Green-Synthesized Silver Nanoparticles in the Prevention of Multidrug-Resistant Proteus mirabilis Infection and Incrustation of Urinary Catheters BioAgNPs Against P. mirabilis Infection.

This study aimed to assess the activity of AgNPs biosynthesized by Fusarium oxysporum (bio-AgNPs) against multidrug-resistant uropathogenic Proteus mirabilis, and to assess the antibacterial activity of catheters coated with bio-AgNPs. Broth microdilution and time-kill kinetics assays were used to determine the antibacterial activity of bio-AgNPs. Catheters were coated with two (2C) and three (3C) bio-AgNPs layers using polydopamine as crosslinker. Catheters were challenged with urine inoculated with P. mirabilis to assess the anti-incrustation activity. MIC was found to be 62.5 µmol l-1, causing total loss of viability after 4 h and bio-AgNPs inhibited biofilm formation by 76.4%. Catheters 2C and 3C avoided incrustation for 13 and 20 days, respectively, and reduced biofilm formation by more than 98%, while the pristine catheter was encrusted on the first day. These results provide evidence for the use of bio-AgNPs as a potential alternative to combat of multidrug-resistant P. mirabilis infections.

Genome Mining Reveals High Biosynthetic Potential of Biocontrol Agent Bacillus velezensis B.BV10.

The present study demonstrates the biocontrol potential of a plant growth-promoting bacterial strain using three different approaches: (i) an in vitro evaluation of antagonistic activity against important phytopathogenic fungi; (ii) an evaluation under greenhouse conditions with strawberry plants to assess the control of gray mold; and (iii) an in silico whole genome sequence mining to assign genetic features such as gene clusters or isolated genes to the strain activity. The in vitro assay showed that the B.BV10 strain presented antagonistic activity, inhibiting the mycelial growth in all the phytopathogenic fungi evaluated. The application of the Bacillus velezensis strain B.BV10 under greenhouse conditions reduced the presence of Botrytis cinerea and increased the mean fruit biomass. The genome of B.BV10 was estimated at 3,917,533 bp, with a GC content of 46.6% and 4088 coding DNA sequences, and was identified as B. velezensis. Biosynthetic gene clusters related to the synthesis of the molecules with antifungal activity were found in its genome. Genes related to the regulation/formation of biofilms, motility, and the important properties for the rhizospheric colonization were also found in the genome. The current study offers a comprehensive understanding of the genomic architecture and control activity of phytopathogenic fungi by the B. velezensis strain B.BV10 that may substantiate the industrialization of this strain in the future.

Antifungal activity and genomic characterization of the biocontrol agent Bacillus velezensis CMRP 4489.

The development of bio-based products has increased in recent years, and species of the Bacillus genus have been widely used for product development due to their elevated production of antimicrobial molecules and resistance to extreme environmental conditions through endospore formation. In this context, the antifungal potential of Bacillus velezensis CMRP 4489 was investigated using in silico predictions of secondary metabolites in its genome and in vitro tests against the following phytopathogenic fungi: Sclerotinia sclerotiorum, Macrophomina phaseolina, and Botrytis cinerea. The in-silico predictions indicated that CMRP 4489 possesses several Biosynthetic Gene Clusters (BGCs) capable of producing molecules with antifungal properties and other non-identified BGCs. The in vitro assay results evidenced strong antifungal activity, inhibiting more than 60% of the tested fungi, and the isolate's molecules were stable under diverse physicochemical conditions. The in vitro assay evidenced significant antifungal activity, deformation of the hyphal structure in SS, biofilm formation capacity, and swarming motility. In the colonization assay, we observed attachment, colonization, and net-shaped biofilm formation, with the strain transitioning from the seeds to nearby structures. Therefore, CMRP 4489 showed to be a potential biocontrol agent against various diseases with agronomic importance and can be used under adverse environmental conditions.

Draft Genome Sequence of Brevibacillus brevis LABIM17, a Biotechnologically Important Antimicrobial-Producing Bacterium.

