Silver nanoparticles obtained from Brazilian pepper extracts with synergistic anti-microbial effect: production, characterization, hydrogel formulation, cell viability, and in vitro efficacy.

The synthesis of silver nanoparticles using plant extracts is known as a green approach, as it does not require the use of high pressure, energy, high temperature, or toxic chemicals. The approach makes use of plant extracts in a process called bioreduction, which is mediated by enzymes, proteins, amino acids, and metabolites found in bark, seed, and leaf extracts, transforming silver ions into metallic silver. This work aimed at developing silver nanoparticles (AgNPs) from Brazilian pepper, applying this green methodology. Hydroalcoholic extract of leaves of Schinus terebinthifolius Raddi was prepared and its concentration of polyphenols, tannins, and saponins quantified. The produced nanoparticles were characterized by UV-Vis spectroscopy, thermogravimetric analysis (TG), dynamic light scattering (DLS), and zeta potential (ZP). AgNPs were formulated in sodium alginate hydrogels to obtain a nano-based semi-solid formulation for skin application. The obtained silver nanoparticles of mean size between 350 and 450 nm showed no cytotoxicity against L929 mouse fibroblasts within the concentration range of 0.025 mg/mL and 10 mg/mL. Schinus terebinthifolius Raddi was found to enhance microbial inhibition against the tested strains, especially against gram-negative bacteria. Its potential use as an alternative to overcome bacterial resistance can be expected.

A selected bacterial strain for the self-healing process in cementitious specimens without cell immobilization steps.

The use of microorganisms capable of mediating the bioprecipitation process can be an important application in the self-healing processes of cement specimens. Thus, the present study identified and evaluated five Bacillus strains for potential application in the protocol of self-healing via bioprecipitation. Cell growth, enzyme production, and kinetic parameters conditions were evaluated during the fermentation process. Based on the analysis of 16S rDNA in conjunction with biochemical testing, results demonstrate that the strains are either Bacillus cereus or Bacillus thuringiensis. Strategically it was found that the addition of glycerol to fermentative medium was essential to increase the bacterial concentration (≈ 4.2 × 107 cells mL-1) and production of the enzyme urease (≈ 3.623,2 U.mL-1). The addition of this medium after 40 days of fermentation promoted the self-healing of cracks and increased compressive strength in ≈ 14.2% of the cementitious specimens; therefore, increasing the sustainability and engineering properties of cement-based materials.

Synthesis of silver-cerium titanate nanotubes and their surface properties and antibacterial applications.

Nano-heterostructures of titanate nanotubes were synthesized and they revealed a complex structure with the formation of TiO2 (anatase), CeO2, Ag2O and metallic silver nanoparticles on the outer walls and intercalation of Ce4+ and Ag+ into the interlayer spaces of the nanotubes by microwave-assisted hydrothermal process and subjected to ion exchange reactions. To the best of our knowledge, this is the first reported silver and cerium co-exchanged titanate nanotubes for bio-applications. The co-ion exchange processes preserved the original tubular structure of titanate nanotubes with significant changes of the superficial as well as interlamellar environment. This study opens up possibility of synthesizing complex, functional nano-heterostructure with the scope of modification of the final structure, especially the amount and oxidation state of the intercalated cation (Ce4+, Ce3+ and Ag+) as well as the quantity and variety of the decorating nanoparticles (CeO2, Ag2O or metallic Ag). The interplay of which, in turn, can lead to important biological properties and applications, owing to their ion-liberation capacity. The samples were tested in antibacterial activity with two different kind of bacteria (gram positive and negative), cell cytotoxicity and adhesion, and it was found that the nano-heterostructure formed shows high antibacterial activity with low cytotoxicity and high cell adhesion, which makes it a promising material for further health applications.

Analysis of potential virulence genes and competence to transformation in Haemophilus influenzae biotype aegyptius associated with Brazilian Purpuric Fever.

