Pseudomonas aeruginosa: study of Antibiotic Resistance and Molecular Typing in Hospital Infection Cases in a Neonatal Intensive Care Unit from Rio de Janeiro City, Brazil

This study had the objective of to analyze the demographic and bacteriologic data of 32 hospitalized newborns in an neonatal intensive care unit of a public maternity hospital in Rio de Janeiro city, Brazil, seized by Pseudomonas aeruginosa sepsis during a period ranged from July 1997 to July 1999, and to determine the antimicrobial resistance percentage, serotypes and pulsed field gel electrophoresis (PFGE) patterns of 32 strains isolated during this period. The study group presented mean age of 12.5 days, with higher prevalence of hospital infection in males (59.4%) and vaginal delivery (81.2%), than females (40.6%) and cesarean delivery (18.8%), respectively. In this group, 20 (62.5%) patients received antimicrobials before positive blood cultures presentation. A total of 87.5% of the patients were premature, 62.5% presented very low birth weight and 40.6% had asphyxia. We detected high antimicrobial resistance percentage to b-lactams, chloramphenicol, trimethoprim/sulfamethoxazole and tetracycline among the isolated strains. All isolated strains were classified as multi-drug resistant. Most strains presented serotype O11 while PFGE analysis revealed seven distinct clones with isolation predominance of a single clone (75%) isolated from July 1997 to June 1998.

Detecció, identificació de Mycobacterium tuberculosis i caracterització molecular de la resistència als principals antituberculosos en mostra clínica directa mitjançant piroseqüenciació: aplicabilitat en l’estudi de la clonalitat de les soques.

L’augment de soques de Mycobacerium tuberculosis multiresistents i l’aparició de soques extremadament resistents, ha posat de manifest la necessitat de disposar de nous mètodes de detecció precoç de M.tuberculosis i les seves resistències als principals fàrmacs antituberculosos, així com l’estudi eficaç dels brots, per a poder dur a terme un control eficient de la malaltia. L’objectiu de l’estudi va ser determinar la capacitat de la piroseqüenciació per a detectar i identificar micobacteris a partir d’aïllats clínics i mostra directa, determinar el seu patró de resistència a Rifampicina, Isoniacida, Etambutol, Fluoroquinolones, Canamicina i Capreomicina i explorar la seva potencialitat per a ser emprada en el tipatge molecular de les soques. Mitjançant la optimització de la tecnologia de la piroseqüenciació pels gens analitzats, i el disseny de primers específics, hem verificat la utilitat de la piroseqüenciació per al maneig de la tuberculosi. S’ha pogut estudiar la presència de mutacions relacionades amb resistències a fàrmacs de primera línia en el 96.5% de les posicions analitzades en soca clínica i en el 70.4% en mostra directa. Van concordar amb el resultats obtinguts per altres mètodes fenotípics i/o fenotípics el 97.1% i el 98.2% del resultats obtinguts en soca clínica i mostra directa respectivament. La piroseqüenciació ens ha permet analitzar la presència de mutacions relacionades amb resistències a antituberculosos de segona línia, servint com a mètode de confirmació en l’anàlisi d’altres mètodes genotípics. La tècnica ens ha permès també identificar els principals micobacteris no tuberculosos implicats en la infecció humana, sòls o en coinfecció. Resultats preliminars mostren que l’anàlisi amb piroseqüenciació pot ser d’utilitat per l’estudi clonal de les soques de M.tuberculosis. Les nostres observacions mostren la piroseqüenciació com una eina valuosa en l’àmbit clínic, ja que permet una reducció de la demora del diagnòstic, estudi filogenètic i detecció de resistències M.tuberculosis, i per tant una millora de l’aplicació del tractament adequat, ajudant al control de la malaltia.

Intergenic and external transcribed spacers of ribosomal RNA genes in lizard-infecting Leishmania: molecular structure and phylogenetic relationship to mammal-infecting Leishmania in the subgenus Leishmania (Leishmania)

To establish the relationships of the lizard- and mammal-infecting Leishmania, we characterized the intergenic spacer region of ribosomal RNA genes from L. tarentolae and L. hoogstraali. The organization of these regions is similar to those of other eukaryotes. The intergenic spacer region was approximately 4 kb in L. tarentolae and 5.5 kb in L. hoogstraali. The size difference was due to a greater number of 63-bp repetitive elements in the latter species. This region also contained another element, repeated twice, that had an inverted octanucleotide with the potential to form a stem-loop structure that could be involved in transcription termination or processing events. The ribosomal RNA gene localization showed a distinct pattern with one chromosomal band (2.2 Mb) for L. tarentolae and two (1.5 and 1.3 Mb) for L. hoogstraali. The study also showed sequence differences in the external transcribed region that could be used to distinguish lizard Leishmania from the mammalian Leishmania. The intergenic spacer region structure features found among Leishmania species indicated that lizard and mammalian Leishmania are closely related and support the inclusion of lizard-infecting species into the subgenus Sauroleishmania proposed by Saf'janova in 1982.

