Caracterização genotípica dos fatores de virulência em amostras de Escherichia coli isoladas de pacientes com cistite

Adhesins (P-fimbriae, S-fimbriae, type 1 fimbriae and afimbrial adhesin), toxins (α-hemolysin and cytotoxic necrotizing factor type 1), iron acquisition systems (aerobactin) and host defense avoidance mechanisms (capsule or lipopolysaccharide) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. In this work, 162 Uropathogenic Escherichia coli (UPEC) strains from patients with cystitis were genotypically characterized by polymerase chain reaction (PCR) assay. We developed three multiplex PCR assays for virulence-related genes papC, papE/F, papG alleles, fimH, sfa/foc, afaE, hly, cnf-1, usp, cdtB, iucD, and kpsMTII, all of them previously identified in UPEC strains. The PCR assay results identified 158 fimH (97.5%), 86 kpsMTII (53.1%), 53 papC/papEF/papG (32.7%), 45 sfa (27.8%), 42 iucD (25.9%), 41 hly (25.3%), 36 usp (22.2%), 30 cnf-1(18.5%) and 10 afa (6.2%) strains. No strain was positive for cdtB. In this work, we also demonstrated that adhesins may be multiple within a single strain and that several virulence genes can occur combined in association.Adesinas (Fímbria P, fímbria S, fímbria do tipo 1 e a adesina afimbrial), toxinas (α-hemolisina e o fator necrosante citotóxico do tipo 1), sistemas de captação de ferro (aerobactina), e mecanismos de defesa do hospedeiro (cápsula ou lipopolissacarídeo) são prevalentes em amostras de Escherichia coli associadas a infecções do trato urinário. O objetivo deste trabalho foi caracterizar genotipicamente 162 amostras de Escherichia coli uropatogênica (UPEC) de pacientes com cistite através do ensaio da reação em cadeia da polimerase. Foram realizados três ensaios de PCR multiplex para os seguintes fatores de virulência: papC, papE/F, alelos de papG, fimH, sfa/foc, afaE, hly, cnf-1, usp, cdtB, iucD, e kpsMTII. Os resultados da PCR identificaram, 158 amostras fimH (97,5%), 86 amostras kpsMTII (53,1%), 53 amostras papC/papEF/papG (32,7%), 45 amostras sfa (27,8%), 42 amostras iucD (25,9%), 41 amostras hly (25,3%), 36 amostras usp (22,2%), 30 amostras cnf-1 (18,5%) e 10 amostras afa (6,2%). Nenhuma amostra foi positiva para o gene cdtB. Neste trabalho, demonstramos que podemos encontrar múltiplas adesinas em uma única amostra e que diferentes genes de fatores de virulência podem ser encontrados em associação

Identification of putative new Escherichia coli flagellar antigens from human origin using serology, PCR-RFlP and DNA sequencing methods

Escherichia coli has been isolated frequently, showing flagellar antigens that are not recognized by any of the 53 antisera, provided by the most important reference center of E. coli, The International Escherichia and Klebsiella Center (WHO) of the Statens Serum Institute, Copenhagen, Denmark. The objective of this study was to characterize flagellar antigens of E. coli that express non-typeable H antigens. The methods used were serology, PCR-RFLP and DNA sequencing. This characterization was performed by gene amplification of the fliC (flagellin protein) by polymerase chain reaction in all 53 standards E.coli strains for the H antigens and 20 E. coli strains for which the H antigen was untypeable. The amplicons were digested by restriction enzymes, and different restriction enzyme profiles were observed. Anti-sera were produced in rabbits, for the non-typeable strains, and agglutination tests were carried out. In conclusion,the results showed that although non-typeable and typable H antigens strains had similar flagellar antigens, the two types of strains were distinct in terms of nucleotide sequence, and did not phenotypically react with the standard antiserum, as expected. Thirteen strains had been characterized as likely putative new H antigen using PCR-RFLP techniques, DNA sequencing and/or serology.14415

Pesticides: A Perspective of Scientific Production in West of Bahia

The use of pesticides has intensified with agricultural modernization process, triggered by the Green Revolution, which brought about changes in the production system, resulting in high social costs, environmental and public health. Brazil is the world's largest consumer of pesticides and assesses that this market will grow even more in the near future. Given the risk that pesticides pose, it was found that there is an urgent need to intensify the studies, as are scarce research in the Northeast and especially in western Bahia. This research aimed to evaluate the scientific literature on pesticides in West of Bahia. Therefore, publications consultation was made in the database Portal.periodicos.Capes with the keywords "West of Bahia" and "Pesticides". The results show that the number of worldwide publications has been growing over the years, representing 98% of the total, while Brazil has only 2% in West of Bahia was not found publication. There are many challenges in front of the dimension that the theme "pesticide" offers. Thus, priorities and strategies for research should be defined, seeking to boost scientific productivity in an attempt to make changes to help chart a way to sustainable development. DOI: http://dx.doi.org/10.17807/orbital.v9i2.90

