Unravelling Pseudomonas aeruginosa and Candida albicans communication in coinfections scenarios: insights through network analysis

The Supplementary Material for this article canbe found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2020.550505/ full#supplementary-materialModern medicine is currently facing huge setbacks concerning infection therapeutics as microorganisms are consistently knocking down every antimicrobial wall set before them. The situation becomes more worrying when taking into account that, in both environmental and disease scenarios, microorganisms present themselves as biofilm communities that are often polymicrobial. This comprises a competitive advantage, with interactions between different species altering host responses, antimicrobial effectiveness, microbial pathogenesis and virulence, usually augmenting the severity of the infection and contributing for the recalcitrance towards conventional therapy. Pseudomonas aeruginosa and Candida albicans are two opportunistic pathogens often co-isolated from infections, mainly from mucosal tissues like the lung. Despite the billions of years of co-existence, this pair of microorganisms is a great example on how little is known about cross-kingdom interactions, particularly within the context of coinfections. Given the described scenario, this study aimed to collect, curate, and analyze all published experimental information on the molecular basis of P. aeruginosa and C. albicans interactions in biofilms, in order to shed light into key mechanisms that may affect infection prognosis, increasing this area of knowledge. Publications were optimally retrieved from PubMed and Web of Science and classified as to their relevance. Data was then systematically and manually curated, analyzed, and further reconstructed as networks. A total of 641 interactions between the two pathogens were annotated, outputting knowledge on important molecular players affecting key virulence mechanisms, such as hyphal growth, and related genes and proteins, constituting potential therapeutic targets for infections related to these bacterial-fungal consortia. Contrasting interactions were also analyzed, and quorum-sensing inhibition approaches were highlighted. All annotated data was made publicly available at www.ceb.uminho.pt/ISCTD, a database already containing similar data for P. aeruginosa and Staphylococcus aureus communication. This will allow researchers to cut on time and effort when studying this particular subject, facilitating the understanding of the basis of the inter-species and inter-kingdom interactions and how it can be modulated to help design alternative and more effective tailored therapies. Finally, data deposition will serve as base for future dataset integration, whose analysis will hopefully give insights into communications in more complex and varied biofilm communities.This work 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 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 Grant of TG [grant number SFRH/BD/136544/2018].info:eu-repo/semantics/publishedVersio

Pitfalls associated with discriminating mixed-species biofilms by flow cytometry

Since biofilms are ubiquitous in different settings and act as sources of disease for humans, reliable methods to characterize and quantify these microbial communities are required. Numerous techniques have been employed, but most of them are unidirectional, labor intensive and time consuming. Although flow cytometry (FCM) can be a reliable choice to quickly provide a multiparametric analysis, there are still few applications on biofilms, and even less on the study of inter-kingdom communities. This work aimed to give insights into the application of FCM in order to more comprehensively analyze mixed-species biofilms, formed by different Pseudomonas aeruginosa and Candida albicans strains, before and after exposure to antimicrobials. For comparison purposes, biofilm culturability was also assessed determining colony-forming units. The results showed that some aspects, namely the microbial strain used, the morphological state of the cells and the biofilm matrix, make the accurate analysis of FCM data difficult. These aspects were even more challenging when double-species biofilms were being inspected, as they could engender data misinterpretations. The outcomes draw our attention towards the need to always take into consideration the characteristics of the biofilm samples to be analyzed through FCM, and undoubtedly link to the need for optimization of the processes tailored for each particular case study.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the UID/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004), which was funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte. The authors also acknowledge COMPETE2020 and FCT under the project POCI-01-0145-FEDER-029841 and FCT for the PhD grant to Tânia Grainha (grant number SFRH/BD/136544/2018).info:eu-repo/semantics/publishedVersio

Modulating an antimicrobial release approach by dopamine chemistryto fight infections associated to orthopedic implants

Alongside with orthopedic implants contribution for modern healthcare improvements, theres the risk associated to their microbial colonization and biofilm formation, compromising the performance of the implant itself and representing niches for infection. This study aimed to engineer an antimicrobial release coating for stainless steel surfaces (SS) to empower them with the ability to prevent Staphylococci colonization. Surface modification was based on dopamine chemistry, which self-polymerization results in the deposition of a thin, adhered film called polydopamine (pDA). Chlorohexidine (CHX) was chosen to confer the antimicrobial features. Its immobilization was performed through a 2-step approach, including pDA formation and immersion in CHX solution, and 1-step strategy, in which dopamine and CHX were dissolved together and SS coupons were immersed in this solution. An additional layer of pDA was also performed for both strategies. SEM and AFM confirmed pDA coating by the presence of self-polymerized pDA particles without altering the roughness of SS surfaces. Immobilization of CHX using a 1-step approach yielded surfaces with a more homogenous coating than the 2-step approach. Different pDA-based strategies yielded different CHX release profiles: the amount of CHX released was higher for the 2-step approach and the addition of another pDA layer reduced the amount of CHX released. The antimicrobial performance of the modified surfaces was evaluated against S. aureus and S. epidermidis and the results showed that all the strategies caused a significant reduction (more than 3 LOG) in the number of cells adhered to the surfaces and in suspension, after 24 h. The 2-step approach was able to impart SS surfaces with antimicrobial activity even after 10 days of exposure. In conclusion, dopamine chemistry can modulate CHX release from the surfaces to obtain an antimicrobial coating strategy with great potential to fight infections associated with orthopedic implants.info: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

Contributos para a compreensão de interações entre espécies, perfil fenotípo e suscetibilidade a antimicrobianos num microbioma artificial da pneumonia associada à ventilação mecânica

Dissertação de mestrado em BioengineeringVentilator associated pneumonia (VAP) is the second most common nosocomial infection in the intensive care units (ICU) and the most common in mechanically ventilated patients. VAP presents a serious problem in ICU due to high mortality and morbidity rates associated, because it is often biofilm-mediated and polymicrobial. Therefore, understanding the impact of microorganisms in VAP and their interaction is a major challenge posed. Additionally, the ineffective current treatment strategies have led to the emergence of new approaches to fight these polymicrobial consortia, with a great number intervening in the quorum-sensing (QS) intercellular communication. This work aimed to give insights into the behavior of bacterial-fungal communities involving Pseudomonas aeruginosa and Candida albicans associated to VAP, when exposed to different antimicrobial approaches. For this, single- and mixed-species biofilms were thoroughly characterized in terms of cultivable cells and biomass after 24 h treatment with conventional drugs (amphotericin B, AmB; polymyxin B, PolyB) and alternative agents, in particular QS inhibitors (QSI) from different sources (commercial drugs: salicylic acid, ciprofloxacin (CIP), azithromycin (AZT); natural sources: chlorogenic acid, farnesol, linalool, patulin) and enzymes (alginate lyase, desoxirribonuclease), tested alone or in combination. Results showed that the combination AmB+PolyB did not affect the pre-established P. aeruginosa and C. albicans consortia. Interestingly, excepting for patulin, QSI agents were effective at reducing biofilm-encased cells, in particular single-species biofilms. CIP showed a great potential to inhibit both single-and mixed-species biofilms. Linalool was also effective in disturbing C. albicans in single and mixed biofilms. Contrariwise, enzymes had no effect against biofilms. Regarding double combinations, the addition of farnesol or linalool to CIP led to similar results from that obtained with CIP alone, with reductions observed in biofilm-encased cells. In general, the addition of a third agent - particularly in the case of chlorogenic acid - did not significantly improve the effect of AmB+PolyB or farnesol/linalool+CIP combinations. Additionally, efforts were made to characterize the un- and treated dual-species biofilms by flow cytometry and RNA sequencing (RNA-seq), however no accurate results were obtained due to unexpected methodological hitches. In conclusion, the use of new approaches seems to be a promise in treating bacterial-fungal consortia often involved in VAP. This work showed that combining different agents from distinct sources is a valuable option to control P. aeruginosa and C. albicans biofilms. Nevertheless, optimization on the antimicrobial doses and further clinical studies are urgently required to improve therapy effectiveness and avoid additional costs.A pneumonia associada à ventilação (PAV) é a segunda infeção nosocomial mais comum em unidades de cuidados intensivos (UCI) e a mais comum em pacientes sob ventilação mecânica. A PAV apresenta um problema grave na UCI devido às elevadas taxas de mortalidade e morbilidade associadas, porque muitas vezes é mediada por biofilmes e tem caráter polimicrobiano. Compreender o impacto dos microrganismos em PAV e suas interações é um grande desafio que se coloca. Além disso, a ineficiência das estratégias de tratamento atuais levaram ao surgimento de novas abordagens para combater esses consórcios polimicrobianos, com um grande número intervindo na comunicação intercelular quorum-sensing (QS). Este trabalho teve como objetivo o conhecimento do comportamento das comunidades bacterianas-fúngicas envolvendo Pseudomonas aeruginosa e Candida albicans associadas a PAV, quando expostas a diferentes estratégias antimicrobianas. Para isso, os biofilmes simples e mistos foram caracterizados em termos de células cultiváveis e biomassa após 24 h de tratamento com medicamentos convencionais (anfotericina B, AmB; polimixina B, PolyB) e com agentes alternativos, em particular, inibidores de QS (QSI) obtidos de diferentes fontes (medicamentos comerciais: ácido salicílico, ciprofloxacina (CIP), azitromicina (AZT); fontes naturais: ácido clorogénico, farnesol, linalol, patulina); enzimas (alginato liase, desoxirribonuclease), testados isoladamente ou em combinação. Os resultados mostraram que a combinação AmB+PolyB não afetou os consórcios préestabelecidos de P. aeruginosa e C. albicans. Curiosamente, com exceção de patulina, os agentes IQS foram eficazes na redução de células de biofilme, em particular nos biofilmes formados por uma única espécie. A CIP mostrou grande potencial para inibir biofilmes simples e mistos. O linalol também foi eficaz contra C. albicans em biofilmes simples e mistos. Pelo contrário, as enzimas não tiveram efeito contra os biofilmes. No que se refere às combinações duplas, a adição de farnesol ou linalol à CIP conduziu a resultados semelhantes obtidos apenas com CIP, com reduções observadas em células envolto-biofilme. Em geral, a adição de um terceiro agente - em particular no caso do ácido clorogénico - não melhorou significativamente o efeito das combinações AmB+PolyB ou farnesol/linalol+CIP. Adicionalmente, foram feitos esforços para caraterizar os biofilmes de dupla espécie, não tratados e tratados, por citometria de fluxo e sequenciação de RNA (RNA-seq), contudo não foram obtidos resultados fiáveis devido a dificuldades metodológicas inesperadas. Em conclusão, o uso de novas estratégias parece ser uma promessa no tratamento de consórcios bacterianos-fúngicos frequentemente envolvidas em PAV. Este trabalho mostrou que a combinação de diferentes agentes obtidos a partir de fontes distintas é uma opção valiosa para controlar biofilmes de P. aeruginosa e C. albicans. No entanto, é necessário a otimização das doses de antimicrobianos e mais estudos clínicos para melhorar a eficácia da terapia e evitar custos adicionais.Fundação para a Ciência e Tecnologia (FCT) - Under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684);FCT and the European Community fund FEDER, through Program COMPETE, under the scope of project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462);This work was supported by ESCMID Research Grants 2014