Cloning and Molecular Characterization of the Schistosoma mansoni Genes RbAp48 and Histone H4

The human nuclear protein RbAp48 is a member of the tryptophan/aspartate (WD) repeat family, which binds to the retinoblastoma (Rb) protein. It also corresponds to the smallest subunit of the chromatin assembly factor and is able to bind to the helix 1 of histone H4, taking it to the DNA in replication. A cDNA homologous to the human gene RbAp48 was isolated from a Schistosoma mansoni adult worm library and named SmRbAp48. The full length sequence of SmRbAp48 cDNA is 1036 bp long, encoding a protein of 308 amino acids. The transcript of SmRbAp48 was detected in egg, cercariae and schistosomulum stages. The protein shows 84% similarity with the human RbAp48, possessing four WD repeats on its C-terminus. A hypothetical tridimensional structure for the SmRbAp48 C-terminal domain was constructed by computational molecular modeling using the b-subunit of the G protein as a model. To further verify a possible interaction between SmRbAp48 and S. mansoni histone H4, the histone H4 gene was amplified from adult worm genomic DNA using degenerated primers. The gene fragment of SmH4 is 294 bp long, encoding a protein of 98 amino acids which is 100% identical to histone H4 from Drosophila melanogaster.

Molecular mechanisms for the transcriptional regulation of the Connexin36 gene expression in insulin-secreting cells

Résumé Régulation de l'expression de la Connexin36 dans les cellules sécrétrices d'insuline La communication intercellulaire est en partie assurée via des jonctions communicantes de type "gap". Dans la cellule ß pancréatique, plusieurs observations indiquent que le couplage assuré par des jonctions gap formées parla Connexine36 (Cx36) est impliqué dans le contrôle de la sécrétion de l'insuline. De plus, nous avons récemment démontré qu'un niveau précis d'expression de la Cx36 est nécessaire pour maintenir une bonne coordination de l'ensemble des cellules ß, et permettre ainsi une sécrétion synchrone et contrôlée d'insuline. Le développement du diabète et du syndrome métabolique est partiellement dû à une altération de la capacité des cellules ß à sécréter de l'insuline en réponse à une augmentation de la glycémie. Cette altération est en partie causée par l'augmentation prolongée des taux circulant de glucose, mais aussi de lipides, sous la forme d'acides gras libres, et de LDL (Low Density Lipoproteins), particules assurant le transport des acides gras et du cholestérol dans le sang. Nous avons étudié la régulation de l'expression de la Cx36 dans différentes conditions reflétant la physiopathologie du diabète de type 2 et du syndrome métabolique et démontré qu'une exposition prolongée à des concentrations élevées de glucose, de LDL, ainsi que de palmitate (acide gras saturé le plus abondant dans l'organisme), inhibent l'expression de la Cx36 dans les cellules ß. Cette inhibition implique l'activation de la PKA (Proteine Kinase A), qui stimule à son tour l'expression du facteur de transcription ICER-1 (Inductible cAMP Early Repressor-1). Ce puissant répresseur se fixe spécifiquement sur un motif CRE (cAMP Response Element), situé dans le promoteur du gène de la Cx36, inhibant ainsi son expression. Nous avons de plus démontré que des cytokines pro-inflammatoires, qui pourraient contribuer au développement du diabète, inhibent également l'expression de la Cx36. Cependant, les cytokines agissent indépendamment du répresseur ICER-1, mais selon un mécanisme requérant l'activation de l'AMPK (AMP dependant protein kinase). Sachant qu'un contrôle précis des niveaux d'expression de la Cx36 est un élément déterminant pour une sécrétion optimale de l'insuline, nos résultats suggèrent que la Cx36 pourrait être impliquée dans l'altération de la sécrétion de l'insuline contribuant à l'apparition du diabète de type 2. Summary A particular way by which cells communicate with each other is mediated by gap junctions, transmembrane structures providing a direct pathway for the diffusion of small molecules between adjacent cells. Gap junctional communication is required to maintain a proper functioning of insulin-secreting ß-cells. Moreover, the expression levels of connexin36 (Cx36), the sole gap junction protein expressed in ß-cells, are critical in maintaining glucose-stimulated insulin secretion. Chronic hyperglycemia and hyperlipidemia exert deleterious effects on insulin secretion and may contribute to the progressive ß-cell failure linked to the development of type 2 diabetes and metabolic syndrome. Since modulations of the Cx36 levels might impair ß-cell function, the general aim of this work was to elucidate wether elevated levels of glucose and lipids affect Cx36 expression. The first part of this work was dedicated to the study of the effect of high glucose concentrations on Cx36 expression. We demonstrated that glucose transcriptionally down-regulates the expression of Cx36 in insulin-secreting cells through activation of the protein kinase A (PKA), which in turn stimulates the expression of the inducible cAMP early repressor-1 (ICER-1). This repressor binds to a highly conserved cAMP response element (CRE) located in the Cx36 promoter, thereby inhibiting Cx36 expression. The second part of this thesis consisted in studying the effects of sustained exposure to free fatty acids (FFA) and human lipoproteins on Cx36 levels. The experiments revealed that the most abundant FFA, palmitate, as well as the atherogenic low density lipoproteins (LDL), also stimulate ICER-1 expression, resulting in Cx36 down-regulation. Finally, the third part of the work focused on the consequences of long-term exposure to proinflammatory cytokines on Cx36 content. Interleukin-1 ß (IL-1 ß) inhibits Cx36 expression and its effect is potentialized by tumor necrosis factor α (TNFα) and interferon γ (IFNγ). We further unveiled that the cytokines effect on Cx36 levels requires activation of the AMP dependent protein kinase (AMPK). Prolonged exposures to glucose, palmitate, LDL, and pro-inflammatory cytokines have all been proposed to contribute to the development of diabetes and metabolic syndrome. Since Cx36 expression levels are critical to maintain ß-cell function, Cx36 down-regulation by glucose, lipids, and cytokines might participate to the ß-cell failure associated with diabetes development.

