Evolution of Drug Resistance: Insight on TEM -Lactamases Structure and Activity and Beta-Lactam Antibiotics

Extended-spectrum β-lactamases (ESBLs) prevalence was studied in the north of Portugal, among 193 clinical isolates belonging to citizens in a district in the boundaries between this country and Spain from a total of 7529 clinical strains. In the present study we recovered some members of Enterobacteriaceae family, producing ESBL enzymes, including Escherichia coli (67.9%), Klebsiella pneumoniae (30.6%), Klebsiella oxytoca (0.5%), Enterobacter aerogenes (0.5%), and Citrobacter freundii (0.5%). β-lactamases genes blaTEM, blaSHV, and blaCTX-M were screened by polymerase chain reaction (PCR) and sequencing approaches. TEM enzymes were among the most prevalent types (40.9%) followed by CTX-M (37.3%) and SHV (23.3%). Among our sample of 193 ESBL-producing strains 99.0% were resistant to the fourth-generation cephalosporin cefepime. Of the 193 isolates 81.3% presented transferable plasmids harboring genes. Clonal studies were performed by PCR for the enterobacterial repetitive intragenic consensus (ERIC) sequences. This study reports a high diversity of genetic patterns. Ten clusters were found for E. coli isolates and five clusters for K. pneumoniae strains by means of ERIC analysis. In conclusion, in this country, the most prevalent type is still the TEM-type, but CTX-M is growing rapidly.

A proteomic approach toward the selection of proteins with enhanced intrinsic conformational stability

Journal of Proteome Research (2006)5: 2720-2726

Transglutaminases do mosquito Anopheles gambiae: Caracterização genética, bioquímica e funcional durante a infecção por Plasmodium berghei

A malária constitui um problema de saúde pública, que tem vindo a agravar-se, sendo crescente a necessidade de estratégias renovadas para o seu controlo, como a interrupção do ciclo esporogónico. Deste modo, é essencial compreender as respostas imunológicas de Anopheles anti-Plasmodium. Demonstrou-se anteriormente, que a inibição de transglutaminases, enzimas que participam em vários processos biológicos ao catalisarem a formação de ligações covalentes entre péptidos, agrava a infecção em mosquitos pelo parasita. O presente trabalho tem por objectivo caracterizar as transglutaminases AGAP009098 e AGAP009100 de Anopheles gambiae. Os métodos utilizados para este efeito foram: a sequenciação de regiões dos genes AGAP009098 e AGAP009100; a clonagem molecular de fragmentos da região codificante do gene AGAP009098, usando o vector plasmídico pET–28a(+) e Escherichia coli como sistema de expressão; e PCR em Tempo Real para analisar a expressão relativa dos genes AGAP009098 e AGAP009100 nos diferentes os estádios de desenvolvimento. AGAP009098 é expressa ubiquamente e AGAP009100 a partir do estádio pupa. Estes resultados apontam para a conclusão de que AGAP009098 e AGAP009100 poderão desempenhar funções em processos biológicos relevantes, por exemplo na defesa imunitária, ou no desenvolvimento. Os péptidos recombinantes, obtidos a partir da clonagem com sucesso de fragmentos da região codificante do gene AGAP009098, constituem uma ferramenta importante para averiguar a função destas TGases, no futuro.

