Analysis of genotypic variation in genes associated with virulence in Aggregatibacter actinomycetemcomitans clinical isolates

Background and Objective: Although certain serotypes of Aggregatibacter actinomycetemcomitans are associated more with aggressive periodontitis than are other serotypes, the correlation between distinct lineages and virulence traits in this species is poorly understood. This study aimed to evaluate the polymorphism of genes encoding putative virulence factors of clinical isolates, and to correlate these findings with A. actinomycetemcomitans serotypes, genotypes and periodontal status of the hosts. Material and Methods: Twenty-six clinical isolates from diverse geographic populations with different periodontal conditions were evaluated. Genotyping was performed using pulse-field gel electrophoresis. Polymorphisms in the genes encoding leukotoxin, Aae, ApaH and determinants for serotype-specific O polysaccharide were investigated. Results: The isolates were classified into serotypes a-f, and exhibited three apaH genotypes, five aae alleles and 25 macrorestriction profiles. Two serotype b isolates (7.7%), obtained from Brazilian patients with aggressive periodontitis, were associated with the highly leukotoxic genotype; these isolates showed identical fingerprint patterns and aae and apaH genotypes. Serotype c, obtained from various periodontal conditions, was the most prevalent among Brazilian isolates, and isolates were distributed in two aae alleles, but formed a genetically distinct group based on apaH analysis. Cluster analysis showed a close relationship between fingerprinting genotypes and serotypes/apaH genotypes, but not with aae genotypes. Conclusion: Apart from the deletion in the ltx promoter region, no disease-associated markers were identified. Non-JP2-like strains recovered from individuals with periodontal disease exhibited considerable genetic variation regarding aae/apaH genotypes, serotypes and XhoI DNA fingerprints.

Construction and preliminary characterization of a river buffalo bacterial artificial chromosome library

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

DNA fragmentation by gamma radiation and electron beams using atomic force microscopy

Double-stranded pBS plasmid DNA was irradiated with gamma rays at doses ranging from 1 to 12 kGy and electron beams from 1 to 10 kGy. Fragment-size distributions were determined by direct visualization, using atomic force microscopy with nanometer-resolution operating in non-tapping mode, combined with an improved methodology. The fragment distributions from irradiation with gamma rays revealed discrete-like patterns at all doses, suggesting that these patterns are modulated by the base pair composition of the plasmid. Irradiation with electron beams, at very high dose rates, generated continuous distributions of highly shattered DNA fragments, similar to results at much lower dose rates found in the literature. Altogether, these results indicate that AFM could supplement traditional methods for high-resolution measurements of radiation damage to DNA, while providing new and relevant information.

Prevalence, populations and pheno- and genotypic characteristics of Listeria monocytogenes isolated from ready-to-eat vegetables marketed in Sao Paulo, Brazil

Listeria monocytogenes is a foodborne pathogen of great concern due to the high fatality rates of listeriosis. The consumption of RTE vegetables has increased in Brazil over the last two decades, but little is known about the risks associated to the consumption of these products. This study evaluated the prevalence and counts of L. monocytogenes in 512 packages of ready-to-eat vegetables marketed in Sao Paulo. The isolates were characterized for their serotypes, ribotypes, positivity for virulence genes inIA, inIC and inIJ, resistance to chlorine, growth rate variability and capability to form biofilm on stainless steel (AISI 304, #4) coupons. L. monocytogenes was detected in 3.1% of the samples. Only five samples presented countable levels, with counts between 1.0x10(1) and 2.6x10(2) CFU/g. Isolates belonged to serotypes 1/2b or 4b and most were positive for genes inIC and inIJ. Ribotypable isolates were grouped into four groups: 1038 (69.4%). 19175 (11.3%), 19191 (17.7%) and 18604 (one isolate). Most isolates survived to exposure to 125 ppm of a chlorine-based disinfectant for 3 min. All isolates were capable to attach to the coupons, reaching counts above 4 log(10) CFU/cm(2) and the growth rate (mu) at 25 degrees C of the majority of the isolates varied between 0.1 and 0.2 log OD/h, but for few strains the mu was as high as 0.26 log OD/h. Results of this survey indicate that RTE vegetables may be vehicles of L. monocytogenes strains with limited variation in serotype, ribotype and virulence factors but varying significantly in resistance to chlorine disinfectants, capability of forming biofilm and growth rate. Data obtained is of foremost importance to serve as baseline for the development of scientific-based policies to control the incidence of L. monocytogenes in RTE vegetables in Brazil. (c) 2012 Elsevier B.V. All rights reserved.

