Human sepsis-associated Escherichia coli (SEPEC) is able to adhere to and invade kidney epithelial cells in culture

The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 x 10(7) CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.

Assessing the diversity of the virulence potential ofEscherichia coli isolated from bacteremia in São Paulo, Brazil

Most of the knowledge of the virulence determinants of extraintestinal pathogenicEscherichia coli (ExPEC) comes from studies with human strains causing urinary tract infections and neonatal meningitis and animal strains causing avian colibacillosis. In this research, we analyzed the phylogenetic background, the presence of 20 ExPEC virulence factors, and the intrinsic virulence potential of 74 E. coli strains isolated in São Paulo, Brazil, from 74 hospitalized patients (43 males and 31 females) with unknown-source bacteremia. Unlike other places in the world, the bacteremic strains originated equally from phylogroups B2 (35%) and D (30%). A great variability in the profiles of virulence factors was noted in this survey. Nevertheless, 61% of the strains were classified as ExPEC, meaning that they possessed intrinsic virulent potential. Accordingly, these strains presented high virulence factor scores (average of 8.7), and were positively associated with 12 of 17 virulence factors detected. On the contrary, the non-ExPEC strains, isolated from 39% of the patients, presented a generally low virulence capacity (medium virulence factor score of 3.1), and were positively associated with only the colicin cvaC gene. These results show the importance of discriminating E. coli isolates that possess characteristics of true pathogens from those that may be merely opportunistic in order to better understand the virulence mechanisms involved in extraintestinalE. coli infections. Such knowledge is essential for epidemiological purposes as well as for development of control measures aimed to minimize the incidence of these life-threatening and costly infections.

From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones

The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia.

Low-level red laser therapy alters effects of ultraviolet C radiation on Escherichia coli cells

Low-level lasers are used at low power densities and doses according to clinical protocols supplied with laser devices or based on professional practice. Although use of these lasers is increasing in many countries, the molecular mechanisms involved in effects of low-level lasers, mainly on DNA, are controversial. In this study, we evaluated the effects of low-level red lasers on survival, filamentation, and morphology of Escherichia colicells that were exposed to ultraviolet C (UVC) radiation. Exponential and stationary wild-type and uvrA-deficientE. coli cells were exposed to a low-level red laser and in sequence to UVC radiation. Bacterial survival was evaluated to determine the laser protection factor (ratio between the number of viable cells after exposure to the red laser and UVC and the number of viable cells after exposure to UVC). Bacterial filaments were counted to obtain the percentage of filamentation. Area-perimeter ratios were calculated for evaluation of cellular morphology. Experiments were carried out in duplicate and the results are reported as the means of three independent assays. Pre-exposure to a red laser protected wild-type and uvrA-deficient E. coli cells against the lethal effect of UVC radiation, and increased the percentage of filamentation and the area-perimeter ratio, depending on UVC fluence and physiological conditions in the cells. Therapeutic, low-level red laser radiation can induce DNA lesions at a sub-lethal level. Consequences to cells and tissues should be considered when clinical protocols based on this laser are carried out.

Low-intensity red and infrared laser effects at high fluences on Escherichia coli cultures

Semiconductor laser devices are readily available and practical radiation sources providing wavelength tenability and high monochromaticity. Low-intensity red and near-infrared lasers are considered safe for use in clinical applications. However, adverse effects can occur via free radical generation, and the biological effects of these lasers from unusually high fluences or high doses have not yet been evaluated. Here, we evaluated the survival, filamentation induction and morphology of Escherichia coli cells deficient in repair of oxidative DNA lesions when exposed to low-intensity red and infrared lasers at unusually high fluences. Cultures of wild-type (AB1157), endonuclease III-deficient (JW1625-1), and endonuclease IV-deficient (JW2146-1) E. coli, in exponential and stationary growth phases, were exposed to red and infrared lasers (0, 250, 500, and 1000 J/cm2) to evaluate their survival rates, filamentation phenotype induction and cell morphologies. The results showed that low-intensity red and infrared lasers at high fluences are lethal, induce a filamentation phenotype, and alter the morphology of the E. coli cells. Low-intensity red and infrared lasers have potential to induce adverse effects on cells, whether used at unusually high fluences, or at high doses. Hence, there is a need to reinforce the importance of accurate dosimetry in therapeutic protocols.

Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α

A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α.

OCORRÊNCIA DE Escherichia coli O157: H7 EM PRODUTOS CÁRNEOS E SENSIBILIDADE DOS MÉTODOS DE DETECÇÃO

Foi verificada a ocorrência de Escherichia coli O157:H7 em 340 amostras de produtos cárneos e ambiente industrial, provenientes de frigoríficos do Sul e Sudeste do Brasil, no período de abril/98 a abril/99. A presença de E.coli O157:H7 não foi detectada em nenhuma das amostras analisadas e os resultados da avaliação da sensibilidade dos métodos de detecção evidenciaram que tanto o método cultural quanto o imunoensaio da Neogem foram capazes de detectar a presença de E.coli O157:H7 em cultura pura em concentrações iniciais de menos de 0,5Log UFC/mL do caldo de enriquecimento.

LISTERIA SPP., COLIFORMES TOTAIS E FECAIS E E.COLI NO LEITE CRU E PASTEURIZADO DE UMA INDÚSTRIA DE LATICÍNIOS, NO ESTADO DA PARAÍBA (BRASIL)

Investigou-se a qualidade microbiológica do leite in natura e na linha de produção (leite recém-pasteurizado e leite ensacado), de uma usina de beneficiamento em Campina Grande-PB. Foi pesquisada a presença de Listeria spp. e sua diversidade de espécies, os níveis de coliformes totais (CT), coliformes fecais (CF) e Escherichia coli. Analisou-se um total de 75 amostras de leite, sendo 45 de leite cru, 15 de leite recém-pasteurizado e 15 de leite ensacado. Os resultados foram reunidos em dois grupos segundo o período de monitoramento: antes e após mudanças no processo de higienização da usina. Foi evidenciada elevada contaminação nas amostras de leite cru nas duas épocas. Na primeira (março-abril/1998), todas as amostras de leite beneficiado estiveram fora dos padrões da legislação vigente para CT e CF; na segunda (maio-agosto/1998), houve acentuada redução dos níveis destas bactérias indicadoras, porém as melhorias na higienização não foram suficientes para solucionar este problema, visto que 11,1% das amostras recém-pasteurizadas estavam fora dos padrões para CT e 33,3% para CF. Das amostras ensacadas, 22,2% estavam fora dos padrões para CT e 44,4% para CF. Comparando-se os resultados de CT, CF, e E.coli nas amostras de leite recém-pasteurizado e não ensacado com as amostras de leite ensacado, foi verificado que as amostras após serem pasteurizadas e ensacadas apresentaram valores de CT e CF levemente mais elevados, sugerindo contaminação durante o processo de ensacamento ou falhas na armazenagem. Observou-se que 33 (73,3%) das amostras de leite cru e 9 (30%) das de leite pasteurizado estavam contaminadas com Listeria spp., sendo identificadas L. monocytogenes em 17 (51,5%) amostras de leite cru e em 9 (100%) de leite beneficiado (4 recém-pasteurizadas e 5 ensacadas). Em relação à diversidade de espécies, nas amostras de leite cru foram encontradas: L. monocytogenes (66,6%), L. innocua (25,3%), L. ivanovii (3,9%), L. welshimeri (2,5%) e L. grayi (1,5%). Nas amostras de leite pasteurizado isolaram-se: L. monocyogenes e L. innocua. O conjunto dos resultados evidenciou deficiências higiênico-sanitárias no leite in natura e ao longo do processo de produção, resultando em porcentagens elevadas de amostras que ultrapassaram os valores padrões de CT e CF além de apresentarem-se contaminadas por Listeria spp., com predominância de L. monocytogenes, sugerindo a existência de uma relação direta entre os altos índices de coliformes e a presença de Listeria spp.