New Strategies on Molecular Biology Applied to Microbial Systematics

Systematics is the study of diversity of the organisms and their relationships comprising classification, nomenclature and identification. The term classification or taxonomy means the arrangement of the organisms in groups (rate) and the nomenclature is the attribution of correct international scientific names to organisms and identification is the inclusion of unknown strains in groups derived from classification. Therefore, classification for a stable nomenclature and a perfect identification are required previously. The beginning of the new bacterial systematics era can be remembered by the introduction and application of new taxonomic concepts and techniques, from the 50’s and 60’s. Important progress were achieved using numerical taxonomy and molecular taxonomy. Molecular taxonomy, brought into effect after the emergence of the Molecular Biology resources, provided knowledge that comprises systematics of bacteria, in which occurs great evolutionary interest, or where is observed the necessity of eliminating any environmental interference. When you study the composition and disposition of nucleotides in certain portions of the genetic material, you study searching their genome, much less susceptible to environmental alterations than proteins, codified based on it. In the molecular taxonomy, you can research both DNA and RNA, and the main techniques that have been used in the systematics comprise the build of restriction maps, DNA-DNA hybridization, DNA-RNA hybridization, sequencing of DNA sequencing of sub-units 16S and 23S of rRNA, RAPD, RFLP, PFGE etc. Techniques such as base sequencing, though they are extremely sensible and greatly precise, are relatively onerous and impracticable to the great majority of the bacterial taxonomy laboratories. Several specialized techniques have been applied to taxonomic studies of microorganisms. In the last years, these have included preliminary electrophoretic analysis of soluble proteins and isoenzymes, and subsequently determination of deoxyribonucleic acid base composition and assessment of base sequence homology by means of DNA-RNA hybrid experiments beside others. These various techniques, as expected, have generally indicated a lack of taxonomic information in microbial systematics. There are numberless techniques and methodologies that make bacteria identification and classification study possible, part of them described here, allowing establish different degrees of subspecific and interspecific similarity through phenetic-genetic polymorphism analysis. However, was pointed out the necessity of using more than one technique for better establish similarity degrees within microorganisms. Obtaining data resulting from application of a sole technique isolatedly may not provide significant information from Bacterial Systematics viewpoint

Rapid Characterization of S. mansoni Expression Library Clones of Potential Interest

A S. mansoni adult worm cDNA expression library was screened with sera from baboons in a early phase after infection. The clones that were positive with the early infection sera were examined for reactivity with pre-infection sera and heterologous infection sera. In order to discriminate a positive antibody reaction from the reactivity due to residual anti-E. coli antibodies, an unrelated cDNA clone was plated with the positive clone. The unrelated clone provided the negative background and the contrast necessary to discern a positive antibody reaction. In this way, we were able to eliminate selected clones that were positive with the pre-infection sera or heterologous infection sera. This characterization of the expression library clones enabled us to quickly target only clones with the desired pattern of antibody reactivity for sequencing, subcloning, and expressing

Genetic diversity of arginine catabolic mobile element in Staphylococcus epidermidis

PLos One, 4(11): ARTe7722

Predictive factors for response to Lamivudine in chronic hepatitis B

BACKGROUND: Lamivudine has been shown to be an efficient drug for chronic hepatitis B (CHB) treatment. AIM: To investigate predictive factors of response, using a quantitative method with high sensitivity. METHODS: We carried out a prospective trial of lamivudine in 35 patients with CHB and evidence for viral replication, regardless to their HBeAg status. Lamivudine was given for 12 months at 300 mg daily and 150 mg thereafter. Response was considered when DNA was undetectable by PCR after 6 months of treatment. Viral replication was monitored by end-point dilution PCR. Mutation associated with resistance to lamivudine was detected by DNA sequencing in non-responder patients. RESULTS: Response was observed in 23/35 patients (65.7%) but only in 5/15 (33.3%) HBeAg positive patients. Only three pre-treatment variables were associated to low response: HBeAg (p = 0.006), high viral load (DNA-VHB > 3 x 10(6) copies/ml) (p = 0.004) and liver HBcAg (p = 0.0028). YMDD mutations were detected in 7/11 non-responder patients. CONCLUSIONS: HBeAg positive patients with high viral load show a high risk for developing drug resistance. On the other hand, HBeAg negative patients show a good response to lamivudine even with high viremia.

A historical perspective and prospects of biomedical research on parasitic diseases

We all hope that biotechnology will answer some social and economical unavoidable requirements of the modern life. It is necessary to improve agriculture production, food abundance and health quality in a sustainable development. It is indeed a hard task to keep the progress on taking into account the rational use of genetic resources and the conservation of biodiversity. In this context, a historical perspective and prospects of the biomedical research on parasitic diseases is described in a view of three generations of investigators. This work begins with a picture of the scientific progress on biomedical research and human health over the last centuries. This black-and-white picture is painted by dissecting current advancements of molecular biology and modern genetics, which are outlined at the meaning of prospecting achievements in health science for this new millenium.

