The Nuanced Effects of Redox-Active Metabolites on Bacterial Physiology and Antibiotic Susceptibility

The production of secondary metabolites is widespread throughout the tree of life. Bacteria, including many relevant opportunistic pathogens, can make redox-active secondary metabolites, both in the environment and while causing infections. Yet, their physiological consequences for the microbial communities exposed to them are much less understood. This thesis investigates the multifaceted and nuanced effects that such metabolites can have on their producers and other bacteria found in the producer's vicinity, focusing on the role these molecules play as modulators of antibiotic susceptibility. I start by presenting a literature review addressing the link between secondary metabolite production and resilience to clinical antibiotics in diverse opportunistic and enteric bacterial pathogens. Next, using Pseudomonas aeruginosa (a widespread opportunistic pathogen) and its endogenously produced metabolite called pyocyanin, I explore the nuanced effects of the metabolite's production throughout the producer's lifecycle. Pyocyanin is part of a class of redox-active molecules made by P. aeruginosa called phenazines. I show that the production of pyocyanin, due to its self-poisoning effects, is a "double-edged sword," where the ultimate consequences for the producer are directly dependent on the physiological and environmental conditions. Carbon source limitation plays a major role in the self-poisoning effect of pyocyanin, a process responsible for killing a subpopulation of cells that, through extracellular DNA release, seems critical for proper biofilm development. Despite pyocyanin's toxicity, P. aeruginosa is remarkably tolerant to its harmful effects. For this reason, I then explore how P. aeruginosa handles the stress caused by the metabolite. I present results using a functional genomics approach (transposon-sequencing) to screen for genes involved in P. aeruginosa tolerance to pyocyanin. Defenses involved in pyocyanin tolerance are similar to ones involved in tolerance to clinical antibiotics. These shared mechanisms lead to testing the hypothesis that defenses induced by the production of or exposure to "natural antibiotics" (such as pyocyanin) may affect the efficacy of treatments with clinical antibiotics. Supporting this hypothesis, exposure to pyocyanin significantly induces tolerance and resistance to certain clinical drugs, both in P. aeruginosa and other opportunistic pathogens within the Burkholderia cepacia complex (Bcc). Pyocyanin and the drugs affected, such as fluoroquinolones, share molecular structure similarities, which is likely responsible for the shared protection. Finally, based on these results, I explore the broader role of redox-active metabolites as modulators of antibiotic resilience in opportunistic pathogens. I show that pyocyanin, another phenazine called phenazine-1-carboxylic acid, and a non-phenazine redox-active molecule called toxoflavin can all modulate antibiotic susceptibility in Bcc species. Depending on the antibiotic's class, the metabolites' presence can either antagonize or potentiate the drug's efficacy. All the studied metabolites are produced by clinical isolates that infect cystic fibrosis and other immunocompromised patients. I demonstrate that the modulator effect of redox-active molecules in the pathogens is dependent on the transcription factor SoxR, which senses the presence of the metabolites and induces specific redox-regulated efflux systems that are effective in transporting both the metabolites and the structurally related drugs. To end, I provide a proof-of-principle that including such metabolites during clinical drug susceptibility tests may lead to a more accurate assessment of pathogens' resistance profile. Taken together, the findings presented in this thesis demonstrate that redox-active secondary metabolites have profound effects on the physiology and antibiotic sensitivity levels of opportunistic pathogens. Their modulator effect on antibiotic susceptibility is likely a widespread phenomenon in polymicrobial communities that has been overlooked and may have direct consequences for the evolution of antibiotic resistance. Understanding the physiological roles of these metabolites at the molecular level is essential for accurate predictions of the drugs and pathogens affected, which may lead to more effective treatment strategies.</p

Fungal communities in pressmud composting harbour beneficial and detrimental fungi for human welfare

