José Nelson Couceiro

Impact of agr dysfunction on virulence profiles and infections associated with a novel methicillin-resistant Staphylococcus aureus (MRSA) variant of the lineage ST1-SCCmec IV

BackgroundA novel variant of the ST1-SCCmecIV methicillin-resistant Staphylococcus aureus (MRSA) lineage, mostly associated with nosocomial bloodstream infections (BSI), has emerged in Rio de Janeiro. Bacterial biofilm has been considered a major virulence factor in central venous catheter-associated BSI. The mechanisms involved in biofilm formation/accumulation are multifactorial and complex. Studies have suggested that biofilm production was affected in vitro and vivo for agr-null mutants of S. aureus.ResultsThe impact of naturally occurring inhibition of agr signaling on virulence profiles and infections associated with the ST1 variant was investigated. agr dysfunction was detected in a significant percentage (13%) of the isolates with concomitant increase in biofilm accumulation in vitro and in vivo, and enhanced ability to adhere to and invade airway cells. The biofilm formed by these ST1 isolates was ica-independent and proteinaceous in nature. In fact, the improved colonization properties were paralleled by an increased expression of the biofilm-associated genes fnbA, spa and sasG. The transcription of sarA, a positive regulator of agr, was two-times reduced for the agr-dysfunctional MRSA. Remarkably, the agr inhibition was genetically stable. Indeed, agr-dysfunctional isolates succeed to colonize and cause both acute and chronic infections in hospitalized patients, and also to effectively accumulate biofilm in a mouse subcutaneous catheter implant model.ConclusionThe ability of agr-dysfunctional isolates to cause infections in humans and to form biofilm in the animal model suggests that therapeutic approaches based on agr-inactivation strategies are unlikely to be effective in controlling human-device infections caused by ST1 isolates. The increased biofilm accumulation associated with the acquisition of multiple antimicrobial resistant traits might have influenced (at least in part) the expansion of this USA400 related clone in our hospitals.

Sialylglycoconjugates and sialyltransferase activity in the fungus Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen associated with systemic mycoses in up to 10% of AIDS patients. C. neoformans yeasts express sialic acids on the cell wall, where they play an anti-phagocytic role, and may represent a virulence factor at the initial phase of infection. Since the nature of the sialic acid-carrying components is undefined in C. neoformans, our aim in the present work was to identify sialylated molecules in this fungus and study the sialylation process. C. neoformans yeast forms were cultivated in a chemically defined medium free of sialic acids, to search for autologous sialylglycoconjugates. Sialylated glycolipids were not detected. Two glycoproteins with molecular masses of 38 and 67 kDa were recognized by Sambucus nigra agglutinin, an α2,6-sialic acid-specific lectin. The 67 kDa glycoprotein also interacted with Influenza C virus, but not with Limax flavus agglutinin, suggesting the presence of the 9-O-acetylated sialic acid derivative as a constituent of the oligosaccharide chains. A partially purified protein fraction from cryptococcal yeast forms was able to transfer sialic acid from CMP-Neu5Ac to both N-(acetyl-1-14C)-lactosamine and asialofetuin. Additional evidence for a sialyltransferase in C. neoformans was obtained through the reactivity of fungal proteins with rabbit anti-rat α2,6 sialyltransferase polyclonal antibody. Our results indicate that sialic acids in C. neoformans are linked to glycoproteins, which are sialylated by the action of a fungal sialyltransferase. This is the first demonstration of this biosynthetic step in pathogenic fungi. Published in 2003.

Cell-surface sialoglycoconjugate structures in wild-type and mutant Crithidia fasciculata.

Abstract The cell-surface expression of sialoglycoconjugate structures in wild-type Crithidia fasciculata and its TFRR1 drug-resistant mutant was analyzed with the aid of an influenza C virus strain, lectin, enzymatic treatment, and flow cytofluorimetry analysis probed with fluorescein isothiocyanate-labeled (FITC) lectins. 9-O-Acetyl-N-acetyl neuraminic acid (Neu5,9Ac2) structures mediate influenza C virus cell-binding. The SAα2,3Gal and SAα2,6Gal sequences are specifically recognized by Maackia amurensis (MAA) and Sambucus nigra (SNA) lectins, respectively. On the basis of these param- eters the TFRR1 mutant strain of C. fasciculata was found to contain exposed sialoglycoconjugates bearing Neu5,9Ac2 surface structures. After the removal of sialic acid residues by neuraminidase activity the marked increases in PNA (peanut agglutinin)-mediated agglutinating activity showed that those acidic units on C. fasciculata cells were glycosidically linked to d-galactose. The bond involves SAα2,6Gal and SAα2,3Gal linkages as suggested by the use of FITC-SNA and FITC-MAA lectins, respectively. Both SAα2,3Gal and SAα2,6Gal sequences were preferentially expressed by the TFRR1 mutant. The SAα2,6 linkage markedly predominated. In the TFRR1 mutant, but not in wild-type cells, two distinct populations of cells were distinguished by reactivity with FITC-SNA, one of which was enriched with surface SAα2,6Gal sequences. These diverse findings suggest that sialoglycoconjugate structures present on the flagellate surface may be associated with mutation and the cell growth cycle in C. fasciculata.

