Bacteria isolated from Aedes aegypti with potential vector control applications.

Highly anthropophilic and adapted to urban environments, Aedes aegypti mosquitoes are the main vectors of arboviruses that cause human diseases such as dengue, zika, and chikungunya fever, especially in countries with tropical and subtropical climates. Microorganisms with mosquitocidal and larvicidal activities have been suggested as environmentally safe alternatives to chemical or mechanical mosquito control methods. Here, we analyzed cultivable bacteria isolated from all stages of the mosquito life cycle for their larvicidal activity against Ae. aegypti. A total of 424 bacterial strains isolated from eggs, larvae, pupae, or adult Ae. aegypti were analyzed for the pathogenic potential of their crude cultures against larvae of this same mosquito species. Nine strains displayed larvicidal activity comparable to the strain AM65-52, reisolated from commercial BTi-based product VectoBac® WG. 16S rRNA gene sequencing revealed that the set of larvicidal strains contains two representatives of the genus Bacillus, five Enterobacter, and two Stenotrophomonas. This study demonstrates that some bacteria isolated from Ae. aegypti are pathogenic for the mosquito from which they were isolated. The data are promising for developing novel bioinsecticides for the control of these medically important mosquitoes.

Yeasts with Fermentative Potential Associated with Fruits of Camu-Camu (Myrciaria dubia, Kunth) from North of Brazilian Amazon.

Considering the high biotechnological potential of yeasts associated to edible fruits, a screening for these microorganisms, capable of alcoholic fermentation, was performed in ripe fruits of camu-camu (Myrciaria dubia, Kunth). The fruits were collected from north of Brazilian Amazon, in the floodplain of the Cauamé River. Yeasts were isolated, and fermentation capability was evaluated using Durham tubes. Quantitative assays were performed to calculate ethanol yield (g g-1), specific growth rate (h-1), and ethanol productivity (g L-1·h-1). Taxonomic identification was performed by ribosomal gene nucleotide sequence analysis by alignment using BLASTN. A total of fifteen yeast colonies were isolated, and three of them presented promising ability to ferment glucose to ethanol. These isolates were identified as Candida orthopsilosis, Pichia kudriavzevii, and Meyerozyma caribbica. When cultured in broth containing 180 g·L-1 of glucose, M. caribbica CC003 reached 91.7 percent of the maximum theoretical ethanol concentration (84.4 g·L-1), presenting an ethanol yield and productivity of 0.4688 g·g-1 and 0.781 g·L-1·h-1, respectively. These results indicate a promising potential of this isolate for bioprocess applications. This paper is a rare report of C. orthopsilosis with endophytic habit because most of the references indicate it as a human pathogen. Besides this, M. caribbica is a promising fermenter for alcoholic beverages due to its osmotolerance and high ethanol yield. This is the first paper reporting endophytic yeasts associated with fruits of Myrciaria dubia.

Endophytic cultivable bacterial community obtained from the Paullinia cupana seed in Amazonas and Bahia regions and its antagonistic effects against Colletotrichum gloeosporioides.

Guarana (Paullinia cupana var. sorbilis) is a plant from the Amazonas region with socio-economic importance. However, guarana production has been increasingly affected by unfavorable conditions resulting from anthracnose, caused by the Colletotrichum fungal genus, which primarily affects mainly the Amazonas region. The aim of the present study was to isolate bacterial endophytes from the seeds of guarana plants obtained from Amazonas region and the Northeast state of Bahia, a region where this disease is not a problem for guarana plantations. The number of bacterial Colony Forming Units (CFU/g seeds) was 2.4 × 10(4) from the Bahia and 2.9 × 10(4) from the Amazonas region. One hundred and two isolated bacteria were evaluated in vitro against the phytopathogenic strain Colletotrichum gloeosporioides L1. These isolates were also analyzed for the enzymatic production of amylase, cellulase, protease, pectinase, lipase and esterase. Approximately 15% of isolates, showing high antagonistic activity, and the production of at least one enzyme were identified through the partial sequencing of 16S rDNA. The genus Bacillus was the most frequently observed, followed by Paenibacillus, Ochrobactrum, Microbacterium and Stenotrophomonas. Proteolytic activity was observed in 24 isolates followed by amylolytic, pectinolytic and cellulolytic activities. No esterase and lipase production was detected. Most of the isolates, showing antagonistic effects against C. gloeosporioides and high enzymatic activities, were isolated from the anthracnose-affected region. A biocontrol method using the endophytes from guarana seeds could be applied in the future, as these bacteria are vertically transferred to guarana seedlings.

