resumo Neste trabalho avaliou-se a atividade antimicrobiana das nanopartículas de prata (AgNPs) incorporadas em membranas de acetato celulose (AC) e blendas de quitosana/poli-óxido de etileno (CTS/PEO) preparadas pelo método de eletrofiação. A formação das AgNPs previamente sintetizadas foi monitorada por UV-Vis e caracterizada por microscopia eletrônica de transmissão (MET). A distribuição de tamanho das AgNPs foi mensurada por espalhamento de luz dinâmico, com tamanho médio em torno de 20 nm. A presença das NPs na superfície das nanofibras eletrofiadas foi observada por microscopia eletrônica com emissão de campo (FEG) e confirmada por MET. A atividade antimicrobiana das membranas nanoestruturadas de AC e CTS/PEO foi avaliada pelo uso de bactérias Gram-positivas, tais como Staphylococcus aureus ATCC 29213 e Propionibacterium acnes ATCC 6919, e Gram-negativas, como Escherichia coli ATCC 25992 e Pseudomonas aeruginosa ATCC 17933. Os resultados microbiológicos mostraram a presença das AgNPs nas membranas de AC e CTS/PEO com significativa atividade antimicrobiana para Escherichia coli e Propionibacterium acnes, respectivamente.

abstract This study examines the antimicrobial activity of silver nanoparticles incorporated into nanostructured membranes made of cellulose acetate (CA) and blends of chitosan/poly-(ethylene oxide, CTS/PEO) and prepared by electrospinning. The formation of chemically synthesized Ag nanoparticles (AgNPs) was monitored by UV-visible spectroscopy (UV-Vis) and characterized by transmission electron microscopy (TEM). The size distribution of the AgNPs was measured by dynamic light scattering (DLS), with an average size of approximately 20 nm. The presence of AgNPs on the surface of electrospun nanofibers was observed by field emission electron microscopy (FEG) and confirmed by TEM. The antimicrobial activity of AgNPs incorporated into nanostructured membranes made of CA and CTS/PEO electrospun nanofibers was evaluated in the presence of both Gram-positive bacteria, such as Staphylococcus aureus ATCC 29213 and Propionibacterium acnes ATCC 6919, and Gram-negative bacteria, such as Escherichia coli ATCC 25992 and Pseudomonas aeruginosa ATCC 17933. Microbiological results showed that the presence of AgNPs in CA and CTS/PEO nanostructured membranes has significant antimicrobial activity for the Gram-positive bacteria Escherichia coli and Propionibacterium acnes.

As propriedades da prata são conhecidas há muitos anos. Recentemente, as nanopartículas de prata têm chamado a atenção por sua atividade antimicrobiana que oferece a possibilidade de uso com propósitos médicos e de higiene. Estas nanopartículas de prata em diferentes formulações, com diferentes formas e tamanhos, exibem atividades antimicrobianas diferentes. Entretanto, os mecanismos da atividade antimicrobiana de íons e de nanopartículas, assim como sua toxicidade em tecidos humanos não estão totalmente esclarecidos. Esta revisão avalia o uso potencial de nanopartículas de prata no controle de patogênicos com ênfase sobre sua ação contra bactérias patogênicas, sua toxicidade e possíveis mecanismos de ação.

The antimicrobial properties of silver have been known for thousands of years. Recently, silver nanoparticles have gained attention because of their antimicrobial activity which offers the possibility of their use for medical and hygiene purposes. Indeed, silver nanoparticles in different formulations and with different shapes and sizes exhibit variable antimicrobial activity. However, the mechanisms of antimicrobial activity of silver ions and silver nanoparticles, and their toxicity to human tissues are not fully characterized. This review evaluates the potential use of silver nanoparticles to control pathogens with emphasis on their action against pathogenic bacteria, their toxicity and possible mechanisms of action.

Linhagens de Escherichia coli patogênicas para aves (APEC) causam uma grande diversidade de doenças em aves e são responsáveis por grandes prejuízos na indústria aviária. Nos últimos anos, vários estudos foram realizados para melhor entender a patogênese de linhagens APEC e para desenvolver ferramentas que podem prevenir as perdas econômicas causadas por estas linhagens. Esta revisão discute os fatores de virulência descritos nestas linhagens e os avanços realizados para entender e identificar os determinantes de virulência presentes em APEC.

