Inhibition of the norA gene expression and the NorA efflux pump by the tannic acid.

The NorA efflux pump of Staphylococcus aureus is known to play a major role in the development of resistance against quinolone drugs by reducing their concentration inside target pathogens. The objective of this study was to evaluate the ability of tannic acid to inhibit the gene expression of the NorA efflux pump in Staphylococcus aureus and to evaluate the in silico effect on the pump. Efflux pump inhibition was evaluated by fluorimetry. The checkerboard method evaluates the effect of the test substance in combination with an antimicrobial at different concentrations. To gene expression evaluation NorA the assay was performed using: a sub-inhibitory concentration preparation (MIC/4) of the antibiotic; a sub-inhibitory concentration preparation (MIC/4) of the antibiotic associated with tannic acid at a sub-inhibitory concentration (MIC/4). In this study, docking simulations were performed by the SWISSDOCK webserver. The ability of tannic acid to inhibit the NorA efflux pump can be related to both the ability to inhibit the gene expression of this protein, acting on signaling pathways involving the ArlRS membrane sensor. As well as acting directly through direct interaction with the NorA protein, as seen in the approach and in silico and in vitro per checkerboard method and fluorimetry of bromide accumulated in the cell.

Antibacterial and Toxic Activity of Geopropolis Extracts from Melipona subnitida (Ducke, 1910) (Hymenoptera: Apidae) and Scaptotrigona depilis (Moure, 1942) (Hymenoptera: Apidae).

Bacteria are associated with many infections that affect humans and present antibiotic resistance mechanisms, causing problems in health organisations and increased mortality rates. Therefore, it is necessary to find new antibacterial agents that can be used in the treatment of these microorganisms. Geopropolis is a natural product from stingless bees, formed by a mixture of plant resins, salivary secretions, wax and soil particles, the chemical composition of this natural product is diverse. Thus, this study aimed to evaluate antibacterial activity, antibiotic modulation and the toxicity of geopropolis extracts from the stingless bees, Melipona subnitida (Ducke, 1910) and Scaptotrigona depilis (Moure, 1942) against standard and multi-resistant Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa bacteria. Geopropolis samples were collected in a meliponary located in Camaragibe, Pernambuco, Brazil. To determine the Minimum Inhibitory Concentration (MIC) and antibiotic modulation we performed broth microdilution tests. Mortality tests were used to verify extract toxicity in the model Drosophila melanogaster. The microbiological tests showing that the M. subnitida extracts had better inhibitory effects compared to S. depilis, presenting direct antibacterial activity against standard and multi-resistant strains. The extracts potentialized antibiotic effects, suggesting possible synergy and did not present toxicity in the model used. The information obtained in this study highlights extracts as promising antibacterial agents and is the first study to evaluate bacterial activity in these extracts, in addition to verifying their modulating effects and determining toxicity in the model used.

Allele Frequencies and Forensic Data of 25 STR Markers for Individuals in Northeast Brazil.

Identifying DNA markers such as Short Tandem Repeats (STR) can be used to investigate genetic diversity based on levels of heterozygosity within and between populations. Allele frequencies and forensic data for STRs were obtained from a sample of 384 unrelated individuals living in Bahia, Northeastern Brazil. Thus, the present study aimed to identify the allele frequency distribution, in addition to the forensic and genetic data, of 25 STR loci in the population of Bahia. Buccal swabs or fingertip punctures were utilized to amplify and detect 25 DNA markers. The most polymorphic loci were SE33 (43), D21S11, and FGA (21). The least polymorphic were TH01 (6), TPOX, and D3S1358 (7). Forensic and statistical data were obtained through data analysis, which revealed a large genetic diversity, with an average value of 0.813 for the analyzed population. The present study was more robust than previous STR marker studies and will contribute to future research on population genetics in Brazil and worldwide. The results of this study allowed the establishment of haplotypes found in the forensic samples of Bahia State to serve as a reference in the elucidation of criminal cases and paternity tests, as well as population and evolutionary investigations.