Brazilian Purpuric Fever (BPF) is a hemorrhagic pediatric illness caused by Haemophilus influenzae biogroup aegyptius (Hae), a bacterium that was formerly associated with self-limited purulent conjunctivitis. BPF is assumed to be eradicated. However, the virulence mechanisms inherent to Hae strains associated with BPF is still a mystery and deficient in studies. Here, we aim to analyze the role of the autotransporter genes related to adherence and colonization las, tabA1, and hadA genes through RT-qPCR expression profiling and knockout mutants. Relative quantification by real-time PCR after infection in human cells and infant rat model suggests that las was initially downregulated probably duo to immune evasion, tabA1, and hadA were overexpressed in general, suggesting an active role of TabA1 and HadA1 adhesins in Hae in vitro and in vivo. Transformation attempts were unsuccessful despite the use of multiple technical approaches and in silico analysis revealed that Hae lacks genes related to competence in Haemophilus, which could be part of the elucidation of the difficulty of genetically manipulating Hae strains.

ACP-TX-I and ACP-TX-II, Two Novel Phospholipases A2 Isolated from Trans-Pecos Copperhead Agkistrodon contortrix pictigaster Venom: Biochemical and Functional Characterization.

This work reports the purification and biochemical and functional characterization of ACP-TX-I and ACP-TX-II, two phospholipases A2 (PLA2) from Agkistrodon contortrix pictigaster venom. Both PLA2s were highly purified by a single chromatographic step on a C18 reverse phase HPLC column. Various peptide sequences from these two toxins showed similarity to those of other PLA2 toxins from viperid snake venoms. ACP-TX-I belongs to the catalytically inactive K49 PLA2 class, while ACP-TX-II is a D49 PLA2, and is enzymatically active. ACP-TX-I PLA2 is monomeric, which results in markedly diminished myotoxic and inflammatory activities when compared with dimeric K49 PLA2s, confirming the hypothesis that dimeric structure contributes heavily to the profound myotoxicity of the most active viperid K49 PLA2s. ACP-TX-II exhibits the main pharmacological actions reported for this protein family, including in vivo local myotoxicity, edema-forming activity, and in vitro cytotoxicity. ACP-TX-I PLA2 is cytotoxic to A549 lung carcinoma cells, indicating that cytotoxicity to these tumor cells does not require enzymatic activity.

Draft Whole-Genome Sequences of Haemophilus influenzae Biogroup aegyptius Strains Isolated from Five Brazilian Purpuric Fever Cases and One Conjunctivitis Case.

Brazilian purpuric fever is a febrile hemorrhagic pediatric disease caused by Haemophilus influenzae biogroup aegyptius, a bacterium which was formerly associated with only self-limited purulent conjunctivitis. Here, we present draft genomes of strains from five Brazilian purpuric fever cases and one conjunctivitis case.

Preparation of Thermosensitive Gel for Controlled Release of Levofloxacin and Their Application in the Treatment of Multidrug-Resistant Bacteria.

Levofloxacin is a synthetic broad-spectrum antibacterial agent for oral or intravenous administration. Chemically, levofloxacin is the levorotatory isomer (L-isomer) of racemate ofloxacin, a fluoroquinolone antibacterial agent. Quinolone derivatives rapidly and specifically inhibit the synthesis of bacterial DNA. Levofloxacin has in vitro activity against a broad range of aerobic and anaerobic Gram-positive and Gram-negative bacteria. However, formulation of combined poloxamers thermoregulated (as Pluronic® F127) and levofloxacin for use in multiresistant bacterial treatment were poorly described in the current literature. Thus, the aim of the present work is to characterize poloxamers for levofloxacin controlled release and their use in the treatment of multidrug bacterial resistance. Micelles were produced in colloidal dispersions, with a diameter between 5 and 100 nm, which form spontaneously from amphiphilic molecules under certain conditions as concentration and temperature. Encapsulation of levofloxacin into nanospheres showed efficiency and enhancement of antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae when compared with only levofloxacin. Furthermore, all formulations were not cytotoxic for NIH/3T3 cell lineage. In conclusion, poloxamers combined with levofloxacin have shown promising results, better than alone, decreasing the minimal inhibitory concentration of the studied bacterial multiresistance strains. In the future, this new formulation will be used after being tested in animal models in patients with resistant bacterial strains.