Avian pathogenic Escherichia coli (APEC) strains cause a great diversity of diseases in birds and are responsible for great economic losses in the avian industry. To date, several studies have been carried out to better understand the APEC pathogenesis for a possible development of tools which could prevent the economics losses caused by these strains. This review discusses the virulence factors described do date to be expressed by these strains and the advances made to understand and identify virulence determinants present in APEC.

One hundred and fifty-one methicillin-resistant z (MRSA) strains have been isolated from patients admitted in tertiary care hospitals in two metropolitan areas (Campinas City and Ribeirão Preto City) in the southeast region of Brazil and analyzed through PCR-based techniques [(PCR amplification of spa, coa, and housekeeping genes (arcC, aroE, gmk, pta, tpi, yqiL)] and further restriction fragment typing of coa and of housekeeping genes. The heterogeneity of spa gene was determined directly by agarose gel electrophoresis migration. The results obtained indicate the existence of three (A, B, C) main clusters. Since the strain distribution in these three clusters is much characteristic, it denotes the existence of three main clones. All strains isolated in Campinas were grouped in clusters A and B, while most of the strains isolated in Ribeirão Preto were grouped in cluster C. This distribution denotes the existence of different founder strains that undergo independent genetic variability. The strains considered representative of the Brazilian Epidemic Clone (BEC) were categorized as cluster A. These results indicate a possible higher variability among Brazilian MRSA strains than currently described and indicate that the techniques herein used can be used as an alternative to Pulsed Field Gel Electrophoresis (PFGE).

A presença de seqüências de DNA associadas à capacidade de captação de ferro (irp-2, fyuA, sitA, fepC, iucA), adesão (iha, lpfA O157/O141, lpfA O157/O154, efa, toxB) e de invasão (inv, ial) e a classificação dentro dos quatro grupos filogenéticos principais de Escherichia coli (Grupos A, B1, B2 e D) foram determinadas, através de PCR, em 30 amostras comensais de E. coli isoladas de frangos e de 49 linhagens APEC (24 isoladas de frangos com septicemia, 14 isoladas de frangos com síndrome da cabeça inchada e 11 isoladas de embriões de galinhas com onfalite). Nenhuma das linhagens apresentou os genes inv, ial, efa, e toxB. Os genes lpfA O157/O154, iucA, fepC e irp-2 foram encontrados em freqüências significativas entre as amostras patogênicas. O gene iucA foi associado com amostras causadoras de septicemia e de síndrome da cabeça inchada. Os genes fepC e irp-2 foram associados a amostras causadoras de síndrome da cabeça inchada. A análise filogenética demonstrou que linhagens comensais e causadoras de onfalite pertenceram principalmente ao Grupo filogenético A, não patogênico. Amostras causadoras de síndrome da cabeça inchada pertenceram, em sua maioria, ao Grupo patogênico D. Linhagens causadoras de septicemia pertenceram aos Grupos A e D. Estes dados sugerem que linhagens APEC causadoras de septicemia provavelmente têm uma origem ancestral múltipla: uma derivada de uma linhagem patogênica e outra de uma linhagem não patogênica que possivelmente evoluiu através da aquisição horizontal de genes de virulência. Amostras causadoras de síndrome da cabeça inchada possivelmente constituem um grupo clonal patogênico. Por outro lado, amostras causadoras de onfalite possivelmente constituem um grupo clonal não patogênico, que, possivelmente causam onfalite devido a uma infecção oportunista. A presença de genes de virulência também encontrados em E. coli de origem humana pode indicar a possível ocorrência de zoonoses causadas por APEC.