Pressmud is a substrate derived from sugarcane juice filtrate, and around 26-40  kg of this residue are produced per ton of sugarcane. It is mainly used as fertilizer in crops, and its application in the field is often made without any prior treatment, but, in this research, it was studied for the risk this practice poses for human health. This research was stimulated by previous results indicating the presence of opportunistic pathogens in residues used in various composting systems and the extensive use of fresh pressmud in agriculture. Here, It was assessed the fungal diversity present in both fresh and composting pressmud using 454 pyrosequencing. In addition, heat-tolerant fungi were isolated and surveyed for their enzymatic repertoire of biomass-degrading enzymes (cellulase, xylanase, laccase and polygalacturonase). A wide range of opportunistic pathogens was found among the most abundant taxa in the fresh pressmud, such as Lomentospora prolificans (43.13 %), Trichosporon sp. (10.07 %), Candida tropicalis (7.91 %), and Hormographiella aspergillata (8.19 %). This indicates that fresh pressmud might be a putative source of human pathogenic fungi, presenting a potential threat to human health if applied as fertilizer without any treatment. With regard to the heat-tolerant fungi found in this substrate, all the 110 isolates screened were able to produce at least one of the tested enzymes. The pressmud composting process not only effectively reduces the load of pathogenic fungi, but also creates an interesting environment for fungi able to produce thermostable hydrolytic and oxidative enzymes with biotechnological applications

Fungal communities in pressmud composting harbour beneficial and detrimental fungi for human welfare

Pressmud is a substrate derived from sugarcane juice filtrate, and around 26-40  kg of this residue are produced per ton of sugarcane. It is mainly used as fertilizer in crops, and its application in the field is often made without any prior treatment, but, in this research, it was studied for the risk this practice poses for human health. This research was stimulated by previous results indicating the presence of opportunistic pathogens in residues used in various composting systems and the extensive use of fresh pressmud in agriculture. Here, It was assessed the fungal diversity present in both fresh and composting pressmud using 454 pyrosequencing. In addition, heat-tolerant fungi were isolated and surveyed for their enzymatic repertoire of biomass-degrading enzymes (cellulase, xylanase, laccase and polygalacturonase). A wide range of opportunistic pathogens was found among the most abundant taxa in the fresh pressmud, such as Lomentospora prolificans (43.13 %), Trichosporon sp. (10.07 %), Candida tropicalis (7.91 %), and Hormographiella aspergillata (8.19 %). This indicates that fresh pressmud might be a putative source of human pathogenic fungi, presenting a potential threat to human health if applied as fertilizer without any treatment. With regard to the heat-tolerant fungi found in this substrate, all the 110 isolates screened were able to produce at least one of the tested enzymes. The pressmud composting process not only effectively reduces the load of pathogenic fungi, but also creates an interesting environment for fungi able to produce thermostable hydrolytic and oxidative enzymes with biotechnological applications

Síndrome de Adie: Adie's Syndrome

Introdução: A Síndrome de Adie-Homes é uma doença caracterizada pela presença de pupila tônica associada à diminuição ou ausência dos reflexos tendíneos profundos. É uma doença considerada típica de adultos jovens e apresenta uma etiologia comumente idiopática.A presença de alteração no tamanho das pupilas é uma das queixas mais comuns.&nbsp; Apresentação do caso: Paciente do sexo feminino, 28 anos, admitida em Hospital Geral de Goiânia, apresentou há 1 mês quadro de cefaleia fronto-orbital a direita, associada a vesículas em mesma região, dolorosas, em queimação, com diagnóstico de herpes zóster. Após 2 semanas, notou que sua acuidade visual à direita para perto estava diminuída associado a fotofobia. Ao avaliar pupilas com lâmpada de fenda, observou-se anisocoria à direita. Ao instilar pilocarpina 0,125% em ambos os olhos, apenas a pupila direita contraiu.. Discussão: Embora a etiologia da doença não esteja totalmente elucidada, acredita-se que possa ocorrer processos imunomediado e infecções por vírus da herpes-zoster, parvovírus B19, Treponema pallidum. Atualmente, o tratamento ainda se baseia no uso de Pilocarpina 0,125% para diminuir o desconforto do paciente, podendo ser usado 2-4 vezes ao junto, em associação ao uso de óculos escuros em ambientes muito iluminados. Conclusão: A síndrome de Adie é rara e encontramos em sua apresentação clínica singularidades que difere de paciente a paciente. Nota-se que a síndrome tem correlação com diversas áreas médicas, o que faz com que seu diagnóstico ocorra através do oftalmologista, podendo ter sua suspeição diagnóstica através de outros especialistas como reumatologistas, infectologistas e até mesmo dermatologistas