Brevibacillus brevis LABIM17 is a bacterial isolate with biotechnological potential. Its draft genome sequence contains a chromosome of 5,950,202 bp, with 5,477 coding sequences, and exhibits 12 clusters involved in the production of secondary metabolites, which are likely responsible for its antimicrobial activity against several human and plant pathogens.

Antibacterial activity of honeys from Amazonian stingless bees of Melipona spp. and its effects on bacterial cell morphology.

BACKGROUND: Stingless bee honey has great therapeutic potential, especially as an antimicrobial agent. In the present study, we evaluated the in vitro antibacterial potential of honey from Melipona spp. with occurrence in Rio Branco-AC and Xapuri-AC from the Amazonian region. Samples were collected from the species Melipona eburnea, Melipona grandis, Melipona flavolineata and Melipona seminigra. The antibacterial activity of the honey samples was tested against standard Gram-positive and Gram-negative bacteria and two strains isolated from bovine mastitis. RESULTS: In the agar diffusion assay, we observed antibacterial activity for the four honeys against the tested strains. The honey from M. flavolineata showed a minimmum inhibitory concentration (MIC) lower than 3.12% (v/v). The minimum bactericidal concentration values were larger than the MIC for most of the microorganisms tested. Scanning electron microscopy (SEM) showed the damaging effect of the honey of M. flavolineata on Staphylococcus aureus cells, as well as its inhibitory effect on cell division. CONCLUSION: The results of the present study demonstrate that the honey from stingless bees possesses in vitro antimicrobial activity against pathogenic bacteria. The effects observed by SEM show that honey from the Amazonian stingless bee M. flavolineata has promising therapeutic potential as a future antimicrobial agent. © 2020 Society of Chemical Industry.

Porcine rotavirus B as primary causative agent of diarrhea outbreaks in newborn piglets.

Rotavirus (RV) is considered a major cause of acute viral gastroenteritis in young animals. RV is classified into nine species, five of which have been identified in pigs. Most studies worldwide have highlighted diarrhoea outbreaks caused by RVA, which is considered the most important RV species. In the present study, we described the detection and characterization of porcine RVB as a primary causative agent of diarrhoea outbreaks in pig herds in Brazil. The study showed a high frequency (64/90; 71.1%) of RVB diagnosis in newborn piglets associated with marked histopathological lesions in the small intestines. Phylogenetic analysis of the VP7 gene of wild-type RVB strains revealed a high diversity of G genotypes circulating in one geographic region of Brazil. Our findings suggest that RVB may be considered an important primary enteric pathogen in piglets and should be included in the routine differential diagnosis of enteric diseases in piglets.

Genomic Insights Into the Antifungal Activity and Plant Growth-Promoting Ability in Bacillus velezensis CMRP 4490.

The main objective of this study was to evaluate Bacillus velezensis strain CMRP 4490 regarding its ability to inhibit soil-borne plant pathogens and to increase plant growth. The study included evaluation of in vitro antifungal control, sequencing the bacterial genome, mining genes responsible for the synthesis of secondary metabolites, root colonization ability, and greenhouse studies for the assessment of plant growth-promoting ability. The strain was obtained from soil samples in the north of Paraná in Brazil and was classified as a B. velezensis, which is considered a promising biological control agent. In vitro assay showed that B. velezensis CMRP 4490 presented antagonistic activity against Sclerotinia sclerotiorum, Macrophomina phaseolina, Botrytis cinerea, and Rhizoctonia solani with a mycelial growth inhibition of approximately 60%, without any significant difference among them. To well understand this strain and to validate its effect on growth-promoting rhizobacteria, it was decided to explore its genetic content through genome sequencing, in vitro, and greenhouse studies. The genome of CMRP 4490 was estimated at 3,996,396 bp with a GC content of 46.4% and presents 4,042 coding DNA sequences. Biosynthetic gene clusters related to the synthesis of molecules with antifungal activity were found in the genome. Genes linked to the regulation/formation of biofilms, motility, and important properties for rhizospheric colonization were also found in the genome. Application of CMRP 4490 as a coating film on soybean increased from 55.5 to 64% on germination rates when compared to the control; no differences were observed among treatments for the maize germination. The results indicated that B. velezensis CMRP 4490 could be a potential biocontrol agent with plant growth-promoting ability.

