Evaluation of a long-lasting microbial larvicide against Culex quinquefasciatus and Aedes aegypti under laboratory and a semi-field trial.

BACKGROUND: Microbial larvicides containing both LysiniBacillus sphaericus and Bacillus thuringiensis svar. israelensis (Bti) insecticidal crystals can display advantages for mosquito control. This includes a broader action against larvae that are refractory to the Binary (Bin) toxin from L. sphaericus, as Bin-resistant Culex quinquefasciatus and Aedes aegypti naturally refractory larvae, which often co-habit urban areas of endemic countries for arboviruses. Our principal goal was to assess the toxicity of a combined L. sphaericus/Bti larvicide (Vectomax FG™) to Cx. quinquefasciatus (susceptible CqS and Bin-resistant CqR) and Ae. aegypti (Rocke) and to determine its persistence in the breeding sites with those larvae. METHODS: The toxicity of a combined L. sphaericus/Bti product (VectoMax FG™) to larvae was performed using bioassays, and persistence was evaluated in simulate field trials carried out under the shade, testing two label concentrations during 12 weeks. A laboratory strain SREC, established with CqS and CqR larvae, was kept during four generations to evaluate the ability of the L. sphaericus/Bti to eliminate resistant larvae. RESULTS: The L. sphaericus/Bti showed toxicity (mg/L) to larvae from all strains with a decreasing pattern for CqS (LC50 = 0.006, LC90 = 0.030), CqR (LC50 = 0.009, LC90 = 0.069), and Rocke (LC50 = 0.042, LC90 = 0.086). In a simulated field trial, the larvicide showed a persistence of 6 weeks and 8 weeks, controlling larvae from all strains in containers with 100 L of water, using 2 g or 4 g per container (100 L), respectively. The treatment of SREC larvae with L. sphaericus/Bti showed its capacity to eliminate the Bin-resistant individuals using suitable concentrations to target those larvae. CONCLUSIONS: Our results showed the high efficacy and persistence of the L. sphaericus/Bti larvicide to control Cx. quinquefasciatus and Ae. aegypti that might cohabit breeding sites. These findings demonstrated that such larvicides can be an effective tool for controlling those species in urban areas with a low potential for selecting resistance.

Síndrome de piel escaldada asociado a estafilococcemia en un neonato: reporte de un caso / Scalded skin syndrome associated with staphylococcemia in a neonate: a case report

RESUMEN El síndrome de piel escaldada estafilocócica es una patología poco frecuente que principalmente afecta a población pediátrica. Si bien en la mayoría de los casos tiene un desenlace favorable, un buen diagnóstico diferencial y manejo antibiótico oportuno son cruciales para un buen pronóstico. Objetivo: Describir un caso de síndrome de piel escaldada con aislamiento en hemocultivos de S. Aureus meticilino resistente en un neonato. Caso clínico: Femenina de 25 días de vida, nacida a término y sin antecedentes perinatales relevantes que cursa con fiebre, dermatosis eritemato-descamativa generalizada, edema palpebral y de miembros inferiores. Se hospitalizó con diagnóstico de sepsis neonatal tardía y sindrome de piel escaldada. Reporte de hemocultivos con crecimiento de S. Aureus meticilino resistente, manejada con vancomicina durante 14 días. Control de hemocultivos negativos, evolución satisfactoria y de alta hospitalaria sin complicaciones. Conclusiones: A pesar de la baja incidencia del síndrome de piel escaldada, es importante incluir dentro del diagnóstico diferencial de la fiebre y las dermatosis tardías del neonato la infecciones por toxinas del S. Aureus, ya que su identificación y tratamiento oportuno son de gran importancia en el pronóstico del menor.

ABSTRACT Staphylococcal scalded skin syndrome is a rare pathology, which mainly affects pediatric population. Although in most cases it has a favorable outcome, making a good differential diagnosis and early antibiotic management are crucial for a good prognosis. Objective: To describe a case of scalded skin syndrome with methicillin-resistant S. aureus bloodstream infection in a newborn. Clinical case: A 25-day-old female, born at term and with no relevant perinatal history, presented with fever, generalized scaly erythematous dermatosis, eyelid and lower limb edema. She is hospitalized with a diagnosis of late neonatal sepsis and scalded skin syndrome. Report of blood cultures with growth of methicillin-resistant S. Aureus, managed with vancomycin for 14 days. Control cultures were negative, satisfactory evolution and hospital discharge without complications. Conclusions: Despite the low incidence of scalded skin syndrome, it is important to include S. aureus toxin infections in the differential diagnosis of fever and late-onset dermatoses in the newborn. The identification and early treatment are of great importance in the prognosis of the child.

Clostridium perfringens phospholipase C, an archetypal bacterial virulence factor, induces the formation of extracellular traps by human neutrophils.