Temporal Changes in Gut Microbiota Composition and Pollen Diet Associated with Colony Weakness of a Stingless Bee.

Compared to honeybees and bumblebees, the effect of diet on the gut microbiome of Neotropical corbiculate bees such as Melipona spp. is largely unknown. These bees have been managed for centuries, but recently an annual disease is affecting M. quadrifasciata, an endangered species kept exclusively by management in Southern Brazil. Here we report the results of a longitudinal metabarcoding study involving the period of M. quadrifasciata colony weakness, designed to monitor the gut microbiota and diet changes preceding an outbreak. We found increasing amounts of bacteria associated to the gut of forager bees 2 months before the first symptoms have been recorded. Simultaneously, forager bees showed decreasing body weight. The accelerated growth of gut-associated bacteria was uneven among taxa, with Bifidobacteriaceae dominating, and Lactobacillaceae decreasing in relative abundance within the bacterial community. Dominant fungi such as Candida and Starmerella also decreased in numbers, and the stingless bee obligate symbiont Zygosaccharomyces showed the lowest relative abundance during the outbreak period. Such changes were associated with pronounced diet shifts, i.e., the rise of Eucalyptus spp. pollen amount in forager bees' guts. Furthermore, there was a negative correlation between the amount of Eucalyptus pollen in diets and the abundance of some bacterial taxa in the gut-associated microbiota. We conclude that diet and subsequent interactions with the gut microbiome are key environmental components of the annual disease and propose the use of diet supplementation as means to sustain the activity of stingless bee keeping as well as native bee pollination services.

Allelic frequencies distribution and forensic parameters of 23 autosomal short tandem repeats in the population of the State of Pernambuco, Brazil.

Located in the Northeast Region, the Pernambuco State is one of the 27 federative units of Brazil. Here, we determined populational data for 23 short tandem repeat (STR) markers - CSF1PO, FGA, TH01, TPOX, vWA, D1S1656, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D22S1045, PENTA D, PENTA E and SE33 - of the Pernambuco population. The sample consisted of 767 healthy, adult, unrelated individuals (437 males, 330 females) born and resident in the State of Pernambuco. STRs were amplified using three multiplex kits, according to the availability: PowerPlex® Fusion 6C System (Promega Corporation), PowerPlex® Fusion System (Promega Corporation) and GlobalFiler™ Express (Thermo Fisher Scientific). Allelic frequencies, forensic parameters and Hardy-Weinberg equilibrium determinations were estimated for all the 23 loci. No deviations from the Hardy-Weinberg equilibrium were observed for any of the markers, after Bonferroni correction. We observed that the most and less informative markers were SE33 and TPOX, respectively. The combined power of discrimination (CPD) was 0.99999999999999999999999999999, and the combined power of exclusion (CPE) was 0.99999999997. The cumulative typical paternity index was 37,919,301,869.3021. Interpopulation analyses (Nei's genetic distance) based on the expanded CODIS core loci was performed between the Pernambuco sample and other global populations. Pernambuco was the closest Brazilian population to African group and stayed distant from the Native American group. This work contributed to show that a panel of 23 autosomal STR loci is very informative, being able for forensic applications related in this population.

Genetic characteristics and phylogenetic analysis of Brazilian clinical strains of Pseudomonas aeruginosa harboring CRISPR/Cas systems.

