Comparação na avaliação microbiológica de dois desinfetantes de uso doméstico no Brasil pelos métodos recomendados pela AOAC e ECN

Surface disinfectants are considered a major tool for preventing the spread of infections in human populations. Changes in the Brazilian law in 2007 allowed the use of various methodologies to evaluate the microbiological quality of surface disinfectants, warranting studies of the efficacy and application of other methodologies, such as those recommended by European Committee of Normalization (ECN). The present study aimed to establish the application of ECN methodologies for assessment of general use disinfectants. The use–dilution (UD) method recommended by the Association of Official Analytical Chemists (AOAC) and the European Standard 1276 from ECN were performed in parallel, and the anti-microbial actions of two quaternary ammonium compounds (products A and B) and 70% ethanol solution were compared using reference microorganisms and two strains isolated from household environments. According to the UD method, all products were effective, except that product B was ineffective against Staphylococcus aureus and gave non-reproducible results. The ECN method indicated that all products were effective, except that product A produced inadequate neutralization of S. aureus. Although the ECN method showed efficacy and applicability to the Brazilian routine, assays with greater number of products are required.Os produtos desinfetantes para superfícies são considerados uma ferramenta da população para prevenção de infecções de natureza domiciliar. Modificações na legislação brasileira em 2007 permitiram a utilização de diferentes metodologias para a avaliação da qualidade microbiológica desses produtos, resultando na necessidade de um maior conhecimento em relação ao comportamento e aplicação de outras metodologias, como as preconizadas pelo Comitê Europeu de Normalização (CEN). Esse trabalho teve como objetivo estabelecer um estudo piloto da aplicação das metodologias do CEN em desinfetantes de uso geral. Foram utilizadas, em paralelo, a metodologia da Diluição de Uso (DU) preconizada pela Association of Official Analytical Chemists (AOAC) e a norma EUROPEAN STANDARD 1276 do CEN, utilizando dois produtos à base de quaternário de amônio (produtos A e B) e o álcool etílico 70% como controle, frente aos micro-organismos de referência e duas amostras de E. cloacae isoladas de ambiente domiciliar. Pela DU, todos os produtos foram eficazes, com exceção do produto B frente a Staphylococcus aureus cujos resultados não foram reprodutíveis. Pelo método do CEN, todos os produtos foram eficazes, com exceção do produto A frente a S. aureus, cuja neutralização não foi adequada. A divergência de resultados obtidos para os dois produtos à base de quaternários de amônio evidencia a necessidade de continuidade na avaliação das duas técnicas, com um maior número de produtos comerciais, considerando a potencialidade do uso do CEN no país

Preparo de Itens de Ensaio de Proficiência em Matriz Queijo para a Pesquisa de Salmonella spp.

The participation of analytical laboratories in proficiency testing allows the verification of the reliability of results in analysis of quality control. The aim of this study was to produce a batch of test items (TI) to be used in a proficiency assay for Salmonella spp. research in a cheese matrix. Ultrafiltered Minas cheese was used as the matrix and spiked with a strain of Salmonella Enteritidis. Trehalose was used as cryoprotectant, and freeze-drying was used as the preservation technique. The batch was evaluated for vacuum verification, homogeneity study, and long-term stability testing at temperatures of -70ºC  (reference temperature) and -20ºC (storage temperature). The batch was also evaluated for short-term stability at temperatures of 4ºC, 25ºC, and 35ºC (transport temperatures). The results showed vacuum in 95.6% of the bottles, and the batch was considered sufficiently homogeneous. TI remained stable at -70ºC for more than 360 days and at -20ºC for more than 160 days. TI was shown to be stable for up 6 days at 4ºC and 25ºC but not at 35ºC. It is concluded that the methodology used was satisfactory for this TI production for Salmonella spp. research in a cheese matrix.A participação de laboratórios analíticos em ensaio de proficiência permite a verificação da confiabilidade dos resultados gerados nas análises de controle da qualidade. O objetivo deste estudo foi produzir um lote de itens de ensaio (IE) a ser utilizado em ensaio de proficiência (EP) para pesquisa de Salmonella spp. em matriz queijo. Queijo Minas Frescal (QMF) Ultrafiltrado foi utilizado como matriz e fortificado com uma cepa de Salmonella Enteritidis. A trealose foi utilizada como crioprotetor e a liofilização como técnica de preservação. O lote foi avaliado quanto a verificação do vácuo, teste da homogeneidade, estudo da estabilidade em longo prazo nas temperaturas de -70oC (referência) e -20oC (armazenamento) e em curto prazo nas temperaturas de 4, 25 e 35ºC (transporte). O lote apresentou presença de vácuo em 95,6% dos frascos e foi considerado suficientemente homogêneo. Os IE apresentaram estabilidade a -70ºC superior a 360 dias e a -20ºC superior a 160 dias. Os IE demonstraram ser estáveis por até seis dias nas temperaturas de 4 e 25ºC, mas não a 35ºC. Conclui-se que a metodologia utilizada foi satisfatória para produção de IE para o ensaio de pesquisa de Salmonella spp. em matriz queijo

Application of different methods in quality control of the antimicrobial activity of sanitizing products

Made available in DSpace on 2015-07-08T12:28:17Z (GMT). No. of bitstreams: 2 24.pdf: 3860047 bytes, checksum: f2f39055c3f43680d80b51fdb2b7cf36 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2012Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em SaúdeMicrorganismos estão presentes em grande escala no ambiente domiciliar e público. Diversos relatos na literatura já demonstraram a existência de bactérias em várias superfícies em domicílios, como pias, geladeiras, toalhas e outros objetos, e em locais de livre circulação, como aeroportos, academias de ginástica, ônibus e banheiros públicos. Com a crescente atenção voltada para a contaminação e risco de infecções causadas por alimentos e produtos manufaturados contaminados, houve um aumento do uso de desinfetantes pelo público em geral, levando a maior comercialização e circulação desses produtos. No Brasil, com a criação do Mercosul, a presença de desinfetantes provenientes de outros países se acentuou, havendo a necessidade de harmonização das normas técnicas. Por essa razão, a legislação brasileira foi alterada em 2007, com a publicação da RDC nº 14, de 28 de fevereiro de 2007, que estabeleceu que para avaliação da qualidade dos produtos desinfetantes poderiam ser aceitas as metodologias preconizadas pela Association of Official Analytical Chemists (AOAC) ou pelo Comitê Europeu de Normalização (CEN). Dessa forma, surgiu a necessidade do Brasil se preparar para essa mudança na legislação, implantando as metodologias preconizadas pelo CEN. Assim, visando a preparação do único laboratório federal oficial do Brasil, o Instituto Nacional de Controle de Qualidade em Saúde, esse trabalho teve como objetivo principal implantar as metodologias baseadas nas normas do CEN para a avaliação da atividade antibacteriana de desinfetantes de uso institucional. Foi avaliado também o comportamento de bactérias isoladas do ambiente domiciliar através da aplicação em paralelo dos métodos da AOAC e do CEN. Os resultados obtidos permitiram a padronização da fase 1 e da fase 2, etapa 1 da metodologia preconizada pelo CEN, assim como os passos iniciais da fase 2, etapa 2.Microorganisms are widely distributed in households and public environments. Several reports in the literature have just demonstrated the existence of bacteria in many households surfaces, as sinks, refrigerators, towels and other objects, and in free circulation places, as airports, gyms, buses and public bathrooms. Greater attention on contamination and infection risk caused by contaminated food and manufactured products led to increasing use of disinfectants by general population and, consequently, more commercialization and circulation of these products. In Brazil, with the foundation of “Mercosul”, the presence of disinfectants from other countries increased, leading to the necessity of technical standards harmonization. For this reason, Brazilian legislation changed in 2007 with the publication of RDC nº 14, from February 28, 2007, which established that the methodologies recommended by Association of Official Analytical Chemists (AOAC) or European Committee of Normalization (CEN) could be accepted for the quality evaluation of disinfectants. Therefore, Brazil needs to be prepared for this law change, implementing CEN methodologies. So, in order to prepare the only official federal laboratory in Brazil, “Instituto Nacional de Controle de Qualidade em Saúde”, this study aimed to implement the CEN methods for antimicrobial activity evaluation of institutional use disinfectants. It was also evaluated the behavior of environment bacteria isolated from households, through application of both AOAC and CEN methods in parallel. The results obtained allowed the standardization of phase 1 and phase 2 step 1, as well as the first steps of phase 2 step 2. It was also observed that ethyl alcohol 70% and product A, based on ammonium quaternary compounds, were considered satisfactory against all microorganisms tested (reference ones and households isolates) in both methods, while product B was not able to eliminate S. aureus by Use-Dilution method. During the study, it was found disinfectant products ready for sale presenting microbial contamination. These bacteria were identified and evidenced the presence of products with serious quality deviations in Brazilian market. These data are corroborated with reports in literature. Complementary studies are required for the complete standardization of phase 2, step 2 from CEN methodology, and for the evaluation of disinfectants based on others actives