Long range physical map of the Wilms' tumor - aniridia region on human chromosome 11

Nephroblastoma or Wilms' tumor is a pediatric renal malignancy that is the most frequently occurring childhood solid tumor. Approximately 1-2% of children with Wilms' tumor also present with aniridia, a congenital absence of all or part of the iris of the eye. These children also have high rates of genitourinary anomalies and mental retardation resulting in what is called the WAGR (Wilms' tumor, aniridia, genitourinary anomaly, mental retardation) syndrome. Cytogenetic analysis of metaphase chromosomes from these patients revealed a consistent deletion of band P13 on chromosome 11. These observations suggest close physical linkage between the disease-related loci, and further imply that development of each phenotype results from the loss of normal gene function.^ The objective of this work is to understand the molecular events at chromosome band 11p13 that are essential to the development of sporadic Wilms' tumor and sporadic aniridia. Two human/hamster somatic cell hybrids have been used to identify sixteen independent DNA probes that map to this segment of the human genome. These newly identified DNA probes and four previously reported probes (CAT, FSHB, D11S16, and HBVIS) have been used to subdivide 11p13 into five intervals defined by overlapping constitutional deletions from several WAGR patients. A long-range physical map of 11p13 has been constructed using each of these probes in Southern blot analysis of genomic DNA after digestion with infrequently cutting restriction enzymes and pulse-field gel electrophoresis. This map, established primarily with MluI and NotI, spans approximately 13 $\times$ 10$\sp{6}$ bp and encompasses deletion and translocation breakpoints associated with genitourinary anomalies, aniridia, and sporadic Wilms' tumor. This complete physical map of human chromosome band 11p13 enables us to localize the genes for sporadic Wilms' tumor and sporadic aniridia to a small number of specific NotI fragments. ^

Characterization of the transposition pattern of the Ac element in Arabidopsis thaliana using endonuclease I-SceI

We have investigated physical distances and directions of transposition of the maize transposable element Ac in Arabidopsis thaliana. We prepared a transferred DNA (T-DNA) construct that carried a non-autonomous derivative of Ac with a site for cleavage by endonuclease I-SceI (designated dAc-I-RS element). Another cleavage site was also introduced into the T-DNA region outside dAc-I-RS. Three transgenic Arabidopsis plants were generated, each of which had a single copy of the T-DNA at a different chromosomal location. These transgenic plants were crossed with the Arabidopsis that carried the gene for Ac transposase and progeny in which dAc-I-RS had been transposed were isolated. After digestion of the genomic DNA of these progeny with endonuclease I-SceI, sizes of segment of DNA were determined by pulse-field gel electrophoresis. We also performed linkage analysis for the transposed elements and sites of mutations near the elements. Our results showed that 50% of all transposition events had occurred within 1,700 kb on the same chromosome, with 35% within 200 kb, and that the elements transposed in both directions on the chromosome with roughly equal probability. The data thus indicate that the Ac–Ds system is most useful for tagging of genes that are present within 200 kb of the chromosomal site of Ac in Arabidopsis. In addition, determination of the precise localization of the transposed dAc-I-RS element should definitely assist in map-based cloning of genes around insertion sites.

Octamer-based genome scanning distinguishes a unique subpopulation of Escherichia coli O157:H7 strains in cattle

Multilocus-genotyping methods have shown that Escherichia coli O157:H7 is a geographically disseminated clone. However, high-resolution methods such as pulse-field gel electrophoresis demonstrate significant genomic diversity among different isolates. To assess the genetic relationship of human and bovine isolates of E. coli O157:H7 in detail, we have developed an octamer-based genome-scanning methodology, which compares the distance between over-represented, strand-biased octamers that occur in the genome. Comparison of octamer-based genome-scanning products derived from >1 megabase of the genome demonstrated the existence of two distinct lineages of E. coli O157:H7 that are disseminated within the United States. Human and bovine isolates are nonrandomly distributed among the lineages, suggesting that one of these lineages may be less virulent for humans or may not be efficiently transmitted to humans from bovine sources. Restriction fragment length polymorphism analysis with lambdoid phage genomes indicates that phage-mediated events are associated with divergence of the lineages, thereby providing one explanation for the degree of diversity that is observed among E. coli O157:H7 by other molecular-fingerprinting methods.

Genomic cloning of methylthioadenosine phosphorylase: a purine metabolic enzyme deficient in multiple different cancers.

5'-Deoxy-5'-methylthioadenosine phosphorylase (methylthioadeno-sine: ortho-phosphate methylthioribosyltransferase, EC 24.2.28; MTAP) plays a role in purine and polyamine metabolism and in the regulation of transmethylation reactions. MTAP is abundant in normal cells but is deficient in many cancers. Recently, the genes for the cyclin-dependent kinase inhibitors p16 and p15 have been localized to the short arm of human chromosome 9 at band p21, where MTAP and interferon alpha genes (IFNA) also map. Homozygous deletions of p16 and p15 are frequent malignant cell lines. However, the order of the MTAP, p16, p15, and IFNA genes on chromosome 9p is uncertain, and the molecular basis for MTAP deficiency in cancer is unknown. We have cloned the MTAP gene, and have constructed a topologic map of the 9p21 region using yeast artificial chromosome clones, pulse-field gel electrophoresis, and sequence-tagged-site PCR. The MTAP gene consists of eight exons and seven introns. Of 23 malignant cell lines deficient in MTAP protein, all but one had complete or partial deletions. Partial or total deletions of the MTAP gene were found in primary T-cell acute lymphoblastic leukemias (T-ALL). A deletion breakpoint of partial deletions found in cell lines and primary T-ALL was in intron 4. Starting from the centromeric end, the gene order on chromosome 9p2l is p15, p16, MTAP, IFNA, and interferon beta gene (IFNB). These results indicate that MTAP deficiency in cancer is primarily due to codeletion of the MTAP and p16 genes.

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis H37Rv, and comparison with Mycobacterium leprae.

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis, was constructed by using a twin-pronged approach. Pulsed-field gel electrophoretic analysis enabled cleavage sites for Asn I and Dra I to be positioned on the 4.4-Mb circular chromosome, while, in parallel, clones from two cosmid libraries were ordered into contigs by means of fingerprinting and hybridization mapping. The resultant contig map was readily correlated with the physical map of the genome via the landmarked restriction sites. Over 165 genes and markers were localized on the integrated map, thus enabling comparisons with the leprosy bacillus, Mycobacterium leprae, to be undertaken. Mycobacterial genomes appear to have evolved as mosaic structures since extended segments with conserved gene order and organization are interspersed with different flanking regions. Repetitive sequences and insertion elements are highly abundant in M. tuberculosis, but the distribution of IS6110 is apparently nonrandom.

Rifampicin and sodium fusidate reduces the frequency of methicillin-resistant Staphylococcus aureus (MRSA) isolation in adults with cystic fibrosis and chronic MRSA infection

Nosocomial transmission of methicillin-resistant Staphylococcus aureus (MRSA) to patients with cystic fibrosis (CF) frequently results in chronic respiratory tract carriage. This is an increasing problem, adds to the burden of glycopeptide antibiotic use in hospitals, and represents a relative contraindication to lung transplantation. The aim of this study was to determine whether it is possible to eradicate MRSA with prolonged oral combination antibiotics, and whether this treatment is associated with improved clinical status. Adult CF patients (six mate, one female) with chronic MRSA infection were treated for six months with rifampicin and sodium fusidate. Outcome data were examined for six months before treatment, on treatment and after treatment. The patients had a mean age of 29.3 (standard deviation = 6.3) years and FEV1 of 36.1% (standard deviation = 12.7) predicted. The mean duration of MRSA isolation was 31 months. MRSA isolates identified in these patients was of the same lineage as the known endemic strain at the hospital when assessed by pulsed-field get electrophoresis. Five of the seven had no evidence of MRSA during and for at [east six months after rifampicin and sodium fusidate. The proportion of sputum samples positive for MRSA was lower during the six months of treatment (0.13) and after treatment (0.19) compared with before treatment (0.85) (P < 0.0001). There was a reduction in the number of days of intravenous antibiotics per six months with 20.3 +/- 17.6 on treatment compared with 50.7 before treatment and 33.0 after treatment (P = 0.02). There was no change in lung function. Gastrointestinal side effects occurred in three, but led to therapy cessation in only one patient. Despite the use of antibiotics with anti-staphylococcal activity for treatment of respiratory exacerbation, MRSA infection persists. MRSA can be eradicated from the sputum of patients with CF and chronic MRSA carriage by using rifampicin and sodium fusidate for six months. This finding was associated with a significant reduction in the duration of intravenous antibiotic treatment during therapy. (C) 2003 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Staphylococcus aureus genotyping using novel real-time PCR formats

One approach to microbial genotyping is to make use of sets of single-nucleotide polymorphisms (SNPs) in combination with binary markers. Here we report the modification and automation of a SNP-plus-binary-marker-based approach to the genotyping of Staphylococcus aureus and its application to 391 S. aureus isolates from southeast Queensland, Australia. The SNPs used were arcC210, tpi243, arcC162, gmk318, pta294, tpi36, tpi241, and pta383. These provide a Simpson's index of diversity (D) of 0.95 with respect to the S. aureus multilocus sequence typing database and define 61 genotypes and the major clonal complexes. The binary markers used were pvl, cna, sdrE, pT181, and pUB110. Two novel real-time PCR formats for interrogating these markers were compared. One of these makes use of light upon extension (LUX) primers and biplexed reactions, while the other is a streamlined modification of kinetic PCR using SYBR green. The latter format proved to be more robust. In addition, automated methods for DNA template preparation, reaction setup, and data analysis were developed. A single SNP-based method for ST-93 (Queensland clone) identification was also devised. The genotyping revealed the numerical importance of the South West Pacific and Queensland community-acquired methicillin-resistant S. aureus (MRSA) clones and the clonal complex 239 Aus-1/Aus-2 hospital-associated MRSA. There was a strong association between the community-acquired clones and pvl.

Cartographie des cassures bicaténaires du remodelage chromatinien du spermatide et développement des outils techniques associés.

