pulsed-field gel electrophoresis

Top PDF pulsed-field gel electrophoresis:

Caracterização de cepas de referência de Leptospira sp utilizando a técnica de pulsed field gel electrophoresis.

Caracterização de cepas de referência de Leptospira sp utilizando a técnica de pulsed field gel electrophoresis.

Introdução: A leptospirose é uma zoonose endêmica, mundialmente distribuída, causada por bactérias do gênero Leptospira. Este gênero compreende espécies patogênicas e saprofíticas, com mais de 200 sorovares distintos, diicultando sua caracterização. A técnica de pulsed ield gel electrophoresis tem sido empregada como uma ferramenta para auxiliar nesta caracterização. Os objetivos deste trabalho foram padronizar a técnica de PFGE, determinar os perfis moleculares das cepas de referência utilizadas pelo Laboratório de Referência Nacional para Leptospirose/Centro Colaborador da Organização Mundial de Saúde para Leptospirose e criar um banco de dados com estes peris. Métodos: Foram analisadas, por PFGE, dezenove cepas utilizando a enzima de restrição NotI. Resultados: Cada cepa apresentou um peril único que pode ser considerado como uma identidade genômica especíica, com exceção dos sorovares Icterohaemorrhagiae e Copenhageni, cujos peris foram indistinguíveis. Conclusões: Dessa forma, foi possível a criação de um banco de peris moleculares que está sendo utilizado no Laboratório para a comparação e identiicação de cepas isoladas de quadros clínicos. Palavras-chaves: Leptospira sp. Pulsed Field Gel Electrophoresis. Leptospirose. Biologia molecular.
Mostrar mais

4 Ler mais

Pseudomonas aeruginosa Endophthalmitis after Penetrating Keratoplasty Transmitted from the Same Donor to Two Recipients Confirmed by Pulsed-Field Gel Electrophoresis

Pseudomonas aeruginosa Endophthalmitis after Penetrating Keratoplasty Transmitted from the Same Donor to Two Recipients Confirmed by Pulsed-Field Gel Electrophoresis

Two devastating cases of multidrug-resistant Pseudomonas aeruginosa endophthalmitis after keratoplasty as the result of transmission from the same donor were confirmed by pulsed-field gel electrophoresis. Strategies for preventing donor-to-host transmission, such as the use of antimicrobial agents of greater efficacy and better methods for detecting microorganisms in preservation medium, could minimize this type of transmission.

2 Ler mais

Molecular analysis of salmonella anteritidis isolates from the Caribbean by pulsed-field gel electrophoresis

Molecular analysis of salmonella anteritidis isolates from the Caribbean by pulsed-field gel electrophoresis

Of the four countries from which we studied S. enteritidis strains, only one, Trinidad and Tobago, recorded a signif- icant increase in the absolute number of S. enteritidis isolates from 1994 to 1996. Barbados, on the other hand, had rela- tively high numbers of S. enteritidis from as early as 1990. The situation in Barba- dos might be explained in part by the country’s heavy dependence on tour- ism. Worldwide, there has been a surge in the involvement of S. enteritidis in TABLE 2. XbaI pulsed-field gel electrophoresis groups of S. enteritidis isolates, by year, four Caribbean countries, 1987–1996
Mostrar mais

6 Ler mais

Genetic heterogeneity of Shiga toxin-producing Escherichia coli strains isolated in São Paulo, Brazil, from 1976 through 2003, as revealed by pulsed-field gel electrophoresis

Genetic heterogeneity of Shiga toxin-producing Escherichia coli strains isolated in São Paulo, Brazil, from 1976 through 2003, as revealed by pulsed-field gel electrophoresis

