A selective membrane-targeting repurposed antibiotic with activity against persistent methicillin-resistant Staphylococcus aureus.

Treatment of Staphylococcus aureus infections is complicated by the development of antibiotic tolerance, a consequence of the ability of S. aureus to enter into a nongrowing, dormant state in which the organisms are referred to as persisters. We report that the clinically approved anthelmintic agent bithionol kills methicillin-resistant S. aureus (MRSA) persister cells, which correlates with its ability to disrupt the integrity of Gram-positive bacterial membranes. Critically, bithionol exhibits significant selectivity for bacterial compared with mammalian cell membranes. All-atom molecular dynamics (MD) simulations demonstrate that the selectivity of bithionol for bacterial membranes correlates with its ability to penetrate and embed in bacterial-mimic lipid bilayers, but not in cholesterol-rich mammalian-mimic lipid bilayers. In addition to causing rapid membrane permeabilization, the insertion of bithionol increases membrane fluidity. By using bithionol and nTZDpa (another membrane-active antimicrobial agent), as well as analogs of these compounds, we show that the activity of membrane-active compounds against MRSA persisters positively correlates with their ability to increase membrane fluidity, thereby establishing an accurate biophysical indicator for estimating antipersister potency. Finally, we demonstrate that, in combination with gentamicin, bithionol effectively reduces bacterial burdens in a mouse model of chronic deep-seated MRSA infection. This work highlights the potential repurposing of bithionol as an antipersister therapeutic agent.

A new method for studying cryptococcosis in a murine model using 99mTc-Cryptococcus gattii.

Cryptococcus gattii is one of the etiologic agents of cryptococcosis, a systemic mycosis that occurs in healthy and immunosuppressed humans and animals worldwide. Primary pulmonary infection caused by C. gattii is usually followed by fungal dissemination to the central nervous system, resulting in high mortality rates. In this context, animal models of cryptococcosis are useful in the study of fungal pathogenesis and host response against the pathogen, and for testing novel therapeutic options. The most frequently applied method to study fungal dissemination from the lungs to other organs is by culturing tissues, which is not accurate for the detection and quantification of fungal load at early stages of the infection. To overcome this problem, the purpose of this study was to develop a new method for the quantification of Cryptococcus dissemination. One C. gattii strain was efficiently radiolabeled with technetium-99m (99mTc), without affecting viability of the cells. Further, the 99mTc-C. gattii (111 MBq) strain was used to infect mice by intratracheal and intravenous route for biodistribution studies. 99mTc-C. gattii was successfully used in detection of the yeast in the brain of mice 6 hours postinoculation, while the detection using colony forming units was possible only 24 hours postinfection. Our results provided an alternative method that could be applied in further investigations regarding the efficacy of antifungals, fungal virulence, and host-pathogen interactions.

In vivo probiotic and antimicrobial photodynamic therapy as alternative therapies against cryptococcosis are ineffective.

Cryptococcosis, an invasive fungal infection distributed worldwide that affects both domestic and wild animals, has incredible rates regarding treatment failure, leading to the necessity of the development of new therapies. In this way, we aimed to evaluate the probiotic (Saccharomyces boulardii, Lactobacillus paracasei ST-11, and Lactobacillus rhamnosus GG) and antimicrobial photodynamic alternative therapies against Cryptococcus gattii in a murine model. Although previous studies suggest that these therapies can be promising against cryptococcosis, our experimental conditions for both probiotic and antimicrobial photodynamic therapies (aPDT) were not able to improve the survival of mice with cryptococcosis, even with the treatment combined with fluconazole. Our results may help other researchers to find the best protocol to test alternative therapies against Cryptococcus gattii.

Atorvastatin as a promising anticryptococcal agent.

Cryptococcosis caused by Cryptococcus gattii leads to pneumonia and meningoencephalitis, and has a high mortality rate worldwide due to the inadequacy of available therapy and increasing drug resistance. There is a need to develop effective treatments, and drug repositioning is an interesting alternative to achieve new strategies to treat cryptococcosis. Atorvastatin (ATO), a statin currently used to treat hypercholesterolaemia, was tested in this study as an adjuvant to control infections caused by C. gattii. Several aspects of the effect of ATO on the host and the yeast were evaluated, with particular focus on the association of ATO with fluconazole (FLC), which (i) reduced ergosterol content in the cell membrane and altered properties of the polysaccharide capsule of C. gattii; (ii) increased the production of reactive oxygen species by macrophages; and (iii) reduced yeast phagocytosis and the intracellular proliferation rate. In an animal model, infected mice treated with ATO + FLC showed increased survival, improved clinical condition, and reduced fungal burden in the lungs and brain. This study is the first to perform in vivo tests with ATO + FLC for the treatment of cryptococcosis. The results suggest that ATO may be an important adjuvant for the treatment of cryptococcosis.