Two New 1,3,4-Oxadiazoles With Effective Antifungal Activity Against Candida albicans.

Candida infections have become a serious public health problem with high mortality rates, especially in immunocompromised patients, since Candida albicans is the major opportunistic pathogen responsible for systemic or invasive candidiasis. Commercially available antifungal agents are restricted and fungal resistance to such drugs has increased; therefore, the development of a more specific antifungal agent is necessary. Using assays for antifungal activity, here we report that two new compounds of 1,3,4-oxadiazoles class (LMM5 and LMM11), which were discovered by in silico methodologies as possible thioredoxin reductase inhibitors, were effective against C. albicans. Both compounds had in vitro antifungal activity with MIC 32 µg/ml. Cytotoxicity in vitro demonstrated that LMM5 and LMM11 were non-toxic in the cell lines evaluated. The kinetic of the time-kill curve suggested a fungistatic profile and showed an inhibitory effect of LMM5 and LMM11 in 12 h that remained for 24 and 36 h, which is better than fluconazole. In the murine systemic candidiasis model by C. albicans, the two compounds significantly reduced the renal and spleen fungal burden. According to the SEM and TEM images, we hypothesize that the mechanism of action of LMM5 and LMM11 is directly related to the inhibition of the enzyme thioredoxin reductase and internally affect the fungal cell. In view of all in vitro and in vivo results, LMM5 and LMM11 are effective therapeutic candidates for the development of new antifungal drugs addressing the treatment of human infections caused by C. albicans.

Implications of the presence of yeasts in tracheobronchial secretions of critically ill intubated patients.

The presence of some microorganisms in the respiratory tract is a known risk factor for the infection of air passages; however, it is not clear whether this holds true for Candida spp. Thus, our objective was to determine the frequency of yeast colonization in the tracheobronchial secretions of critically ill intubated patients and to assess the presence of these yeasts in the infra-cuff region of the endotracheal tube (ET). Patients aged 18 years or older who had been using an endotracheal tube for 48 hours were recruited. Tracheal secretions were collected; after extubation, the ETs were cut into two fragments in the infra-cuff region. One of these fragments was placed in a solution containing antibiotics and sent to the lab for culture and identification of yeasts. The remaining fragment was fixed and subjected to scanning electron microscopy (SEM). In total, 20 patients with an average age of 73.3 years (± 13.1) participated in this study. These patients remained under endotracheal intubation and invasive mechanical ventilation for an average of 6.4 (± 1.8) and 13.5 days (± 15), respectively. Of these patients, 45 % showed respiratory tract colonization by yeasts of the Candida genus, with C. albicans being the most frequently isolated species (66.7 %). Moreover, in almost 90 % of these patients, blastoconidia of the same yeast were found in the infra-cuff portion of the ET, as evidenced by SEM, strongly fixed on the ET surface. Yeasts isolated from both the infra-cuff region and the tracheobronchial secretions were susceptible to amphotericin B and fluconazole. In conclusion, our results show that the frequency of colonization by yeasts of the Candida genus in the tracheobronchial secretions of intubated patients within 48 hours is high, and that these species can also be found as a biofilm on the ET surface.

Phenotypic characteristics and transcriptome profile of Cryptococcus gattii biofilm.