Neutrophil extracellular traps (NETs) are networks of DNA and various microbicidal proteins released to kill invading microorganisms and prevent their dissemination. However, a NETs excess is detrimental to the host and involved in the pathogenesis of various inflammatory and immunothrombotic diseases. Clostridium perfringens is a widely distributed pathogen associated with several animal and human diseases, that produces many exotoxins, including the phospholipase C (CpPLC), the main virulence factor in gas gangrene. During this disease, CpPLC generates the formation of neutrophil/platelet aggregates within the vasculature, favoring an anaerobic environment for C. perfringens growth. This work demonstrates that CpPLC induces NETosis in human neutrophils. Antibodies against CpPLC completely abrogate the NETosis-inducing activity of recombinant CpPLC and C. perfringens secretome. CpPLC induces suicidal NETosis through a mechanism that requires calcium release from inositol trisphosphate receptor (IP3) sensitive stores, activation of protein kinase C (PKC), and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathways, as well as the production of reactive oxygen species (ROS) by the metabolism of arachidonic acid. Proteomic analysis of the C. perfringens secretome identified 40 proteins, including a DNAse and two 5´-nucleotidases homologous to virulence factors that could be relevant in evading NETs. We suggested that in gas gangrene this pathogen benefits from having access to the metabolic resources of the tissue injured by a dysregulated intravascular NETosis and then escapes and spreads to deeper tissues. Understanding the role of NETs in gas gangrene could help develop novel therapeutic strategies to reduce mortality, improve muscle regeneration, and prevent deleterious patient outcomes.

Estratégias de clonagem de proteínas de alta massa molecular: α-hemolisina e CNF1 produzidas por Escherichia coli uropatogênica como modelos

Uropathogenic Escherichia coli (UPEC) is the main cause of urinary tract infection (UTI). Among the virulence factors produced by UPEC, alpha hemolysin (HlyA) and cytotoxic necrotizing factor 1 (CNF1) stand out. Keeping in mind that up to 60% of UPEC strains produce HlyA and about 40% express CNF1, these toxins become good targets for diagnosis and therapeutic interventions. The importance of HlyA and CNF1 toxins in the cases of UTI caused by UPEC and the enormous challenges in the production of recombinant proteins, led to this work proposal to obtain different structural forms of these toxins through the methodology of cloning and heterologous expression, of integral toxins, low molecular mass and intermediate immunogenic toxins. Different structural forms of these high molecular weight toxins have been proposed, from which the production integrates to low molecular weight proteins. Recombinant toxins were analyzed in silico, expressed and purified. Among the cloning and expression approaches, we highlight for HlyA the strategy to obtain the intermediate immunogen of this toxin. For CNF1, both the construction of the full protein and the recombinant protein using only its catalytic portion. In addition, a bacterial collection of UPEC was analyzed to define local epidemiological data on the prevalence of toxin genes and the ability to cause hemolysis and multinucleation. The hlyA and cnf1 genes were present in the isolates in 36% and 23%, respectively, and the hemolysis phenotype occurred in 33% and the multinucleation in 23% of the bacterial isolates.

Escherichia coli uropatogênica (UPEC) é a principal causa de infecção do trato urinário (ITU). Entre os fatores de virulência produzidos pela UPEC, destacam- se a alfa hemolisina (HlyA) e o fator necrosante citotóxico 1 (CNF1). Tendo em vista que até 60% das cepas de UPEC produzem HlyA e cerca de 40% expressam CNF1, estas toxinas se tornam bons alvos para o diagnóstico e intervenções terapêuticas. A importância das toxinas HlyA e CNF1 nos casos de ITU causados por UPEC e os enormes desafios na produção de proteínas recombinantes, levou a este trabalho que se propôs a obter diferentes formas estruturais destas toxinas pela metodologia de clonagem e expressão heteróloga. Diferentes formas estruturais destas toxinas de alta massa molecular foram propostas, deste a produção integra a proteínas de baixo peso molecular. As toxinas recombinantes foram analisadas in silico, expressas e purificadas. Dentre as abordagens de clonagem e expressão destacamos para HlyA a estratégia de obtenção do imunógeno intermediário dessa toxina. Já para CNF1, tanto a construção da proteína integra quanto a proteína recombinante utilizando apenas sua porção catalítica. Além disso, analisou-se uma coleção bacteriana de UPEC para definir dados epidemiológicos locais da prevalência dos genes das toxinas e capacidade de causar hemólise e multinucleação. A genes hlyA e cnf1 estavam presentes nos isolados em 36% e 23%, respectivamente e o fenótipo de hemólise ocorreu em 33% e a multinucleação em 23% dos isolados bacterianos.

In Silico Prediction and Design of Uropathogenic Escherichia coli Alpha-Hemolysin Generate a Soluble and Hemolytic Recombinant Toxin.

The secretion of α-hemolysin by uropathogenic Escherichia coli (UPEC) is commonly associated with the severity of urinary tract infections, which makes it a predictor of poor prognosis among patients. Accordingly, this toxin has become a target for diagnostic tests and therapeutic interventions. However, there are several obstacles associated with the process of α-hemolysin purification, therefore limiting its utilization in scientific investigations. In order to overcome the problems associated with α-hemolysin expression, after in silico prediction, a 20.48 kDa soluble α-hemolysin recombinant denoted rHlyA was constructed. This recombinant is composed by a 182 amino acid sequence localized in the aa542-723 region of the toxin molecule. The antigenic determinants of the rHlyA were estimated by bioinformatics analysis taking into consideration the tertiary form of the toxin, epitope analysis tools, and solubility inference. The results indicated that rHlyA has three antigenic domains localized in the aa555-565, aa600-610, and aa674-717 regions. Functional investigation of rHlyA demonstrated that it has hemolytic activity against sheep red cells, but no cytotoxic effect against epithelial bladder cells. In summary, the results obtained in this study indicate that rHlyA is a soluble recombinant protein that can be used as a tool in studies that aim to understand the mechanisms involved in the hemolytic and cytotoxic activities of α-hemolysin produced by UPEC. In addition, rHlyA can be applied to generate monoclonal and/or polyclonal antibodies that can be utilized in the development of diagnostic tests and therapeutic interventions.

