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 S��o 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 S��o 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.

OCORR��NCIA DE Escherichia coli O157: H7 EM PRODUTOS C��RNEOS E SENSIBILIDADE DOS M��TODOS DE DETEC����O

Foi verificada a ocorr��ncia de Escherichia coli O157:H7 em 340 amostras de produtos c��rneos e ambiente industrial, provenientes de frigor��ficos do Sul e Sudeste do Brasil, no per��odo de abril/98 a abril/99. A presen��a de E.coli O157:H7 n��o foi detectada em nenhuma das amostras analisadas e os resultados da avalia����o da sensibilidade dos m��todos de detec����o evidenciaram que tanto o m��todo cultural quanto o imunoensaio da Neogem foram capazes de detectar a presen��a de E.coli O157:H7 em cultura pura em concentra����es 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 IND��STRIA DE LATIC��NIOS, NO ESTADO DA PARA��BA (BRASIL)

Investigou-se a qualidade microbiol��gica do leite in natura e na linha de produ����o (leite rec��m-pasteurizado e leite ensacado), de uma usina de beneficiamento em Campina Grande-PB. Foi pesquisada a presen��a de Listeria spp. e sua diversidade de esp��cies, os n��veis 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 rec��m-pasteurizado e 15 de leite ensacado. Os resultados foram reunidos em dois grupos segundo o per��odo de monitoramento: antes e ap��s mudan��as no processo de higieniza����o da usina. Foi evidenciada elevada contamina����o nas amostras de leite cru nas duas ��pocas. Na primeira (mar��o-abril/1998), todas as amostras de leite beneficiado estiveram fora dos padr��es da legisla����o vigente para CT e CF; na segunda (maio-agosto/1998), houve acentuada redu����o dos n��veis destas bact��rias indicadoras, por��m as melhorias na higieniza����o n��o foram suficientes para solucionar este problema, visto que 11,1% das amostras rec��m-pasteurizadas estavam fora dos padr��es para CT e 33,3% para CF. Das amostras ensacadas, 22,2% estavam fora dos padr��es para CT e 44,4% para CF. Comparando-se os resultados de CT, CF, e E.coli nas amostras de leite rec��m-pasteurizado e n��o ensacado com as amostras de leite ensacado, foi verificado que as amostras ap��s serem pasteurizadas e ensacadas apresentaram valores de CT e CF levemente mais elevados, sugerindo contamina����o 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 rec��m-pasteurizadas e 5 ensacadas). Em rela����o �� diversidade de esp��cies, 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 defici��ncias higi��nico-sanit��rias no leite in natura e ao longo do processo de produ����o, resultando em porcentagens elevadas de amostras que ultrapassaram os valores padr��es de CT e CF al��m de apresentarem-se contaminadas por Listeria spp., com predomin��ncia de L. monocytogenes, sugerindo a exist��ncia de uma rela����o direta entre os altos ��ndices de coliformes e a presen��a de Listeria spp.

Ocorr��ncia de Escherichia coli 0157:H7 em vegetais e resist��ncia aos agentes de desinfec����o de verduras

Foi feito um estudo da ocorr��ncia de E.coli O157:H7 em vegetais que s��o normalmente consumidos crus no Brasil e uma avalia����o da sua resist��ncia aos sanitizantes dispon��veis no mercado para desinfec����o de verduras, equipamentos e utens��lios, incluindo compostos clorados e compostos de am��nio quatern��rio. Na avalia����o da ocorr��ncia em vegetais foram analisadas 869 amostras, n��o sendo detectada a presen��a do pat��geno. Os imunoensaios enzim��ticos (ELISA) utilizados nas an��lises (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, n��o confirmados como E.coli O157 nos testes bioqu��micos posteriores. Na avalia����o da resist��ncia aos sanitizantes pelo m��todo 960.9 da AOAC, observou-se que os tratamentos com 100 e 200ppm de hipoclorito de s��dio, dicloroisocianurato de s��dio e cloreto de benzalc��nio/30s se mostraram eficazes contra E.coli ATCC 11229 e E.coli O157:H7 ATCC 43890, promovendo mais de 5 redu����es decimais nas popula����es alvo.

Avalia����o do padr��o coliformes a 45��C e compara����o da efici��ncia das t��cnicas dos tubos m��ltiplos e Petrifilm EC na detec����o de coliformes totais e Escherichia coli em alimentos

Coliformes fecais s��o definidos como coliformes capazes de fermentar a lactose com produ����o de g��s em 48 h a 45��C. Escherichia coli, juntamente com algumas cepas de Enterobacter e Klebsiella, podem apresentar essas caracter��sticas. Entretanto, apenas a presen��a de Escherichia coli em alimentos indica contamina����o fecal por ser encontrada em grande quantidade no trato gastrointestinal do homem e animais de sangue quente, n��o sendo isolada normalmente em outros nichos. A denomina����o cl��ssica de coliformes fecais foi alterada para coliformes a 45��C, na Resolu����o n�� 12 da Ag��ncia Nacional de Vigil��ncia Sanit��ria. O objetivo deste trabalho foi avaliar a presen��a de E. coli entre os coliformes a 45��C e comparar a efici��ncia das t��cnicas dos tubos m��ltiplos e Petrifilm EC na detec����o de coliformes totais e E. coli em queijo Minas, ling��i��a frescal, hortali��as e fub��. Petrifilm EC mostrou-se mais sens��vel na detec����o de E. coli em rela����o ao m��todo de tubos m��ltiplos, 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 pr��tico, sendo um m��todo alternativo adequado para a enumera����o de coliformes totais e E. coli em alimentos.

Inibi����o e inativa����o de Escherichia coli por extratos de plantas com indicativo etnogr��fico medicinal ou condimentar

Determinou-se in vitro a Intensidade de Atividade de Inibi����o Bacteriana (IINIB) e a Intensidade de Atividade de Inativa����o Bacteriana (IINAB), atrav��s de Testes de Dilui����o e Suspens��o em Sistema de Tubos M��ltiplos, de diferentes extratos aquosos ou alco��licos/hidroalco��licos de 59 plantas com indicativo etnogr��fico medicinal ou condimentar acessadas na regi��o metropolitana de Porto Alegre/RS/BR, frente �� Escherichia sp. (ou E. coli ATCC n�� 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 etnogr��fica na prospec����o de fatores de prote����o antibacteriana em plantas, bem como a influ��ncia da inibi����o/inativa����o na preditividade do diagn��stico de E. coli.

Avalia����o da sensibilidade de cepas de Escherichia coli isoladas de mexilh��es (Perna perna linnaeus, 1758) �� antimicrobianos

Foram avaliadas 44 cepas de Escherichia coli isoladas de amostras c��rneas de mexilh��es capturados no munic��pio de Niter��i, Estado do Rio de Janeiro, quanto a sua sensibilidade a antimicrobianos. Vinte e quatro antimicrobianos foram testados e padr��es vari��veis 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 resist��ncia total a 4,16% dos antimicrobianos (Ca). A cepa n��mero 18 apresentou sensibilidade a 95,83% dos antimicrobianos, enquanto que a cepa n��mero 30 aduziu resist��ncia a 41,66% dos antimicrobianos. Frente aos resultados obtidos �� importante ponderarmos sobre o risco �� Sa��de P��blica associado ao h��bito de ingerir pescado cru ou insuficientemente cozido, especialmente bivalves filtradores contaminados por bact��rias com comprovada resist��ncia a diferentes antimicrobianos, alimentos potencialmente envolvidos em processos de reinfec����o 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 Cuiab��-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.