Molecular typing and virulence markers of Yersinia enterocolitica strains from human, animal and food origins isolated between 1968 and 2000 in Brazil

Molecular typing and virulence markers were used to evaluate the genetic profiles and virulence potential of 106 Yersinia enterocolitica strains. of these strains, 71 were bio-serotype 4/O: 3, isolated from human and animal clinical material, and 35 were of biotype 1 A or 2 and of diverse serotypes, isolated from food in Brazil between 1968 and 2000. Drug resistance was also investigated. All the strains were resistant to three or more drugs. The isolates showed a virulence-related phenotype in the aesculin, pyrazinamidase and salicin tests, except for the food isolates, only two of which were positive for these tests. For the other phenotypic virulence determinants (autoagglutination, Ca++ dependence and Congo red absorption), the strains showed a diverse behaviour. The inv, ail and ystA genes were detected in all human and animal strains, while all the food isolates were positive for inv, and 3% of them positive for ail and ystA. The presence of virF was variable in the three groups of strains. The strains were better discriminated by PFGE than by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). A higher genomic similarity was observed among the 4/O: 3 strains, isolated from human and animal isolates, than among the food strains, with the exception of two food strains possessing the virulence genes and grouped close to the 4/O: 3 strains by ERIC-PCR. Unusually, the results revealed the virulence potential of a bio-serotype 1 A/O: 10 strain, suggesting that food contaminated with Y. enterocolitica biotype 1 A may cause infection. This also suggests that ERIC-PCR may be used as a tool to reveal clues about the virulence potential of Y. enterocolitica strains. Furthermore, the results also support the hypothesis that animals may act as reservoirs of Y. enterocolitica for human infections in Brazil, an epidemiological aspect that has not been investigated in this country, confirming data from other parts of the world.

Polyclonal endemicity of Pseudomonas aeruginosa in a teaching hospital from Brazil: molecular typing of decade-old strains

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

First identification of Sarcocystis tenella (Railliet, 1886) Moule, 1886 (Protozoa: Apicomplexa) by PCR in naturally infected sheep from Brazil

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

Practical considerations of embryo manipulation: Preimplantation genetic typing

In the past years, research in embryo technologies is moving to the establishment of preimplantation genetic typing or also denominated preimplantation genetic diagnosis (PGD). The objectives of these tests are the prevention of genetic diseases transmission and the prediction of phenotypic characteristics, as well as sex determination, genetic disorders and productive and reproductive profiles, prior to the embryo transfer or freezing, during early stages of development. This paper points out the state-of-the-art of PGD, mainly in cattle and discuss the perspectives of multiloci genetic analysis of embryos. (C) 2001 by Elsevier B.V.

Genotyping of Brazilian Giardia duodenalis human axenic isolates

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

Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)

The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP Two coding substitutions were found in comparison With the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429] PLAP, into wildtype Chinese hamster ovary cells. We determined the k(cat) and K-m, of the PLAP S, F. and D allozymes using the non,physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5'-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples. Hum Mutat 19:258-267, 2002. (C) 2002 Wiley-Liss, Inc.

Loss of imprinting and loss of heterozygosity on 11p15.5 in head and neck squamous cell carcinomas

Background. IGF2 and H19 are reciprocal imprinted genes with paternal and maternal monoallelic expression, respectively. This is interesting, because IGF2 is known as a growth factor, and H19 encodes a RNA with putative tumor suppressor action. Furthermore, IGF2 and H19 are linked genes located on chromosome 11p15.5, a common site of loss of heterozygosity in human cancers.Methods. We performed an allelic-typing assay using a PCR-RFLP-based method for identification of heterozygous Informative cases in head and neck squamous cell carcinomas. Tumoral total RNA was extracted from each of the heterozygotes and further studied by RT-PCR analysis.Results. We detected the expression of the IGF2 gene in 10 of 10 informative cases. Two cases exhibited LOI of the IGF2 gene as evidenced by biallelic expression, and in another case, LOH was coupled with monoallelic expression of this growth factor. LOI for the H19 gene was observed in 1 of 14 informative samples analyzed. In this case, we also detected parallel mono-allelic expression of the IGF2 gene. Down-regulation of the H19 gene was observed in 10 of 14 cases.Conclusion. These findings support the hypothesis that H19 may be a tumor suppressor gene involved In head and neck carcinogenesis. Furthermore, our data showed that genetic and epigenetic chances at 11p15.5 could lead to abnormal expression of imprinted genes in HNSCC. (C) 2001 John Wiley & Sons, Inc.

A single multiplex PCR and SNaPshot minisequencing reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups

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

Comparison of a multiplex-PCR assay with mycolic acids analysis and conventional methods for the identification of Mycobacteria

A fast, sensitive and cost-effective multiplex-PCR assay for Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium (M. avium) identification for routine diagnosis was evaluated. A total of 158 isolates of mycobacteria from 448 clinical specimens from patients with symptoms of mycobacterial disease were analyzed. By conventional biochemical methods 151 isolates were identified as M. tuberculosis, five as M. avium and two as Mycobacterium chelonae (M. chelonae). Mycolic acid patterns confirmed these results. Multiplex-PCR detected only IS6110 in isolates identified as MTC, and IS1245 was found only in the M. avium isolates. The method applied to isolates from two patients, identified by conventional methods and mycolic acid analysis, one as M. avium and other as M. chelonae, resulted positive for IS6110, suggesting co-infection with M. tuberculosis. These patients were successfully submitted to tuberculosis treatment. The multiplex-PCR method may offer expeditious identification of MTC and M. avium, which may minimize risks for active transmission of these organisms and provide useful treatment information.