The presence of iron uptake (irp-2, fyuA, sitA, fepC, iucA), adhesion (iha, lpfA O157/O141, lpfA O157/O154, efa, toxB) and invasion (inv, ial-related DNA sequences and assignment to the four main Escherichia coli phylogenetic groups (A, B1, B2 e D) were determined in 30 commensal E. coli strains isolated from healthy chickens and in 49 APEC strains isolated from chickens presenting clinical signs of septicemia (n=24) swollen head syndrome (n=14) and omphalitis (n=11) by PCR. None of the strains presented DNA sequences related to the inv, ial, efa, and toxB genes. DNA sequences related to lpfA O157/O154, iucA, fepC, and irp-2 genes were significantly found among pathogenic strains, where iucA gene was associated with septicemia and swollen head syndrome and fepC and irp-2 genes were associated with swollen head syndrome strains. Phylogenetic typing showed that commensal and omphalitis strains belonged mainly to phylogenetic Group A and swollen head syndrome to phylogenetic Group D. Septicemic strains were assigned in phylogenetic Groups A and D. These data could suggest that clonal lineage of septicemic APEC strains have a multiple ancestor origin; one from a pathogenic bacteria ancestor and other from a non-pathogenic ancestor that evolved by the acquisition of virulence related sequences through horizontal gene transfer. Swollen head syndrome may constitute a pathogenic clonal group. By the other side, omphalitis strains probably constitute a non-pathogenic clonal group, and could cause omphalitis as an opportunistic infection. The sharing of virulence related sequences by human pathogenic E. coli and APEC strains could indicate that APEC strains could be a source of virulence genes to human strains and could represent a zoonotic risk.

A relação clonal entre linhagens de Escherichia coli de origem aviária e sua proximidade genética com E. coli patogênica para humanos, Salmonella enterica, Yersinia enterocolitica e Proteus mirabilis foi determinada através da utilização das seqüências conservadas 5' e 3' do gene fliC (responsável pela codificação da flagelina). Entre as 30 linhagens comensais de E. coli aviária e as 49 linhagens patogênicas de E. coli para aves (APEC), 24 linhagens comensais e 39 APEC apresentaram o gene fliC, que foi encontrado em tamanhos que variam de 670pb a 1900pb. Um dendrograma representando similaridade genética foi obtido a partir do seqüenciamento das regiões 5' e 3' conservadas do gene fliC das linhagens de E. coli de origem aviária, das seqüências dos antígenos H de E. coli de origem humana, de S. enterica, Y. enterocolitica e de P. mirabilis. A análise do dendrograma demonstrou que este apresenta dois grupos principais: um composto principalmente por isolados APEC e por antígenos H de E. coli de origem humana e outro formado por isolados comensais de E. coli aviária, S. enterica e por antígenos H de E. coli. No geral, o presente trabalho demonstrou que as regiões conservadas do gene fliC podem estar associadas à diferenciação clonal de linhagens de E. coli aviária, e que existe uma grande similaridade genética entre estas linhagens e antígenos H de E. coli humana. Estes dados podem adicionar evidências de que linhagens APEC podem apresentar riscos zoonóticos.

The clonal relationship among avian Escherichia coli strains and their genetic proximity with human pathogenic E. coli, Salmonela enterica, Yersinia enterocolitica and Proteus mirabilis, was determined by the DNA sequencing of the conserved 5' and 3'regions fliC gene (flagellin encoded gene). Among 30 commensal avian E. coli strains and 49 pathogenic avian E. coli strains (APEC), 24 commensal and 39 APEC strains harbored fliC gene with fragments size varying from 670bp to 1,900bp. The comparative analysis of these regions allowed the construction of a dendrogram of similarity possessing two main clusters: one compounded mainly by APEC strains and by H-antigens from human E. coli, and another one compounded by commensal avian E. coli strains, S. enterica, and by other H-antigens from human E. coli. Overall, this work demonstrated that fliC conserved regions may be associated with pathogenic clones of APEC strains, and also shows a great similarity among APEC and H-antigens of E. coli strains isolated from humans. These data, can add evidence that APEC strains can exhibit a zoonotic risk.

Forty-five Haemophilus influenzae strains isolated from patients were characterized based on biochemical characteristics. Their capsular types were determined by polymerase chain reaction (PCR); they were compared, using two molecular methods [ribotyping with a specific DNA probe amplified from the 16S rDNA region from H. influenzae and through restriction fragment length polymorphism (RLFP) of an amplified 16S DNA region]. The strains were better discriminated by the ribotyping technique that used the 16S probe and by the combination of both techniques. Biotypes I and IV were the most common, followed by biotypes VI, VIII and III. Biotypes II and VII were not found. Most of the capsular samples were nontypable (89%), with capsular types a and b found in 2 and 9% of the samples, respectively. We concluded that there is a very close genetic identity among pathogenic and non-pathogenic strains.