Blood-sucking lice may disseminate Trypanosoma cruzi infection in baboons

Trypanosoma cruzi (Schyzotrypanum, Chagas, 1909), and Chagas disease are endemic in captive-reared baboons at the Southwest Foundation for Biomedical Research, San Antonio, Texas. We obtained PCR amplification products from DNA extracted from sucking lice collected from the hair and skin of T. cruzi-infected baboons, with specific nested sets of primers for the protozoan kinetoplast DNA, and nuclear DNA. These products were hybridized to their complementary internal sequences. Selected sequences were cloned and sequencing established the presence of T. cruzi nuclear DNA, and minicircle kDNA. Competitive PCR with a kDNA set of primers determined the quantity of approximately 23.9 ± 18.2 T. cruzi per louse. This finding suggests that the louse may be a vector incidentally contributing to the dissemination of T. cruzi infection in the baboon colony.

High prevalence of GB Virus C/Hepatitis G Virus RNA among Brazilian blood donors

The aim of this study was to investigate the presence of the Hepatitis G Virus on a population of blood donors from São Paulo, Brazil and to evaluate its association to sociodemographic variables. Two RT-PCR systems targeting the putative 5'NCR and NS3 regions were employed and the former has shown a higher sensitivity. The observed prevalence of HGV-RNA on 545 blood donors was 9.7% (CI 95% 7.4;12.5). Statistical analysis depicted an association with race/ethnicity, black and mulatto donors being more frequently infected; and also with years of education, less educated donors presenting higher prevalences. No association was observed with other sociodemographic parameters as age, gender, place of birth and of residence. DNA sequencing of nine randomly chosen isolates demonstrated the presence of genotypes 1, 2 and 3 among our population but clustering of these Brazilian isolates was not detected upon phylogenetic analysis.

Determination of human cytomegalovirus genetic diversity in different patient populations in Costa Rica

Seroprevalence of HCMV in Costa Rica is greater than 95% in adults; primary infections occur early in life and is the most frequent congenital infection in newborns. The objectives of this study were to determine the genetic variability and genotypes of HCMV gB gene in Costa Rica. Samples were collected from alcoholics, pregnant women, blood donors, AIDS patients, hematology-oncology (HO) children and HCMV isolates from neonates with cytomegalic inclusion disease. A semi-nested PCR system was used to obtain a product of 293-296 bp of the gB gene to be analyzed by Single Stranded Conformational Polymorphism (SSCP) and sequencing to determine the genetic polymorphic pattern and genotypes, respectively. AIDS patients showed the highest polymorphic diversity with 14 different patterns while fifty-six percent of HO children samples showed the same polymorphic pattern, suggesting in this group a possible nosocomial infection. In neonates three genotypes (gB1, gB2 and gB3), were determined while AIDS patients and blood donors only showed one (gB2). Of all samples analyzed only genotypes gB1, 2 and 3 were determined, genotype gB2 was the most frequent (73%) and mixed infections were not detected. The results of the study indicate that SSCP could be an important tool to detect HCMV intra-hospital infections and suggests a need to include additional study populations to better determine the genotype diversity and prevalence.

Mapping of a Leishmania major gene/locus that confers pentamidine resistance by deletion and insertion of transposable element

Pentamidine (PEN) is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK) into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.