Análise do sistema toxina-antitoxina tipo II na fisiologia bacteriana de uma cepa híbrida: Escherichia coli enteropatogênica atípica e E. coli extraintestinal

The toxin-antitoxin (TA) systems are genetic modules associated with some bacterial processes, such as cell formation persistence, biofilm formation, protection against bacteriophages and responses to stressful environments. In these systems, the toxin is related to the inhibition of physiological processes and the antitoxin provides protection to the cell against the toxin. Five TA type II systems present in the genome of the hybrid strain BA1250 (atypical enteropathogenic E. coli and extraintestinal E. coli strain) were analyzed. The hybrid strain BA1250 can colonize different host niches and can face different stress environments, therefore, through transcription analysis under stress conditions such as nutritional, oxidative, osmotic and acid, the CcdB-CcdA, YhaV-PrlF, MazF-MazE, YoeB-YefM and PasI-PasT were evaluated both in the log and stationary phases of BA1250 strain. We observed significant differences in the expression levels of the CcdB-CcdA, YhaV-PrlF, YoeB-YefM and PasT-PasI systems in the presence of nutritional stress in the stationary phase. In acid stress, an increase in the gene expression of YhaV, YefM and PasT toxins in stationary phase was observed. In contrast, the analysis of the MazF-MazE system did not show any change in expression levels under the stress conditions evaluated. These data indicate that these four TA systems seem to be involved in the stress response of the BA1250 strain, therefore involved in the physiological processes of BA1250.

Os sistemas toxina-antitoxina (TA) são módulos genéticos que estão associados a alguns processos das bactérias, como formação de células persistentes, formação de biofilme, proteção contra bacteriófagos e respostas a ambientes estressantes. Nesses sistemas, a toxina está relacionada à inibição de processos fisiológicos e a antitoxina fornece proteção à célula contra a toxina. Cinco sistemas TA tipo II presentes no genoma da cepa híbrida BA1250 (E. coli enteropatogênica atípica e cepa de E. coli extraintestinal) foram analisados. A cepa híbrida BA1250 pode colonizar diferentes nichos do hospedeiro e pode enfrentar diferentes ambientes de estresse, por tanto, por meio de análises de transcrição sob condições de estresse como nutricional, oxidativo, osmótico e ácido, os sistemas CcdB-CcdA, YhaV-PrlF, MazF-MazE, YoeB-YefM e PasI-PasT foram avaliados tanto na fase log quanto estacionária do cultivo da cepa BA1250. Observamos diferenças significativas nos níveis de expressão dos sistemas CcdB-CcdA, YhaV-PrlF, YoeB-YefM e PasT-PasI na presença de estresse nutricional na fase estacionária. No estresse ácido foi observado um aumento de expressão gênica das toxinas YhaV, YefM e PasT em fase estacionária. Em contraste, a análise do sistema MazF-MazE não apresentou nenhuma alteração nos níveis de expressão nas condições de estresse avaliadas. Esses dados indicam que esses quatro sistemas TA parecem estar envolvidos na resposta ao estresse da cepa BA1250, portanto envolvidos em processos fisiológicos da BA1250.

Análise do sistema toxina-antitoxina tipo II na fisiologia bacteriana de uma cepa híbrida: Escherichia coli enteropatogênica atípica e E. coli extraintestinal

The toxin-antitoxin (TA) systems are genetic modules associated with some bacterial processes, such as cell formation persistence, biofilm formation, protection against bacteriophages and responses to stressful environments. In these systems, the toxin is related to the inhibition of physiological processes and the antitoxin provides protection to the cell against the toxin. Five TA type II systems present in the genome of the hybrid strain BA1250 (atypical enteropathogenic E. coli and extraintestinal E. coli strain) were analyzed. The hybrid strain BA1250 can colonize different host niches and can face different stress environments, therefore, through transcription analysis under stress conditions such as nutritional, oxidative, osmotic and acid, the CcdB-CcdA, YhaV-PrlF, MazF-MazE, YoeB-YefM and PasI-PasT were evaluated both in the log and stationary phases of BA1250 strain. We observed significant differences in the expression levels of the CcdB-CcdA, YhaV-PrlF, YoeB-YefM and PasT-PasI systems in the presence of nutritional stress in the stationary phase. In acid stress, an increase in the gene expression of YhaV, YefM and PasT toxins in stationary phase was observed. In contrast, the analysis of the MazF-MazE system did not show any change in expression levels under the stress conditions evaluated. These data indicate that these four TA systems seem to be involved in the stress response of the BA1250 strain, therefore involved in the physiological processes of BA1250.