Characterization of Scedosporium apiospermum glucosylceramides and their involvement in fungal development and macrophage functions.

Scedosporium apiospermum is an emerging fungal pathogen that causes both localized and disseminated infections in immunocompromised patients. Glucosylceramides (CMH, GlcCer) are the main neutral glycosphingolipids expressed in fungal cells. In this study, glucosylceramides (GlcCer) were extracted and purified in several chromatographic steps. Using high-performance thin layer chromatography (HPTLC) and electrospray ionization mass spectrometry (ESI-MS), N-2'-hydroxyhexadecanoyl-1-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine was identified as the main GlcCer in S. apiospermum. A monoclonal antibody (Mab) against this molecule was used for indirect immunofluorescence experiments, which revealed that this CMH is present on the surface of the mycelial and conidial forms of S. apiospermum. Treatment of S. apiospermum conidia with the Mab significantly reduced fungal growth. In addition, the Mab also enhanced the phagocytosis and killing of S. apiospermum by murine cells. In vitro assays were performed to evaluate the CMHs for their cytotoxic activities against the mammalian cell lines L.929 and RAW, and an inhibitory effect on cell proliferation was observed. Synergistic in vitro interactions were observed between the Mab against GlcCer and both amphotericin B (AmB) and itraconazole. Because Scedosporium species develop drug resistance, the number of available antifungal drugs is limited; our data indicate that combining immunotherapy with the available drugs might be a viable treatment option. These results suggest that in S. apiospermum, GlcCer are most likely cell wall components that are targeted by antifungal antibodies, which directly inhibit fungal development and enhance macrophage function; furthermore, these results suggest the combined use of monoclonal antibodies against GlcCer and antifungal drugs for antifungal immunotherapy

Glucosylceramide Plays a Role in Fungal Germination, Lipid Raft Organization and Biofilm Adhesion of the Pathogenic Fungus Scedosporium aurantiacum

Infections caused by Scedosporium species present a wide range of clinical manifestations, from superficial to disseminated, especially in immunocompromised patients. Glucosylceramides (GlcCer) are glycosphingolipids found on the fungal cell surface and play an important role in growth and pathogenicity processes in different fungi. The present study aimed to evaluate the structure of GlcCer and its role during growth in two S. aurantiacum isolates. Purified GlcCer from both isolates were obtained and its chemical structure identified by mass spectrometry. Using ELISA and immunofluorescence techniques it was observed that germination and NaOH-treatment of conidia favor GlcCer exposure. Monoclonal anti-GlcCer antibody reduced germination when cultivated with the inhibitor of melanin synthesis tricyclazole and also reduced germ tube length of conidia, both cultivated or not with tricyclazole. It was also demonstrated that anti-GlcCer altered lipid rafts organization, as shown by using the fluorescent stain filipin, but did not affect the susceptibility of the cell surface to damaging agents. Anti-GlcCer reduced total biomass and viability in biofilms formed on polystyrene plates. In the presence of anti-GlcCer, germinated S. aurantiacum conidia and biofilms could not adhere to polystyrene with the same efficacy as control cells. These results highlight the relevance of GlcCer in growth processes of S. aurantiacum