Human vaccinia-like virus outbreaks in São Paulo and Goiás States, Brazil: virus detection, isolation and identification

Since October 2001, the Adolfo Lutz Institute has been receiving vesicular fluids and scab specimens of patients from Paraíba Valley region in the São Paulo and Minas Gerais States and from São Patricio Valley, in the Goiás State. Epidemiological data suggested that the outbreaks were caused by Cowpox virus or Vaccinia virus. Most of the patients are dairy milkers that had vesiculo-pustular lesions on the hands, arms, forearms, and some of them, on the face. Virus particles with orthopoxvirus morphology were detected by direct electron microscopy (DEM) in samples of 49 (66.21%) patients of a total of 74 analyzed. Viruses were isolated in Vero cell culture and on chorioallantoic membrane (CAM) of embryonated chicken eggs. Among 21 samples submitted to PCR using primers for hemagglutinin (HA) gene, 19 were positive. Restriction digestion with TaqI resulted in four characteristic Vaccinia virus fragments. HA nucleotide sequences showed 99.9% similarity with Cantagalo virus, described as a strain of Vaccinia virus. The only difference observed was the substitution of one nucleotide in the position 616 leading to change in one amino acid of the protein in the position 206. The phylogenetic analysis showed that the isolates clustered together with Cantagalo virus, other Vaccinia strains and Rabbitpox virus.

Inoculation with Metal-Mobilizing Plant-GrowthPromoting Rhizobacterium Bacillus sp. SC2b and Its Role in Rhizoremediation

A plant growth-promoting bacterial (PGPB) strain SC2b was isolated from the rhizosphere of Sedum plumbizincicola grown in lead (Pb)/zinc (Zn) mine soils and characterized as Bacillus sp. based on (1) morphological and biochemical characteristics and (2) partial 16S ribosomal DNA sequencing analysis. Strain SC2b exhibited high levels of resistance to cadmium (Cd) (300 mg/L), Zn (730 mg/L), and Pb (1400 mg/L). This strain also showed various plant growth-promoting (PGP) features such as utilization of 1-aminocyclopropane-1-carboxylate, solubilization of phosphate, and production of indole-3-acetic acid and siderophore. The strain mobilized high concentration of heavy metals from soils and exhibited different biosorption capacity toward the tested metal ions. Strain SC2b was further assessed for PGP activity by phytagar assay with a model plant Brassica napus. Inoculation of SC2b increased the biomass and vigor index of B. napus. Considering such potential, a pot experiment was conducted to assess the effects of inoculating the metal-resistant PGPB SC2b on growth and uptake of Cd, Zn and Pb by S. plumbizincicola in metal-contaminated agricultural soils. Inoculation with SC2b elevated the shoot and root biomass and leaf chlorophyll content of S. plumbizincicola. Similarly, plants inoculated with SC2b demonstrated markedly higher Cd and Zn accumulation in the root and shoot system, indicating that SC2b enhanced Cd and Zn uptake by S. plumbizincicola through metal mobilization or plant-microbial mediated changes in chemical or biological soil properties. Data demonstrated that the PGPB Bacillus sp. SC2b might serve as a future biofertilizer and an effective metal mobilizing bioinoculant for rhizoremediation of metal polluted soils.

Analysis of HIV- type 1 protease and reverse transcriptase in Brazilian children failing highly active antiretroviral therapy (HAART)

The aim of this study was to evaluate the genotypic resistance profiles of HIV-1 in children failing highly active antiretroviral therapy (HAART). Forty-one children (median age = 67 months) receiving HAART were submitted to genotypic testing when virological failure was detected. cDNA was extracted from PBMCs and amplified by nested PCR for the reverse transcriptase and protease regions of the pol gene. Drug resistance genotypes were determined from DNA sequencing. According to the genotypic analysis, 12/36 (33.3%) and 6/36 (16.6%) children showed resistance and possible resistance, respectively, to ZDV; 5/36 (14%) and 4/36 (11.1%), respectively, showed resistance and possible resistance to ddI; 4/36 (11.1%) showed resistance to 3TC and D4T; and 3/36 (8.3%) showed resistance to Abacavir. A high percentage (54%) of children exhibited mutations conferring resistance to NNRTI class drugs. Respective rates of resistance and possible resistance to PIs were: RTV (12.2%, 7.3%); APV (2.4%, 12.1%); SQV(0%, 12.1%); IDV (14.6%, 4.9%), NFV (22%, 4.9%), LPV/RTV (2.4%, 12.1%). Overall, 37/41 (90%) children exhibited virus with mutations related to drug resistance, while 9% exhibited resistance to all three antiretroviral drug classes.