Tuberculose e o estudo molecular da sua epidemiologia

Systems that can distinguish epidemiologically-related Mycobacterium tuberculosis strains from unrelated ones are extremely valuable. Molecular biology techniques have allowed a great deal of information to be acquired about the infectious disease tuberculosis (TB) that was very hard or impossible to obtain by conventional epidemiology. A typing method based on bacterial DNA genome differences, known as RFLP (restriction fragment length polymorphism), is widely used to discriminate strains in the epidemiologic study of TB. However, RFLP is laborious and there is a tendency to replace it by other methods. Thus, other DNA sequences have been employed as epidemiological markers, as in Spoligotyping, a fast technique based on PCR followed by differential hybridization of amplified products. The polymorphism observed among different isolates is probably the product of strain-dependent recombination. MIRU (mycobacterial interspersed repetitive unit) typing is a reproducible and fast assay, involving the generation of genotypes based on the study of 12 loci containing VNTRs (variable-number tandem repeats) in strains of the M. tuberculosis complex. It compares strains from different geographic areas and allows the movement of individual lineages to be tracked, as in RFLP. This approach enables a greater number of isolates to be analyzed, leading to the identification of a larger number of foci of transmission within the population and thus to improved ways of slowing the progress of the disease.

Usefulness of Mycobacterium tuberculosis molecular typing in a tuberculosis low-endemic agro-industrial setting of Brazil

To highlight the transmission and major phylogenetic clades of Mycobacterium tuberculosis, a retrospective study was carried out at two health facilities in a small agro-industrial area in São Paulo, Brazil, that has a low tuberculosis incidence rate. IS6110-RFLP and spoligotyping were performed on the isolates, with the former revealing that 31.3% (35/112) of strains were clustered. Epidemiological links were found in 16 of the 35 clustered patients and were associated with transmission among patients living in public housing. Spoligotyping grouped 62.8% of the strains. The T genetic family predominated among the isolates. Of interest is that five strains had a pattern characteristic of African or Asian origin (ST535), and two others were of the rare localized type ST1888 (BRA, VEN). In addition, three new types-1889, 1890, and 1891-were identified. Spoligotyping showed that some ST may be circulating to or from Brazil, and RFLP revealed ongoing transmission in inadequately ventilated public-housing buildings. This may point to a failure in tuberculosis control policy.

Tipagem molecular, perfis de sensibilidade e caracterização de transcritos diferencialmente expressos durante a infecção de Cryptococcus neoformans

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Genotipagem do Mycobacterium tuberculosis utilizando RFLP e Spoligotyping em associação com Miru para avaliar a epidemiologia molecular da tuberculose no município de Araraquara-SP

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

Discriminação alélica do fator V da coagulação por PCR em tempo real: diagnóstico simples e preciso

Dentre as doenças cardiovasculares, a trombose venosa (TV) destaca-se pela associação entre fatores de riscos adquiridos e fatores genéticos. A resistência hereditária à proteína C ativada tem sido identificada como a principal causa dos casos de trombose venosa, sendo frequentemente associada à mutação fator V Leiden (G1694A). Em indivíduos homozigotos, o risco de trombose venosa é 50 a 100 vezes maior que em pacientes homozigotos normais, enquanto em pacientes heterozigotos o risco é de 5 a 10 vezes. Baseado na necessidade de avaliação e acompanhamento de pacientes com casos de trombose venosa e prevenção de seus respectivos familiares, foi desenvolvido um método simples de discriminação alélica do fator V da coagulação utilizando PCR em tempo real. Foram selecionados 67 pacientes com histórico de TV e 51 indivíduos sem histórico de TV. Primeiramente, a discriminação alélica do fator V foi realizada através de PCR convencional seguida de digestão enzimática (Mnl). Posteriormente, o diagnóstico foi realizado por PCR em tempo real. Ambos os métodos foram baseados no polimorfismo G1691A, sendo no segundo utilizado fluoróforos VIC e FAM para marcar os nucleotídeos G e A, respectivamente. A técnica de PCR-RFLP foi utilizada para diagnosticar 95 indivíduos homozigotos normais, 21 heterozigotos e 2 homozigotos FVL. Utilizando PCR em tempo real foram obtidos os mesmos resultados. A máxima similaridade entre os resultados obtidos por PCR em tempo real e PCR-RFLP indicou precisão significativa do novo método de discriminação e visualização alélica do fator V.

Staphylococcus aureus: resistência de virulência e tipagem de MRSA pelas técnicas de MLST e spa typing

Pós-graduação em Doenças Tropicais - FMB