Os sistemas toxina-antitoxina (TA) são módulos genéticos que estão associados a alguns processos das bactérias, como formação de células persistentes, formação de biofilme, proteção contra bacteriófagos e respostas a ambientes estressantes. Nesses sistemas, a toxina está relacionada à inibição de processos fisiológicos e a antitoxina fornece proteção à célula contra a toxina. Cinco sistemas TA tipo II presentes no genoma da cepa híbrida BA1250 (E. coli enteropatogênica atípica e cepa de E. coli extraintestinal) foram analisados. A cepa híbrida BA1250 pode colonizar diferentes nichos do hospedeiro e pode enfrentar diferentes ambientes de estresse, por tanto, por meio de análises de transcrição sob condições de estresse como nutricional, oxidativo, osmótico e ácido, os sistemas CcdB-CcdA, YhaV-PrlF, MazF-MazE, YoeB-YefM e PasI-PasT foram avaliados tanto na fase log quanto estacionária do cultivo da cepa BA1250. Observamos diferenças significativas nos níveis de expressão dos sistemas CcdB-CcdA, YhaV-PrlF, YoeB-YefM e PasT-PasI na presença de estresse nutricional na fase estacionária. No estresse ácido foi observado um aumento de expressão gênica das toxinas YhaV, YefM e PasT em fase estacionária. Em contraste, a análise do sistema MazF-MazE não apresentou nenhuma alteração nos níveis de expressão nas condições de estresse avaliadas. Esses dados indicam que esses quatro sistemas TA parecem estar envolvidos na resposta ao estresse da cepa BA1250, portanto envolvidos em processos fisiológicos da BA1250.

Modelos in vitro e in vivo e anticorpos recombinantes para o estudo da toxina termolábil produzida por Escherichia coli enterotoxigênica

Enterotoxigenic Escherichia coli (ETEC) is one of the main bacterial pathotypes in- volved in diarrhea, mainly affecting children under 5 years old and travelers to areas where this pathogen is endemic. Heat-labile type I toxin (LT-I), one of the main virulence factors of this pathotype, is associated with diarrhea in humans and is described as a potent mucosal and systemic immune adjuvant. Early diagnosis, therapeutic approaches, and in vitro and in vivo models are the main concerns in diarrhea caused by ETEC. Different in vitro and in vivo models have already been described, however, its use does not always allow screening of biomolecules and more systemic studies with the LT toxin. In addition, the generation of specific and high-affinity recombinant antibodies for diagnosis and therapy are of great importance. Thus, the present work aimed to establish strategies for the generation of recombinant antibodies such as scFv and Fab and evaluate and validate Caco-2 cells and zebrafish as models for studies with LT-I. Caco-2 cells were seeded in the 62nd passage and at different cells density per microplate well. The internalization of LT-I conjugated to FITC was visualized in Caco-2 cells at a density of 3x104 cells per well, both associated with the cell membrane and with the nucleus, thus demonstrating its retrograde transport and validating the use of this model. In a zebrafish, the fish embryo test revealed the sensitivity of embryos to different concentrations of LT-I, as well as evident malformation phenotypes, mainly in the pericardial and yolk region. Systemically, migration of the FITC labeled toxin was observed in the cardiac region, yolk, and intestine, suggesting the observed phenotypes of cardiac edema (100%), absence of swim bladder (100%), yolk edema (80%), in addition to growth delay in larvae, were caused by the toxin. None of these phenotypes were observed in the control group of animals. There was also a decrease in heart rate during the larvae' survival kinetics, showing the cardiotoxic effect of LT-I. In ELISA assays the scFv-LT obtained in the present study was reactive against the purified antigen as well to LT-I producing strains supernatant, but not to LT-I-non-producing strains. For the Fab-LT, a synthetic library was constructed, but no LT-I-reactive clones were generated. Therefore, herein we established in vitro and in vivo models that allowed us to validate and demonstrate unknown characteristics of the LT-I concerning its relationship with the host. Moreover, the generation of scFv-LT will allows us to employ recombinant antibodies for ETEC diagnosis avoiding animals immunization.

Escherichia coli enterotoxigênica (ETEC), é um dos principais patotipos bacterianos causadores de diarreia, afetando principalmente crianças menores de cinco anos e viajantes em áreas onde esse patógeno é endêmico. A toxina termolábil do tipo I (LT-I) é um dos principais fatores de virulência deste patotipo e está associada à diarreia em humanos; é também descrita como potente adjuvante de mucosa e sistêmico. O diagnóstico precoce, a abordagem terapêutica e os modelos experimentais são importantes aspectos que merecem atenção em relação à diarreia causada por ETEC. Diferentes modelos in vitro e in vivo já foram descritos, no entanto, seu uso nem sem- pre permite a triagem de biomoléculas e estudos mais sistêmicos com a toxina LT. Além disso, a geração de anticorpos específicos e de alta afinidade para uso no diagnóstico e terapia são de grande importância. Assim, o presente trabalho propôs estabelecer estratégias para geração de fragmentos de anticorpos do tipo scFv e Fab; bem como avaliar e validar as células Caco-2 e o zebrafish como modelos para estudos da LT-I. As células Caco-2 foram utilizadas na 62a passagem e em diferentes densidades de célula por poço da microplaca de cultivo. A internalização da LT-I conjugada ao FITC foi visualizada com 3x104 células Caco-2, tanto associada à membrana celular, quanto ao núcleo, evidenciando seu transporte retrógrado e validando o uso deste modelo. Em zebrafish, pelo teste de sensibilidade em embriões, foi observada sensibilidade dos embriões frente a diferentes concentrações de LT-I e fenótipos de malformações, principalmente na região pericárdica e no vitelo. Por via sistêmica, a migração da LT-I foi observada na região cardíaca, vitelo e intestino, sugerindo que os fenótipos observados de edema cardíaco (100%), ausência de bexiga natatória (100%), edema de vitelo (80%), além de retardo no crescimento nas larvas, tenham sido ocasionados pela toxina. Nenhum desses efeitos foi encontrado no grupo controle de animais. Houve também diminuição dos batimentos cardíacos durante a cinética de sobrevivência, mostrando o efeito cardiotóxico da LT-I. Em testes por técnica de ELISA o scFv-LT obtido no presente estudo mostrou-se reativo contra a toxina LT purificada, reconheceu as cepas produtoras de LT-I e não reconheceu cepas não produtoras da toxina. Para o Fab-LT foi estabelecida a biblioteca sintética, mas não foram gerados clones reativos contra a toxina LT-I. Assim, podemos afirmar que os modelos in vitro e in vivo utilizados permitiram-nos validar e demonstrar características até então não exploradas da LT-I na sua relação com o hospedeiro. A geração do scFv-LT nos permitirá utilizar anticorpos recombinantes no diagnóstico de ETEC sem depender da imunização de animais.