The CRISPR-Cas are adaptive immune systems found in archaea and bacteria, responsible for providing sequence-specific resistance against foreign DNA. Strains of Pseudomonas aeruginosa may carry CRISPR/Cas system types I-F, I-E and/or I-C; however, several aspects related to the epidemiology and functionality of these systems have not yet been revealed. Here, we report 13 genomes of clinical strains of P. aeruginosa from Brazil that were positive for CRISPR/Cas system types I-F and I-E, a rare feature in this species. The phylogenetic tree, which was constructed with 161 other publicly available genomes, suggested no direct relationship between positive strains, and the various types of CRISPR/Cas systems were spread throughout the tree. Comparative analysis of the genetic locations of type I-F and a specific orphan CRISPR array (without cas genes), named the LES locus, showed sequence similarities between this orphan locus and type I-F, but these LES loci were inserted in a different genomic location. We also report the presence of prophages, the presence of anti-CRISPR genes, and possibly the presence of self-targeting spacers. Here, we conclude that CRISPR/Cas is highly associated with certain lineages and is spread throughout the phylogenetic tree, showing no clear pattern of evolutionary distribution. Moreover, the LES locus might be a CRISPR1 locus related to type I-F that may have been misplaced and maintained over time. Furthermore, strains carrying I-F and I-E are rare, and not all of them are closely related. Further functional work is needed to better comprehend if aspects reported in this study are functional, including the LES locus, self-targeting spacers, anti-CRISPR protection, and I-F/I-E-carrying lineages.

Helicobacter pylori cagE, cagG, and cagM can be a prognostic marker for intestinal and diffuse gastric cancer.

It is known that Helicobacter pylori is the main cause of peptic ulceration and gastric cancer. However, there is a lack of information on whether H. pylori strains may differ in gastric cancer histological subtypes. This study aimed to investigate different H. pylori strains considering six cag Pathogenicity Island - cagPAI genes (cagA, cagE, cagG, cagM, cagT, and virb11), and vacuolating cytotoxin - vacA alleles, and their relation to gastric cancer histologic subtypes. For this purpose, tumor samples from 285 patients with gastric carcinoma were used. H. pylori infection and genotypes were determined by polymerase chain reaction (PCR). H. pylori was detected in 93.9% of gastric tumors. For comparative analyzes between histopathological subtypes considering H. pylori cagPAI genes the strains were grouped according to the vacA s1/s2 alleles. In the vacAs1 group, the strains cagA(-)cagE(+), cagA(+)cagE(+)cagG(+), cagA(+)cagM(+), or only cagE(+) strains were more frequent in the intestinal subtype (P = .009; P = .024; P = .046, respectively). In contrast, cagM(+)cagG(+)cagA(-) and cagE(-) were associated with diffuse tumors (P = .036), highlighting the presence of cagE in the development of intestinal tumors, and the presence of cagG and absence of cagE in diffuse tumors. Furthermore, WEKA software and Decision Tree (CART) analyses confirmed these findings, in which cagE presence was associated with intestinal tumors, and cagE absence and cagG(+) with diffuse tumors. In conclusion our results showed that vacAs1 (cagG + cagM) strains, mainly cagG positive with cagE absence, were relevant in the studied population for the diffuse outcome, while the presence of cagE was relevant for the intestinal outcome. These findings suggest the relevance of these H. pylori genes as potential markers for gastric cancer histological outcomes.

Comparative Analysis of the Antibacterial Activity and HPLC Phytochemical Screening of the Brazilian Red Propolis and the Resin of Dalbergia ecastaphyllum.

The aim of this study was to investigate the antibacterial activity of red propolis and resin and their association with standard antibiotics to evaluate possible differences of activity. We also submitted red propolis and the resin to a HPLC analysis to confirm the botanical origin. The extracts were tested against P. aeruginosa and S. aureus alone and in association with gentamicin and imipenem. The HPLC analysis identified seven compounds with six of them present in both substances. The lowest MIC values obtained in this study were observed against S. aureus. In general, MIC values showed to be lower for red propolis against all species tested in comparison to resin. Despite the synergistic behavior to be similar for both substances, we observed that inhibitory concentrations of drugs were lower when associated with red propolis in comparison to resin.

Antitrypanosomal, antileishmanial and cytotoxic activities of Brazilian red propolis and plant resin of Dalbergia ecastaphyllum (L) Taub.