Evaluation of an oil emulsified vaccine against bovine colibacillosis using semi-purified K99-F41 adhesins

The K99 and F41 fimbrial antigens were extracted from Escherichia coli B41 strain by heating, and semi-purified by precipitation with ammonium sulfate and treatment with sodium desoxicolate (DOC). The semi-purified antigens K99 and F4I were used to prepare an oil emulsified vaccine against bovine colibacillosis. The vaccine was made up containing in each dose 1,500, 750 or 380 HU (Haemagglutinating Units). The test of the vaccine efficiency was measured by gel immunodiffusion and ELISA test. Challenge of calves from vaccinated and control cows was carried out with the virulent Escherichia coli B4I. Our findings suggested that 750 HU vaccine, induces better production of anti-K99-F4l antibodies in vaccinated cows, and the transference of these antibodies to calves through the colostrum, gave full protection to challenge. Furthermore no adverse effects were observed in any of the vaccinated cows.Os antígenos K99-F41 foram extraídos da amostra de Escherichia coli B41 por aquecimento e semi-purificados pela precipitação com sulfato de amônio e tratamento com desoxicolato de sódio (DOC). Os antígenos semi-purificados foram utilizados na produção de uma vacina oleosa contra a colibacilose bovina. Foram preparadas vacinas contendo em cada dose 1.500 HU (Unidades Hemaglutinantes), 750 HU e 380 HU. A eficiência da vacina foi avaliada através do ensaio de imunodifusão dupla, ensaio imunoenzimático (ELISA) e por um desafio, em que a amostra de Escherichia coli virulenta foi inoculada nos bezerros nascidos de vacas vacinadas e não vacinadas. Observamos que a vacina contendo 750 HU foi a que melhor induziu a produção de anticorpos nas vacas vacinadas, e que estes mostraram-se protetores, uma vez que os bezerros nascidos de vacas vacinadas e que mamaram o colostro, nada sofreram no desafio. Não se verificou nenhum efeito colateral nas vacas vacinadas

Long-Term Habitat Fragmentation Is Associated With Reduced MHC IIB Diversity and Increased Infections in Amphibian Hosts

Habitat fragmentation and wildlife disease are two widespread drivers of biodiversity loss, yet few empirical studies have explored their interactions. In this study, we utilized a naturally fragmented island system to examine the impacts of fragmentation on genetic diversity and amphibian infection dynamics. We determined the impacts of fragmentation on genetic diversity at the immunity locus MHC IIB, a hypothesized predictor of disease susceptibility. Contrary to the expectation that MHC diversity would remain high due to balancing selection, island populations lost genetic diversity at this locus while simultaneously experiencing positive selection at MHC IIB. We then used Next-Generation Sequencing to identify a variety of potential eukaryotic parasites from amphibian skin swabs. Island populations exhibited higher potential parasite richness (proportion of eukaryotic microbe operational taxonomic units or OTUs from parasitic taxa) relative to mainland populations. MHC homozygotes hosted a lower diversity of potential parasites, and population-level MHC diversity was negatively associated with parasite richness. Our results show that genetic erosion can occur at the MHC IIB locus following fragmentation, which may contribute to increased susceptibility to parasites

Prevalence of Avian Pathogenic Escherichia coli (APEC) Clone Harboring sfa Gene in Brazil

Escherichia coli sfa+ strains isolated from poultry were serotyped and characterized by polymerase chain reaction (PCR) and amplified fragment length polymorphism (AFLP). Isolates collected from 12 Brazilian poultry farms mostly belonged to serogroup O6, followed by serogroups O2, O8, O21, O46, O78, O88, O106, O111, and O143. Virulence genes associated were: iuc 90%, fim 86% neuS 60%, hly 34%, tsh 28%, crl/csg 26%, iss 26%, pap 18%, and 14% cnf. Strains from the same farm presented more than one genotypic pattern belonging to different profiles in AFLP. AFLP showed a clonal relation between Escherichia coli sfa+ serogroup O6. The virulence genes found in these strains reveal some similarity with extraintestinal E. coli (ExPEC), thus alerting for potential zoonotic risk