Standardization of denaturing gradient gel electrophoresis for mutant screening of influenza A (H3N2) virus samples.

Because of the extensive genetic variability of the influenza viruses, new virus mutants arise worldwide. In the human population, some strains may become potentially epidemic after evading the immune response of the host. At present, molecular methods have made it possible to identify these variants. However, if a large number of samples need to be analyzed the identification of randomly mutated nucleotides cannot be achieved by sequencing analysis or restriction fragment length polymorphism (RFLP). In order to improve this process, a denaturing gradient gel electrophoresis (DGGE) protocol capable of discriminating between reference strains representative of different influenza seasons, some mutant strains, and five clinical isolates was standardized Ribonudeic acid (RNA) was isolated and submitted to a one-step RT-PCR that amplified the region codifying for the globular domain of the Haemagglutinin (HA) molecule. The amplicons were analyzed by electrophoresis in 6% polyacrylamide gel at 60 degreeC/150 V for 8 h, using a 31--41% urea--formamide gradient. This method was able to distinguish between closely related nucleotide sequences, confirming its suitability as screening methodology for the analysis of influenza virus epidemiology, by allowing a faster and more extensive evaluation of a large number of the variant strains detected in a specific region of the world.

Characterization of the hemagglutinin receptor specificity and neuraminidase substrate specificity of clinical isolates of human influenza A viruses

Six clinical isolates of influenza A viruses were examined for hemagglutinin receptor specificity and neuraminidase substrate specificity. All of the viral isolates minimally passaged in mammalian cells demonstrated preferential agglutination of human erythrocytes enzymatically modified to contain NeuAc alpha 2,6Gal sequences, with no agglutination of cells bearing NeuAc alpha 2,3Gal sequences. This finding is consistent with the hemagglutination receptor specificity previously demonstrated for laboratory strains of influenza A viruses. The neuraminidase substrate specificities of the clinical isolates examined were also identical to that described for the N2 neuraminidase of recent laboratory strains of human influenza viruses. The H3N2 viruses all displayed the ability to release sialic acid from both alpha 2, 3 and alpha 2, 6 linkages. In addition, two clinical isolates of H1N1 viruses also demonstrated this dual neuraminidase substrate specificity, a characteristic which has not been previously described for the N1 neuraminidase. These results demonstrate that complementary hemagglutinin and neuraminidase specificities are found in recent isolates of both H1N1 and H3N2 influenza viruses.

Virulence characteristics of genetically related isolates of group B streptococci from bovines and humans.

The present study had the objective of evaluating the pathogenic potential of the genetically related strains of Streptococcus agalactiae no. 80427 (human origin) and no. 87159 (bovine origin), and comparing the results with two other strains isolated from bovine mastitis (no. 87244) and invasive human infection (no. 90356), with no genetic or epidemiologic relationship between them or with the first 2 isolates. Virulence genes hylB (hyaluronidase) and lmb (laminin-binding protein) were detected in the 4 strains, and genes bac (beta protein) and bca (alpha protein) were only detected in human strains. The protein profile obtained using SDS-PAGE did not indicate any differences between the 4 strains. No significant difference was detected between human and bovine strains in the assays of adherence to and invasion of 16HBe cells, as well as in the resistance assay for intracellular bacterial survival in macrophages. However, the strain 87159 exhibited a greater survival in the killing test with whole human blood and was more virulent in newborn mice than the 80427 strain. The strain 87244 was not virulent in mice. These data suggest that isolates of human and bovine origins may express similar virulence attributes, leading to a possible, however limited, dissemination.