Conservation implications of the mating system of the Pampa Hermosa landrace of peach palm analyzed with microsatellite markers.

Peach palm (Bactris gasipaes) is cultivated by many indigenous and traditional communities from Amazonia to Central America for its edible fruits, and is currently important for its heart-of-palm. The objective of this study was to investigate the mating system of peach palm, as this is important for conservation and breeding. Eight microsatellite loci were used to genotype 24 open-pollinated progenies from three populations of the Pampa Hermosa landrace maintained in a progeny trial for genetic improvement. Both the multi-locus outcrossing rates (0.95 to 0.99) and the progeny level multi-locus outcrossing rates (0.9 to 1.0) were high, indicating that peach palm is predominantly allogamous. The outcrossing rates among relatives were significantly different from zero (0.101 to 0.202), providing evidence for considerable biparental inbreeding within populations, probably due to farmers planting seeds of a small number of open-pollinated progenies in the same plot. The correlations of paternity estimates were low (0.051 to 0.112), suggesting a large number of pollen sources (9 to 20) participating in pollination of individual fruit bunches. Effective population size estimates suggest that current germplasm collections are insufficient for long-term ex situ conservation. As with most underutilized crops, on farm conservation is the most important component of an integrated conservation strategy.

Preliminary profile of the gut microbiota from amerindians in the Brazilian amazon experiencing a process of transition to urbanization.

The Yanomami are one of the oldest indigenous tribes in the Amazon and are direct descendants of the first people to colonize South America 12,000 years ago. They are located on the border between Venezuela and Brazil, with the Venezuelan side remaining uncontacted. While they maintain a hunter-gatherer society, they are currently experiencing contact with urbanized populations in Brazil. The human gut microbiota of traditional communities has become the subject of recent studies due to the Westernization of their diet and the introduction of antibiotics and other chemicals, which have affected microbial diversity in indigenous populations, thereby threatening their existence. In this study, we preliminarily characterized the diversity of the gut microbiota of the Yanomami, a hunter-gatherer society from the Amazon, experiencing contact with urbanized populations. Similarly, we compared their diversity with the population in Manaus, Amazonas. A metabarcoding approach of the 16 S rRNA gene was carried out on fecal samples. Differences were found between the two populations, particularly regarding the abundance of genera (e.g., Prevotella and Bacteroides) and the higher values of the phyla Bacteroidetes over Firmicutes, which were significant only in the Yanomami. Some bacteria were found exclusively in the Yanomami (Treponema and Succinivibrio). However, diversity was statistically equal between them. In conclusion, the composition of the Yanomami gut microbiota still maintains the profile characteristic of a community with a traditional lifestyle. However, our results suggest an underlying Westernization process of the Yanomami microbiota when compared with that of Manaus, which must be carefully monitored by authorities, as the loss of diversity can be a sign of growing danger to the health of the Yanomami.

Using an Aluminum Hydroxide-Chitosan Matrix Increased the Vaccine Potential and Immune Response of Mice against Multi-Drug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative, immobile, aerobic nosocomial opportunistic coccobacillus that causes pneumonia, septicemia, and urinary tract infections in immunosuppressed patients. There are no commercially available alternative antimicrobials, and multi-drug resistance is an urgent concern that requires emergency measures and new therapeutic strategies. This study evaluated a multi-drug-resistant A. baumannii whole-cell vaccine, inactivated and adsorbed on an aluminum hydroxide-chitosan (mAhC) matrix, in an A. baumannii sepsis model in immunosuppressed mice by cyclophosphamide (CY). CY-treated mice were divided into immunized, non-immunized, and adjuvant-inoculated groups. Three vaccine doses were given at 0D, 14D, and 28D, followed by a lethal dose of 4.0 × 108 CFU/mL of A. baumannii. Immunized CY-treated mice underwent a significant humoral response, with the highest IgG levels and a higher survival rate (85%); this differed from the non-immunized CY-treated mice, none of whom survived (p < 0.001), and from the adjuvant group, with 45% survival (p < 0.05). Histological data revealed the evident expansion of white spleen pulp from immunized CY-treated mice, whereas, in non-immunized and adjuvanted CY-treated mice, there was more significant organ tissue damage. Our results confirmed the proof-of-concept of the immune response and vaccine protection in a sepsis model in CY-treated mice, contributing to the advancement of new alternatives for protection against A. baumannii infections.