Sorogrupos e genes de virulência em Escherichia coli isoladas de psitacídeos

Escherichia coli isolates from 24 sick psittacine birds were serogrouped and investigated for the presence of genes encoding the following virulence factors: attaching and effacing (eae), enteropathogenic E. coli EAF plasmid (EAF), pili associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin (afa), capsule K1 (neu), curli (crl, csgA), temperature-sensitive hemagglutinin (tsh), enteroaggregative heat-stable enterotoxin-1 (astA), heat-stable enterotoxin -1 heat labile (LT) and heat stable (STa and STb) enterotoxins, Shiga-like toxins (stx1 and stx2), cytotoxic necrotizing factor 1 (cnf1), haemolysin (hly), aerobactin production (iuc) and serum resistance (iss). The results showed that the isolates belonged to 12 serogroups: O7; O15; O21; O23; O54; O64; O76; O84; O88; O128; O152 and O166. The virulence genes found were: crl in all isolates, pap in 10 isolates, iss in seven isolates, csgA in five isolates, iuc and tsh in three isolates and eae in two isolates. The combination of virulence genes revealed 11 different genotypic patterns. All strains were negative for genes encoding for EAF, EAEC, K1, sfa, afa, hly, cnf, LT, STa, STb, stx1 and stx2. Our findings showed that some E. coli isolated from psittacine birds present the same virulence factors as avian pathogenic E. coli (APEC), uropathogenic E. coli (UPEC) and Enteropathogenic E. coli (EPEC) pathotypes.Amostras de Escherichia coli isoladas de 24 psitacídeos doentes foram sorogrupadas e investigadas para a presença de genes que codificam os seguintes fatores de virulência: attaching e effacing (eae), plasmídeo EAF (EAF), pili associado à pielonefrite (pap), fímbria S (sfa), adesina afimbrial (afa), cápsula K1 (neu), curli (crl, csgA), hemaglutinina termosensível (tsh), enterotoxina termo-estável 1 de E. coli enteroagregativa (astA), toxina termolábil (LT) e toxina termoestável (STa e STb), Shiga-like toxinas (stx1 e stx2), fator citotóxico necrotizante 1 (cnf1), hemolisina (hly), produção de aerobactina (iuc) e resistência sérica (iss). Os resultados mostraram que os isolados pertenciam a 12 sorogrupos: O7; O15; O21; O23; O54; O64; O76; O84; O88; O128; O152 e O166. Os genes de virulência encontrados foram: crl em todos os isolados, pap em 10 isolados, iss em sete isolados, csgA em cinco isolados, iuc e tsh em três isolados e eae em dois isolados. A combinação dos genes de virulência revelou 11 perfis genotípicos distintos. Todas as amostras foram negativas para os genes que codificam EAF, EAEC, K1, sfa, afa, hly, cnf, LT, STa, STb, stx1 e stx2. Estes resultados demonstraram que algumas amostras de E. coli isoladas de psitacídeos apresentam os mesmos fatores de virulência presentes nos patotipos de E. coli patogênicas para aves (APEC), uropatogênicas (UPEC) e E. coli enteropatogênicas (EPEC).Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Faculdades Metropolitanas Unidas Laboratório de Epidemiologia e Doenças InfecciosasUniabc Faculdade de Medicina VeterináriaUniversidade de São Paulo Faculdade de Medicina Veterinária e ZootecniaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de MedicinaUniversidade Estadual de Campinas Instituto de BiologiaUniversidade de Santiago de Compostela Facultad de Veterinaria Departamento de Microbiologia e ParasitologíaUNIFESP, EPMSciEL

Genes de enterotoxinas, multirresistência a antimicrobianos e caracterização molecular de espécies de Staphylococcus spp. isoladas de leite bovino orgânico

The multidrug resistant and the emergence of methicillin-resistant staphylococci isolated from animals, food, and humans are public health concern. These microorganisms produce different toxins related to food poisoning in humans. This study aimed to characterize Staphylococcus spp. isolated from two organic milk farms in Brazil. A total of 259 milk samples were collected, from which 58 (22.4%) Staphylococcus spp. were isolated. The highest sensibility to ceftiofur and sulfamethoxazole/trimethoprim was observed in 96.6% of Staphylococcus spp., and whereas 89% were resistant to penicillin G. The mecA gene was detected in 13.8% of the isolates. SEA and SEC were the most common enterotoxins detected. PFGE revealed genetic heterogeneity from S. intermedius and S. warneri analyzed, while S. aureus presented similar profiles among isolates from the two studied herds. To the best of our knowledge, the current study describes for the first time presence of enterotoxins, mecA gene, and genetic diversity of staphylococci isolated from organic dairy farms in Brazil.A emergência de estafilococos multirresistentes e resistentes à meticilina, isolados de animais, alimentos e humanos é uma preocupação em saúde pública. Esses micro-organismos produzem diferentes toxinas relacionadas à intoxicação alimentar em humanos. Este estudo caracterizou Staphylococcus spp. isolados em duas fazendas orgânicas no Brasil. Foram coletadas 259 amostras de leite em duas propriedades leiteiras orgânicas, nas quais 58 (22,4%) estirpes de Staphylococcus spp. foram isoladas. A maior sensibilidade dos isolados foi observada para ceftiofur e sulfametoxazol/trimetoprim em 96,6%. Em contraste, acima de 89% de resistência dos estafilicocos foi encontrada para penicilina G. O gene mecA foi identificado em 13,8% dos isolados. SEA e SEC foram as enterotoxinas mais comumente detectadas. PFGE revelou heterogeneidade genética entre S. intermedius e S. warneri, enquanto S. aureus demonstraram perfis semelhantes entre isolados dos dois rebanhos estudados. Relata-se pela primeira vez no Brasil a detecção de enterotoxinas, o gene mecA e diversidade genética em estafilococos isolados de vacas em produção orgânica