from 24- up to 72-h of growth in dual-species biofilms with P. aeruginosa, suggesting a clear competitive advantage for P. aeruginosa after 24-h. The gene expression profile showed that P. aeruginosa PA14 significantly overexpressed several key virulence-related genes (pqsE, rhlR, pvdE and lasI) after 24-h of growth with S. aureus ATCC 25923, comparatively with singles-pecies biofilms, which could justify the rapid decline of S. aureus population after 24-h of co-culture with P. aeruginosa. Nevertheless, the isolated strain P. aeruginosa 362668 non-mucoid exhibited a reverse pattern by downregulated the expression of those genes during dual-species growth. As P. aeruginosa exoproducts in addition to cause S. aureus lysis or inhibition may also induce biofilm dispersion, the bulk-fluid of 48-h biofilms was analysed by culture counts and flow-cytometry. Interestingly, while no S. aureus cells were detected by CFU, flow-cytometry data revealed the presence of S. aureus in high numbers (~6 Log). The metabolic activity of S. aureus cells was confirmed by measure the expression levels of sodA gene. In conclusion, the presence of S. aureus in a viable but non-cultivable state within dual-species biofilms with P. aeruginosa underscores the impact of interspecies interactions on antibiotic efficacy in the context of polymicrobial infections.Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit; European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte for the BioTecNorte operation (NORTE-01-0145-FEDER-000004); COMPETE2020 and FCT for the project POCI-01-0145-FEDER-029841. The authors also thank FCT for the PhD Grant of Andreia Patrícia Magalhães [grant number SFRH/BD/132165/2017]info:eu-repo/semantics/publishedVersio

Covid-19 - Morbimortalidade pela COVID-19 segundo raça/cor/etnia: a experiência do Brasil e dos Estados Unidos

This study sought to describe the experience of Brazil and the United States in relation to COVID-19 morbidity and mortality data according to race/skin color/ethnicity.  Toward this end, it seeks to describe the factors involved in the treatment and dissemination of the morbimortality data for this pathology in the two countries. The analysis includes epidemiological bulletins released by the Brazilian Ministry of Health, partial results from Brazil’s National Household Survey (PNAD) for COVID-19 collected by the Brazilian Institutes of Geography and Statistics (IBGE), and state-of-the-art health data about the impact of the pandemic in the United States, from the perspective of race/skin color/ethnicity. Despite the low quality of health information on the COVID-19 morbidity and mortality of the black population, the results corroborate racial inequities in health for COVID-19, confirming the existence of structural and institutional racism in both countries.  This article highlights the need to qualify data about race/skin color/ethnicity, by relating them to age, place of residence, type of residence, access to basic sanitation, and occupation, among other social determinants, that impact how individuals become ill and die from COVID-19, in order to enact strategies and public policies that truly promote equity.Este estudo objetivou descrever a experiência do Brasil e dos Estados Unidos em relação aos dados de morbimortalidade por COVID-19, segundo a raça/cor/etnia. Para isto, procurou-se descrever os fatores envolvidos no tratamento e divulgação dos dados de morbimortalidade por esta patologia nos dois países. Foram analisados boletins epidemiológicos divulgados pelo Ministério da Saúde, resultados parciais da Pesquisa Nacional por Amostragem de Domicílio (PNAD) COVID-19 realizada pelo IBGE no Brasil e o estado da arte em saúde sobre os impactos da pandemia nos Estados Unidos, sob a perspectiva de raça/cor/etnia. Apesar da baixa qualidade da informação em saúde referente à morbimortalidade da população negra por COVID-19, os resultados desvelam iniquidades raciais em saúde para a COVID-19, ratificando o racismo estrutural/institucional em ambos países. Como contribuição, enfatiza-se a necessidade de qualificar os dados sobre raça/cor/etnia, relacionando-os à idade, local de moradia, tipo de residência, acesso a saneamento básico, ocupação, dentre outros determinantes sociais, que sabidamente impactam no modo de adoecer e morrer pela COVID-19, a fim de viabilizar estratégias e políticas públicas verdadeiramente promotoras da equidade