The pulsed-field gel electrophoresis (PFGE) patterns of 46 Shiga toxin-producing Escherichia coli (STEC) strains isolated in Sa ˜o Paulo, Brazil, during the period from 1976 to 2003 were compared with those found among 30 non-STEC strains that carried eae and that belonged to the same serogroups as the STEC strains. All except two of the STEC and non-STEC strains of human origin were from sporadic and unrelated cases of infection; two O111 strains originated from the same patient. Multiple PFGE patterns were found among STEC strains of distinct serotypes. Moreover, the PFGE restriction patterns of STEC strains differed sub- stantially from those observed among non-STEC strains of the same serogroup except serotype O26 strains. Based on the indistinguishable PFGE pattern for two O157:H7 STEC strains isolated in the same geographic area at an interval of approximately 15 days and toxin profile data, the first occurrence of an O157:H7 outbreak in Brazil during that period can be suggested. In general, a close relationship between types of intimin, serotypes, and diarrheagenic groups of E. coli was observed. This is the first time that a large collection of STEC strains from Brazil has been analyzed, and a great genetic diversity was shown among O157:H7 and non-O157:H7 STEC strains isolated in Sa ˜o Paulo, Brazil.
Mostrar mais

7 Ler mais

Diversity of Pulsed-Field Gel Electrophoresis Patterns of Mycobacterium abscessus Type 2 Clinical Isolates

Diversity of Pulsed-Field Gel Electrophoresis Patterns of Mycobacterium abscessus Type 2 Clinical Isolates

Outbreaks of infections caused by rapidly growing mycobac- teria (RGM) related to surgical and cosmetic procedures have concerned Brazilian health care authorities since 2003 (5, 12, 19, 22–24, 28). More than 2,000 notifications received between 2004 and 2008 by the Brazilian National Health Surveillance Agency (ANVISA) reported cases of RGM surgical-site infec- tions after laparoscopic, arthroscopic, or plastic surgeries (4, 19). Most isolates recovered from surgical patients studied so far showed three common features: an uncommon pattern when they were submitted to PCR-restriction enzyme analysis of the hsp65 gene (PRA-hsp65), called Mycobacterium absces- sus type 2 by Devallois et al. (10) and others (19); two char- acteristic substitutions [C(2683)T and T(2874)C] in the se- quence of region V of the rpoB gene (2, 19, 28); and two highly similar patterns with one band difference when they were typed by pulsed-field gel electrophoresis (PFGE) using DraI and AseI restriction enzymes (5, 12, 19, 28). The Brazilian National Institute for Health Quality Control (INCQS) deposited one of these isolates under number INCQS 594.
Mostrar mais

7 Ler mais

Avaliação da disseminação de Salmonella Senftenberg isoladas de diversos ambientes avícolas utilizando a técnica de Pulsed Field Gel Electrophoresis

Avaliação da disseminação de Salmonella Senftenberg isoladas de diversos ambientes avícolas utilizando a técnica de Pulsed Field Gel Electrophoresis

Nos últimos anos, a emergência de Salmonella Senftenberg no ambiente de granjas de frango tornou-se causa de preocupação, pois este sorotipo, além de ser um potencial agente de paratifo aviário em condições de estresse, tem sido associado a diversos casos de infecções humanas. Acredita-se que sua ampla disseminação seja resultado da capacidade de formar biofilmes, resistir à desidratação, acidez, temperaturas elevadas e radiação, além de permanecer por longos períodos no ceco das aves, na ração e no ambiente. Devido a estas peculiaridades, o controle de Salmonella Senftenberg é um desafio para a avicultura e exige a adoção de medidas de controle baseadas no conhecimento das rotas de disseminação e na biologia do micro-organismo. Neste estudo, a disseminação de Salmonella Senftenberg em granjas de frango, localizadas na região noroeste do estado de São Paulo, foi avaliada usando a tipagem molecular pela técnica de Pulsed Field Gel Electrophoresis (PFGE). Os dados epidemiológicos foram utilizados como ferramenta auxiliar para a comparação dos perfis de PFGE das cepas. As Salmonella Senftenberg isoladas de ingredientes de ração, ambiente de fábrica de ração, aves, ambiente dos aviários e de animais provenientes dos arredores de granjas avícolas, apresentaram diferentes identidades genéticas, indicando a transmissão de genótipos distintos entre as várias localidades.
Mostrar mais

61 Ler mais

Genetic profiling of Klebsiella pneumoniae: comparison of pulsed field gel electrophoresis and random amplified polymorphic DNA