Helicobacter pylori is considered a significant agent in the development of various gastric diseases. However, the diseases caused by this bacterium are known as being multi-factorial, with the genotype, immune system and life habits of the host playing important roles in the establishment of the clinical outcome. Also, H. pylori exhibit a high degree of genetic variability, contributing to the complexity of the host-pathogen relationship. These observations, considered together with the widely-varying origins and social habits of the Brazilian population, lead us to speculate about the influence of these life habits on H. pylori infection and the clinical outcome. Therefore, in this study we evaluated the relationship between H. pylori infection and certain diseases in 172 patients treated at the Hospital das Clínicas of Ribeirão Preto (HCRP), Brazil, taking into account their different life habits, such as non-steroidal anti-inflammatory drugs and alcohol ingestion, and smoking habit. Our analysis indicated that H. pylori infection is not affected by any of the life habits evaluated but is associated with the development of peptic ulcers (gastric and duodenal ulcer) and inverse correlate with gastroesophageal reflux disease (GERD). No correlation was found between the infection with this bacterium and gastritis or intestinal metaplasia. However, gastritis and erosive gastritis were directly correlated with non-steroidal anti-inflammatory drugs (NSAID) ingestion. Moreover, ingestion of alcohol beverages exhibited a protective effect on gastritis development in men. Our data also indicated that to achieve reliable detection of this bacterium in biopsies, two or three detection methods should be used.

Restriction and Modification (R-M) systems are present in all Mycoplasma species sequenced so far. The presence of these genes poses barriers to gene transfer and could protect the cell against phage infections. The number and types of R-M genes between different Mycoplasma species are variable, which is characteristic of a polymorphism. The majority of the CDSs code for Type III R-M systems and particularly for methyltransferase enzymes, which suggests that functions other than the protection against the invasion of heterologous DNA may exist. A possible function of these enzymes could be the protection against the invasion of other but similar R-M systems. In Mycoplasma hyopneumoniae strain J, three of the putative methyltransferase genes were clustered in a region forming a genomic island. Many R-M CDSs were mapped in the vicinity of transposable elements suggesting an association between these genes and reinforcing the idea of R-M systems as mobile selfish DNA. Also, many R-M genes present repeats within their coding sequences, indicating that their expression is under the control of phase variation mechanisms. Altogether, these data suggest that R-M systems are a remarkable characteristic of Mycoplasma species and are probably involved in the adaptation of these bacteria to different environmental conditions.

Diversification of bacterial species and pathotypes is largely caused by lateral gene transfer (LGT) of diverse mobile DNA elements such as plasmids, phages, transposons and genomic islands. Thus, acquisition of new phenotypes by LGT is very important for bacterial evolution and relationship with hosts. This paper reports a 23 kb region containing fourteen CDSs with similarity to the previous described Integrative Conjugal Element of Mycoplasma fermentans (ICEF). This element, named ICEH, is present as one copy at distinct integration sites in the chromosome of 7448 and 232 pathogenic strains and is absent in the type strain J (non-pathogenic). Notable differences in the nucleotide composition of the insertion sites were detected, and could be correlated to a lack of specificity of the ICEH integrase. Although present in strains of the same organism, the ICEH elements are more divergent than the typical similarity between other chromosomal locus of Mycoplasma hyopneunomiae, suggesting an accelerated evolution of these constins or an ongoing process of degeneration, while maintaining conservation of the tra genes. An extrachromosomal form of this element had been detected in the 7448 strain, suggesting a possible involvement in its mobilization and transference of CDSs to new hosts.