Antigen-Specific Antibody Signature Is Associated with COVID-19 Outcome.

Numerous studies have focused on inflammation-related markers to understand COVID-19. In this study, we performed a comparative analysis of spike (S) and nucleocapsid (N) protein-specific IgA, total IgG and IgG subclass response in COVID-19 patients and compared this to their disease outcome. We observed that the SARS-CoV-2 infection elicits a robust IgA and IgG response against the N-terminal (N1) and C-terminal (N3) region of the N protein, whereas we failed to detect IgA antibodies and observed a weak IgG response against the disordered linker region (N2) in COVID-19 patients. N and S protein-specific IgG1, IgG2 and IgG3 response was significantly elevated in hospitalized patients with severe disease compared to outpatients with non-severe disease. IgA and total IgG antibody reactivity gradually increased after the first week of symptoms. Magnitude of RBD-ACE2 blocking antibodies identified in a competitive assay and neutralizing antibodies detected by PRNT assay correlated with disease severity. Generally, the IgA and total IgG response between the discharged and deceased COVID-19 patients was similar. However, significant differences in the ratio of IgG subclass antibodies were observed between discharged and deceased patients, especially towards the disordered linker region of the N protein. Overall, SARS-CoV-2 infection is linked to an elevated blood antibody response in severe patients compared to non-severe patients. Monitoring of antigen-specific serological response could be an important tool to accompany disease progression and improve outcomes.

An Electrochemical Immunosensor Based on Carboxylated Graphene/SPCE for IgG-SARS-CoV-2 Nucleocapsid Determination.

The COVID-19 pandemic has emphasized the importance and urgent need for rapid and accurate diagnostic tests for detecting and screening this infection. Our proposal was to develop a biosensor based on an ELISA immunoassay for monitoring antibodies against SARS-CoV-2 in human serum samples. The nucleocapsid protein (N protein) from SARS-CoV-2 was employed as a specific receptor for the detection of SARS-CoV-2 nucleocapsid immunoglobulin G. N protein was immobilized on the surface of a screen-printed carbon electrode (SPCE) modified with carboxylated graphene (CG). The percentage of IgG-SARS-CoV-2 nucleocapsid present was quantified using a secondary antibody labeled with horseradish peroxidase (HRP) (anti-IgG-HRP) catalyzed using 3,3',5,5'-tetramethylbenzidine (TMB) mediator by chronoamperometry. A linear response was obtained in the range of 1:1000-1:200 v/v in phosphate buffer solution (PBS), and the detection limit calculated was 1:4947 v/v. The chronoamperometric method showed electrical signals directly proportional to antibody concentrations due to antigen-antibody (Ag-Ab) specific and stable binding reaction.

Bacillus subtilis spores as delivery system for nasal Plasmodium falciparum circumsporozoite surface protein immunization in a murine model.

Malaria remains a widespread public health problem in tropical and subtropical regions around the world, and there is still no vaccine available for full protection. In recent years, it has been observed that spores of Bacillus subtillis can act as a vaccine carrier and adjuvant, promoting an elevated humoral response after co-administration with antigens either coupled or integrated to their surface. In our study, B. subtillis spores from the KO7 strain were used to couple the recombinant CSP protein of P. falciparum (rPfCSP), and the nasal humoral-induced immune response in Balb/C mice was evaluated. Our results demonstrate that the spores coupled to rPfCSP increase the immunogenicity of the antigen, which induces high levels of serum IgG, and with balanced Th1/Th2 immune response, being detected antibodies in serum samples for 250 days. Therefore, the use of B. subtilis spores appears to be promising for use as an adjuvant in a vaccine formulation.