Ocorrência de Escherichia coli 0157:H7 em vegetais e resistência aos agentes de desinfecção de verduras

Foi feito um estudo da ocorrência de E.coli O157:H7 em vegetais que são normalmente consumidos crus no Brasil e uma avaliação da sua resistência aos sanitizantes disponíveis no mercado para desinfecção de verduras, equipamentos e utensílios, incluindo compostos clorados e compostos de amônio quaternário. Na avaliação da ocorrência em vegetais foram analisadas 869 amostras, não sendo detectada a presença do patógeno. Os imunoensaios enzimáticos (ELISA) utilizados nas análises (Reveal E.coli O157 Neogem e EHEC Test Kit 3M Company) apresentaram uma taxa de falsos resultados presuntivos de 13,6 e 11,8%, respectivamente, não confirmados como E.coli O157 nos testes bioquímicos posteriores. Na avaliação da resistência aos sanitizantes pelo método 960.9 da AOAC, observou-se que os tratamentos com 100 e 200ppm de hipoclorito de sódio, dicloroisocianurato de sódio e cloreto de benzalcônio/30s se mostraram eficazes contra E.coli ATCC 11229 e E.coli O157:H7 ATCC 43890, promovendo mais de 5 reduções decimais nas populações alvo.

Avaliação do padrão coliformes a 45ºC e comparação da eficiência das técnicas dos tubos múltiplos e Petrifilm EC na detecção de coliformes totais e Escherichia coli em alimentos

Coliformes fecais são definidos como coliformes capazes de fermentar a lactose com produção de gás em 48 h a 45ºC. Escherichia coli, juntamente com algumas cepas de Enterobacter e Klebsiella, podem apresentar essas características. Entretanto, apenas a presença de Escherichia coli em alimentos indica contaminação fecal por ser encontrada em grande quantidade no trato gastrointestinal do homem e animais de sangue quente, não sendo isolada normalmente em outros nichos. A denominação clássica de coliformes fecais foi alterada para coliformes a 45ºC, na Resolução nº 12 da Agência Nacional de Vigilância Sanitária. O objetivo deste trabalho foi avaliar a presença de E. coli entre os coliformes a 45ºC e comparar a eficiência das técnicas dos tubos múltiplos e Petrifilm EC na detecção de coliformes totais e E. coli em queijo Minas, lingüiça frescal, hortaliças e fubá. Petrifilm EC mostrou-se mais sensível na detecção de E. coli em relação ao método de tubos múltiplos, o qual apresentou resultados falso-negativos ou contagens subestimadas de E. coli, principalmente para amostras de alimentos de origem animal. Petrifilm EC foi o mais eficiente e prático, sendo um método alternativo adequado para a enumeração de coliformes totais e E. coli em alimentos.

Inibição e inativação de Escherichia coli por extratos de plantas com indicativo etnográfico medicinal ou condimentar

Determinou-se in vitro a Intensidade de Atividade de Inibição Bacteriana (IINIB) e a Intensidade de Atividade de Inativação Bacteriana (IINAB), através de Testes de Diluição e Suspensão em Sistema de Tubos Múltiplos, de diferentes extratos aquosos ou alcoólicos/hidroalcoólicos de 59 plantas com indicativo etnográfico medicinal ou condimentar acessadas na região metropolitana de Porto Alegre/RS/BR, frente à Escherichia sp. (ou E. coli ATCC nº 11229 ou E. coli p.16 CPVDF - SAA/RS), em doses-desafio < 10(8) UFC.mL-1. Trinta plantas apresentaram alguma atividade seletiva antiescherichia coli, enquanto as restantes 29 apresentaram nenhuma atividade. Discute-se a validade da ferramenta etnográfica na prospecção de fatores de proteção antibacteriana em plantas, bem como a influência da inibição/inativação na preditividade do diagnóstico de E. coli.

Avaliação da sensibilidade de cepas de Escherichia coli isoladas de mexilhões (Perna perna linnaeus, 1758) à antimicrobianos

Foram avaliadas 44 cepas de Escherichia coli isoladas de amostras cárneas de mexilhões capturados no município de Niterói, Estado do Rio de Janeiro, quanto a sua sensibilidade a antimicrobianos. Vinte e quatro antimicrobianos foram testados e padrões variáveis de comportamento frente aos mesmos foram observados. Todas as cepas avaliadas apresentaram sensibilidade total a apenas 41,66% dos antimicrobianos (R, Ac, C, To, Fx, Cz, Ct, Nt, Cp, Ge) e resistência total a 4,16% dos antimicrobianos (Ca). A cepa número 18 apresentou sensibilidade a 95,83% dos antimicrobianos, enquanto que a cepa número 30 aduziu resistência a 41,66% dos antimicrobianos. Frente aos resultados obtidos é importante ponderarmos sobre o risco à Saúde Pública associado ao hábito de ingerir pescado cru ou insuficientemente cozido, especialmente bivalves filtradores contaminados por bactérias com comprovada resistência a diferentes antimicrobianos, alimentos potencialmente envolvidos em processos de reinfecção do homem, no qual desencadeiam quadros de gastroenterite.