In this study, we characterized Cryptococcus gattii biofilm formation in vitro. There was an increase in the density of metabolically active sessile cells up to 72 h of biofilm formation on polystyrene and glass surfaces. Scanning electron microscopy and confocal laser scanning microscopy analysis revealed that in the early stage of biofilm formation, yeast cells adhered to the abiotic surface as a monolayer. After 12 h, extracellular fibrils were observed projecting from C. gattii cells, connecting the yeast cells to each other and to the abiotic surface; mature biofilm consisted of a dense network of cells deeply encased in an extracellular polymeric matrix. These features were also observed in biofilms formed on polyvinyl chloride and silicone catheter surfaces. We used RNA-Seq-based transcriptome analysis to identify changes in gene expression associated with C. gattii biofilm at 48 h compared to the free-floating planktonic cells. Differential expression analysis showed that 97 and 224 transcripts were up-regulated and down-regulated in biofilm, respectively. Among the biological processes, the highest enriched term showed that the transcripts were associated with cellular metabolic processes, macromolecule biosynthetic processes and translation.

Draft Genome Sequence of Vancomycin-Resistant Enterococcus faecium UEL170 (Sequence Type 412), Isolated from a Patient with Urinary Tract Infection in a Tertiary Hospital in Southern Brazil.

Enterococcus faecium is a leading cause of health care-associated infections, with specific lineages circulating in hospital settings worldwide. Here, we report the draft genome sequence of the multidrug-resistant and biofilm-producing E. faecium UEL170, sequence type 412 (ST412), isolated from an inpatient with a urinary tract infection. This strain is a member of clonal complex 17 (CC17), a globally hospital-associated clone.

In vitro interaction of Candida tropicalis biofilm formed on catheter with human cells.

Candida tropicalis has emerged as one of the major Candida non-C. albicans species, in terms of epidemiology and virulence. Despite its virulence, C. tropicalis pathogenic mechanism has yet not been fully defined. The current study aimed to demonstrate the interaction of mature C. tropicalis ATCC 750 biofilm formed on catheter with different human cell lines. In vitro mature (72 h) C. tropicalis biofilms were produced on small catheter fragments (SCF) and were mainly composed by blastoconidia. Then, migration of yeast cells from mature biofilm to human cell surfaces (HeLa and HUVEC) was investigated. After contact with both cell lines, the surface of SCF, containing mature C. tropicalis biofilm, exhibited predominantly the filamentous form. Meanwhile, fresh biofilm formed on human cell surfaces also revealed mainly of blastoconidia involved by extracellular matrix. Total biomass and metabolic activity from the remaining biofilm on SCF surface, after direct contact with human cells, exhibited a significant reduction. Mature C. tropicalis biofilm modified its extracellular matrix components, after contact with human cells. Thus, we described for the first time an easy and simple in vitro model with catheter, which could be a powerful tool for future studies that desires to elucidate the mechanisms involved in C. tropicalis biofilm.

RNAm expression profile of cancer marker genes in HepG2 cells treated with different concentrations of a new indolin-3-one from Pseudomonas aeruginosa.

The present study tested the effects of a newly identified indolin-3-one compound (compound 1), produced by Pseudomonas aeruginosa, on HepG2 cells. The MTT assays demonstrated decreased metabolic activities in HepG2 cells treated with compound 1, with dose- and time-dependent intensifying effect, starting at a concentration of 40 µM. The IC50 after 24, 48, 72, and 96 h treatments were 41.35, 52.7, 92.79 and 66.65 µM of compound 1, respectively. Below 80 µM, no significative damage on erythrocytes membranes was observed by the hemolytic assays. The RT-qPCR revealed that the compound modulated key genes involved in carcinogenesis process, indicating possible indolin-3-one mechanisms of action. The data showed that gene expression alterations promoted by compound 1, in concentrations up to 60 µM after 48 h, led to a decrease in cellular progression and there was no direct cellular damage. In addition, non-cytotoxic concentrations of compound 1 halved the concentration of the chemotherapeutic doxorubicin, maintaining similar therapeutic effect against HepG2 cells. The novelty of the molecule and the biological activities observed in the present study emphasize the potential of the compound 1 in cancer therapy research.

Complete Genome Sequence of Bacillus velezensis LABIM40, an Effective Antagonist of Fungal Plant Pathogens.