In silico prediction and design of uropathogenic Escherichia coli alpha-hemolysin generate a soluble and hemolytic recombinant toxin

The secretion of α-hemolysin by uropathogenic Escherichia coli (UPEC) is commonly associated with the severity of urinary tract infections, which makes it a predictor of poor prognosis among patients. Accordingly, this toxin has become a target for diagnostic tests and therapeutic interventions. However, there are several obstacles associated with the process of α-hemolysin purification, therefore limiting its utilization in scientific investigations. In order to overcome the problems associated with α-hemolysin expression, after in silico prediction, a 20.48 kDa soluble α-hemolysin recombinant denoted rHlyA was constructed. This recombinant is composed by a 182 amino acid sequence localized in the aa542–723 region of the toxin molecule. The antigenic determinants of the rHlyA were estimated by bioinformatics analysis taking into consideration the tertiary form of the toxin, epitope analysis tools, and solubility inference. The results indicated that rHlyA has three antigenic domains localized in the aa555–565, aa600–610, and aa674–717 regions. Functional investigation of rHlyA demonstrated that it has hemolytic activity against sheep red cells, but no cytotoxic effect against epithelial bladder cells. In summary, the results obtained in this study indicate that rHlyA is a soluble recombinant protein that can be used as a tool in studies that aim to understand the mechanisms involved in the hemolytic and cytotoxic activities of α-hemolysin produced by UPEC. In addition, rHlyA can be applied to generate monoclonal and/or polyclonal antibodies that can be utilized in the development of diagnostic tests and therapeutic interventions.

Bacterial phospholipases C with dual activity: phosphatidylcholinesterase and sphingomyelinase.

Bacterial phospholipases and sphingomyelinases are lipolytic esterases that are structurally and evolutionarily heterogeneous. These enzymes play crucial roles as virulence factors in several human and animal infectious diseases. Some bacterial phospholipases C (PLCs) have both phosphatidylcholinesterase and sphingomyelinase C activities. Among them, Listeria monocytogenes PlcB, Clostridium perfringens PLC, and Pseudomonas aeruginosa PlcH are the most deeply understood. In silico predictions of substrates docking with these three bacterial enzymes provide evidence that they interact with different substrates at the same active site. This review discusses structural aspects, substrate specificity, and the mechanism of action of those bacterial enzymes on target cells and animal infection models to shed light on their roles in pathogenesis.

Adjuvants: friends in vaccine formulations against infectious diseases.

Infectious diseases represent a major cause of deaths worldwide. No vaccine or effective treatment exists nowadays, especially against intracellular pathogens. The increase in multiple drug and superbug antibiotic resistance strains, excessive medication, or misuse of drugs has prompted the search for other safe and effective alternatives. Consistent with this, adjuvants (Latin word "adjuvare": "help or aid") co-administered (Exo) in vaccines have emerged as a promising alternative to initiate and boost an innate, downstream signal that led to adaptative immune response. Nowadays, a promising model of strong immunogens and adjuvants at mucosal sites are the microbial bacterial toxins. Other adjuvants that are also used and might successfully replace aluminum salts in combination with nanotechnology are CpG-ODN, poly IC, type I IFNs, mRNA platforms. Therefore, in the present review, we focused to revisit the old to the new adjuvants compounds, the properties that make them friends in vaccine formulations against infectious diseases.

Use of Short-Chain Fatty Acids for the Recovery of the Intestinal Epithelial Barrier Affected by Bacterial Toxins.

Short-chain fatty acids (SCFAs) are carboxylic acids produced as a result of gut microbial anaerobic fermentation. They activate signaling cascades, acting as ligands of G-protein-coupled receptors, such as GPR41, GPR43, and GPR109A, that can modulate the inflammatory response and increase the intestinal barrier integrity by enhancing the tight junction proteins functions. These junctions, located in the most apical zone of epithelial cells, control the diffusion of ions, macromolecules, and the entry of microorganisms from the intestinal lumen into the tissues. In this sense, several enteric pathogens secrete diverse toxins that interrupt tight junction impermeability, allowing them to invade the intestinal tissue and to favor gastrointestinal colonization. It has been recently demonstrated that SCFAs inhibit the virulence of different enteric pathogens and have protective effects against bacterial colonization. Here, we present an overview of SCFAs production by gut microbiota and their effects on the recovery of intestinal barrier integrity during infections by microorganisms that affect tight junctions. These properties make them excellent candidates in the treatment of infectious diseases that cause damage to the intestinal epithelium.