The treatment for leishmaniasis and Chagas disease can be hard and painful, such that many patients give up on the treatment. In order to find an alternative path for the treatment of these diseases, researchers are using natural products to fight these parasites. The aim of this study was to evaluate the antiprotozoan and cytotoxic activities of red propolis samples collected from different Brazilian states and seasons whilst searching for possible activity differences. We also compared the red propolis results with the ones obtained for the plant resin extract collected from Dalbergia ecastaphyllum trees. The hydroethanolic red propolis extracts from Pernambuco and Alagoas, and the D. ecastaphyllum resin were evaluated regarding their antileishmanial, antitrypanosomal and cytotoxic activity. All extracts showed antiprotozoan and cytotoxic activity. RP-PER showed to be more cytotoxic against protozoan parasites and fibroblast cells. All propolis extracts showed a higher cytotoxic activity when compared to resin extracts. The propolis sample collected in Pernambuco during the rainy season killed the parasites with lower concentrations than the sample collected in the dry season. The IC50 observed against the parasites could be used without high fibroblast cell damage.

New Records of Phlebotomine Sand Flies (Diptera: Psychodidae) From the State of Alagoas, Northeast of Brazil.

Phlebotomine sand flies are small insects of great medical importance. This study presents new records of phlebotomine sand flies, which augment the list of species, recorded in the state of Alagoas, Brazil. Sand flies were collected using CDC miniature light traps. Collections were made between November 2008 and April 2016, in 47 municipalities within the state of Alagoas. Twenty-seven thousand two-hundred forty-four sand flies were collected, representing 18 species in seven genera. Lutzomyia longipalpis was the predominant species (85.4% of total), and it was recorded in all biomes. Three species (Evandromyia evandroi (Costa Lima and Antunes 1936), Evandromyia tupynambai (Mangabeira 1942), and Pressatia choti (Floch and Abonnenc 1941)) were collected in coastal and Atlantic forest biomes; five species (Lutzomyia villelai (Mangabeira 1942), Micropygomyia oswaldoi (Mangabeira 1942), Nyssomyia umbratilis (Ward and Frahia 1977), Nyssomyia intermedia (Lutz and Neiva 1912), and Nyssomyia whitmani (Antunes and Coutinho 1939)) were collected in Atlantic forest and Caatinga biomes. The species Evandromyia corumbaensis (Galati, Nunes, Oshiro and Rego 1989) and Psathyromyia naftalekatzi (Falcão, Andrade-Filho, Almeida and Brandão-Filho 2000) were found only in the coastal zones. This work provides an updated list of the phlebotomine sand flies of Alagoas, with some notes on their taxonomy, ecology, distribution, and epidemiological relevance.

Seasonal variation of Brazilian red propolis: Antibacterial activity, synergistic effect and phytochemical screening.

The aim of this study was to investigate the effect of the dry and rainy season on the antibacterial activity and chemical composition of the Brazilian red propolis. The samples were collected in rainy (RP-PER) and dry (RP-PED) seasons and analyzed by HPLC-DAD. The extracts were tested alone and in association with antibiotics against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The HPLC analysis identified luteolin and quercetin as the main compounds. Seasonal variation was observed according to concentrations of the compounds. The MIC values against E. coli ranged from 128 µg/mL to 512 µg/mL (EC 06 and EC ATCC). The red propolis showed MIC values of 512 µg/mL against both strains of P. aeruginosa used in our study (PA03 and PA24) and against strains of Gram-positive bacteria S. aureus the MICs ranged from 64 µg/mL to ≥1024 µg/mL (SA10). A synergistic effect was observed when we combined the RP-PED with gentamicin against all the strains tested. When we combined the RP-PED with Imipenem, we only observed synergistic effect against P. aeruginosa. According to our synergistic activity results, the utilization of red propolis collected in the drier periods can be used as an adjuvant against multiresistant bacterial infections.

Association of genetic polymorphisms of IL1ß -511 C>T, IL1RN VNTR 86 bp, IL6 -174 G>C, IL10 -819 C>T and TNFα -308 G>A, involved in symptomatic patients with dengue in Brazil.