Criptococose pulmonar: Pulmonary cryptococcosis

Introdução: A criptococose é uma micose sistêmica causada pela inalação de esporos viáveis do fungo Cryptococcus spp. As principais espécies em humanos são Cryptococcus neoformans e Cryptococcus gattii, que se associam, respectivamente, às condições de imunodepressão celular e à infecção primária de indivíduos imunocompetentes. O pulmão é o sítio primário mais comum, logo após o sistema nervoso central.&nbsp; Apresentação do caso: JMF, masculino, 39 anos, apresentava queixa de tosse produtiva associada a desconforto respiratório aos moderados esforços, há cerca de 04 meses. Portador de HIV, sem outras comorbidades. Ao exame físico, sinais vitais estáveis, ausculta pulmonar com diminuição do murmúrio vesicular à direita. Na tomografia computadorizada (TC) de tórax evidenciou-se múltiplas opacidades nodulares difusas e de tamanhos variados, com presença de broncograma aéreo. Paciente foi submetido a biópsia por agulha percutânea e a análise do anatomopatológico constatou Criptococose Pulmonar. Discussão: A Criptococose pulmonar era considerada uma afecção rara, quando a prevalência de imunossuprimidos aumentou consideravelmente no século XX, sobretudo devido a Síndrome da Imunodeficiência Adquirida (SIDA). As manifestações clínicas são inespecíficas, variam desde infecção autolimitada até a disseminada, com meningoencefalite. O estado imunológico do paciente é o fator de risco e de prognóstico mais importante, influenciando tanto na evolução clínica quanto na abordagem terapêutica. Conclusão: Tendo em vista a similaridade dos sinais e sintomas com outras infecções pulmonares e ao potencial de gravidade da doença, principalmente em portadores de imunodeficiência, é de suma importância o diagnóstico precoce e o uso apropriado dos agentes antifúngicos para a redução da morbimortalidade

Antagonismo de actinobactérias isoladas de Trachymyrmex frente ao fungo parasita Escovopsis

As formigas da tribo Attini mantêm uma associação com actinobactérias encontradas nos biofilmes presentes no integumento desses insetos. Dentre as actinobactérias associadas às formigas Attini, os gêneros Pseudonocardia e Streptomyces são considerados prevalentes. Estudos indicam que essas bactérias auxiliam na defesa do jardim contra patógenos através da secreção de compostos antimicrobianos. Recentemente foi sugerido que tais bactérias podem ser importantes na defesa do próprio inseto, especialmente contra entomopatógenos. Duas teorias principais foram propostas para explicar as interações entre actinobactérias, formigas Attini e o microfungo parasita Escovopsis. Uma delas defende a coevolução entre esses organismos e a outra rejeita a existência da mesma, argumentando que a interação formiga-actinobactéria é uma simbiose aberta. Neste trabalho, avaliamos a interação entre actinobactérias e Escovopsis em experimentos in vitro. Um total de 14 linhagens de actinobactérias, compreendendo 12 de Pseudonocardia, uma de Streptomyces e uma de Actinoplanes, isoladas de Trachymyrmex (um gênero de formiga Attini não cortadeira) foram testadas frente a quatro linhagens de Escovopsis. Os Escovopsis foram isolados tanto de jardins de fungos de formigas Trachymyrmex quanto de cortadeiras de folhas (Acromyrmex e Atta). O crescimento micelial de Escovopsis (em cm²) foi medido na presença e na ausência (controle) das actinobactérias. Os resultados demonstraram que todas as actinobactérias testadas inibiram Escovopsis, mas as taxas de inibição foram variáveis dependendo da linhagem da actinobactéria (p < 0,05). A linhagem de Streptomyces foi a mais efetiva, corroborando dados da literatura que indicam que outros micro-organismos, além de Pseudonocardia, também apresentam ação inibitória frente à Escovopsis... (Resumo completo, clicar acesso eletrônico abaixo

Sistemática e filogenia do fungo parasita Escovopsis associado às formigas Attini