Recent Advances in Anti-virulence Therapeutic Strategies With a Focus on Dismantling Bacterial Membrane Microdomains, Toxin Neutralization, Quorum-Sensing Interference and Biofilm Inhibition.

Antimicrobial resistance constitutes one of the major challenges facing humanity in the Twenty-First century. The spread of resistant pathogens has been such that the possibility of returning to a pre-antibiotic era is real. In this scenario, innovative therapeutic strategies must be employed to restrict resistance. Among the innovative proposed strategies, anti-virulence therapy has been envisioned as a promising alternative for effective control of the emergence and spread of resistant pathogens. This review presents some of the anti-virulence strategies that are currently being developed, it will cover strategies focused on quench pathogen quorum sensing (QS) systems, disassemble of bacterial functional membrane microdomains (FMMs), disruption of biofilm formation and bacterial toxin neutralization.

The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins.

Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs) released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.

Influência da ativação ultrassônica do gel de hipoclorito de sódio 3 por cento, e da medicação intracanal com hidróxido de cálcio sobre micro-organismos e ácido lipoteicóico / Influence of activation by ultrasound on sodium hypochlorite 3% gel and an intracanal medication of calcium hydroxide on microrganisms and lipotheicoic acid

O objetivo deste estudo foi avaliar in vitro a ação do gel de hipoclorito de sódio (NaOCl) 3 % e da medicação intracanal (MIC) de hidróxido de cálcio (CaOH2), agitados ou não por ultrassom sobre Enterococcus faecalis, Escherichia coli e ácido lipoteicóico (LTA). Para isso 80 dentes humanos unirradiculares tiveram suas coroas removidas padronizando seu comprimento em 16 mm +- 0,5 mm, sendo seus canais instrumentados inicialmente até instrumento R25 (Reciproc). Os canais foram contaminados com suspensões de E. faecalis e E. coli. Os canais foram instrumentados utilizando-se instrumento R40, sob irrigação com 2 ml de NaOCl 3% gel seguida de irrigação com 10 ml de solução salina estéril e apirogênica. Após os canais foram irrigados com EDTA 17%, seguido de irrigação com 5 ml de solução salina estéril e apirogênica, sendo por último preenchidos com medicação intracanal de hidróxido de cálcio mantida durante 7 ou 14 dias. Os espécimes foram divididos inicialmente em 2 grupos (n=40) de acordo com a agitação ultrassônica (US) ou não da substância química auxiliar. Sendo novamente divididos de acordo com a agitação ultrassônica ou não da medicação intracanal (MIC) e o tempo de ação desta (n=10): 1) NaOCl + Ca(OH)2 (7 dias). 2) NaOCl + Ca(OH)2 (14 dias). 3) NaOCl + Ca(OH)2 com US (7 dias) 4) NaOCl + Ca(OH)2 com US (14 dias). 5) NaOCl com US + Ca(OH)2 (7 dias). 6) NaOCl com US + Ca(OH)2 (14dias) 7) NaOCl com US + Ca(OH)2 com US (7dias). 8) NaOCl com US + Ca(OH)2 com US (14 dias). Foram realizadas coletas do canal radicular 28 dias após o início da contaminação dos espécimes (1ª coleta), logo após o preparo biomecânico (PBM) (2ª coleta), logo após o preenchimento com EDTA (3ª coleta) e após o tempo de ação da MIC (4ª coleta). Para todas as coletas foram avaliadas a atividade antimicrobiana (UFC/ml) e quantificação de LTA pelo teste de Elisa. Os resultados foram submetidos aos testes estatísticos Kruskal-Wallis e Dunn (5%). Verificou-se pela análise microbiana e quantificação de LTA, que o NaOCl 3% gel foi capaz de eliminar quase completamente os micro-organismos dos canais radiculares, mas não o ácido lipoteicóico, independente da ativação ultrassônica. A ativação da MIC de Ca(OH)2 não exerceu efeito sobre micro-organismos. Sobre LTA, a ativação da MIC de Ca(OH)2 não foi eficaz, sendo os grupos com maior percentual de redução os que não sofreram agitação da MIC, nem do NaOCl. Conclui-se que o NaOCl 3% gel boa tem capacidade antimicrobina, independente da ativação ultrassônica. O PBM não foi capaz de detoxificar o LTA dos canais radiculares. A MIC com ativação ultrassônica não foi eficiente na redução de LTA(AU)

