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POTENSI GEN dtx DAN dtxR SEBAGAI MARKER UNTUK DETEKSI DAN PEMERIKSAAN TOKSIGENISITAS Corynebacterium diphtheriae  [cached]
Sunarno Sunarno,Kambang Sariadji,Holly Arif Wibowo
Bulletin of Health Research , 2013,
Abstract: . Corynebacterium diphtheriae is the causative agent of diphtheria. The main virulence determinant of the bacteria is diphtheria toxin, the cause of the systemic complication seen with diphtheria. Production of diphtheria toxin by toxigenic strain encoded by dtx/tox gene and repressed by dtxR gene. Gold standard for bacterial toxigenicity test carried out by conventional methods (Elek test, Guinea pig and vero cell cytotoxicity). However, Elek test have variety result, time consume and problem of the reagent availability. On the other hand, the animal (Guinea pig) testing was opposed by many animal lovers and the vero cell cytotoxicity test require high cost. The study purposed to evaluate the using of dtx and dtxR genes as a detection marker of C.diphtheriae and bacterial toxigenicity test simultaneusly by Multiplex PCR. The study examined 44 bacterial and fungal isolates, included 22 C.diphtheriae (4 reference strains and 18 clinical isolates), 5 other specieses of Corynebacterium (reference strains) and 17 non-Corynebacterium (10 reference strains and 7 stock cultures ). All of sample were examined by Multiplex PCR for 2 primer pairs targeted dtx and dtxR genes. The study showed that the Multiplex PCR for dtx and dtxR as target genes able to detect all of sample correctly thus concluded that dtx and dtxR genes could be used as a marker for alternative detection and toxigenicity test of C.diphtheriae by Multiplex PCR rapidly and accuratelly. Key words: Corynebacterium diphtheriae, dtx, dan dtxR Abstrak. Corynebacterium diphtheriae merupakan agen penyebab penyakit difteri.. Faktor virulensi utama C. diphtheriae adalah toksigenisitas (kemampuan memproduksi toksin) bakteri toxin. Produksi toksin diatur seperangkat gen yang disebut gen tox/dtx dan diregulasi oleh gen dtxR. Gold standard untuk pemeriksaan toksigenisitas C.diphtheriae adalah dengan metode konvensional (Elek test, Guinea pig dan vero cell cytotoxigenicity),namun Elek test mempunyai variasi hasil yang cukup beragam, membutuhkan waktu yang cukup lama, serta masalah ketersediaan reagen standard. Di sisi lain pemeriksaan dengan hewan coba banyak ditentang oleh para pecinta satwa. Sementara itu, pemeriksaan dengan vero cell membutuhkan biaya yang sangat tinggi. Penelitian ini bertujuan untuk mengevaluasi potensi gen dtx dan dtxR sebagai marker deteksi C.diphtheriae sekaligus pemeriksaan toksigenisitas bakteri menggunakan PCR Multipleks. Jumlah sampel penelitian sebanyak 44 isolat, termasuk 22 C.diphtheriae (4 strain referensi dan 18 isolat klinik), 5 spesies lain Corynebacterium (
Purification and Structural Characterization of Siderophore (Corynebactin) from Corynebacterium diphtheriae  [PDF]
Sheryl Zajdowicz, Jon C. Haller, Amy E. Krafft, Steve W. Hunsucker, Colin T. Mant, Mark W. Duncan, Robert S. Hodges, David N. M. Jones, Randall K. Holmes
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0034591
Abstract: During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(β) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin.
Diphtheria due to non-toxigenic corynebacterium diphtheriae: A report of two cases  [cached]
Kanungo R,Vijayalakshmi N,Nalini P,Bhattacharya S
Indian Journal of Medical Microbiology , 2002,
Abstract: Diseases due to non-toxigenic strains of Corynebacterium diphtheriae are being increasingly reported. These diseases have been found to occur in vaccinated individuals. We report two cases of diphtheria with myocarditis and polyneuritis caused by non-toxigenic strains of C. diphtheriae. The virulence factors of this organism and the pitfalls in diagnosis have also been discussed.