Effects of hyperbaric oxygen on Pseudomonas aeruginosa susceptibility to imipenem and macrophages.

BACKGROUND: The seriousness to treat burn wounds infected with Pseudomonas aeruginosa led us to examine whether the effect of the carbapenem antibiotic imipenem is enhanced by hyperbaric oxygen (HBO). MATERIALS & METHODS: The effects of HBO (100% O2, 3 ATA, 5 h) in combination with imipenen on bacterial counts of six isolates of P. aeruginosa and bacterial ultrastructure were investigated. Infected macrophages were exposed to HBO (100% O2, 3 ATA, 90 min) and the production of reactive oxygen species monitored. RESULTS: HBO enhanced the effects of imipenen. HBO increased superoxide anion production by macrophages and likely kills bacteria by oxidative mechanisms. CONCLUSION: HBO in combination with imipenem can be used to kill P. aeruginosa in vitro and such treatment may be beneficial for the patients with injuries containing the P. aeruginosa.

Inflammatory response of Haemophilus influenzae biotype aegyptius causing Brazilian Purpuric Fever

The Brazilian Purpuric Fever (BPF) is a systemic disease with many clinical features of meningococcal sepsis and is usually preceded by purulent conjunctivitis. The illness is caused by Haemophilus influenza biogroup aegyptius, which was associated exclusively with conjunctivitis. In this work construction of the las gene, hypothetically responsible for this virulence, were fusioned with ermAM cassette in Neisseria meningitidis virulent strains and had its DNA transfer to non BPF H. influenzae strains. The effect of the las transfer was capable to increase the cytokines TNFα and IL10 expression in Hec-1B cells line infected with these transformed mutants (in eight log scale of folding change RNA expression). This is the first molecular study involving the las transfer to search an elucidation of the pathogenic factors by horizontal intergeneric transfer from meningococci to H. influenzae.

Haemophilus influenzae porine ompP2 gene transfer mediated by graphene oxide nanoparticles with effects on transformation process and virulence bacterial capacity.

BACKGROUND: H. influenzae is a natural competent bacterium that can uptake DNA from the environment and recombine into bacterial genome. The outbreaks of Brazilian purpuric fever, heavily polluted areas of a different H. influenzae biogroup - aegyptius - as well as gene transference between Neisseria meningitis make the transformation process an important evolutionary factor. This work studied the horizontal transference of the ompP2 gene from a multiresistant strain of H. influenzae 07 (NTHi), under the influence of graphene oxide nanoparticles in order to mimic an atmosphere rich in suspended particles and this way verify if the CFU transformants number was increased. MATERIAL AND METHODS: In this article the gene ompP2 was transformed into different strains of H. influenzae mediated or not by graphene oxide nanoparticles in suspension, followed by the adhesion tests in Hec-1B (human endometrium adenocarcinoma) and A549 (pulmonary epithelial carcinoma) cells lines. The transformation frequency and the adhesion capacity were determined in all the mutants to which the ompP2 gene was transferred and compared to their wild type strains. RESULTS: The nanoparticles increased the transformation ratio of one particular strain isolated from a pneumonia case. The adhesion patterns to A549 and Hec1b cell lines of these mutated bacteria has their capacity increased when compared to the wild type. CONCLUSIONS: Graphene oxide nanoparticles aid the transformation process, helping to increase the number of CFUs, and the mutants generated with the ompP2 gene from a H. influenzae resistant strain not only present a chloramphenicol resistance but also have an increased adherence patterns in A549 and Hec1B cell lines.

Inflammatory response of Haemophilus influenzae biotype aegyptius causing Brazilian Purpuric Fever.

The Brazilian Purpuric Fever (BPF) is a systemic disease with many clinical features of meningococcal sepsis and is usually preceded by purulent conjunctivitis. The illness is caused by Haemophilus influenza biogroup aegyptius, which was associated exclusively with conjunctivitis. In this work construction of the las gene, hypothetically responsible for this virulence, were fusioned with ermAM cassette in Neisseria meningitidis virulent strains and had its DNA transfer to non BPF H. influenzae strains. The effect of the las transfer was capable to increase the cytokines TNFα and IL10 expression in Hec-1B cells line infected with these transformed mutants (in eight log scale of folding change RNA expression). This is the first molecular study involving the las transfer to search an elucidation of the pathogenic factors by horizontal intergeneric transfer from meningococci to H. influenzae.

