O26 Polysaccharides as Key Players in Enteropathogenic E. coli Immune Evasion and Vaccine Development.

Enteropathogenic Escherichia coli (EPEC) produce a capsule of polysaccharides identical to those composing the O-antigen polysaccharide of its LPS (lipopolysaccharide) molecules. In light of this, the impact of O26 polysaccharides on the immune evasion mechanisms of capsulated O26 EPEC compared to non-capsulated enterohemorrhagic Escherichia coli (EHEC) was investigated. Our findings reveal that there was no significant difference between the levels in EPEC and EHEC of rhamnose (2.8:2.5), a molecule considered to be a PAMP (Pathogen Associated Molecular Patterns). However, the levels of glucose (10:1.69), heptose (3.6:0.89) and N-acetylglucosamine (4.5:2.10), were significantly higher in EPEC than EHEC, respectively. It was also observed that the presence of a capsule in EPEC inhibited the deposition of C3b on the bacterial surface and protected the pathogen against lysis by the complement system. In addition, the presence of a capsule also protected EPEC against phagocytosis by macrophages. However, the immune evasion provided by the capsule was overcome in the presence of anti-O26 polysaccharide antibodies, and additionally, these antibodies were able to inhibit O26 EPEC adhesion to human epithelial cells. Finally, the results indicate that O26 polysaccharides can generate an effective humoral immune response, making them promising antigens for the development of a vaccine against capsulated O26 E. coli.

O26 Polysaccharides as key players in enteropathogenic E. coli immune evasion and vaccine development

Enteropathogenic Escherichia coli (EPEC) produce a capsule of polysaccharides identical to those composing the O-antigen polysaccharide of its LPS (lipopolysaccharide) molecules. In light of this, the impact of O26 polysaccharides on the immune evasion mechanisms of capsulated O26 EPEC compared to non-capsulated enterohemorrhagic Escherichia coli (EHEC) was investigated. Our findings reveal that there was no significant difference between the levels in EPEC and EHEC of rhamnose (2.8:2.5), a molecule considered to be a PAMP (Pathogen Associated Molecular Patterns). However, the levels of glucose (10:1.69), heptose (3.6:0.89) and N-acetylglucosamine (4.5:2.10), were significantly higher in EPEC than EHEC, respectively. It was also observed that the presence of a capsule in EPEC inhibited the deposition of C3b on the bacterial surface and protected the pathogen against lysis by the complement system. In addition, the presence of a capsule also protected EPEC against phagocytosis by macrophages. However, the immune evasion provided by the capsule was overcome in the presence of anti-O26 polysaccharide antibodies, and additionally, these antibodies were able to inhibit O26 EPEC adhesion to human epithelial cells. Finally, the results indicate that O26 polysaccharides can generate an effective humoral immune response, making them promising antigens for the development of a vaccine against capsulated O26 E. coli.

O55 Polysaccharides Are Good Antigen Targets for the Formulation of Vaccines against O55 STEC and Capsulated aEPEC Strains.

The serogroup O55 of E. coli is composed of strains whose mechanisms of virulence are different from each other. Since the O55 polysaccharides are present in all E. coli O55 strains, and so are the polymers that compose the capsule of O55 atypical enteropathogenic E. coli (aEPEC), it was investigated whether anti-O55 antibodies were able to help the innate immune system to eliminate capsulated aEPEC and Shiga toxin-producing E. coli (STEC) belonging to the serogroup O55. The results demonstrate that the capsule of EPEC was able to inhibit the deposition of C3b on the bacterial surface and, as a consequence, their lysis by the alternative pathway of the complement system. However, in the presence of antibodies, the ability of the complement to lyse these pathogens was restored. It was also observed that macrophages were able to ingest EPEC and STEC, but they were only able to kill the ingested pathogens in the presence of antibodies. Anti-O55 antibodies were also able to inhibit aEPEC and STEC O55 adherence to human epithelial cells. In summary, the results demonstrated that the O55 polysaccharides have the potential to induce an effective humoral immune response against STEC and EPEC, indicating that they are good antigen targets to be used in vaccine formulations against these pathogens.

Escherichia coli Strains Responsible for Cystitis in Female Pediatric Patients with Normal and Abnormal Urinary Tracts Have Different Virulence Profiles.

The role of uropathogenic Escherichia coli (UPEC) in colonization and infection of female patients with anatomical and functional abnormalities of the urinary system is elusive. In this study, the phenotype, genotype and the phylogeny of UPEC strains isolated from the urine of pediatric female patients with cystitis of normal and abnormal urinary tract were determined. Multiplex PCR results demonstrated that 86% of the strains isolated from female patients with normal urinary tract (NUT), belonged to the phylo-groups B2 and D. Their prevalence decreased to 23% in strains isolated from patients with abnormal urinary tract (AUT). More of the isolates from AUT patients produced a biofilm on polystyrene and polyvinyl chloride (PVC), adhered to epithelial cells, and encoded pap and sfa genes than strains isolated from female patients with NUT. In contrast, a higher number of hemolysin-producing strains with serogroups associated with UPEC were isolated from patients with NUT. In summary, the results suggest that cystitis in female patients with NUT is associated with ExPEC, whereas cystitis in female patients with AUT is associated with pathogenic intestinal E. coli strains that have acquired the ability to colonize the bladder.

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.

O55 polysaccharides are good antigen targets for the formulation of vaccines against O55 STEC and capsulated aEPEC strains

The serogroup O55 of E. coli is composed of strains whose mechanisms of virulence are different from each other. Since the O55 polysaccharides are present in all E. coli O55 strains, and so are the polymers that compose the capsule of O55 atypical enteropathogenic E. coli (aEPEC), it was investigated whether anti-O55 antibodies were able to help the innate immune system to eliminate capsulated aEPEC and Shiga toxin-producing E. coli (STEC) belonging to the serogroup O55. The results demonstrate that the capsule of EPEC was able to inhibit the deposition of C3b on the bacterial surface and, as a consequence, their lysis by the alternative pathway of the complement system. However, in the presence of antibodies, the ability of the complement to lyse these pathogens was restored. It was also observed that macrophages were able to ingest EPEC and STEC, but they were only able to kill the ingested pathogens in the presence of antibodies. Anti-O55 antibodies were also able to inhibit aEPEC and STEC O55 adherence to human epithelial cells. In summary, the results demonstrated that the O55 polysaccharides have the potential to induce an effective humoral immune response against STEC and EPEC, indicating that they are good antigen targets to be used in vaccine formulations against these pathogens.

Escherichia coli strains responsible for cystitis in female pediatric patients with normal and abnormal urinary tracts have different virulence profiles

The role of uropathogenic Escherichia coli (UPEC) in colonization and infection of female patients with anatomical and functional abnormalities of the urinary system is elusive. In this study, the phenotype, genotype and the phylogeny of UPEC strains isolated from the urine of pediatric female patients with cystitis of normal and abnormal urinary tract were determined. Multiplex PCR results demonstrated that 86% of the strains isolated from female patients with normal urinary tract (NUT), belonged to the phylo-groups B2 and D. Their prevalence decreased to 23% in strains isolated from patients with abnormal urinary tract (AUT). More of the isolates from AUT patients produced a biofilm on polystyrene and polyvinyl chloride (PVC), adhered to epithelial cells, and encoded pap and sfa genes than strains isolated from female patients with NUT. In contrast, a higher number of hemolysin-producing strains with serogroups associated with UPEC were isolated from patients with NUT. In summary, the results suggest that cystitis in female patients with NUT is associated with ExPEC, whereas cystitis in female patients with AUT is associated with pathogenic intestinal E. coli strains that have acquired the ability to colonize the bladder.

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.

A new class of antimicrobial molecules derived from kefir, effective against Pseudomonas aeruginosa and methicillin resistant Staphylococcus aureus (MRSA) strains

Many studies have linked the antimicrobial properties of kefir with the presence of bacteriocins and organic acids. In the present work, results obtained from bacteriostatic and bactericidal studies, and from RP-HPLC, Mass Spectrometry and proton NMR analysis, show that a sample of milk kefir grains is able to produce an antimicrobial fraction, denoted FK-1000, composed of sugars and amino acids, predominantly polymers of alanine, doublets of tyrosine and phenylalanine. Since this fraction is a lyophilized product whose molecular profile is different from bacteriocins and simple carboxylic acids, its antimicrobial effect cannot be attributed to these molecules, or to alcohols or hydrogen peroxide. The fraction is bactericidal against weak-acid-resistant MRSA and weak-acid resistant P. aeruginosa at pH 5, and is bacteriostatic against both pathogens at pH 7. In combination formulation, the FK-1000 fraction is able to increase fivefold the effect of streptomycin against P. aeruginosa and it is not toxic to human epithelial cells at antimicrobial concentrations. 16 S rRNA microbiota analysis of antimicrobial-producing and non-producing kefir grains demonstrated that they are distinct. In summary, the results indicate that milk kefir grains can produce different classes of molecules with potent antibiotic activity against resistant bacteria.