Genetic profiling of Klebsiella pneumoniae: comparison of pulsed field gel electrophoresis and random amplified polymorphic DNA

pulsed field gel electrophoresis (PFGE) has been consid- ered as the gold standard for typing of K. pneumoniae (Arlet et al., 1994; Li et al., 2009). Since PFGE is techni- cally demanding, time consuming and expensive, interests have been raised in easier, faster and more economical typ- ing methods such as random amplified polymorphic DNA (RAPD) (Gori et al., 1996). Despite the economical and practical merits offered by RAPD, the reproducibility of this method remains a challenge (Atienzar and Jha, 2006). After systematic optimization, RAPD has been suggested as a reliable, sensitive and reproducible assay for molecular typing of bacteria (Atienzar and Jha, 2006; Blixt et al., 2003). In this study, 54 nosocomial isolates of K. pneumoniae were fingerprinted using an optimized RAPD protocol and a PFGE method. Typeability, repeatability, reproducibility and discriminatory power of the optimized RAPD protocol were compared with the PFGE method.
Mostrar mais

6 Ler mais

Associated Risk Factors and Pulsed Field Gel Electrophoresis of Nasal Isolates of Staphylococcus aureus from Medical Students in a Tertiary Hospital in Lagos, Nigeria

Associated Risk Factors and Pulsed Field Gel Electrophoresis of Nasal Isolates of Staphylococcus aureus from Medical Students in a Tertiary Hospital in Lagos, Nigeria

Macrorestriction and Pulsed field gel electrophoresis is well known in being superior in typeability and reproducibility to any typing methods and allows an excellent discrimination between strains [24]. Sixteen of the twenty- six strains were investigated genotypically using PFGE. The results generated clearly support other studies [10] and show that hospital personnel particularly medics appear to be a major reservoir for staphylococcal infections and indeed important vector for transmission of S.aureus within the hospital setting. Among the sixteen strains, a major clone (PFGE type B) containing about 44% of the isolates was identified. This suggests the possible transmission of a single strain among the group and also strengthens the hypothesis that S.aureus is clonal in nature. It might be further interesting to know whether nasal strains from hospital personnel would be genetically identical to clinical strains circulating within the same hospital.
Mostrar mais

7 Ler mais

Single-cell pulsed-field gel electrophoresis to detect the early stage of DNA fragmentation in human sperm nuclei.

Single-cell pulsed-field gel electrophoresis to detect the early stage of DNA fragmentation in human sperm nuclei.

Single-cell pulsed-field gel electrophoresis (SCPFGE) with dual electrode pairs was developed to detect the early stage of DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugation technique and subsequent swim-up. The sperm were embedded in a thin film of agarose containing bovine trypsin (20 m g/mL) and were then lysed. Prior to SCPFGE, proteolysis of DNA-binding components, such as protamine and the nuclear matrix was essential to separate the long chain fibers from the fibrous and granular fragments derived from a single nucleus. The overall electrophoretic profiles elucidated the course of DNA fragmentation. A few large fibrous fragments were observed at the beginning of the process, however, as the fragmentation advanced, the long chain fibers decreased and shortened, and, conversely, the granular fragments increased until finally almost all the DNA was shredded. Although the ejaculate contained sperm with heterogeneous stages, the purified motile sperm exhibited several dozens of uniformly elongated fibers arising from the tangled DNA at the origin, whereas a part of these fibers gave rise to fibrous fragments beyond the tip of the elongated fibers, and their numbers and sizes varied among the sperm. Conventional intra- cytoplasmic sperm injection (ICSI) usually depends on intra-operative light microscopic observation to select a sperm for injection. The present results revealed that sperm motility could not give full assurance of DNA integrity. SCPFGE is likely to serve an important role in the preoperative differential diagnosis to determine the competence of the sperm population provided for injection.
Mostrar mais

8 Ler mais

Genetic diversity of Xanthomonas axonopodis pv. citri based on plasmid profile and pulsed field gel electrophoresis