A técnica de REP (Repetitive extragenic palindrome)-PCR foi utilizada para avaliar a variabilidade genética de 49 amostras de Escherichia coli patogênicas para aves (APEC), isoladas de aves de corte (frangos) em diferentes surtos de septicemia (n=24), síndrome da cabeça inchada (n=14) e onfalite (n=11). Trinta amostras comensais, isoladas de frangos sem sinais de doença, foram utilizadas como controle. A análise do perfil eletroforético obtido por reação de REP-PCR utilizando DNA purificado das amostras evidenciou a amplificação de 0 a 15 bandas de DNA com pesos moleculares variando entre 100 pb e 6.1 Kb. A análise deste padrão permitiu a construção de um dendrograma demonstrando o agrupamento das 79 amostras em 49 perfis distintos. Embora a técnica de REP-PCR tenha apresentado grande poder discriminatório, as amostras patogênicas e não patogênicas não foram discriminadas entre si assim como não foi observado o agrupamento de amostras causadoras do mesmo tipo de doença. Por outro lado, demonstramos recentemente que outras técnicas tais como ERIC-PCR e a análise de isoenzimas foram eficientes quando utilizadas para esta mesma finalidade. Concluindo, REP-PCR parece não ser uma técnica eficiente e universal para discriminar entre amostras APEC. Porém, a estrutura clonal populacional obtida com o uso de REP-PCR não deve ser desprezada, particularmente se considerarmos que os mecanismos de patogenicidade de APEC ainda não são completamente conhecidos.

In the present study the repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) technique was used to establish the clonal variability of 49 avian Escherichia coli (APEC) strains isolated from different outbreak cases of septicemia (n=24), swollen head syndrome (n=14) and omphalitis (n=11). Thirty commensal strains isolated from poultry with no signs of these illnesses were used as control strains. The purified DNA of these strains produced electrophoretic profiles ranging from 0 to 15 bands with molecular sizes varying from 100 bp to 6.1 kb, allowing the grouping of the 79 strains into a dendrogram containing 49 REP-types. Although REP-PCR showed good discriminating power it was not able to group the strains either into specific pathogenic classes or to differentiate between pathogenic and non-pathogenic strains. On the contrary, we recently demonstrated that other techniques such as ERIC-PCR and isoenzyme profiles are appropriate to discriminate between commensal and APEC strains and also to group these strains into specific pathogenic classes. In conclusion, REP-PCR seems to be a technique neither efficient nor universal for APEC strains discrimination. However, the population clonal structure obtained with the use of REP-PCR must not be ignored particularly if one takes into account that the APEC pathogenic mechanisms are not completely understood yet.

O objetivo deste trabalho foi estudar características biológicas tais como a expressão de fímbrias e adesinas, capacidade de absorção do corante Vermelho Congo, produção de hemolisina e aerobactina, adesão e invasão a células HeLa e adesão a células do epitélio urinário em amostras de Eschericia coli isoladas de pacientes com sinais clínicos de infecção do trato urinário (UTI). A presença dos genes responsáveis pela expressão de fímbrias (apa, afa e spa) e das Citotoxinas Necrotizantes CNF1, CNF2 foi avaliada por PCR. Esses dados foram comparados com a estrutura clonal das amostras obtidas por análises de isoenzimas (MLEE), Ribotipagem (RFLP) e ERIC-PCR. Com uma única exceção, os isolados apresentaram combinação de ao menos duas das características estudadas, fato que sugere a existência de Ilhas de Patogenicidade (PAIs). A maioria das amostras apresentaram um padrão difuso de aderência a células HeLa. Os resultados indicam que a capacidade de adesão a células epiteliais do sistema urinário poderia ser um teste mais específico e correlacionado à patogenicidade. Embora os estudos com microscopia óptica indicassem que quatro linhagens pudessem ser invasivas, dados de microscopia eletrônica não confirmaram tais achados. As técnicas de MLEE, Ribotipagem e ERIC-PCR separaram os isolados em diferentes grupos principais mas estes não foram correlacionados à patogenicidade.

The aim of the present study was to determine biological characteristics such as expression of fimbriae, Congo red binding, production of hemolysin and aerobactin, adhesion to HeLa and uroepithelial cells and invasion of HeLa cells by Escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (UTI). Also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (CNF1, CNF2) was assayed using specific primers in PCR. The data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP) of the rDNA (ribotyping) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). All isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (PAIs). Diffuse adherence type to HeLa cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. Although four strains seemed to be able to invade HeLa cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. MLEE, RFLP and ERIC-PCR were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits.