Navegando por Palavras-chave "PFGE"

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    Características fenotípicas e genotípicas de amostras de Escherichia coli 0157 produtoras de toxina Shiga
    (Universidade Federal de São Paulo (UNIFESP), 2006-01-01) Bastos, Flavia Correa [UNIFESP]; Guth, Beatriz Ernestina Cabilio [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Shiga toxin-producing O157:H7 Escherichia coli has been identified as an important cause of hemorrhagic colites and Hemolytic Uremic Syndrome in many countries. Some studies conducted in Brazil have shown the presence of this serotype in human infections and in the animal reservoir. In the present study, a total of 38 E. coli O157 strains isolated in Brazil, Argentina, Chile, Colombia, United States and Uruguay from different sources was studied in regard to several phenotypic and genotypic characteristics. The detection of the stx genotype, the presence of other toxins (ehx and cdtV) and putative adhesin gene sequences (efa1, iha, lpfO113, lpfO157 saa and toxB) were identified by PCR. The stx variants were determined by RFLP-PCR. Several biochemical characteristics and the susceptibility to antimicrobial agents were identified by standard methods. Most (97.3%) of the strains carried stx2 alone or in association with stx1. Many combinations of stx genes occurred among strains of different sources. stx2stx2c was more frequently found among human strains isolated in Brazil and Argentina, while stx2c was more frequently found among animal strains. In the other hand, stx1 alone or in combination with stx2 was identified in 18.4% of strains isolated from animal and food. All strains were positive for eae-γ , efa1, ehx, iha, lpfO157 and toxB genes independent of their source and country, and presented similar biochemical behaviour, except for three strains that displayed urease activity, and one strain that did not decarboxylate lysine. All the strains carried the H7 flagellar antigen, thus belonging to serotype O157:H7. Different biotypes were identified among the strains, but 61% of them belong o only four biotypes. Fermentation of sorbitol was only observed in four of the 38 strains analysed. Most of the strains was sensitive to the 10 antimicrobial agents tested, but resistance to at least one antimicrobial agent was more commonly observed among bovine strains. A diversity of PFGE patterns was observed among the O157:H7 STEC strains by macrorestriction analysis of genomic DNA with XbaI. However, a high degree of similarity and many closely related subgroups (more than 80% of similarity) was identified among strains isolated from different origins and countries. Therefore, O157:H7 STEC strains isolated from human infections, animal reservoir and from food in Brazil and in different countries showed very similar profiles related to the phenotypic and genotypic characteristics analysed in the present study, and although some particular differences were detected, the presence of some clones was also identified among the strains.
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    Escherichia coli produtora de toxina Shiga ( STEC): marcadores de Virulência e análise clonal
    (Universidade Federal de São Paulo (UNIFESP), 2006-12-31) Vaz, Tânia Mara Ibelli [UNIFESP]; Guth, Beatriz Ernestina Cabilio [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Forty eigth Shiga toxin-producing Escherichia coli (STEC) strains, the majority from human origin, and thirty non-STEC strains carrying eae and belonging to the same serogroups of STEC strains, and isolated between 1976 and 2003 were studied. Phenotypical traits, virulence characteristics and genetic diversity were evaluated. The majority of STEC strains was isolated in two distinct periods: some strains were recovered from a retrospective study (1976-99), and other strains from a prospective study (2000-03). STEC and non-STEC strains from human origin were from sporadic and unrelated cases of infection, except for two strains isolated from one patient. STEC strains belonging to serotypes O111:H8(H-), O26:H11 e O157:H7 prevailed. Differences on the prevalence of serotypes during the two periods were observed; while O111:H8(H-) e O26:H11 STEC strains prevailed during the period 1976-99, only one O111:H- STEC strain was identified during the period 2000-03. The inability to ferment rhamnose and dulcitol was mostly associated with O26 and O118 strains, whereas O111 STEC strains failed to decarboxylate lysine. The majority of the STEC strains was susceptible to all drugs; however, multi-resistant strains were detected mainly among O111:H- and O111:H8 STEC strains. All O157:H7 STEC strains carried stx2. Strains belonging to O93:H19 and O77:H18 harbored stx1 and stx2 sequences, and the remaining STEC strains carried only stx1. Except for O93:H19, O77:H18 e O55:H19 serotypes, all STEC strains carried eae. A close relationship was seen between intimin types, serotypes and diarrheagenic groups of E. coli. The presence of ehxA gene varied according to the serotypes. Multiple PFGE patterns were found among STEC strains of distinct serotypes. Moreover, PFGE restriction patterns of STEC strains differed substantially from those observed among non-STEC strains of the same serogroup except for O26 strains. Based on the indistinguishable PFGE pattern seen in two O157:H7 STEC strains, it can be suggested the first probable occurrence of an O157:H7 outbreak in Brazil. Human infections caused by STEC strains of distinct phenotypical and genotypical features occurred in our setting since the late 1970.