Attine ant nests harbor a diverse and complex microbiota. Such insects have a mutualistic relationship with mutualistic fungus, cultured as food source. However, several microorganisms are also present in attine nests. Within this microbiota, fungi in the genus Escovopsis are considered specific parasites of the mutualistic fungus. Escovopsis is only reported associated with attine ant nests. Furthermore, this parasite coevolved with attine ants and their mutualistic fungi in a tripartite relationship (tripartite coevolution model). Although several studies aimed to understand the genetic diversity of Escovopsis in Central America (mostly Panama), no comprehensive reports considering large Escovopsis samples are known from South America. There is also few taxonomic work onr this fungus; as a result, after two decades of the description of the first species, up to the present work, only six Escovopsis species have been formally described. In this study, we investigated the systematics, phylogeny and evolution of Escovopsis and separated our results in two chapters. In the first part, we describe a new species, E. kreiselii, first species belonging to basal attine ants. We also discuss the evolution of Escovopsis, indicating a correlation between phylogeny and morphological characters, where less derived strains shows absence of vesicles (structures supporting spore bearing-cells). In the second chapter, we exploit the phylogenetic diversity of Escovopsis associated with higher-attine ants in different countries of the American continent, especially Brazil. Contrary to previous resports, we show that higher attine ants share Escovopsis infections, indicating no strict co-cladogenesis between ant-cultivar-Escovopsis. The description of a new species and the extensive Escovopsis phylogeny presented here provide a first step towards a better understanding of the evolution and taxonomy of this group of fungi, which has potential for biological ...Ninhos de formigas Attini são conhecidos por abrigar uma diversa e complexa microbiota. Esses insetos possuem uma relação de mutualismo com seu fungo simbionte, utilizado como fonte de alimento. Porém, muitos micro-organismos também são encontrados nos ninho dessas formigas. Dentre eles, destacam-se fungos do gênero Escovopsis, considerados parasitas específicos do fungo cultivado por esses insetos. Somente encontrado associado aos ninhos de formigas Attini, Escovopsis coevoluiu com as formigas e seus fungos mutualistas em uma relação tripartida. Apesar dos diversos estudos voltados para entender a variedade genética de Escovopsis realizados na América Central (especialmente Panamá), não há relatos de grandes amostragens do parasita presente na América do Sul. São também escassos trabalhos taxonômicos para esse grupo de fungo; como resultado, após mais de duas décadas da descrição da primeira espécie do gênero, até o presente trabalho, apenas seis espécies foram formalmente descritas. Nesse contexto, estudamos a filogenia e evolução de Escovopsis e separamos nossos resultados em dois capítulos. No primeiro deles, descrevemos uma nova espécie, E. kreiselii, primeira a ser descrita pertencente ao clado das formigas Attini basais. Discutimos também a evolução do fungo, indicando que parece haver correlação entre filogenia e características morfológias, com linhagens menos derivadas apresentando ausência de vesícula (célula que suporta as fiálides, produtoras de conídios). Já no segundo capítulo, exploramos a diversidade filogenética de Escovopsis associados às formigas Attini derivadas, em diferentes países do continente Americano, especialmente o Brasil. Contrariamente ao que foi previamente proposto, nossos resultados sugerem que as formigas Attini derivadas podem compartilhar linhagens de Escovopsis, indicando ausência de co-cladogênese estrita entre formiga-Escovopsis-fungo mutualista. A...Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Unraveling Trichoderma species in the attine ant environment: description of three new taxa

Fungus-growing “attine” ants forage diverse substrates to grow fungi for food. In addition to the mutualistic fungal partner, the colonies of these insects harbor a rich microbiome composed of bacteria, filamentous fungi and yeasts. Previous work reported some Trichoderma species in the fungus gardens of leafcutter ants. However, no studies systematically addressed the putative association of Trichoderma with attine ants, especially in non-leafcutter ants. Here, a total of 62 strains of Trichoderma were analyzed using three molecular markers (ITS, tef1 and rpb2). In addition, 30 out of 62 strains were also morphologically examined. The strains studied correspond to the largest sampling carried out so far for Trichoderma in the attine ant environment. Our results revealed the richness of Trichoderma in this environment, since we found 20 Trichoderma species, including three new taxa described in the present work (Trichoderma attinorum, Trichoderma texanum and Trichoderma longifialidicum spp. nov.) as well as a new phylogenetic taxon (LESF 545). Moreover, we show that all 62 strains grouped within different clades across the Trichoderma phylogeny, which are identical or closely related to strains derived from several other environments. This evidence supports the transient nature of the genus Trichoderma in the attine ant colonies. The discovery of three new species suggests that the dynamic foraging behavior of these insects might be responsible for accumulation of transient fungi into their colonies, which might hold additional fungal taxa still unknown to science