Genetic diversity of Xanthomonas axonopodis pv. citri based on plasmid profile and pulsed field gel electrophoresis

b) Pulsed Field Gel Electrophoresis (PFGE): Total genomic DNA of X. axonopodis pv. citri strains was ex- tracted by using the protocol described by Cooksey and Graham (1989) and Egel et al. (1991), with few modifica- tions. The X. axonopodis pv. citri strains were grown on NA (Nutrient Agar) medium for 48 h at 28 °C. Pure colonies were transferred to test tubes containing NB (Nutrient Broth) and kept on a rotatory shaker (150 rpm) for 24 h at 28 °C. Cells (5 x 10 9 CFU/mL) were washed in 1 mL of SE buffer (75 mM NaCl, 25 mM EDTA, pH 8.0) and resus-

6 Ler mais

Pulsed Field Gel Electrophoresis Reveals Chromosome Length and Number Differences in Brazilian Strains of Metarhizium Anisopliae

Pulsed Field Gel Electrophoresis Reveals Chromosome Length and Number Differences in Brazilian Strains of Metarhizium Anisopliae

among soil isolates, suggesting that this fungus was developed a certain degree of host specificity. An effective technique designated pulsed-field gel electrophoresis (PFGE), was used to separate chromosome-sized DNA molecules of M. anisopliae (Shimizu et al., 1992 and Valadares- Inglis and Peberdy, 1998). However, so far only a few isolates have been tested by this technique and most of them derived from a single species host. The present work was carried out in an attempt to improve the conditions used in the chromosome separation for this fungus; to study the possible chromosome-sized DNA molecules differences in number and size of eight isolates of M. anisopliae var. anisopliae from different insect-hosts and regions and find some correlation between the occurrence of parasexual cycle described by other authors and the karyotype found in the present work using the same strains.
Mostrar mais

6 Ler mais

Multilocus Sequence Typing Scheme versus Pulsed-Field Gel Electrophoresis for Typing Mycobacterium abscessus Isolates

Multilocus Sequence Typing Scheme versus Pulsed-Field Gel Electrophoresis for Typing Mycobacterium abscessus Isolates

Molecular typing of outbreak isolates helps epidemiological investigation and the identification of possible sources of infection (26). Ultimately, typing can contribute to outbreak control. Pulsed-field gel electrophoresis (PFGE) is usually performed when specific methods are not available for the species being stud- ied. PFGE has good discriminatory power for separating M. ab- scessus subsp. abscessus and M. abscessus subsp. bolletii strains (24, 27) but is a cumbersome and time-consuming technique, requir- ing dedicated equipment that is not widely available. Moreover, DNA degradation associated with PFGE can occur with M. absces- sus isolates (28). Schemes for MST and multilocus sequence typ- ing (MLST) were recently developed for M. abscessus (20, 29). The MST scheme analyzes intergenic sequences, which, as a conse- quence of less evolutionary pressure, can show high variability (20). In the MLST scheme, single-copy housekeeping genes are sequenced, and the combination of the different gene alleles gen- erates a sequence type (ST) and defines a strain (29). A site for the interpretation and comparison of MLST profiles is available at http: //www.pasteur.fr/recherche/genopole/PF8/mlst/Myco-abscessus .html.
Mostrar mais

11 Ler mais

Typing of Enterococcus faecium by polymerase chain reaction and pulsed field gel electrophoresis

Typing of Enterococcus faecium by polymerase chain reaction and pulsed field gel electrophoresis

Polymerase chain reaction (PCR) with JB1 or REP consensus oligo- nucleotides and pulsed field gel electrophoresis (PFGE) were used to study genomic DNA extracted from 31 strains of enterococci. Eleven ATCC strains, representative of 11 species of Enterococcus, were initially tested by JB1-PCR, revealing that Enterococcus malodoratus and Enterococcus hirae presented identical banding patterns. Eight Enterococcus faecium isolates from Stanford University and 12 from São Paulo Hospital were studied by JB1-PCR, REP-PCR 1/2R and PFGE. Among the isolates from Stanford University, 5 genotypes were defined by JB1-PCR, 7 by REP-PCR 1/2R and 4 by PFGE. Among the isolates from São Paulo Hospital, 9 genotypes were identified by JB1-PCR, 6 by REP-PCR and 5 by PFGE. The three methods identified identical genotypes, but there was not complete agreement among them.
Mostrar mais

6 Ler mais

PODER DISCRIMINATÓRIO DAS TÉCNICAS DE RAPD (Random Amplified Polymorphic DNA) e PFGE (Pulsed-field gel electrophoresis) PARA DIFERENCIAR Campylobacter jejuni ISOLADAS DE CARCAÇAS DE FRANGOS PHELIPE AUGUSTO BORBA MARTINS PERES DAISE APARECIDA ROSSI FAMEV

PODER DISCRIMINATÓRIO DAS TÉCNICAS DE RAPD (Random Amplified Polymorphic DNA) e PFGE (Pulsed-field gel electrophoresis) PARA DIFERENCIAR Campylobacter jejuni ISOLADAS DE CARCAÇAS DE FRANGOS PHELIPE AUGUSTO BORBA MARTINS PERES DAISE APARECIDA ROSSI FAMEV

PFGE é uma técnica de alta reprodutibilidade, que permite a diferenciação eficiente de genótipos. Nesta técnica, o material genético é armazenado em blocos de agarose, mantendo- se intacto para a ação das enzimas de restrição (SmaI, SalI, KpnI, ApaI e BssHII) específicas para cada espécie, e posteriormente, é submetido a eletroforese em gel utilizando campos pulsados, que permite a separação de grandes fragmentos. É uma técnica morosa e trabalhosa, sendo os padrões variáveis de acordo com a enzima empregada, sendo necessária a correta escolha da enzima e das condições de restrição de eletroforese, que devem ser otimizadas para cada espécie (WASSENAAR; NEWELL, 2000).
Mostrar mais

31 Ler mais

Ana Beatriz de A Corrêa, Ivi Cristina M de Oliveira, Tatiana de CA Pinto, Marcos C de Mattos, Leslie C Benchetrit

Ana Beatriz de A Corrêa, Ivi Cristina M de Oliveira, Tatiana de CA Pinto, Marcos C de Mattos, Leslie C Benchetrit

Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Pers- ing DH, Swaminathan B 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33: 2233-2239. Uh Y, Jang IH, Yoon KJ, Song W 2001. Emerging erythromycin resis- tance among group B streptococci in Korea. Eur J Clin Microbiol Infect Dis 20: 52-54.

5 Ler mais

Susceptibility of clinical isolates of multiresistant Pseudomonas aeruginosa to a hospital disinfectant and molecular typing

Susceptibility of clinical isolates of multiresistant Pseudomonas aeruginosa to a hospital disinfectant and molecular typing

clinical and industrial environment (Mc Bain et al. 2004). They have been used for a variety of medical, pharmaceu- tical, and other purposes (Hugo & Russell 1992). They are in general low-level disinfectants, showing activity to Gram-positive and Gram-negative bacteria (Romão 1996). There are some papers that deal with microbial resis- tance to biocides and antibiotics (Murtough et al. 2001) but beside the aspects about cross-resistance and co- resistance between antibiotics and disinfectants some questions should be investigated: are the hospital iso- lates being eliminated by commercial hospital disinfec- tants as expected? Are the hospital isolates as suscep- tible as the reference microorganisms used in disinfectant testing? Health-care professionals frequently ask us about these issues since data about these subjects are scarce in our country. According to Herruzo-Cabrera et al. (2004), the increased susceptibility of laboratory-adapted organ- isms may allow any disinfectant product to pass the stan- dard tests but the product may fail in clinical settings. On the other hand, it has been observed that strains with the same PFGE (pulsed field gel electrophoresis) pattern or belonging to the same clonal group showed differences in the susceptibility to antimicrobial agents (Harris et al. 1999) but very few data are available concerning the be- havior of strains from the same clonal group regarding susceptibility to disinfectants. Is PFGE a reliable marker to disinfectant susceptibility?
Mostrar mais

8 Ler mais

Molecular Techniques for MRSA Typing: Current Issues and Perspectives

Molecular Techniques for MRSA Typing: Current Issues and Perspectives

Staphylococcus aureus has long been recognised as an important pathogen in human disease. Serious staphylococcal infections can frequently occur in inpatients and may lead to dire consequences, especially as to therapy with antimicrobial agents. The increase in the frequency of Methicillin-Resistant Staphylococcus aureus (MRSA) as the causal agent of nosocomial infection and the possibility of emergence of resistance to vancomycin demands a quick and trustworthy characterization of isolates and identification of clonal spread within hospitals. Enough information must be generated to permit the implementation of appropriate measures for control of infection, so that outbreaks can be contained. Molecular typing techniques reviewed in this manuscript include: plasmid profile analysis, analysis of chromosomal DNA after enzymatic restriction, Southern blotting, pulsed field gel electrophoresis (PFGE), techniques involving polymerase chain reaction and multilocus sequence typing (MLST). Repetitive DNA Sequence PCR (rep- PCR) may be used for screening due to its practicality, low cost and reproducibility. Because of its high discriminatory power Pulsed-Field Gel Electrophoresis (PFGE) still remains the gold standard for MRSA typing. New techniques with higher reproducibility and discriminatory power, such as Multi-Locus Sequence Typing (MLST), are appearing. These are mostly useful for global epidemiology studies. Molecular typing techniques are invaluable tools for the assessment of putative MRSA outbreaks and so should be extensively used for this purpose.
Mostrar mais

12 Ler mais

Avaliação comparativa de protocolos rápidos versus protocolos convencionais para tipagem molecular de amostras clinicas de Staphylococcus aureus, Escherichia coli e Pseudomonas aeruginosa pela técnica de pulsed fiedl gel electrophoresis

Avaliação comparativa de protocolos rápidos versus protocolos convencionais para tipagem molecular de amostras clinicas de Staphylococcus aureus, Escherichia coli e Pseudomonas aeruginosa pela técnica de pulsed fiedl gel electrophoresis

A técnica de tipagem molecular avaliada neste estudo foi o "Pulsed Field Gel Electrophoresis" (PFGE), que vem demonstrando ser uma excelente técnica para a tipagem molecular da maioria das espécies bacterianas (OLIVE & BEAN, 1999; SWAMINATHAN et al., 2001). O PFGE caracteriza-se por separar fragmentos grandes de DNA, que variam de 10 a 800kb e, é considerado o método mais discriminatório entre todos os métodos de tipagem molecular utilizados em estudos epidemiológicos (GRUNDMANN et al., 1995; PFALLER et al., 1996; TENOVER et al., 1997; LIU & WU, 1997; HOLLIS et al., 1999; OLIVE & BEAN, 1999; PFALLER et al., 2001). A técnica supracitada tem sido utilizada com sucesso para traçar surtos hospitalares e, por meio de análises epidemiológicas, pode auxiliar na detecção da causa de infecções relacionadas à assistência à saúde, que envolvem um grande número de diferentes patógenos, incluindo Staphylococcus aureus, Escherichia coli e Psedomonas aeruginosa (GAUTON, 1997).
Mostrar mais

113 Ler mais

Analysis of the clonal diversity of Staphylococcus aureus methicillin-resistant strains isolated at João Pessoa, state of Paraíba, Brazil

Analysis of the clonal diversity of Staphylococcus aureus methicillin-resistant strains isolated at João Pessoa, state of Paraíba, Brazil

cell wall formation in concentrations of drugs that render the other PBPs inactive (Hartman & Tomasz 1984). The PBP 2a and the gene encoding it (mecA) have been found in all resistant staphylo- cocci (De Lencastre et al. 1991), and their detec- tion may be used as reference procedure when other tests provide ambiguous results (Richard et al. 1993). The Crystal MRSA system is a qualitative screening method for the rapid detection of intrin- sic methicillin resistance and there was a close cor- relation between the presence of gene mec and a positive reaction with this test (Knapp et al. 1994). Traditional and molecular analysis have been extensively used for epidemiologic evaluation of MRSA infections (Struellens et al. 1992, Schilichting et al. 1993). The similarity among epidemiologic unrelated strains make their analy- sis very difficult. Several studies have recom- mended the use of a molecular technique, such as pulsed-field gel electrophoresis (PFGE). This methodology is reported to be very sensitive, hightly reproducible and to have a very good dis- criminatory power (Prevost et al. 1992).
Mostrar mais

6 Ler mais

Show all 4462 documents...