Résumé : La phase haploïde de la spermatogenèse (spermiogenèse) est caractérisée par une modification importante de la structure de la chromatine et un changement de la topologie de l’ADN du spermatide. Les mécanismes par lesquels ce changement se produit ainsi que les protéines impliquées ne sont pas encore complètement élucidés. Mes travaux ont permis d’établir la présence de cassures bicaténaires transitoires pendant ce remodelage par l’essai des comètes et l’électrophorèse en champ pulsé. En procédant à des immunofluorescences sur coupes de tissus et en utilisant un extrait nucléaire hautement actif, la présence de topoisomérases ainsi que de marqueurs de systèmes de réparation a été confirmée. Les protéines de réparation identifiées font partie de systèmes sujets à l’erreur, donc cette refonte structurale de la chromatine pourrait être génétiquement instable et expliquer le biais paternel observé pour les mutations de novo dans de récentes études impliquant des criblages à haut débit. Une technique permettant l’immunocapture spécifique des cassures bicaténaires a été développée et appliquée sur des spermatides murins représentant différentes étapes de différenciation. Les résultats de séquençage à haut débit ont montré que les cassures bicaténaires (hotspots) de la spermiogenèse se produisent en majorité dans l’ADN intergénique, notamment dans les séquences LINE1, l’ADN satellite et les répétions simples. Les hotspots contiennent aussi des motifs de liaisons des protéines des familles FOX et PRDM, dont les fonctions sont entre autres de lier et remodeler localement la chromatine condensée. Aussi, le motif de liaison de la protéine BRCA1 se trouve enrichi dans les hotspots de cassures bicaténaires. Celle-ci agit entre autres dans la réparation de l’ADN par jonction terminale non-homologue (NHEJ) et dans la réparation des adduits ADN-topoisomérase. De façon remarquable, le motif de reconnaissance de la protéine SPO11, impliquée dans la formation des cassures méiotiques, a été enrichi dans les hotspots, ce qui suggère que la machinerie méiotique serait aussi utilisée pendant la spermiogenèse pour la formation des cassures. Enfin, bien que les hotspots se localisent plutôt dans les séquences intergéniques, les gènes ciblés sont impliqués dans le développement du cerveau et des neurones. Ces résultats sont en accord avec l’origine majoritairement paternelle observée des mutations de novo associées aux troubles du spectre de l’autisme et de la schizophrénie et leur augmentation avec l’âge du père. Puisque les processus du remodelage de la chromatine des spermatides sont conservés dans l’évolution, ces résultats suggèrent que le remodelage de la chromatine de la spermiogenèse représente un mécanisme additionnel contribuant à la formation de mutations de novo, expliquant le biais paternel observé pour certains types de mutations.

Separation and Mapping of Chromosomes of Parasitic Protozoa

Many protozoan parasites represent an important group of human pathogens. Pulsed Field Gradient Gel Electrophoresis (PFGE) analysis has been an important tool for fundamental genetic studies of parasites like Trypanosoma, Leishmania, Giardia or the human malaria parasite Plasmodium falciparum. We present PFGE conditions allowing a high resolution separation of chromosomes ranging from 500 to 4000 kb within a two day electrophoresis run. In addition, we present conditions for separating large chromosomes (2000-6000 kb) within 36 hr. We demontrate that the application of two dimentional PFGE (2D-PFGE) technique to parasite karyotypes is a very useful method for the analysis of dispersed gene families and comparative studies of the intrachomosomal genome organization

Bacterial soil community in a Brazilian sugarcane field

The assessment of bacterial communities in soil gives insight into microbial behavior under prevailing environmental conditions. In this context, we assessed the composition of soil bacterial communities in a Brazilian sugarcane experimental field. The experimental design encompassed plots containing common sugarcane (variety SP80-1842) and its transgenic form (IMI-1 - imazapyr herbicide resistant). Plants were grown in such field plots in a completely randomized design with three treatments, which addressed the factors transgene and imazapyr herbicide application. Soil samples were taken at three developmental stages during plant growth and analyzed using 16S ribosomal RNA (rRNA)-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and clone libraries. PCR-DGGE fingerprints obtained for the total bacterial community and specific bacterial groups - Actinobacteria, Alphaproteobacteria and Betaproteobacteria - revealed that the structure of these assemblages did not differ over time and among treatments. Nevertheless, slight differences among 16S rRNA gene clone libraries constructed from each treatment could be observed at particular cut-off levels. Altogether, the libraries encompassed a total of eleven bacterial phyla and the candidate divisions TM7 and OP10. Clone sequences affiliated with the Proteobacteria, Actinobacteria, Firmicutes and Acidobacteria were, in this order, most abundant. Accurate phylogenetic analyses were performed for the phyla Acidobacteria and Verrucomicrobia, revealing the structures of these groups, which are still poorly understood as to their importance for soil functioning and sustainability under agricultural practices.

Characterization of sol-gel matrices with entrapped cutinase

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica