Danielle Jannuzzi Madureira | Swiss Federal Institute of Technology (ETH) (original) (raw)
Papers by Danielle Jannuzzi Madureira
Advanced Materials Research, 2007
Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans are involved in bioleaching, a p... more Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans are involved in bioleaching, a process that has been successfully used in copper recovery. Apparently, A. ferrooxidans can tolerate 55 g/L of Cu 2+ in the growth medium while A. thiooxidans tolerates only 5 g/L. In bacteria many proteins involved in metal tolerance are members of a large class of membrane transporters called ATP Binding Cassette (ABC proteins). A gene that encodes for an ABC transporter protein was isolated from A. ferrooxidans LR, by RNA Arbitrarily Primed PCR (RAP-PCR), after the bacteria were kept for 24 hours in the presence of 2.5 g/ 100 mL of covellite (CuS). In comparison to ferrous iron, this gene was up-regulated in the presence of covellite. The objective of this work was to investigate the expression of transporter protein genes, in the presence of covellite, in A. thiooxidans. Three genes, two that encode for ABC transporter proteins and one that encodes for an efflux transporter protein had their relative expression pattern analyzed in this bacterium using realtime PCR. For this, A. thiooxidans FG01 was cultivated in the presence of sulphur (10 g/L) pH 1.8 at 250 rpm, 30°C, until an optical density of 0.11 was reached. The cells were collected and then kept in contact with covellite during 24 hours. The 16S rRNA gene was used as a reference standard and transcripts isolated from sulphur grown cells were used as the calibration parameter. The primers used to amplify the target genes were based on the A. ferrooxidans ATCC 23270 T complete genome sequence made available by TIGR (www.tigr.org). The real-time PCR quantification was performed twice, using RNA samples from independent cultures. In comparison to sulphur, the three genes analyzed were up-regulated in the presence of covellite. Experiments with A. ferrooxidans LR are in progress in order to compare the results among these two species. Further analyses will correlate the obtained data with bacterial colonization and adherence capability using scanning electron microscopy (SEM).
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2010
firm basis for the inclusion of gender and stage of the reproductive cycle in evaluating mussel r... more firm basis for the inclusion of gender and stage of the reproductive cycle in evaluating mussel responses to environmental stressors and may account for some of the inter-individual variation that complicates interpretation of such studies.
Orientadore: Laura Maria Mariscal Ottoboni, Fernanda de Castro ReisDissertação (mestrado) - Unive... more Orientadore: Laura Maria Mariscal Ottoboni, Fernanda de Castro ReisDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Acidithiobacillus ferrooxidans e Acidithiobacillus thiooxidans são espécies bacterianas acidofilicas, quimiolitotróficas e mesofilicas, encontradas em ambientes de biolixiviação. A. ferrooxidans utiliza íons ferrosos e compostos que contém enxofte como doadores de elétrons enquanto que A. thiooxidans utiliza apenas enxofte e compostos contendo enxofte. Ambas as espécies utilizam principalmente o oxigênio como receptor final de elétrons, contudo, podem também crescer em ambientes de anaerobiose. A. thiooxidans possui maior resistência ao pH podendo ser encontrada em ambientes com pH variando de 0,5 a 5,0, enquanto que A.ferrooxidans é encontrada em pHs que variam de 1,0 a 2,5. Devido a essas características e devido a capacidade dessas bactérias de solubilizar metais, A. ferrooxidans e A. thiooxidans vem sendo utilizadas em experi...
World Journal of Microbiology and Biotechnology, 2010
Gene expression in response to the copper sulfide, covellite (CuS), in Acidithiobacillus ferrooxi... more Gene expression in response to the copper sulfide, covellite (CuS), in Acidithiobacillus ferrooxidans LR was investigated by using RNA arbitrarily primed polymerase chain reaction (RAP-PCR). Seven genes that encode for proteins involved with transport and binding were up-regulated in the presence of CuS. The differential expression of the seven genes was confirmed by real-time PCR. An atomic absorption analysis of the covellite samples showed that the quantity of copper(II) ions in solution changed from zero to approximately 1.11 g/l after 24 h, and the pH changed from 1.8 to 4.0, suggesting that the copper ions in solution and the pH alteration may be responsible, at least in part, for the up-regulation of the transporter genes in the presence of covellite. An in silico protein-protein interaction analysis of the proteins encoded by the seven transporter protein genes, as well as of proteins encoded by genes of the same functional category that are adjacent to the seven genes identified by RAP-PCR, showed that besides the correlated function, the transporter proteins may act in different steps of the bacterial response to covellite.