The aim of this study was to evaluate in vitro the action of sodium hypochlorite (NaOCl) 3% gel and calcium hydroxide as an intracanal medication, both activated by ultrasound on the reduction of E. faecalis e E. coli and lipotheicoic acid (LTA). Eight human single-rooted teeth with standardized size of 16 mm were prepared initially to R25 instrument (Reciproc) and distributed in microplate (n = 10). After sterilization (Co60 gamma radiation), the infection was carried out 8 microlitres of E. coli suspension, and after 7 days 8 microlitres of E. faecalis suspension, maintained for 21 days. Following the collection confirmation was performed (1st collection), then the root were instrumented using R40 instrument, irrigation with 2 ml of NaOCl 3% gel followed by washing with 10 ml of saline. The specimens were divided into 8 groups (n = 10) according to different ultrasonic irrigation protocols and different periods exposed to intracanal medication: 1) NaOCl + Ca(OH)2(7 days). 2) NaOCl + Ca(OH)2 (14 days). 3) NaOCl + Ca(OH)2 with PUI (7 dias) 4) NaOCl + Ca(OH)2 with PUI (14 days). 5) NaOCl with PUI + Ca(OH)2 (7 days). 6) NaOCl with PUI + Ca(OH)2 (14 days) 7) NaOCl with PUI + Ca(OH)2 (7 days). 8) NaOCl with PUI + Ca(OH)2 with PUI (14 days). Were made to sample colections of content of root canals, after instrumentation (2nd sample), after the use of EDTA (3rd sample) and after the Ca(OH)2 (4th Collection) . Microbiological culture and LTA quantification revealed that 3 % NaOCl gel was capable to eliminate E. faecalis and E. coli almost completely from root canals, but not lipotheicoic acid, regardless the use of ultrasonic activation. The ultrasonic activation of intracanal medication (Ca(OH)2) was not effective in removing neither microorganisms nor LTA. In addition, the groups with greatest reduction were the ones with no ultrasonic activation either of intracanal medication or NaOCl. It was concluded that 3% NaOCl gel is effective on antimicrobial activity regardless the use of ultrasonic activation. Biomechanical preparation was not capable to detoxify LTA from root canals. Ultrasonic activation of intracanal medication was not effetive on LTA reduction(AU)

Staphylococcal superantigen-specific IgE antibodies: degree of sensitization and association with severity of asthma / Anticorpos IgE específicos para superantígenos estafilocócicos: grau de sensibilização e associação com a gravidade da asma

ABSTRACT Objective: To determine the presence of staphylococcal superantigen-specific IgE antibodies and degree of IgE-mediated sensitization, as well as whether or not those are associated with the severity of asthma in adult patients. Methods: This was a cross-sectional study involving outpatients with asthma under treatment at a tertiary care university hospital in the city of Rio de Janeiro, Brazil. Consecutive patients were divided into two groups according to the severity of asthma based on the Global Initiative for Asthma criteria: mild asthma (MA), comprising patients with mild intermittent or persistent asthma; and moderate or severe asthma (MSA). We determined the serum levels of staphylococcal toxin-specific IgE antibodies, comparing the results and performing a statistical analysis. Results: The study included 142 patients: 72 in the MA group (median age = 46 years; 59 females) and 70 in the MSA group (median age = 56 years; 60 females). In the sample as a whole, 62 patients (43.7%) presented positive results for staphylococcal toxin-specific IgE antibodies: staphylococcal enterotoxin A (SEA), in 29 (20.4%); SEB, in 35 (24.6%); SEC, in 33 (23.2%); and toxic shock syndrome toxin (TSST), in 45 (31.7%). The mean serum levels of IgE antibodies to SEA, SEB, SEC, and TSST were 0.96 U/L, 1.09 U/L, 1.21 U/L, and 1.18 U/L, respectively. There were no statistically significant differences between the two groups in terms of the qualitative or quantitative results. Conclusions: Serum IgE antibodies to SEA, SEB, SEC, and TSST were detected in 43.7% of the patients in our sample. However, neither the qualitative nor quantitative results showed a statistically significant association with the clinical severity of asthma.

RESUMO Objetivo: Determinar a presença de anticorpos IgE específicos para superantígenos estafilocócicos e o grau de sensibilização mediada por esses, assim como se esses estão associados à gravidade da asma em pacientes adultos. Métodos: Estudo transversal incluindo asmáticos adultos em acompanhamento ambulatorial em um hospital universitário terciário no Rio de Janeiro (RJ). Os pacientes foram alocados consecutivamente em dois grupos de gravidade da asma segundo critérios da Global Initiative for Asthma: asma leve (AL), com asmáticos leves intermitentes ou persistentes, e asma moderada ou grave (AMG). Foram determinados os níveis séricos de anticorpos IgE antitoxinas estafilocócicas, e os resultados foram comparados por análise estatística. Resultados: Foram incluídos 142 pacientes no estudo: 72 no grupo AL (mediana de idade = 46 anos; 59 do sexo feminino) e 70 do grupo AMG (mediana de idade = 56 anos; 60 do sexo feminino). Na amostra geral, 62 pacientes (43,7%) apresentaram resultados positivos para dosagens de anticorpos IgE antitoxinas estafilocócicas: enterotoxina (TX) A, em 29 (20,4%); TXB, em 35 (24,6%); TXC, em 33 (23,2%); e toxic shock syndrome toxin (TSST), em 45 (31,7%). As médias das dosagens séricas de anticorpos IgE específicos anti-TXA, TXB, TXC e TSST foram, respectivamente, de 0,96 U/l, 1,09 U/l, 1,21 U/l, e 1,18 U/l. Não houve diferença estatisticamente significativa dos resultados qualitativos ou quantitativos entre os grupos. Conclusões: A presença de anticorpos IgE séricos anti-TXA, TXB, TXC e TSST, foi detectada em 43,7% nessa amostra de pacientes, mas não houve associação estatisticamente significativa entre seus resultados qualitativos ou quantitativos e gravidade clínica da asma.

High frequency of Panton-Valentine leukocidin in Staphylococcus aureus causing pediatric infections in the city of Cartagena-Colombia.