Structural Differences Influence Biological Properties of Glucosylceramides from Clinical and Environmental Isolates of Scedosporium aurantiacum and Pseudallescheria minutispora

Scedosporium/Lomentospora complex is composed of filamentous fungi, including some clinically relevant species, such as Pseudallescheria boydii, Scedosporium aurantiacum, and Scedosporium apiospermum. Glucosylceramide (GlcCer), a conserved neutral glycosphingolipid, has been described as an important cell surface molecule playing a role in fungal morphological transition and pathogenesis. The present work aimed at the evaluation of GlcCer structures in S. aurantiacum and Pseudallescheria minutispora, a clinical and an environmental isolate, respectively, in order to determine their participation in fungal growth and host-pathogen interactions. Structural analysis by positive ion-mode ESI-MS (electrospray ionization mass spectrometer) revealed the presence of different ceramide moieties in GlcCer in these species. Monoclonal antibodies against Aspergillus fumigatus GlcCer could recognize S. aurantiacum and P. minutispora conidia, suggesting a conserved epitope in fungal GlcCer. In addition, these antibodies reduced fungal viability, enhanced conidia phagocytosis by macrophages, and decreased fungal survival inside phagocytic cells. Purified GlcCer from both species led to macrophage activation, increasing cell viability as well as nitric oxide and superoxide production in different proportions between the two species. These results evidenced some important properties of GlcCer from species of the Scedosporium/Lomentospora complex, as well as the effects of monoclonal anti-GlcCer antibodies on fungal cells and host-pathogen interaction. The differences between the two species regarding the observed biological properties suggest that variation in GlcCer structures and strain origin could interfere in the role of GlcCer in host-pathogen interaction

Biofilm Formation by Pseudallescheria/Scedosporium Species: A Comparative Study

https://digitalcommons.nyls.edu/newspapers/1167/thumbnail.jp

Biofilm Formation by Pseudallescheria/Scedosporium Species: A Comparative Study

Pseudallescheria/Scedosporium species are medically important fungi that are present in soil and human impacted areas and capable of causing a wide spectrum of diseases in humans. Although little is known about their pathogenesis, their growth process and infection routes are very similar to those of Aspergillus species, which grow as biofilms in invasive infections. All nine strains tested here displayed the ability to grow as biofilms in vitro and to produce a dense network of interconnected hyphae on both polystyrene and the surfaces of central venous catheters, but with different characteristics. Scedosporium boydii and S. aurantiacum clinical isolates were able to form biofilms faster than the corresponding environmental strains, as evidenced in kinetic assays for S. boydii and CLSM for S. aurantiacum. Biofilms formed by Pseudallescheria/Scedosporium species had significantly higher resistance to the class of antifungal azole than was observed in planktonic cells, indicating a protective role for this structure. In addition, the clinical S. aurantiacum isolate that formed the most robust biofilms was also more virulent in a larvae Galleria mellonella infection model, suggesting that the ability to form biofilms enhances virulence in Pseudallescheria/Scedosporium species

ESI-MS (positive ion mode, Li+ adducts) analysis of the GlcCer species of <i>S. apiospermum</i>.

<p>(A) MS1 spectrum. (B) ESI-MS2 of the ions species with <i>m/z</i> 734.9 observed in (A) and proposed structures for the major GlcCer species in <i>S. apiospermum.</i> (C) HPTLC plate of monosaccharides from <i>S. apiospermum</i> CMH. 1. Galactose, glucose, mannose and rhamnose standards; 2. Glucose from CMH. The sugars were detected using the orcinol-sulfuric acid spray reagent.</p

Cytotoxic activity of <i>S. apiospermum</i> CMH.

<p>Cytotoxic assay of <i>S. apiospermum</i> CMH on L929 and RAW cells during 48 h of incubation. Cell viability was measured by adding a neutral-red solution and measuring the absorbance at 490 nm using a spectrophotometer. The presence of <i>S. apiospermum</i> CMH in the culture supernatant was analyzed using TLC.</p