Chemical modifications of PhTX-I myotoxin from Porthidium hyoprora snake venom: effects on structural, enzymatic, and pharmacological properties.

We recently described the isolation of a basic PLA2 (PhTX-I) from Porthidium hyoprora snake venom. This toxin exhibits high catalytic activity, induces in vivo myotoxicity, moderates footpad edema, and causes in vitro neuromuscular blockade. Here, we describe the chemical modifications of specific amino acid residues (His, Tyr, Lys, and Trp), performed in PhTX-I, to study their effects on the structural, enzymatic, and pharmacological properties of this myotoxin. After chemical treatment, a single His, 4 Tyr, 7 Lys, and one Trp residues were modified. The secondary structure of the protein remained unchanged as measured by circular dichroism; however other results indicated the critical role played by Lys and Tyr residues in myotoxic, neurotoxic activities and mainly in the cytotoxicity displayed by PhTX-I. His residue and therefore catalytic activity of PhTX-I are relevant for edematogenic, neurotoxic, and myotoxic effects, but not for its cytotoxic activity. This dissociation observed between enzymatic activity and some pharmacological effects suggests that other molecular regions distinct from the catalytic site may also play a role in the toxic activities exerted by this myotoxin. Our observations supported the hypothesis that both the catalytic sites as the hypothetical pharmacological sites are relevant to the pharmacological profile of PhTX-I.

Opsonization and transformation: effects of anticapsular sera on the DNA transfer in Neisseria meningitidis

The capsular switching process indicates the action of specific capsular antibodies on the meningococcal strains adaptation. Different antibodies were employed for assessing the effect of opsonization on the transformation of Neisseria meningitidis serogroups C and W135. These analyses showed the blocking action of the specific capsular antibodies on the meningococcal transformation capacity. Thus, the blocking effect of these antibodies on N. meningitidis transformation process was demonstrated. This effect could be involved in the capsular switching process and the found data might open new subjects for scientific exploratio.

Effects of multi-walled carbon nanotubes (MWCNT) under Neisseria meningitidis transformation process.

BACKGROUND: This study aimed at verifying the action of multi-walled carbon nanotubes (MWCNT) under the naturally transformable Neisseria meningitidis against two different DNA obtained from isogenic mutants of this microorganism, an important pathogen implicated in the genetic horizontal transfer of DNA, causing the escape of the principal vaccination measured worldwide by the capsular switching process. MATERIALS AND METHODS: The bacterium receptor strain C2135 was cultivated and had its mutant DNA donor M2 and M6, which received a receptor strain and MWCNT at three different concentrations. The inhibition effect of DNAse on the DNA in contact with nanoparticles was evaluated. RESULTS: The results indicated an in increase in the transformation capacity of N. meninigtidis in different concentrations of MWCNT when compared with negative control without nanotubes. A final analysis of the interaction between DNA and MWCNT was carried out using Raman Spectroscopy. CONCLUSION: These increases in the transformation capacity mediated by MWCNT, in meningococci, indicate the interaction of these particles with the virulence acquisition of these bacteria, as well as with the increase in the vaccination escape process.

Effect of mesoporous silica under Neisseria meningitidis transformation process: environmental effects under meningococci transformation.

BACKGROUND: This study aimed the use of mesoporous silica under the naturally transformable Neisseria meningitidis, an important pathogen implicated in the genetic horizontal transfer of DNA causing a escape of the principal vaccination measures worldwide by the capsular switching process. This study verified the effects of mesoporous silica under N. meningitidis transformation specifically under the capsular replacement. METHODS: we used three different mesoporous silica particles to verify their action in N. meningitis transformation frequency. RESULTS: we verified the increase in the capsular gene replacement of this bacterium with the three mesoporous silica nanoparticles. CONCLUSION: the mesouporous silica particles were capable of increasing the capsule replacement frequency in N. meningitidis.