Hygienic-sanitary quality of vegetables and evaluation of treatments for the elimination of indigenous E. coli and E. coli O157:H7 from the surface of leaves of lettuce (Lactuca sativa L.)

The purpose of this study is to evaluate the hygienic-sanitary quality of vegetables and irrigation water and assess the effectiveness of lemon juice and vinegar in reducing E. coli strains inoculated on lettuce. One hundred and forty samples of vegetables and 45 samples of irrigation water were investigated for thermotolerant coliforms and Salmonella spp. In order to verify the effectiveness of natural household sanitizers in reducing E. coli in inoculated lettuce, four treatment solutions were tested: fresh lemon juice, alcohol vinegar, lemon juice-vinegar mixture, and lemon juice-vinegar-water mixture. The microbiological analysis revealed high rates of contamination by thermotolerant coliforms and identified the presence of E. coli in 32% of the tested vegetable samples and 56% of the water samples. While no significant statistical difference (p < 0, 05) was identified in the tested solutions, the treatment with a combination of lemon juice and vinegar resulted in the highest Decimal Reductions (DR) of E. coli O157: H7 while the treatment with vinegar alone was the most effective against the indigenous E. coli strain

Analysis of virulence genes in Escherichia coli isolated from grated cheese

This research aimed to verify the presence of virulence genes in strains of Escherichia coli isolated from grated cheese sold in farmers' markets of Cuiabá-MT, Brazil. Forty samples of this food were submitted for microbiological analysis and 22 (55%) tested positive for E. coli. Next, 64 strains of E. coli were isolated from the positive samples and screened by the polymerase chain reaction (PCR) for the presence of the genes encoding the following virulence factors: stx1 and stx2 (verotoxin types 1 and 2), eae (intimin), lt1 (heat-labile toxin type 1), st1 (heat-stable toxin type 1), cnf1 and cnf2 (cytotoxic necrozing factor types 1 and 2), and cdtB (cytolethal distending toxin). All the isolates were negative for the genes stx1, stx2, eae, lt1, st1, cnf1, and cdtB, and five strains (7.81%) were positive for cnf2. A low prevalence of E. coli positive for virulence factors associated with the pathogenesis of diarrhoea was observed in this study. However, the presence of CNF-2 producing strains and the possibility of occurrence and scattering of other virulence factors that were not surveyed in the work indicate the risk related to the consumption of grated cheese from farmers' markets.

Molecular characterization and evaluation of antimicrobial susceptibility of enteropathogenic E. coli (EPEC) isolated from minas soft cheese

Foodborne disease caused by microorganisms is a problem of public health. Minas soft cheese is a national product manufactured using simple technology; it has high level of acceptance in the country making its production an important economic activity. Many microorganisms may be present in foods including the bacterium Escherichia coli (E. coli). Overall, E. coli is a harmless commensal bacterium; however, some strains may have a pathogenic potential. Several outbreaks of foodborne diseases associated with consumption of contaminated cheese have been reported, and the presence of pathogenic strains of E. coli has increased. The objective of this study was to isolate, evaluate the antimicrobial susceptibility, and characterize, by Multiplex PCR, the pathogenic E. coli strains isolated from Minas cheese commercialized in Rio de Janeiro. Thirty samples were analyzed and five strains of E. coli (EPEC) were identified. The assessment of antimicrobial susceptibility revealed 40% of the isolates resistant to ampicillin and 40% with intermediate resistance to ampicillin-sulbactam combination. These findings are a warning signal to health authorities since Minas cheese is a ready to eat food product, and therefore should not pose health risks to the population.