Partial molecular characterization of Sm8, a tegumental antigen of Schistosoma mansoni

Sm8 is a major tegumental antigen of Schistosoma mansoni. The partial cDNA was isolated and analyzed. Sequence analysis revealed transmembrane compatible hydrophobic domains and a putative leucine zipper pattern. The mRNA and the protein are predominantly expressed in adult worms.

Comprehensive clinical and molecular analysis of 12 families with type 1 recessive cutis laxa.

Autosomal recessive cutis laxa type I (ARCL type I) is characterized by generalized cutis laxa with pulmonary emphysema and/or vascular complications. Rarely, mutations can be identified in FBLN4 or FBLN5. Recently, LTBP4 mutations have been implicated in a similar phenotype. Studying FBLN4, FBLN5, and LTBP4 in 12 families with ARCL type I, we found bi-allelic FBLN5 mutations in two probands, whereas nine probands harbored biallelic mutations in LTBP4. FBLN5 and LTBP4 mutations cause a very similar phenotype associated with severe pulmonary emphysema, in the absence of vascular tortuosity or aneurysms. Gastrointestinal and genitourinary tract involvement seems to be more severe in patients with LTBP4 mutations. Functional studies showed that most premature termination mutations in LTBP4 result in severely reduced mRNA and protein levels. This correlated with increased transforming growth factor-beta (TGFβ) activity. However, one mutation, c.4127dupC, escaped nonsense-mediated decay. The corresponding mutant protein (p.Arg1377Alafs(*) 27) showed reduced colocalization with fibronectin, leading to an abnormal morphology of microfibrils in fibroblast cultures, while retaining normal TGFβ activity. We conclude that LTBP4 mutations cause disease through both loss of function and gain of function mechanisms.

Molecular epidemiology of human immunodeficiency virus-1 in the state of Ceará, Northeast, Brazil

We analyzed, by env and gag heteroduplex mobility assay, 149 human immunodeficiency virus (HIV-1) positive samples collected in Ceará during the year 2000. The prevalence of subtype B was 81.2% and the prevalence of subtype F and B/F recombinants were both 2.7%. Eight (5.4%) and 12 (8%) out of 149 samples showed indeterminate results in the env and gag analysis respectively. By FokI restriction fragment length polymorphism, 34% of the subtype B samples were identified as the typical Brazilian subtype B.In the present study, we identified HIV-1 subtype F and B/F in Ceará for the first time. Our results contribute to the understanding of HIV in Brazil, and may prove useful for the development of vaccine candidates.

