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

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