Prevalence of diphtheria and tetanus antibodies and circulation of Corynebacterium diphtheriae in S?o Paulo, Brazil
Divino-Goes, K.G.;Moraes-Pinto, M.I. de;Dinelli, M.I.S.;Casagrande, S.T.;Bonetti, T.C.S.;Andrade, P.R.;Weckx, L.Y.;
Brazilian Journal of Medical and Biological Research , 2007, DOI: 10.1590/S0100-879X2006005000184
Abstract: the introduction of routine vaccination against tetanus and diphtheria in brazil has decreased the incidence and changed the epidemiology of both diseases. we then investigated the prevalence of corynebacterium diphtheriae carrier status and diphtheria and tetanus immunity in s?o paulo, brazil. from november 2001 to march 2003, 374 individuals were tested for the presence of c. diphtheriae in the naso-oropharynx and of serum diphtheria and tetanus antibodies. participants were all healthy individuals without acute or chronic pathologies and they were stratified by age as follows: 0-12 months and 1-4, 5-9, 10-14, 15-24, 25-39, 40-59, and 360 years. antibodies were assessed using a double-antigen elisa. c. diphtheriae species were identified by biochemical analysis and toxigenicity was assessed by the elek test. for diphtheria, full protection (antibodies 30.1 iu/ml) was present in 84% of the individuals, 15% had basic protection (antibodies 30.01 and <0.1 iu/ml) and 1% were susceptible (antibodies <0.01 iu/ml). full tetanus protection (antibodies 30.1 iu/ml) was present in 79% of the participants, 18% had basic protection (antibodies 30.01 and <0.1 iu/ml) and 3% were susceptible (antibodies <0.01 iu/ml). the geometric mean of diphtheria and tetanus antibodies reached the highest values at 5-9 years and decreased until the 40-59-year age range, increasing again in individuals over 60 years. three participants (0.8%) were carriers of c. diphtheriae, all non-toxigenic strains. the present results demonstrate the clear need of periodic booster for tetanus and diphtheria vaccine in adolescents and adults after primary immunization in childhood.
Molecular epidemiology of C. diphtheriae strains during different phases of the diphtheria epidemic in Belarus
Valentina Kolodkina, Leonid Titov, Tatyana Sharapa, Francine Grimont, Patrick AD Grimont, Androulla Efstratiou
BMC Infectious Diseases , 2006, DOI: 10.1186/1471-2334-6-129
Abstract: The biotype and toxigenicity status of 3513 C. diphtheriae strains isolated from all areas in Belarus during a declining period of diphtheria morbidity (1996–2005) was undertaken. Of these, 384 strains were isolated from diphtheria cases, 1968 from tonsillitis patients, 426 from contacts and 735 from healthy carriers. Four hundred and thirty two selected strains were ribotyped.The C diphtheriae gravis biotype, which was prevalent during 1996–2000, was "replaced" by the mitis biotype during 2001–2005. The distribution of toxigenic C. diphtheriae strains also decreased from 47.1% (1996) to 5.8% (2005). Changes in the distribution of the epidemic ribotypes Sankt-Peterburg and Rossija were also observed. During 2001–2005 the proportion of the Sankt-Peterburg ribotype decreased from 24.3% to 2.3%, in contrast to the Rossija ribotype, that increased from 25.1% to 49.1%. The circulation of other toxigenic ribotypes (Otchakov, Lyon, Bangladesh), which were prevalent during the period of high diphtheria incidence, also decreased. But at the same time, the proportion of non-toxigenic strains with the Cluj and Rossija ribotypes dramatically increased and accounted for 49.3% and 30.1%, respectively.The decrease in morbidity correlated with the dramatic decrease in the isolation of the gravis biotype and Sankt Peterburg ribotype, and the prevalence of the Rossija ribotype along with other rare ribotypes associated with non-toxigenic strains (Cluj and Rossija, in particular).The diphtheria epidemic, which emerged in the 1990's in the newly independent states (NIS) of the former Soviet Union, was also reported from Belarus. In Belarus, 794 diphtheria cases were identified during 1990–1995, of which 25 were fatal. As a consequence of mass immunization the morbidity stabilized in 1996. In 2005 the morbidity decreased to 0.11 per 100 000 population with a morbidity index of 0.1/100 000 population in advance of the WHO target for 2010. However, the diphtheria incidence in Belarus rema
Genetic relationships of Corynebacterium diphtheriae strains isolated from a diphtheria case and carriers by restriction fragment length polymorphism of rRNA genes
Sacchi, Claudio Tavares;Lemos, Ana Paula Silva de;Casagrande, Silvana Tadeu;Mori, Alice Massumi;Almeida, Carmecy Lopes de;
Revista do Instituto de Medicina Tropical de S?o Paulo , 1995, DOI: 10.1590/S0036-46651995000400002
Abstract: in the present study we report the results of an analysis, based on ribotyping of corynebacterium diphtheriae intermedius strains isolated from a 9 years old child with clinical diphtheria and his 5 contacts. quantitative analysis of rflps of rrna was used to determine relatedness of these 7 c.diphtheriae strains providing support data in the diphtheria epidemiology. we have also tested those strains for toxigenicity in vitro by using the elek's gel diffusion method and in vivo by using cell culture method on cultured monkey kidney cell (vero cells). the hybridization results revealed that the 5 c.diphtheriae strains isolated from contacts and one isolated from the clinical case (nose case strain) had identical rflp patterns with all 4 restriction endonucleases used, ribotype b. the genetic distance from this ribotype and ribotype a (throat case strain), that we initially assumed to be responsible for the illness of the patient, was of 0.450 showing poor genetic correlation among these two ribotypes. we found no significant differences concerned to the toxin production by using the cell culture method. in conclusion, the use of rflps of rrna gene was successful in detecting minor differences in closely related toxigenic c.diphtheriae intermedius strains and providing information about genetic relationships among them.
Reannotation of the Corynebacterium diphtheriae NCTC13129 genome as a new approach to studying gene targets connected to virulence and pathogenicity in diphtheria
D'Afonseca V, Soares SC, Ali A, Santos AR, Pinto AC, Magalh es AAC, Faria CJ, Barbosa E, Guimar es LC, Eslab o M, Almeida SS, Abreu VAC, Zerlotini A, Carneiro AR, Cerdeira LT, Ramos RTJ, Hirata Jr R, Mattos-Guaraldi AL, Trost E, Tauch A, Silva A, Schneider MP, Miyoshi A, Azevedo V
Open Access Bioinformatics , 2012, DOI: http://dx.doi.org/10.2147/OAB.S25500
Abstract: nnotation of the Corynebacterium diphtheriae NCTC13129 genome as a new approach to studying gene targets connected to virulence and pathogenicity in diphtheria Original Research (2640) Total Article Views Authors: D'Afonseca V, Soares SC, Ali A, Santos AR, Pinto AC, Magalh es AAC, Faria CJ, Barbosa E, Guimar es LC, Eslab o M, Almeida SS, Abreu VAC, Zerlotini A, Carneiro AR, Cerdeira LT, Ramos RTJ, Hirata Jr R, Mattos-Guaraldi AL, Trost E, Tauch A, Silva A, Schneider MP, Miyoshi A, Azevedo V Published Date February 2012 Volume 2012:4 Pages 1 - 13 DOI: http://dx.doi.org/10.2147/OAB.S25500 Received: 25 August 2011 Accepted: 14 October 2011 Published: 03 February 2012 Vívian D’Afonseca1,*, Siomar C Soares1,*, Amjad Ali1, Anderson R Santos1, Anne C Pinto1, Aryane AC Magalh es1, Cássio de Jesus Faria1, Eudes Barbosa1, Luis C Guimar es1, Marcus Eslab o2, Sintia S Almeida1, Vinicius AC Abreu1, Adhemar Zerlotini3,4, Adriana R Carneiro5, Louise T Cerdeira5, Rommel TJ Ramos5, Raphael Hirata Jr6, Ana L Mattos-Guaraldi6, Eva Trost7, Andreas Tauch7, Artur Silva5, Maria P Schneider5, Anderson Miyoshi1, Vasco Azevedo1 1Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; 2Federal University of Pelotas, Pelotas, Rio Grande do Sul, Brazil; 3FIOCRUZ - CEBIO, Belo Horizonte, Minas Gerais, Brazil; 4EMBRAPA - CNPTIA, Campinas, S o Paulo, Brazil; 5Federal University of Pará, Belém, Pará, Brazil; 6Rio de Janeiro State University, Rio de Janeiro, Brazil; 7Center for Biotechnology, Bielefeld University, Bielefeld, Germany *These authors contributed equally to this work Background: The reannotation of genomes already on file is a new approach to discovering new genetic elements and to make the genomes more descriptive and current with relevant features regarding the organism’s lifestyle. Within this approach, the present study aimed to reannotate the genome of the Gram-positive human pathogen Corynebacterium diphtheriae, which causes diphtheria. The deposit of massive amounts of information linked to other species of the genus Corynebacterium has facilitated the updating of the genomic interpretation of this microorganism. Additionally, the emergence of invasive disease by nontoxigenic strains of C. diphtheriae and the reemergence of diphtheria in partially immunized populations have given impetus to new studies in relation to its structural and functional genome. Results: In relation to structural genomics, 23 coding regions (coding sequences) were deleted and 71 new genes were added to the genome annotation. Nevertheless, all the pseudogenes were validated and ten new pseudogenes were created. In relation to functional genomics, about 57% of the genome annotation was updated and became functionally more informative. The product descriptions of 41% (973 proteins) were updated. Among them, 370 that were previously annotated as “hypothetical proteins,” now have more informative descriptions. With the new annotation, the plasticity of the genome became e
Corynebacterium ulcerans 0102 carries the gene encoding diphtheria toxin on a prophage different from the C. diphtheriae NCTC 13129 prophage
Tsuyoshi Sekizuka, Akihiko Yamamoto, Takako Komiya, Tsuyoshi Kenri, Fumihiko Takeuchi, Keigo Shibayama, Motohide Takahashi, Makoto Kuroda, Masaaki Iwaki
BMC Microbiology , 2012, DOI: 10.1186/1471-2180-12-72
Abstract: We determined the whole genome sequence of the toxigenic C. ulcerans 0102 isolated in Japan. The genomic sequence showed a striking similarity with that of Corynebacterium pseudotuberculosis and, to a lesser extent, with that of C. diphtheriae. The 0102 genome contained three distinct prophages. One of these, ΦCULC0102-I, was a tox-positive prophage containing genes in the same structural order as for tox-positive C. diphtheriae prophages. However, the primary structures of the individual genes involved in the phage machinery showed little homology between the two counterparts.Taken together, these results suggest that the tox-positive prophage in this strain of C. ulcerans has a distinct origin from that of C. diphtheriae NCTC 13129.
The DtxR protein acting as dual transcriptional regulator directs a global regulatory network involved in iron metabolism of Corynebacterium glutamicum
Iris Brune, Hendrikje Werner, Andrea T Hüser, J?rn Kalinowski, Alfred Pühler, Andreas Tauch
BMC Genomics , 2006, DOI: 10.1186/1471-2164-7-21
Abstract: A deletion of the dtxR gene of C. glutamicum ATCC 13032 led to the mutant strain C. glutamicum IB2103 that was able to grow in minimal medium only under low-iron conditions. By performing genome-wide DNA microarray hybridizations, differentially expressed genes involved in iron metabolism of C. glutamicum were detected in the dtxR mutant. Bioinformatics analysis of the genome sequence identified a common 19-bp motif within the upstream region of 31 genes, whose differential expression in C. glutamicum IB2103 was verified by real-time reverse transcription PCR. Binding of a His-tagged DtxR protein to oligonucleotides containing the 19-bp motifs was demonstrated in vitro by DNA band shift assays. At least 64 genes encoding a variety of physiological functions in iron transport and utilization, in central carbohydrate metabolism and in transcriptional regulation are controlled directly by the DtxR protein. A comparison with the bioinformatically predicted networks of C. efficiens, C. diphtheriae and C. jeikeium identified evolutionary conserved elements of the DtxR network.This work adds considerably to our currrent understanding of the transcriptional regulatory network of C. glutamicum genes that are controlled by DtxR. The DtxR protein has a major role in controlling the expression of genes involved in iron metabolism and exerts a dual regulatory function as repressor of genes participating in iron uptake and utilization and as activator of genes responsible for iron storage and DNA protection. The data suggest that the DtxR protein acts as global regulator by controlling the expression of other regulatory proteins that might take care of an iron-dependent regulation of a broader transcriptional network of C. glutamicum genes.One of the major challenges in post-genomic research is to decipher and reconstruct the complete connectivity of transcriptional regulatory networks encoded by a bacterial genome sequence [1,2]. The key components in regulation of bacterial gen
DIPHTHERIA IN A VACCINATED ADULT IN RIO DE JANEIRO, BRAZIL
Mattos-Guaraldi, Ana Luiza;Formiga, Luiz Carlos Duarte;Marques, Elizabeth Andrade;Pereira, Gabriela Andrade;Moreira, Lílian Oliveira;Pimenta, Fabrícia Pires;Camello, Thereza Cristina Ferreira;Oliveira, Elsa Fuchshuber;
Brazilian Journal of Microbiology , 2001, DOI: 10.1590/S1517-83822001000300015
Abstract: in 1999, a case of diphtheria in a 32-year-old woman was reported. the patient developed a sore throat immediately after participating of a five-day meeting with european workers in rio de janeiro. her history included complete pediatric immunization (dtp) and three doses of adult formulation tetanus and diphtheria toxoid (dt) two years earlier. clinical diagnosis of diphtheria was not made until microbiologic examination of specimens confirmed toxigenicity of corynebacterium diphtheriae var. gravis, a biotype currently found circulating within europe where diphtheria remains epidemic. this case reinforces the potential susceptibility of brazilian adults to epidemic diphtheria in the vaccine era.
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