Pathogenic and opportunistic respiratory bacteria-induced apoptosis

Several pathogenic or opportunistic bacteria have the ability to either induce or inhibit host cell apoptosis. The capacity to modulate cell pathways that result in the induction or delay of host cell apoptosis is considered to be an important bacterial virulence mechanism. These processes could be mediated by different host cell signaling pathways that are subverted by the bacteria. Pathogens are able to activate apoptotic proteins, such as caspases, or inactivate anti-apoptotic proteins, such as NFkB and the MAPKKs, or even up-regulate the endogenous receptor/ligand system that induces apoptosis, generally when the bacteria are bound to the host cell surface. The bacteria-induced apoptotic or anti-apoptotic processes are often related with the fact that the bacteria acquire the ability to reach the host tissues. However, apoptosis is also considered to be a host defense mechanism against infectious agents. Thus, the apoptosis phenomenon plays a central role in host-pathogen interactions.

Pathogenic and opportunistic respiratory bacteria-induced apoptosis.

Several pathogenic or opportunistic bacteria have the ability to either induce or inhibit host cell apoptosis. The capacity to modulate cell pathways that result in the induction or delay of host cell apoptosis is considered to be an important bacterial virulence mechanism. These processes could be mediated by different host cell signaling pathways that are subverted by the bacteria. Pathogens are able to activate apoptotic proteins, such as caspases, or inactivate anti-apoptotic proteins, such as NFkB and the MAPKKs, or even up-regulate the endogenous receptor/ligand system that induces apoptosis, generally when the bacteria are bound to the host cell surface. The bacteria-induced apoptotic or anti-apoptotic processes are often related with the fact that the bacteria acquire the ability to reach the host tissues. However, apoptosis is also considered to be a host defense mechanism against infectious agents. Thus, the apoptosis phenomenon plays a central role in host-pathogen interactions.

An inherited mutation leading to production of only the short isoform of GATA-1 is associated with impaired erythropoiesis.

Acquired somatic mutations in exon 2 of the hematopoietic transcription factor GATA-1 have been found in individuals with Down syndrome with both transient myeloproliferative disorder and acute megakaryoblastic leukemia. These mutations prevent the synthesis of the full-length protein but allow the synthesis of its short isoform, GATA-1s. Experiments in mice suggest that GATA-1s supports normal adult megakaryopoiesis, platelet formation and erythropoiesis. Here we report a mutation, 332G --> C, in exon 2 of GATA1, leading to the synthesis of only the short isoform in seven affected males from two generations of a family. Hematological profiles of affected males demonstrate macrocytic anemia, normal platelet counts and neutropenia in most cases. Altogether, data suggest that GATA-1s alone, produced in low or normal levels, is not sufficient to support normal erythropoiesis. Moreover, this is the first study to indicate that a germline splicing mutation does not lead to leukemia in the absence of other cooperating events, such as Down syndrome.

Biological characteristics and pathogenicity of avian Escherichia coli strains.

Fifty avian (chicken) pathogenic Escherichia coli strains (APEC) isolated from individuals suffering from omphalitis, septicaemia and swollen head syndrome, and 30 strains isolated from healthy chickens were studied regarding their biological characteristics such as serogroups, haemolysin, colicin, cytotoxin, toxin and siderophore production, adhesion capacity to in vitro cultivated cells, and absorption of Congo red dye. Serotyping demonstrated that most of the omphalitis and normal strains were untypable, whereas most of the septicaemic strains were either untypable or rough. There was no prevalent serogroup among the pathogenic strains studied. The capacity for adhesion and invasion of in vitro cultured cells (HeLa, HEp-2, KPCC), as well as the agglutination of different types of red blood cells and the LD50 of each strain were also evaluated. No correlation was observed between the biological characteristics and pathogenicity, except that colicin was characteristically produced by swollen head syndrome E. coli strains. No correlation was found between adhesion or haemagglutination patterns and pathogenicity. Only six of the 50 strains revealed invasive capacity and the strain that best invaded the cell lines was the one with the lowest LD50.