Serological analysis reveals circulation of influenza C viruses, Brazil

The circulation of influenza C viruses in Rio de Janeiro, Brazil, was studied when significant levels of antibodies were detected (56. 7%) with hemagglutination inhibition test, used as a standard methodology for influenza virus studies.

Homeopathic medicines for prevention of influenza and acute respiratory tract infections in children: blind, randomized, placebo-controlled clinical trial.

BACKGROUND Influenza and its complications are common at all ages, especially in children. Vaccines and anti-influenza drugs aim to prevent it. Preventative approaches with favorable risk profiles should be considered for flu, particularly since the evidence of the efficacy of anti-viral drugs is debated. METHODS This pragmatic clinical trial was conducted in the Brazilian Public Health System in Petrópolis (BPHSP) with children aged from 1 to 5 years old. The medications used were mainly selected based on in vitro experiments (InfluBio), and in successful qualitative clinical experiences (Homeopathic Complex). Following informed parental consent, subjects were randomly distributed, in a blind manner, to three experimental groups: Homeopathic Complex, Placebo, and InfluBio. BPHSP health agents collected flu and acute respiratory infection symptomatic episodes monthly following the established protocol. The number of these episodes was registered in one year (2009-2010). RESULTS Out of the 600 children recruited, 445 (74.17%) completed the study (149: Homeopathic complex; 151: Placebo; 145: InfluBio). The number of flu and acute respiratory infection symptomatic episodes detected in this clinical trial was low; however, it was different between homeopathic groups and placebo (p < 0.001). In the first year post-intervention, 46/151 (30.5%) of children in the placebo group developed 3 or more flu and acute respiratory infection episodes, while there was no episode in the group of 149 children who used Homeopathic Complex, and only 1 episode in the group of 145 (1%) children who received InfluBio. CONCLUSION These results suggested that the use of homeopathic medicines minimized the number of flu and acute respiratory infection symptomatic episodes in children, signalizing that the homeopathic prophylactic potential should be investigated in further studies.

Detection of paramyxoviruses in Magellanic penguins (Spheniscus magellanicus) on the Brazilian tropical coast

Aquatic migratory birds are a major vectors by which influenza viruses and paramyxoviruses are spread in nature. Magellanic penguins (Spheniscus magellanicus) are usually present on the southern shores of South America and can swim as far as the southern coast of Brazil in winter. In 2008, however, several Magellanic penguins were observed on the northeastern coast of Brazil. Paramyxoviruses were isolated from Magellanic penguins on the Espírito Santo state coast, approximately 4000 km from their breeding colonies, although influenza viruses were not detected. Among the paramyxoviruses, five Avulavirus isolates belonging to serotype APMV-2 and the serotype APMV-10, which was proposed by Miller et al. (2010), were identified. These results highlight the risks associated with the spread of paramyxoviruses between natural to non-natural habitats by birds exhibiting unusual migration patterns, and they document for the first time the presence of the APMV-2 and APMV-10 serotypes on penguins in Brazil. The local avifauna may become infected with these viruses through close contact between migratory and resident birds. Continued surveillance of virus incidence in these migratory populations of penguins is necessary to detect and prevent the potential risks associated with these unusual migration patterns.

Full Inactivation of Human Influenza Virus by High Hydrostatic Pressure Preserves Virus Structure and Membrane Fusion While Conferring Protection to Mice against Infection

Whole inactivated vaccines (WIVs) possess greater immunogenicity than split or subunit vaccines, and recent studies have demonstrated that WIVs with preserved fusogenic activity are more protective than non-fusogenic WIVs. In this work, we describe the inactivation of human influenza virus X-31 by high hydrostatic pressure (HHP) and analyze the effects on the structure by spectroscopic measurements, light scattering, and electron microscopy. We also investigated the effects of HHP on the glycoprotein activity and fusogenic activity of the viral particles. The electron microscopy data showed pore formation on the viral envelope, but the general morphology was preserved, and small variations were seen in the particle structure. The activity of hemagglutinin (HA) during the process of binding and fusion was affected in a time-dependent manner, but neuraminidase (NA) activity was not affected. Infectious activity ceased after 3 hours of pressurization, and mice were protected from infection after being vaccinated. Our results revealed full viral inactivation with overall preservation of viral structure and maintenance of fusogenic activity, thereby conferring protection against infection. A strong response consisting of serum immunoglobulin IgG1, IgG2a, and serum and mucosal IgA was also detected after vaccination. Thus, our data strongly suggest that applying hydrostatic pressure may be an effective method for developing new vaccines against influenza A as well as other viruses.