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    Evaluation of the Susceptibility profiles, genetic similarity and presence of qnr gene in Escherichia coli resistant to ciprofloxacin isolated in Brazilian hospitals
    (Brazilian Society of Infectious Diseases, 2007-02-01) Pereira, Andrea dos Santos [UNIFESP]; Andrade, Soraya Sgambatti [UNIFESP]; Monteiro, Jussimara [UNIFESP]; Sader, Helio Silva [UNIFESP]; Pignatari, Antonio Carlos Campos [UNIFESP]; Gales, Ana Cristina [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); JMI Laboratories
    Increasing quinolone resistance has been reported worldwide, mainly among clinical isolates of Escherichia coli. The objectives of this study were to determine the susceptibility profile, the genetic relatedness, and the prevalence of the qnr gene among ciprofloxacin-resistant Escherichia coli isolated from distinct Brazilian hospitals. A total of 144 ciprofloxacin-resistant Escherichia coli were isolated from 17 Brazilian hospitals between January/2002 and June/2003. The antimicrobial susceptibility testing was performed by microdilution according to NCCLS. The presence of the qnr gene was initially screened by colony blotting, and then confirmed by PCR followed by DNA sequencing. Ninety-five urinary ciprofloxacin-resistant Escherichia coli were further selected for molecular typing by pulsed-field gel electrophoresis (PFGE). Imipenem and meropenem showed the highest susceptibility rates (100.0% for both compounds) followed by amikacin (91.0%) and piperacillin/tazobactan (84.8%). A single ciprofloxacin-resistant Escherichia coli isolate was positive for qnr among the 144 ciprofloxacin-resistant Escherichia coli. Forty-six PFGE patterns were observed among the 95 ciprofloxacin-resistant Escherichia coli type. This study shows that therapeutic options are limited for treatment of ciprofloxacin-resistant Escherichia coli due to the presence of additional mechanisms of antimicrobial resistance, such as ESBL production. The qnr gene was uncommon among ciprofloxacin-resistant Escherichia coli clinical isolates, but its identification might indicate the emergence of this mechanism of quinolone resistance in Brazil. The great genomic variability found among the ciprofloxacin-resistant Escherichia coli highlights the importance of the appropriate use of quinolone to restrict the selection of resistant isolates.
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    Genetic relatedness among clinical strains of Stenotrophomonas maltophilia in tertiary care hospital settings in São Paulo State, Brazil
    (Sociedade Brasileira de Microbiologia, 2007-06-01) Almeida, Margarete Teresa Gottardo; Rubio, Fernando Gongora [UNIFESP]; Garcia, Doroti Oliveira; Pavarino-Bertelli, Érika Cristina; Rossit, Andrea Regina Baptista; Bando, Silvia Yano; Silbert, Suzane [UNIFESP]; Goloni-Bertollo, Eny Maria; Soares, Márcia Maria Costa Nunes; Martinez, Marina Baquerizo; Faculdade de Medicina de São José do Rio Preto Departamento de Doenças Dermatológicas, Infecciosas e Parasitárias; Universidade de São Paulo (USP); Faculdade de Medicina de São José do Rio Preto Departamento de Biologia Molecular; Universidade Federal de São Paulo (UNIFESP); Instituto Adolfo Lutz Seção de Bacteriologia; Faculdade de Medicina de São José do Rio Preto
    Stenotrophomonas maltophilia is a Gram-negative bacillus, which is becoming widely recognized as an important nosocomial pathogen. The main objective of this study was to evaluate the genetic relatedness, by random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) of 86 clinical isolates of S. maltophilia (colonization 22, infection 64) obtained from 79 hospitalized patients, from different geographic regions of São Paulo State. The genotypic analysis performed by RAPD and PFGE was used in 24 isolates for genetic identity confirmation. The results were congruent between the two methods but it was not possible to link genetic profiles with the studied variables, clinical state and geographic area, probably due to the great variability among the strains. The analyses by PFGE confirmed identity in 5 pairs of microorganisms and RAPD, in this study, showed to be a useful tool for investigation of diversity leading the identification of 85 genetic profiles. The genetic diversity shown may be due to re-infection by different strains or co-infection by multiple strains which suggests multiple entry sources of the bacterium in the hospital setting or of acquisition by patient. In this setting, colonization, infection and re-infection occur with unknown frequency, raising the need for the establishment of specific control measures.