Panton-Valentine leucocidin (PVL) is a pore-forming toxin that has been epidemiologically associated with CA-MRSA infections. However, its role in the pathogenicity of Staphylococcus aureus is still unclear. We evaluated the prevalence of PVL-coding genes in methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) isolates that cause infections in pediatric patients in the city of Cartagena, Colombia. We obtained S. aureus isolates from patients at the Napoleon Franco Pareja Children's Hospital in Cartagena. Then, we evaluated the presence of the nuc, mecA, and PVL genes in these isolates by multiplex PCR and determined the antibiotic susceptibility profiles using CLSI standards. We further correlated methicillin susceptibility and the presence of PVL genes with clinical variables. Overall PVL prevalence in S. aureus isolates was 73.91%, with a frequency of 80.92% among MRSA isolates and 67.59% among MSSA. We found a correlation between erythromycin resistance and lack of PVL and found that PVL+ cases were more common in older patients. We found a high PVL prevalence in both MRSA and MSSA isolates, in concordance with previous regional reports.

Anticorpos recombinantes contra as toxinas Stx1 e Stx2 produzidas por Escherichia coli produtora da toxina de Shiga: obtenção e caracterização dessas ferramentas para o imunodiagnóstico e terapia / Recombinant antibodies against Stx1 and Stx2 toxins: obtainment and characterization of these tools for Shiga toxin-producing Escherichia coli (STEC) immunodiagnosis and therapy

Shiga toxin-producing Escherichia coli (STEC) causes hemorrhagic colitis and hemolytic uremic syndrome by producing Shiga toxin (Stx), a AB5 type toxin. The B subunit binds to ganglioside receptors, and the active A subunit is translocated into the cell, where it inhibits protein synthesis by acting on rRNA, leading to cell death. Diagnosis of these strains is not commonly done in the routine laboratory of low-income countries. Besides, therapy for intoxication is based on minimizing the symptoms, since no toxin antidote is available. Thus, the development of effective tools for toxin detection and therapy is highly relevant. Antibodies exhibit excellent high affinity for their specific antigens. To maintain the homogeneity and specificity of monoclonal antibodies, together with largescale production and low cost, genetic engineering has been used to develop recombinant antibodies. These include single chain variable fragments and antigenbinding fragments, which consist of antibody fragments that have antigen recognition regions. In the present study we obtained and characterized two different recombinant antibody fragments (Fab and scFv), produced in bacteria, against Stx toxins from STEC isolates. Furthermore, for the first time a human monovalent fragment was constructed (Fab), which was able to recognize and neutralize Stx toxins. These fragments obtained are promising tools for its use in the diagnosis and therapy of intoxication by STEC.

Linhagens de Escherichia coli produtoras da toxina de Shiga (STEC) causam colite hemorrágica e síndrome hemolítica urêmica pela produção da citotoxina de Shiga (Stx), uma toxina do tipo AB5, a subunidade B se liga ao receptor, e transloca a subunidade A ativa que inibe a síntese proteica por agir no rRNA levando à morte celular. O diagnóstico dessas linhagens não é realizado na rotina laboratorial de países em desenvolvimento e a terapia da intoxicação se baseia em tratar os sintomas, pois não há antídoto para a toxina. A obtenção de ferramentas para a detecção e terapia destas toxinas são de grande importância. Os anticorpos tem se mostrado moléculas excelentes como reagentes ligantes de alta afinidade à proteínas. Com o intuito de manter a homogeneidade e a especificidade dos anticorpos monoclonais, aliados ainda à produção em larga escala com baixo custo, a engenharia genética tem sido utilizada para obtenção de anticorpos recombinantes como os fragmentos variáveis de cadeia única e os fragmentos de ligação ao antígeno, que são fragmentos de anticorpos que possuem as regiões de reconhecimento ao antígeno. No presente trabalho foram obtidos e caracterizados dois tipos diferentes de fragmentos de anticorpos recombinantes (scFv e Fab), produzidos em bactérias, contra as toxinas Stx produzidas por isolados de STEC. Além disso, pela primeira vez, foi construído um fragmento monovalente humano (Fab), capaz de reconhecer e neutralizar as toxinas Stx. Estes fragmentos obtidos são promissores para seu uso como ferramentas tanto no diagnóstico como na terapia das intoxicações por STEC.

Impedimetric sensor of bacterial toxins based on mixed (Concanavalin A)/polyaniline films.

In this paper, we report the use of Concanavalin A (ConA) and electrosynthesized polyaniline (PANI) thin films for the development of a new electrochemical sensor that allows the specific detection of two bacterial toxins: lipopolysaccharide (LPS) from Escherichia coli and lipoteichoic acid from Staphylococcus aureus. The impedimetric sensor is fabricated by using glutaraldehyde to self-assemble ConA lectin on PANI-modified steel electrodes through covalent binding. ConA acts as a recognition element for bacterial toxins. Electrical impedance spectroscopy (EIS) and scanning electron microscope (SEM) were applied to characterize the assembly process on the modified electrode. The EIS measurements revealed that the resistance charge transfer (RCT) of the electrode/electrolyte interface increases considerably after the ConA lectin interacts with specific carbohydrate moieties present in the molecule of the bacterial toxin. Our results showed that the ConA lectin retained its activity after immobilization on the PANI surface and also the existence of electrochemical impedance response of the bioelectrode which is linear to the extent of the lectin-toxin interaction, with maximum values of RCT for E. coli (14.40 kΩ), and S. aureus (17.80 kΩ). We have observed that electrosynthesized PANI is an excellent support layer for the covalent binding of lectins on the electrode surface. Thus, the recognition system provides an appropriate biomimetic interface for detection of specific constituents of gram-positive and gram-negative bacteria.