Bacillus velezensis strain LABIM40 holds high potential for biological control of plant pathogens. Its complete genome contains one chromosome of 3,972,310 bp with 3,777 DNA coding sequences and displays 33 gene clusters potentially involved in the suppression of fungal pathogens.

Extracellular Vesicles Shed By Trypanosoma cruzi Potentiate Infection and Elicit Lipid Body Formation and PGE2 Production in Murine Macrophages.

During the onset of Trypanosoma cruzi infection, an effective immune response is necessary to control parasite replication and ensure host survival. Macrophages have a central role in innate immunity, acting as an important trypanocidal cell and triggering the adaptive immune response through antigen presentation and cytokine production. However, T. cruzi displays immune evasion mechanisms that allow infection and replication in macrophages, favoring its chronic persistence. One potential mechanism is the release of T. cruzi strain Y extracellular vesicle (EV Y), which participate in intracellular communication by carrying functional molecules that signal host cells and can modulate the immune response. The present work aimed to evaluate immune modulation by EV Y in C57BL/6 mice, a prototype resistant to infection by T. cruzi strain Y, and the effects of direct EV Y stimulation of macrophages in vitro. EV Y inoculation in mice prior to T. cruzi infection resulted in increased parasitemia, elevated cardiac parasitism, decreased plasma nitric oxide (NO), reduced NO production by spleen cells, and modulation of cytokine production, with a reduction in TNF-α in plasma and decreased production of TNF-α and IL-6 by spleen cells from infected animals. In vitro assays using bone marrow-derived macrophages showed that stimulation with EV Y prior to infection by T. cruzi increased the parasite internalization rate and release of infective trypomastigotes by these cells. In this same scenario, EV Y induced lipid body formation and prostaglandin E2 (PGE2) production by macrophages even in the absence of T. cruzi. In infected macrophages, EV Y decreased production of PGE2 and cytokines TNF-α and IL-6 24 h after infection. These results suggest that EV Y modulates the host response in favor of the parasite and indicates a role for lipid bodies and PGE2 in immune modulation exerted by EVs.

Galleria mellonella hemocytes: A novel phagocytic assay for Leishmania (Viannia) braziliensis.

Galleria mellonella is an excellent invertebrate model for the study of diseases that involve interactions with cells from the innate immune system, since they have an innate immune system capable of recognizing the pathogens. Here we present for the first time, an alternative model for an in vitro phagocytic assay using hemocytes of G. mellonella larvae to study infection by Leishmania (Viannia) braziliensis. We showed that the insect phagocytic cells were able to engulf promastigotes. Furthermore, this infective form differentiated into the amastigote form inside those cells. However, the cells in this model seem resistant to the parasite, since amastigotes were depleted after 24h and NO levels were maintained after infection. Our model opens an avenue of possibilities for new investigations regarding other Leishmania species, mechanisms of invasion and evasion, receptors involved, release of signaling molecules and, above all, it is a novel infection model using invertebrate animals.

Guaraná (Paullinia cupana) seeds: Selective supercritical extraction of phenolic compounds.

Approximately 70% of the Brazilian production of guaraná (Paullinia cupana) seeds is absorbed by the beverage industries. Guaraná has several pharmacological properties: energy stimulant, antimicrobial, chemoprophylactic, antigenotoxic, antidepressive, anxiolytic, and anti-amnesic effects. Supercritical carbon dioxide extraction of bioactive compounds from guaraná seeds was carried out and optimized by an orthogonal array design (OA9(3(4))). The factors/levels studied were: modifier(s) (ethanol and/or methanol), extraction time (20, 40, and 60min), temperature (40, 50, and 60°C), and pressure (100, 200, and 300bar). The statistical design was repeated with increasing proportions of modifiers. The percentage of modifier used was proportional to the amount of polar compounds extracted. The best conditions for the supercritical extraction, based on the content of polyphenols, epicatechin/catechin quantification, yield and operating cost, proved to be: 40% ethanol:methanol during 40min, under 40°C, and 100bar. The temperature had a significant effect on the total phenolic content.