Microbial additives in the composting process / Aditivos microbianos no processo de compostagem

ABSTRACT Composting is the process of natural degradation of organic matter carried out by environmental microorganisms whose metabolic activities cause the mineralization and partial humification of substances in the pile. This compost can be beneficially applied to the soil as organic fertilizer in horticulture and agriculture. The number of studies involving microbial inoculants has been growing, and they aim to improve processes such as composting. However, the behavior of these inoculants and other microorganisms during the composting process have not yet been described. In this context, this work aimed to investigate the effects of using a microbial inoculum that can improve the composting process and to follow the bacterial population dynamics throughout the process using the high-resolution melt (HRM) technique. To do so, we analysed four compost piles inoculated with Bacillus cereus, Bacillus megaterium, B. cereus + B. megaterium and a control with no inoculum. The analyses were carried out using samples collected at different stages of the process (5th to 110th days). The results showed that the bacterial inocula influenced the process of composting, altering the breakdown of cellulose and hemicelluloses and causing alterations to the temperature and nitrogen levels throughout the composting process. The use of a universal primer (rDNA 16S) allowed to follow the microbial succession during the process. However, the design of a specific primer is necessary to follow the inoculum throughout the composting process with more accuracy.

RESUMO A compostagem é um processo de degradação natural da matéria orgânica realizado por microrganismos presentes no ambiente, levando a mineralização e humificação parcial das substâncias presentes na pilha, esse composto formado pode ser beneficamente aplicado ao solo como fertilizante orgânico na horticultura e agricultura. O número de estudos envolvendo inoculantes microbianos é crescente, os quais tem por objetivo a otimização de processos de compostagem. Contudo, o comportamento desses inoculantes e da microbiota ao longo do processo não tem sido caracterizado. Nesse contexto, este trabalho foi realizado com o objetivo de avaliar o efeito da utilização de um inóculo bacteriano que promova melhorias no processo de compostagem, bem como o de acompanhar a dinâmica populacional bacteriana ao longo de todo o processo através da técnica de High Resolution Melt (HRM). Para isso foram analisados quatro pilhas de compostagem inoculadas com Bacillus cereus, Bacillus megaterium, B. cereus + B. megaterium e o controle sem adição de inóculo. Foram realizadas análises químicas e moleculares (HRM) das amostras coletadas em diferentes períodos da compostagem (5º ao 110º dias). Os resultados mostraram que os inóculos bacterianos influenciaram no processo de compostagem com alteração na degradação de celulose, hemicelulose bem como alteração da temperatura e níveis de nitrogênio ao longo da compostagem. A utilização de um primer universal (rDNA 16S) permitiu acompanhar a sucessão bacteriana ao longo do processo, nos tratamentos. Contudo a construção de um primer específico é necessário para acompanhar de maneira mais precisa o inóculo durante o desenvolvimento da compostagem.

A subdose of fluconazole alters the virulence of Cryptococcus gattii during murine cryptococcosis and modulates type I interferon expression.

Cryptococcosis is an invasive infection caused by yeast-like fungus of the genera Cryptococcus spp. The antifungal therapy for this disease provides some toxicity and the incidence of infections caused by resistant strains increased. Thus, we aimed to assess the consequences of fluconazole subdoses during the treatment of cryptococcosis in the murine inflammatory response and in the virulence factors of Cryptococcus gattii. Mice infected with Cryptococcus gattii were treated with subdoses of fluconazole. We determined the behavior of mice and type 1 interferon expression during the treatment; we also studied the virulence factors and susceptibility to fluconazole for the colonies recovered from the animals. A subdose of fluconazole prolonged the survival of mice, but the morbidity of cryptococcosis was higher in treated animals. These data were linked to the increase in: (i) fluconazole minimum inhibitory concentration, (ii) capsule size and (iii) melanization of C. gattii, which probably led to the increased expression of type I interferons in the brains of mice but not in the lungs. In conclusion, a subdose of fluconazole altered fungal virulence factors and susceptibility to this azole, leading to an altered inflammatory host response and increased morbidity.