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    Improving typeability of multiple bacterial species using pulsed-field gel electrophoresis and thiourea
    (Elsevier B.V., 2003-12-01) Silbert, S.; Boyken, L.; Hollis, R. J.; Pfaller, M. A.; Universidade Federal de São Paulo (UNIFESP); Univ Iowa
    Although pulsed-field gel electrophoresis is considered the gold standard technique for molecular typing, typeability may not be excellent for some bacterial species because of DNA degradation. Previous reports suggest that the addition of thiourea in the gel buffer can improved the typeability for some species. in the present study, 66 Gram-negative strains (seven species) known to be affected by DNA degradation and four control strains were evaluated by PFGE with and without the addition of 50 mug/M of thiourea to the buffer used in the electrophoresis. Macrorestriction patterns were obtained for all K. pneumoniae, S. marcescens, P. aeruginosa, and Salmonella spp., for 95.4% of E. coli, and for 50% of E. cloacae strains from the gels performed in the buffer with throurea. However, typeability was not improved for Acinetobacter spp. the range of non-typeable species for which thiourea can limit the problem of DNA degradation is considerably wider than described in previous publications. (C) 2003 Elsevier Inc. All rights reserved.
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    The Influence of Genetic Stability on Aspergillus fumigatus Virulence and Azole Resistance
    (Genetics Society America, 2018) dos Reis, Thaila Fernanda; Silva, Lilian Pereira; de Castro, Patricia Alves; Almeida de Lima, Pollyne Borborema; do Carmo, Rafaela Andrade [UNIFESP]; Marini, Marjorie Mendes [UNIFESP]; da Silveira, Jose Franco [UNIFESP]; Ferreira, Beatriz Henriques; Rodrigues, Fernando; Malavazi, Iran; Goldman, Gustavo H.
    Genetic stability is extremely important for the survival of every living organism, and a very complex set of genes has evolved to cope with DNA repair upon DNA damage. Here, we investigated the Aspergillus fumigatus AtmA (Ataxia-telangiectasia mutated, ATM) and AtrA kinases, and how they impact virulence and the evolution of azole resistance. We demonstrated that A. fumigatus atmA and atrA null mutants are haploid and have a discrete chromosomal polymorphism. The Delta atmA and Delta atrA strains are sensitive to several DNA-damaging agents, but surprisingly both strains were more resistant than the wild-type strain to paraquat, menadione, and hydrogen peroxide. The atmA and atrA genes showed synthetic lethality emphasizing the cooperation between both enzymes and their consequent redundancy. The lack of atmA and atrA does not cause any significant virulence reduction in A. fumigatus in a neutropenic murine model of invasive pulmonary aspergillosis and in the invertebrate alternative model Galleria mellonela. Wild-type, Delta atmA, and Delta atrA populations that were previously transferred 10 times in minimal medium (MM) in the absence of voriconazole have not shown any significant changes in drug resistance acquisition. In contrast, Delta atmA and Delta atrA populations that similarly evolved in the presence of a subinhibitory concentration of voriconazole showed an similar to 5-10-fold increase when compared to the original minimal inhibitory concentration (MIC) values. There are discrete alterations in the voriconazole target Cyp51A/Erg11A or cyp51/erg11 and/or Cdr1B efflux transporter overexpression that do not seem to be the main mechanisms to explain voriconazole resistance in these evolved populations. Taken together, these results suggest that genetic instability caused by Delta atmA and Delta atrA mutations can confer an adaptive advantage, mainly in the intensity of voriconazole resistance acquisition.
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    Oxacilin-resistant Coagulase-negative staphylococci (CoNS) bacteremia in a general hospital at São Paulo city, Brasil
    (Sociedade Brasileira de Microbiologia, 2008-12-01) d'Azevedo, Pedro Alves [UNIFESP]; Secchi, C.; Antunes, A.l.s.; Sales, T.; Silva, F.m. [UNIFESP]; Tranchesi, R.; Pignatari, Antonio Carlos Campos [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade Federal de Ciências da Saúde de Porto Alegre Laboratório de Cocos Gram Positivos; Hospital 9 de Julho
    In the last decades, coagulase-negative staphylococci (CoNS), especially Staphylococcus epidermidis have become an important cause of bloodstream infections. In addition, rates of methicillin-resistance among CoNS have increased substantially, leading to the use of glicopeptides for therapy. The objective of this study was to evaluate eleven consecutives clinically relevant cases of oxacillin-resistant CoNS bacteremia in a general hospital localized in São Paulo city, Brazil. Five different species were identified by different phenotypic methods, including S. epidermidis (5), S. haemolyticus (3), S. hominis (1), S. warneri (1) and S. cohnii subsp urealyticus (1). A variety of Pulsed Field Gel Electrophoresis profiles was observed by macrorestriction DNA analysis in S. epidermidis isolates, but two of three S. haemolyticus isolates presented the same profile. These data indicated the heterogeneity of the CoNS isolates, suggesting that horizontal dissemination of these microorganisms in the investigated hospital was not frequent. One S. epidermidis and one S. haemolyticus isolates were resistant to teicoplanin and susceptible to vancomycin. The selective pressure due to the use of teicoplanin in this hospital is relevant.