High prevalence detection of Chlamydia trachomatis by polymerase chain reaction in endocervical samples of infertile women attending university hospital in Manaus-Amazonas, Brazil.

BACKGROUND: We established for the first time the prevalence of Chlamydia trachomatis infection among infertile women in Manaus Amazonas Brazil using the polymerase chain reaction (PCR) technique. METHODS: 106 women were studied at a public university hospital fertility clinic for infertility problems correlated with chlamydia infection. Social-economic and clinical information was obtained before medical examination to obtain samples for the amplification of C. trachomatis DNA plasmid. RESULTS: The prevalence of chlamydial infection among infertile women was 52.8, and 51.8% of the positive participants were older than 30 years of age (p = 0.8697). Of the 56 women positive for C. trachomatis, 31 (55.4%) had never given birth, while 9 (16%) had a pregnancy resulting in fetal death. Our findings also revealed that these patients had a low socio-economic status and high unemployment (p = 0.0274), and a significant association of 5% (p < 0.05) of chlamydial infection with family income. CONCLUSIONS: Due to the high prevalence of C. trachomatis, concerns about effects on reproductive health and fertility are undertaken. Therefore, we believe it is extremely necessary to implement large-scale PCR-based screening as part of routine clinical detection programs concerning preventive effects of chlamydial infection among this population.

Enzymatic Hydrolysis of Lignocellulosic Biomass Using an Optimized Enzymatic Cocktail Prepared from Secretomes of Filamentous Fungi Isolated from Amazonian Biodiversity.

The use of lignocellulosic biomass (LCB) has emerged as one of the main strategies for generating renewable biofuels. For the efficient use of such feedstock, pre-treatments are essential. The hydrolysis of cellulose - major component of LCB - demands enzymatic cocktails with improved efficiency to generate fermentable sugars. In this scenario, lignocellulolytic fungi have enormous potential for the development of efficient enzyme platforms. In this study, two enzymatic cocktails were developed for hydrolysis of two lignocellulosic biomasses: industrial cellulose pulp and cassava peel. The solid biomass ratio in relation to the protein content of the enzyme cocktail was performed by experimental design. The optimized cocktail for the hydrolysis of cellulose pulp (AMZ 1) was composed, in protein base, by 43% of Aspergillus sp. LMI03 enzyme extract and 57% of T. reesei QM9414, while the optimal enzyme cocktail for cassava peel hydrolysis (AMZ 2) was composed by 50% of Aspergillus sp. LMI03 enzyme extract, 25% of the extract of P. citrinum LMI01 and 25% of T. reesei. The ratio between solids and protein loading for AMZ 1 cocktail performance was 52 g/L solids and 30 mg protein/g solids, resulting in a hydrolytic efficiency of 93%. For the AMZ 2 cocktail, the hydrolytic efficiency was 78% for an optimized ratio of 78 g/L solids and 19 mg protein/g solids. These results indicate that cocktails formulated with enzymatic extracts of P. citrinum LMI01, Aspergillus sp. LMI03, and T. reesei QM9414 are excellent alternatives for efficient hydrolysis of plant biomass and for other processes that depend on biocatalysis.

Physical Mapping of the Anopheles (Nyssorhynchus) darlingi Genomic Scaffolds.

The genome assembly of Anopheles darlingi consists of 2221 scaffolds (N50 = 115,072 bp) and has a size spanning 136.94 Mbp. This assembly represents one of the smallest genomes among Anopheles species. Anopheles darlingi genomic DNA fragments of ~37 Kb were cloned, end-sequenced, and used as probes for fluorescence in situ hybridization (FISH) with salivary gland polytene chromosomes. In total, we mapped nine DNA probes to scaffolds and autosomal arms. Comparative analysis of the An. darlingi scaffolds with homologous sequences of the Anopheles albimanus and Anopheles gambiae genomes identified chromosomal rearrangements among these species. Our results confirmed that physical mapping is a useful tool for anchoring genome assemblies to mosquito chromosomes.

Anticoagulant activity in salivary gland homogenates of Thyrsopelma guianense (Diptera: Simuliidae), the primary vector of onchocerciasis in the Brazilian Amazon.

In this study, anticoagulant activity was detected in salivary gland homogenates (SGHs) of Thyrsopelma guianense (Diptera: Simuliidae). The SGH yielded 1.07 microg +/- 0.03 (n = 15) of total soluble protein per pair of glands. In addition, following SDS-PAGE (12.5% gel) and silver nitrate staining, 12 polypeptides with molecular weights ranging from 14-69 kDa were detected in all physiological ages analyzed (12 h, 24 h, 48 h and 72 h following emergence). Coagulation bioassays showed that the SGHs had activities that interacted at all levels of coagulation (the intrinsic, extrinsic and common pathways), by extending the plasma recalcification time, prothrombin time, thrombin time. This is the first report on the activity of salivary gland proteins from the main vector of onchocerciasis in Brazil. We also suggest detailed studies on the morphology and function of the salivary glands in order to understand the role of these proteins in host/vector interactions.

Mutational data and population profiling of 23 Y-STRs in three Brazilian populations.

Y-chromosomal STRs are important markers in forensic genetics, due to some peculiar characteristics. The absence of recombination makes them a useful tool to infer kinship in complex cases involving distant paternal relatives, or to infer paternal bio-geographic ancestry. The presence of a single copy, being transmitted from father to son, allow tracing mutational events in Y-STRs without ambiguity. For the statistical interpretation of forensic evidences based on Y-STR profiles, it is necessary to have estimates on both mutation rates and haplotype frequencies. In this work, 407 father-son duos from São Paulo and Rio de Janeiro states and 204 unrelated individuals from Manaus were analyzed. Haplotype frequencies and mutation rates for the Y-STRs from the PowerPlex Y23 commercial kit were estimated. Thirty-six mutations were observed in 15 of the 22 Y-STRs analyzed, for an average mutation rate of 3.84 × 10-3 (95 % CI 2.69 × 10-3 to 5.32 × 10-3). All mutations in GAAA repeats occurred in alleles with 13 or more uninterrupted units. Mutations in GATA repeats were observed in alleles with 9-17 uninterrupted units. An analysis carried out in different father's age groups showed an increase of 2.48 times the mutation rate in the age group of 40-50 years, when compared to the 20-30 age group, in agreement with the described for autosomal STRs. A high haplotype diversity was found in the three Brazilian populations. Pairwise genetic distance analysis (FST) showed no significant differences between the three populations in this study, which were also close to populations with strong European influence. The highest distances among the Brazilian populations were with São Gabriel da Cachoeira, which has a high Native American ancestry.

Draft Genome Sequence of Burkholderia gladioli Coa14, a Bacterium with Petroleum Bioremediation Potential Isolated from Coari Lake, Amazonas, Brazil.

Burkholderia gladioli Coa14 is a bacterium isolated from water collected from Coari Lake (Amazonas, Brazil) that shows a capacity for survival in a medium containing only oil as a carbon source. Here, we report its draft genome sequence, highlighting some genes involved with petroleum derivative degradation.

Draft Genome Sequence of the Novel Enterobacter cloacae Strain amazonensis, a Highly Heavy Metal-Resistant Bacterium from a Contaminated Stream in Amazonas, Brazil.

Here, we report the draft genome of the Enterobacter cloacae strain amazonensis, a bacterium highly resistant to mercury that was isolated from a metal- and sewage-contaminated stream in Amazonas, Brazil. The exploration of the 5.0-Mb genome revealed 104 genes encoding resistance to toxic compounds and heavy metals, highlighting the potential biotechnological applications of this strain.

Analysis of Chromobacterium sp. natural isolates from different Brazilian ecosystems.

BACKGROUND: Chromobacterium violaceum is a free-living bacterium able to survive under diverse environmental conditions. In this study we evaluate the genetic and physiological diversity of Chromobacterium sp. isolates from three Brazilian ecosystems: Brazilian Savannah (Cerrado), Atlantic Rain Forest and Amazon Rain Forest. We have analyzed the diversity with molecular approaches (16S rRNA gene sequences and amplified ribosomal DNA restriction analysis) and phenotypic surveys of antibiotic resistance and biochemistry profiles. RESULTS: In general, the clusters based on physiological profiles included isolates from two or more geographical locations indicating that they are not restricted to a single ecosystem. The isolates from Brazilian Savannah presented greater physiologic diversity and their biochemical profile was the most variable of all groupings. The isolates recovered from Amazon and Atlantic Rain Forests presented the most similar biochemical characteristics to the Chromobacterium violaceum ATCC 12472 strain. Clusters based on biochemical profiles were congruent with clusters obtained by the 16S rRNA gene tree. According to the phylogenetic analyses, isolates from the Amazon Rain Forest and Savannah displayed a closer relationship to the Chromobacterium violaceum ATCC 12472. Furthermore, 16S rRNA gene tree revealed a good correlation between phylogenetic clustering and geographic origin. CONCLUSION: The physiological analyses clearly demonstrate the high biochemical versatility found in the C. violaceum genome and molecular methods allowed to detect the intra and inter-population diversity of isolates from three Brazilian ecosystems.

Isolation and Characterization of Yeasts Able to Assimilate Sugarcane Bagasse Hemicellulosic Hydrolysate and Produce Xylitol Associated with Veturius transversus (Passalidae, Coleoptera, and Insecta).

Yeasts are an important component of insect gut microbial content, playing roles such as degradation of polymers and toxic compounds, biological control, and hormone, vitamin, and digestive enzyme production. The xylophagous beetle gut is a hyperdiverse habitat and a potential source of new species with industrial abilities such as enzyme production, pentose fermentation, and biodetoxification. In this work, samples of Veturius transversus (Passalidae, Coleoptera, and Insecta) were collected from the Central Amazon Rainforest. Their guts were dissected and a total of 20 microbial colonies were isolated using sugarcane bagasse hemicellulosic hydrolysate. They were identified as having 10 distinct biochemical profiles, and genetic analysis allowed identification as three clades in the genera Candida, Williopsis, and Geotrichum. All colonies were able to assimilate D-xylose and 18 were able to produce xylitol, especially a strain of Geotrichum, with a maximum yield of 0.502 g·g-1. These results agree with a previous prediction that the microbial community associated with xylophagous insects is a promising source of species of biotechnological interest.

Oral focal epithelial hyperplasia: report of five cases.

Focal epithelial hyperplasia or Heck's disease is a rare contagious disease caused by human papillomavirus types 13 or 32, initially described among Native American populations. This condition is characterized by the occurrence of multiple small papules or nodules in oral cavity, especially on labial and buccal mucosa and tongue. This report describes the diagnosis of focal epithelial hyperplasia in five Central Amazonian Indians who sought treatment at the Amazonas State Foundation of Tropical Medicine (FMT-AM), using clinical criteria, polymerase chain reaction (PCR) and DNA sequencing.

Molecular characterization of the gene feminizer in the stingless bee Melipona interrupta (Hymenoptera: Apidae) reveals association to sex and caste development.

In highly eusocial insects, development of reproductive traits are regulated not only by sex determination pathway, but it also depends on caste fate. The molecular basis of both mechanisms in stingless bees and possible interaction with each other is still obscure. Here, we investigate sex determination in Melipona interrupta, focusing on characterization and expression analysis of the feminizer gene (Mi-fem), and its association to a major component of caste determination, the juvenile hormone (JH). We present evidence that Mi-fem mRNA is sex-specifically spliced in which only the female splice variant encodes the full length protein, following the same principle known for other bee species. We quantified Mi-fem expression among developmental stages, sexes and castes. Mi-fem expression varies considerably throughout development, with higher expression levels in embryos. Also, fem levels in pupae and newly emerged adults were significantly higher in queens than workers and males. Finally, we ectopically applied JH in cocoon spinning larvae, which correspond to the time window where queen/worker phenotypes diverge. We observed a significantly increase in Mi-fem expression compared to control groups. Since up to 100% of females turn into queens when treated with JH (while control groups are composed mainly of workers), we propose that fem might act to regulate queens' development. Our findings provide support for the conserved regulatory function of fem in Melipona bees and demonstrate a significant correlation between key elements of sex and caste determination pathways, opening the avenue to further investigate the molecular basis of these complex traits.