OBJECTIVE: In this study, single nucleotide polymorphisms (SNP) of interleukin (IL) 1ß -511C>T, IL1RN VNTR 86 bp, IL6 -174G>C, IL10 -819C>T and TNFα -308G>A were analyzed by PCR-RFLP with symptoms of dengue with the clinical features. SUBJECTS: 196 individuals admitted to the São José Infectious Diseases Hospital with suspected dengue infection. Dengue was confirmed in 111 of the patients. The control group consisted of 85 other individuals confirmed without dengue. RESULTS: It was demonstrated that the presence the T allele of IL1ß (P < 0.05) was associated with susceptibility to developing the disease. Other results also suggested that the polymorphism in the combinations IL6 × IL1ß (C and T alleles, respectively), IL1ß (T allele) × IL1RN (*2/*2 genotype), IL6 (C allele) × TNFα (A allele), IL10 (C/T genotype) × TNFα (A/A genotype) (P < 0.01, P = 0.01, P < 0.05 and P = 0.03, respectively) were associated with predisposition to developing the disease and its symptoms. CONCLUSIONS: In summary, the findings of this study in a Brazilian population point out the importance of studies of combinations of polymorphisms in the development of dengue, which can increase the risk of dengue infection and its severity.

Phenotypic and genotypic variations among three allopatric populations of Lutzomyia umbratilis, main vector of Leishmania guyanensis.

BACKGROUND: In South America, Lutzomyia umbratilis is the main vector of Leishmania guyanensis, one of the species involved in the transmission of American tegumentary leishmaniasis. In Brazil, L. umbratilis has been recorded in the Amazon region, and in the state of Pernambuco, Northeastern region, where an isolated population has been identified. This study assessed the phylogeographic structure and size and shape differences of the wing of three Brazilian populations. METHODS: Samples of L. umbratilis were collected from Rio Preto da Eva (north of the Amazon River, Amazonas), from Manacapuru (south of the Amazon River), and from the isolated population in Recife, Pernambuco state. These samples were processed to obtain sequences of the Cytochrome Oxidase I mitochondrial gene. Geometrics morphometry analysis of the right wing shape of the three populations was made using discriminate canonical analysis. RESULTS: Phylogenetic analysis revealed the presence of two distinct monophyletic clades: one clade comprised of the Recife and Rio Preto da Eva samples, and the other clade comprised of the Manacapuru samples. Comparing the Manacapuru population with the Recife and Rio Preto da Eva populations generated high indices of interpopulational divergence. Geometric morphometry analysis indicated two distinct groups between the studied populations. Canonical variate analysis of wing shape indicated that Rio Preto da Eva population is significantly closer to Recife population, and both populations were genetically distant from Manacapuru. CONCLUSION: The polymorphic sites and geometric morphometry analysis indicate that the distance, lack of continuity and environmental differences have not modified the ancestral relationship between Recife and Rio Preto da Eva populations. The genetic and morphological similarities shared by the Recife and Rio Preto da Eva populations suggest that these populations are more closely related evolutionarily. These results confirm the existence of an L. umbratilis species complex in the North and Northeast regions.

Genetic structure of natural populations of the sand fly Lutzomyia longipalpis (Diptera: Psychodidae) from the Brazilian northeastern region.

In Latin America, Lutzomyia longipalpis is the principal vector of Leishmania chagasi, and is associated with the majority of active foci of visceral leishmaniasis. In spite of the fact that this sand fly is spread practically throughout the entire Neotropical Region, its distribution is not uniform due to geographic and environmental barriers. Geographic isolation coupled with reduced flight abilities may contribute to the appearance of cryptic species of Lutzomyia longipalpis, which may differ in their capacity to transmit L. chagasi. In this work, we describe the genetic structuring patterns based on polymorphism analysis of 24 RAPD-PCR loci of 7 natural populations of Lutzomyia longipalpis obtained from Brazil's northeastern region. The estimated degree of genetic differentiation between populations, based on the population subdivision index theta(ST) (0.136), suggests a moderate degree of genetic structuring as a result of geographical isolation and restricted gene flow. Genetic distances were found to be compatible with those found between members of a single species, suggesting a taxonomic uniformity of Lutzomyia longipalpis in the region studied.