The potential of Loxosceles gaucho spider venom to regulate Pseudomonas aeruginosa mechanisms of virulence

Loxosceles venom is a potential source of bioactive molecules which may be transformed into antimicrobial products against multi-resistant bacteria. Here, it was investigated whether Loxosceles gaucho spider had any influence on the proliferation, enzyme release and biofilm formation of a Pseudomonas aeruginosa strain resistant to two different classes of antibiotic. The results demonstrated that L. gaucho whole venom has no influence on P. aeruginosa proliferation. However, it increases P. aeruginosa production of gelatinase, caseinase and biofilm formation. The same effects were noted when P. aeruginosa was exposed to a L. gaucho venom molecular fraction with mass lower than 1 kDa. Separation of this molecular fraction into different subsets by RP-HPLC demonstrated that, among the molecules with the ability to increase the production of enzymes and biofilm formation, there are some with antimicrobial activities whose effects are not observed in the whole venom. In summary, the results obtained herein indicate that L. gaucho venom has a variety of low molecular mass bioactive components that influence the mechanisms of virulence of P. aeruginosa in different ways.

The potential of Loxosceles gaucho spider venom to regulate Pseudomonas aeruginosa mechanisms of virulence

Loxosceles venom is a potential source of bioactive molecules which may be transformed into antimicrobial products against multi-resistant bacteria. Here, it was investigated whether Loxosceles gaucho spider had any influence on the proliferation, enzyme release and biofilm formation of a Pseudomonas aeruginosa strain resistant to two different classes of antibiotic. The results demonstrated that L. gaucho whole venom has no influence on P. aeruginosa proliferation. However, it increases P. aeruginosa production of gelatinase, caseinase and biofilm formation. The same effects were noted when P. aeruginosa was exposed to a L. gaucho venom molecular fraction with mass lower than 1 kDa. Separation of this molecular fraction into different subsets by RP-HPLC demonstrated that, among the molecules with the ability to increase the production of enzymes and biofilm formation, there are some with antimicrobial activities whose effects are not observed in the whole venom. In summary, the results obtained herein indicate that L. gaucho venom has a variety of low molecular mass bioactive components that influence the mechanisms of virulence of P. aeruginosa in different ways.

VaxcineTM: an oil-based adjuvant for influenza vaccines

Vaccination is the method of choice for the prevention of influenza infection. However, the quantity of the antigen available, especially in the case of pandemics, often fails to meet the global demand. However, improved adjuvants can overcome this problem. Preliminary results obtained in this study revealed that one year after a single subcutaneous immunisation with influenza A H3N2 virus in an oil-based carrier, VaxcineTM, outbreed mice produced a high immunoglobulin G response that lasted for up to one year and exhibited less variation in titre compared with the response of the control group treated with alum. The haemagglutination-inhibition titres induced by VaxcineTM were also higher than those generated by alum. These data indicate that VaxcineTM is a good adjuvant candidate for seasonal influenza vaccines.

Vaxcine(TM): an oil-based adjuvant for influenza vaccines.

Vaccination is the method of choice for the prevention of influenza infection. However, the quantity of the antigen available, especially in the case of pandemics, often fails to meet the global demand. However, improved adjuvants can overcome this problem. Preliminary results obtained in this study revealed that one year after a single subcutaneous immunisation with influenza A H3N2 virus in an oil-based carrier, Vaxcine(TM), outbreed mice produced a high immunoglobulin G response that lasted for up to one year and exhibited less variation in titre compared with the response of the control group treated with alum. The haemagglutination-inhibition titres induced by Vaxcine(TM) were also higher than those generated by alum. These data indicate that Vaxcine(TM) is a good adjuvant candidate for seasonal influenza vaccines.