Hepatitis B and C in the hemodialysis unit of Tocantins, Brazil: serological and molecular profiles

A survey was conducted in the hemodialysis population of the state of Tocantins, Brazil, aiming to assess the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, to analyze associated risk factors, and also to investigate these viruses genotypes distribution. During January and March 2001, all patients (n = 100) were interviewed at the unique dialysis unit in Tocantins. Blood samples were collected and serum samples were screened for HBV serological markers. Hepatitis B surface antigen positive samples were tested for HBV DNA. All samples were also tested for anti-HCV antibodies and HCV RNA. An overall prevalence of 45% was found for HBV infection (4% were HBsAg/anti-HBc positive, 2% were anti-HBc only and 39% had anti-HBc/anti-HBs markers). Concerning HCV infection, anti-HCV and HCV RNA were detected in 13% and 14% of the subjects, respectively. Three patients were HCV RNA positive and anti-HCV negative, resulting in an overall HCV prevalence of 16%. Univariate analysis of risk factors showed that only shift and length of time on hemodialysis were associated with HBV and HCV positivity, respectively. Among the four HBsAg-positive samples, HBV DNA was detected in three of them, which were identified as genotype A by restriction fragment length polymorphism (RFLP) analysis. All 14 HCV RNA-positive samples were genotyped by INNO-LiPA. Genotypes 1a and 3a were found in 85% and 15%, respectively. The present data show low HBsAg and HCV prevalence rates. The risk factors associated with HBV and HCV positivity suggest that nosocomial transmission may influence in spreading these viruses in the dialysis unit studied.

Phenotypic and molecular characterization of proliferating and differentiated GnRH-expressing GnV-3 cells.

GnRH neurons provide the primary driving force upon the neuroendocrine reproductive axis. Here we used GnV-3 cells, a model of conditionally immortalized GnRH-expressing neurons, to perform an analysis of cell cycle and compare the gene expression profile of proliferating cells with differentiated cells. In the proliferation medium, 45 ± 1.5% of GnV-3 cells are in S-phase by FACS analysis. In the differentiation medium, only 9 ± 0.9% of them are in S-phase, and they acquire the characteristic bipolar shape displayed by preoptic GnRH neurons in vivo. In addition, GnV-3 cells in the differentiated state exhibit electrophysiological properties characteristic of neurons. Transcriptomic analysis identified up-regulation of 1931 genes and down-regulation of 1270 genes in cells grown in the differentiation medium compared to cells in the proliferation medium. Subsequent gene ontology study indicated that genes over-expressed in proliferating GnV-3 cells were mainly involved in cell cycle regulations, whereas genes over-expressed in differentiated cells were mainly involved in processes of differentiation, neurogenesis and neuronal morphogenesis. Taken together, these data demonstrate the occurrence of morphological and physiological changes in GnV-3 cells between the proliferating and the differentiated state. Moreover, the genes differentially regulated between these two different states are providing novel pathways potentially important for a better understanding of the physiology of mature GnRH neurons.

Use of molecular methods in identification of Candida Species and evaluation of fluconazole resistance

The aim of this study was to evaluate the use of one of the molecular typing methods such as PCR (polymerase chain reaction) following by RFLP (restriction fragment length polymorphism) analysis in the identification of Candida species and then to differentiate the identified azole susceptible and resistant Candida albicans strains by using AP-PCR (arbitrarily primed-polymerase chain reaction). The identification of Candida species by PCR and RFLP analysis was based on the size and primary structural variation of rDNA intergenic spacer regions (ITS). Forty-four clinical Candida isolates comprising 5 species were included to the study. The amplification products were digested individually with 3 different restriction enzymes: HaeIII, DdeI, and BfaI. All the isolates tested yielded the expected band patterns by PCR and RFLP analysis. The results obtained from this study demonstrate that Candida species can be differentiated as C. albicans and non-C. albicans strains only by using HaeIII restriction enzyme and BfaI maintains the differentiation of these non-C. albicans species. After identification Candida species with RFLP analysis, C. albicans strains were included to the AP-PCR test. By using AP-PCR, fluconazole susceptible and resistant strains were differentiated. Nine fluconazole susceptible and 24 fluconazole resistant C. albicans were included to the study. Fluconazole resistant strains had more bands when evaluating with the agarose gel electrophoresis but there were no specific discriminatory band patterns to warrant the differentiation of the resistance. The identification of Candida species with the amplification of intergenic spacer region and RFLP analysis is a practical, short, and a reliable method when comparing to the conventional time-consuming Candida species identification methods. The fluconazole susceptibility testing with AP-PCR seems to be a promising method but further studies must be performed for more specific results.

Molecular differentiation of Angiostrongylus costaricensis, A. cantonensis, and A. vasorum by polymerase chain reaction- restriction fragment length polymorphism

Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment length polymorphism was carried out. The rDNA second internal transcribed spacer (ITS2) and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study.