The Journal of Microbiology, 2009
Plasmid pRJ6 is the first known bacteriocinogenic mobilizable (Mob) plasmid of Staphylococcus aur... more Plasmid pRJ6 is the first known bacteriocinogenic mobilizable (Mob) plasmid of Staphylococcus aureus. Its Mob region is composed of four mob genes (mobCDAB) arranged as an operon, a genetic organization uncommon among S. aureus Mob plasmids. oriT (pRJ6) was detected in a region of 431 bp, positioned immediately upstream of mobC. This region, when cloned into pCN37, was able to confer mobilization to the re-combinant plasmid only in the presence of pRJ6. The entire Mob region, including oriT (pRJ6), is much more similar to Mob regions from several coagulase-negative staphylococci plasmids, although some remarkable similarities with S. aureus Mob plasmids can also be noted. These similarities include the presence within oriT (pRJ6) of the three mcb (MobC binding sites), firstly described in pC221 and pC223, an identical nick site also found in these same plasmids, and a nearly identical sra(pC223) site (sequence recognized by MobA). pRJ6 was successfully transferred to S. epidermidis by conjugation in the presence of the conjugative plasmid pGOl. Altogether these findings suggest that pRJ6 might have been originally a coagulase-negative staphylococci plasmid that had been transferred successfully to S. aureus.
Scandinavian Journal of Infectious Diseases, 2007
Fever and a petechial rash are strongly associated with meningococcal disease in the city of Rio ... more Fever and a petechial rash are strongly associated with meningococcal disease in the city of Rio de Janeiro. Early antibiotic therapy is indicated and, consequently, a reduction of confirmed cases by culture, Gram stain, and latex agglutination test is expected. We evaluated a multiplex PCR assay to identify Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae in biological samples from cases of non-culture proven meningitis with a petechial rash at presentation. To detect DNA in cerebrospinal fluid (n = 71) or blood (n = 5), a PCR screen was performed, based on the crgA, ply and bexA targets, respectively. Of the total, 70 CSF and 3 blood samples (96%) were positive by PCR for the presence of N. meningitidis DNA. Another PCR assay predicted in 82% of these samples N. meningitidis serogroups A (2%), B (60%), C (7%), X (3%), Y (2%), 29E (2%) or W135 (24%). In non-culture proven meningitis, PCR was found to be a valuable adjunct for the demonstration of meningococcal aetiology.
Research in Microbiology, 2007
The inhibitory activity of seven bacteriocins produced by Staphylococcus aureus (aureocins A70, A... more The inhibitory activity of seven bacteriocins produced by Staphylococcus aureus (aureocins A70, A53, and 215FN) and Staphylococcus epidermidis (Pep5, epidermin, epilancin K7 and epicidin 280) was tested against strains of both S. aureus (165 strains) and Streptococcus agalactiae (74 strains) isolated from udders of cows suffering from bovine mastitis. Most strains of the two species were inhibited by epidermin (>85%), aureocin A53 (>67%) and by a combination of aureocins A70 and A53 (>91%), co-expressed in the genetic background of strain A70, the native producer of aureocin A70. Synergy between aureocins A70 and A53 was also demonstrated, which broadened the spectrum of strains inhibited. The remaining staphylococcins inhibited either none of, or a lower percentage (<48%) of, the mastitis-causing pathogens tested. Our results therefore show that the use of epidermin and/or a combination of aureocins A53 and A70 may represent a new non-antibiotic alternative for successfully inhibiting both mastitic staphylococci and streptococci.
Memórias do Instituto Oswaldo Cruz, 2007
This study analyzed the involvement of tetA and tetE genes in the tetracycline resistance of 16 s... more This study analyzed the involvement of tetA and tetE genes in the tetracycline resistance of 16 strains of genus Aeromonas, isolated from clinical and food sources. Polymerase chain reactions revealed that 37.5% of the samples were positive for tetA, and also 37.5% were tetE positive. One isolate was positive for both genes. Only the isolate A. caviae 5.2 had its resistance associated to the presence of a plasmid, pSS2. The molecular characterization of pSS2 involved the construction of its restriction map and the determination of its size. The digestion of pSS2 with HindIII originated two fragments (A and B) that were cloned separately into the pUC18 vector. The tetA gene was shown to be located on the HindIII-A fragment by PCR. After transforming a tetracycline-sensitive strain with pSS2, the transformants expressed the resistance phenotype and harbored a plasmid whose size was identical to that of pSS2. The results confirmed the association between pSS2 and the tetracycline resistance phenotype, and suggest a feasible dissemination of tetA and tetE among strains of Aeromonas. This study suggests the spreading tetA and tetE genes in Aeromonas in Brazil and describes a resistance plasmid that probably contributes to the dissemination of the resistance.
Memórias do Instituto Oswaldo Cruz, 2006
Diagnosis of bacterial meningitis has long been based on classical methods of Gram stain, serolog... more Diagnosis of bacterial meningitis has long been based on classical methods of Gram stain, serological tests, and culture of cerebrospinal fluid (CSF). The performance of these methods, especially culture and direct smear, is thwarted by failure to detect bacteria following administration of antimicrobial agents and reluctance to performance lumbar punctures at admission. Indeed, patients with meningitis frequently receive antibiotics orally or by injection before the diagnosis is suspected or established. Thus an alternative method has become necessary to help clinicians and epidemiologists to management and control of bacterial meningitis. We evaluate the application of a polymerase chain reaction-based (PCR) assay for amplification of pneumolysin gene (ply) to diagnosis of Streptococcus pneumoniae meningitis. The PCR assay sensitivity for CSF was 96% (95% confidence interval, CI, 90-99%) compared to a sensitivity of 59% for culture (95% CI 49-69%), 66% for Gram stain (95% CI 56-74%), and 78% for latex agglutination test (95% CI 69-86%); PCR specificity was 100% (95% CI 83-100%). PCR results were available within 4 h of the start of the assay. This molecular approach proved to be reliable and useful to identify this bacterium compared with other classical laboratory methods for identification of bacterial meningitis pathogens.
Current Microbiology, 2008
Acidithiobacillus ferrooxidans is a gram-negative bacterium that obtains energy from the oxidatio... more Acidithiobacillus ferrooxidans is a gram-negative bacterium that obtains energy from the oxidation of ferrous iron or reduced sulfur compounds. In this bacterium, the proteins encoded by the rus operon are involved in electron transfer from Fe(II) to O(2), and the first two proteins in this pathway also participate in the electron transfer pathway from Fe(II) to NAD(P). In this work we analyzed the expression, by real-time PCR, of the eight genes from the rus operon when A. ferrooxidans LR was grown in the presence of iron (control) and then kept in contact with chalcopyrite (CuFeS(2)) and covellite (CuS). A small decrease in rus operon gene expression was observed in the presence of chalcopyrite, while in the presence of covellite the expression of these genes showed a remarkable decrease. These results can be explained by the absence of ferrous iron in covellite. To explain the expression difference observed between the gene cyc1 and the gene rus, we investigated the information content presented at the Translation Initiation Site (TIS) of both genes. cyc1 showed a highly information content (8.4 bits) that can maximize translation, and rus showed a less favorable context (5.5 bits). Our hypothesis is that the energetic metabolism in A. ferrooxidans may be controlled at the transcriptional and posttranscriptional level by different mechanisms.
![Research paper thumbnail of Transcriptional signatures of regulatory and toxic responses to benzo-[a]-pyrene exposure](https://attachments.academia-assets.com/87420757/thumbnails/1.jpg)
](BMC Genomics, 2011
Background Small molecule ligands often have multiple effects on the transcriptional program of a... more Background Small molecule ligands often have multiple effects on the transcriptional program of a cell: they trigger a receptor specific response and additional, indirect responses ("side effects"). Distinguishing those responses is important for understanding side effects of drugs and for elucidating molecular mechanisms of toxic chemicals. Results We explored this problem by exposing cells to the environmental contaminant benzo-[a]-pyrene (B[a]P). B[a]P exposure activates the aryl hydrocarbon receptor (Ahr) and causes toxic stress resulting in transcriptional changes that are not regulated through Ahr. We sought to distinguish these two types of responses based on a time course of expression changes measured after B[a]P exposure. Using Random Forest machine learning we classified 81 primary Ahr responders and 1,308 genes regulated as side effects. Subsequent weighted clustering gave further insight into the connection between expression pattern, mode of regulation, and b...
![Research paper thumbnail of Transcriptional signatures of regulatory and toxic responses to benzo-[a]-pyrene exposure](https://attachments.academia-assets.com/87420687/thumbnails/1.jpg)
](BMC Genomics, 2011
Background Small molecule ligands often have multiple effects on the transcriptional program of a... more Background Small molecule ligands often have multiple effects on the transcriptional program of a cell: they trigger a receptor specific response and additional, indirect responses ("side effects"). Distinguishing those responses is important for understanding side effects of drugs and for elucidating molecular mechanisms of toxic chemicals. Results We explored this problem by exposing cells to the environmental contaminant benzo-[a]-pyrene (B[a]P). B[a]P exposure activates the aryl hydrocarbon receptor (Ahr) and causes toxic stress resulting in transcriptional changes that are not regulated through Ahr. We sought to distinguish these two types of responses based on a time course of expression changes measured after B[a]P exposure. Using Random Forest machine learning we classified 81 primary Ahr responders and 1,308 genes regulated as side effects. Subsequent weighted clustering gave further insight into the connection between expression pattern, mode of regulation, and b...
Chemical Research in Toxicology, 2014
Cell-based models are important for deriving mechanistic information about stress response pathwa... more Cell-based models are important for deriving mechanistic information about stress response pathways that have evolved to protect cells from toxic insult, such as exposure to environmental pollutants. One determinant of the stress response is the amount of chemical entering the cell and the cell's ability to detoxify and remove the chemical. If the stress response is overwhelmed, an adverse outcome will ensue. It was the goal of our study to quantify uptake and elimination rates of benzo(a)pyrene (BaP), a ubiquitous environmental pollutant, in a murine liver cell line. We evaluated the kinetic behavior in the context of BaP uptake, biotransformation, DNA adduct formation and repair along with the transcriptional and cell proliferation response. A low (50 nM) and a high (5 μM) BaP concentration were chosen in order to differentiate the role of exposure concentration in the time-resolved interaction of BaP with cells. While rates of uptake and the initial transcriptional response were similar for both BaP concentrations, cells exposed to 50 nM BaP completely recovered from exposure within 24 h, whereas cells exposed to 5 μM BaP did not. Biotransformation proceeded faster on 50 nM BaP, and the few DNA adducts formed were completely repaired after transient cell cycle arrest. In contrast, DNA adducts greatly accumulated in cells exposed to 5 μM BaP, despite significant biotransformation; complete cell cycle arrest and toxicity evolved. On the basis of the kinetic rate constants and cellular response, we conclude that at least short-term, pulsed exposures to 50 nM BaP, which we consider environmentally relevant, can be handled by cells without adverse outcome. Further studies are needed to determine the ability of cells to recover from repeated exposure. Our study emphasizes the importance of quantifying chemical uptake and fate in cell models to differentiate a stress response from an adverse outcome for better risk assessment.
Benzo(a)pyrene (BaP) is a ubiquitous pollutant derived by incomplete combustion of organic matter... more Benzo(a)pyrene (BaP) is a ubiquitous pollutant derived by incomplete combustion of organic matter. BaP is classified by the International Agency of Cancer (IARC) as carcinogenic to humans and is controlled in drinking water in the United States and Europe.
Analytical and Bioanalytical Chemistry, Apr 1, 2010
The emergence of analytical tools for highthroughput screening of biomolecules has revolutionized... more The emergence of analytical tools for highthroughput screening of biomolecules has revolutionized the way in which toxicologists explore the impact of chemicals or other stressors on organisms. One of the most developed and routinely applied high-throughput analysis approaches is transcriptomics, also often referred to as gene expression profiling. The transcriptome represents all RNA molecules, including the messenger RNA (mRNA), which constitutes the building blocks for translating DNA into amino acids to form proteins. The entirety of mRNA is a mirror of the genes that are actively expressed in a cell or an organism at a given time. This in turn allows one to deduce how organisms respond to changes in the external environment. In this article we explore how transcriptomics is currently applied in ecotoxicology and highlight challenges and trends.
Cell-based models are important for deriving mechanistic information about stress response pathwa... more Cell-based models are important for deriving mechanistic information about stress response pathways that have evolved to protect cells from toxic insult, such as exposure to environmental pollutants. One determinant of the stress response is the amount of chemical entering the cell and the cell's ability to detoxify and remove the chemical. If the stress response is overwhelmed, an adverse outcome will ensue. It was the goal of our study to quantify uptake and elimination rates of benzo(a)pyrene (BaP), a ubiquitous environmental pollutant, in a murine liver cell line. We evaluated the kinetic behavior in the context of BaP uptake, biotransformation, DNA adduct formation and repair along with the transcriptional and cell proliferation response. A low (50 nM) and a high (5 μM) BaP concentration were chosen in order to differentiate the role of exposure concentration in the time-resolved interaction of BaP with cells. While rates of uptake and the initial transcriptional response were similar for both BaP concentrations, cells exposed to 50 nM BaP completely recovered from exposure within 24 h, whereas cells exposed to 5 μM BaP did not. Biotransformation proceeded faster on 50 nM BaP, and the few DNA adducts formed were completely repaired after transient cell cycle arrest. In contrast, DNA adducts greatly accumulated in cells exposed to 5 μM BaP, despite significant biotransformation; complete cell cycle arrest and toxicity evolved. On the basis of the kinetic rate constants and cellular response, we conclude that at least short-term, pulsed exposures to 50 nM BaP, which we consider environmentally relevant, can be handled by cells without adverse outcome. Further studies are needed to determine the ability of cells to recover from repeated exposure. Our study emphasizes the importance of quantifying chemical uptake and fate in cell models to differentiate a stress response from an adverse outcome for better risk assessment.
![Research paper thumbnail of Transcriptional signatures of regulatory and toxic responses to benzo-[a]-pyrene exposure](https://attachments.academia-assets.com/30999472/thumbnails/1.jpg)
](Background: Small molecule ligands often have multiple effects on the transcriptional program of ... more Background: Small molecule ligands often have multiple effects on the transcriptional program of a cell: they trigger a receptor specific response and additional, indirect responses ("side effects"). Distinguishing those responses is important for understanding side effects of drugs and for elucidating molecular mechanisms of toxic chemicals.
Advanced Materials Research, 2007
Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans are involved in bioleaching, a p... more Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans are involved in bioleaching, a process that has been successfully used in copper recovery. Apparently, A. ferrooxidans can tolerate 55 g/L of Cu 2+ in the growth medium while A. thiooxidans tolerates only 5 g/L. In bacteria many proteins involved in metal tolerance are members of a large class of membrane transporters called ATP Binding Cassette (ABC proteins). A gene that encodes for an ABC transporter protein was isolated from A. ferrooxidans LR, by RNA Arbitrarily Primed PCR (RAP-PCR), after the bacteria were kept for 24 hours in the presence of 2.5 g/ 100 mL of covellite (CuS). In comparison to ferrous iron, this gene was up-regulated in the presence of covellite. The objective of this work was to investigate the expression of transporter protein genes, in the presence of covellite, in A. thiooxidans. Three genes, two that encode for ABC transporter proteins and one that encodes for an efflux transporter protein had their relative expression pattern analyzed in this bacterium using realtime PCR. For this, A. thiooxidans FG01 was cultivated in the presence of sulphur (10 g/L) pH 1.8 at 250 rpm, 30°C, until an optical density of 0.11 was reached. The cells were collected and then kept in contact with covellite during 24 hours. The 16S rRNA gene was used as a reference standard and transcripts isolated from sulphur grown cells were used as the calibration parameter. The primers used to amplify the target genes were based on the A. ferrooxidans ATCC 23270 T complete genome sequence made available by TIGR (www.tigr.org). The real-time PCR quantification was performed twice, using RNA samples from independent cultures. In comparison to sulphur, the three genes analyzed were up-regulated in the presence of covellite. Experiments with A. ferrooxidans LR are in progress in order to compare the results among these two species. Further analyses will correlate the obtained data with bacterial colonization and adherence capability using scanning electron microscopy (SEM).
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2010
firm basis for the inclusion of gender and stage of the reproductive cycle in evaluating mussel r... more firm basis for the inclusion of gender and stage of the reproductive cycle in evaluating mussel responses to environmental stressors and may account for some of the inter-individual variation that complicates interpretation of such studies.
Orientadore: Laura Maria Mariscal Ottoboni, Fernanda de Castro ReisDissertação (mestrado) - Unive... more Orientadore: Laura Maria Mariscal Ottoboni, Fernanda de Castro ReisDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Acidithiobacillus ferrooxidans e Acidithiobacillus thiooxidans são espécies bacterianas acidofilicas, quimiolitotróficas e mesofilicas, encontradas em ambientes de biolixiviação. A. ferrooxidans utiliza íons ferrosos e compostos que contém enxofte como doadores de elétrons enquanto que A. thiooxidans utiliza apenas enxofte e compostos contendo enxofte. Ambas as espécies utilizam principalmente o oxigênio como receptor final de elétrons, contudo, podem também crescer em ambientes de anaerobiose. A. thiooxidans possui maior resistência ao pH podendo ser encontrada em ambientes com pH variando de 0,5 a 5,0, enquanto que A.ferrooxidans é encontrada em pHs que variam de 1,0 a 2,5. Devido a essas características e devido a capacidade dessas bactérias de solubilizar metais, A. ferrooxidans e A. thiooxidans vem sendo utilizadas em experi...
World Journal of Microbiology and Biotechnology, 2010
Gene expression in response to the copper sulfide, covellite (CuS), in Acidithiobacillus ferrooxi... more Gene expression in response to the copper sulfide, covellite (CuS), in Acidithiobacillus ferrooxidans LR was investigated by using RNA arbitrarily primed polymerase chain reaction (RAP-PCR). Seven genes that encode for proteins involved with transport and binding were up-regulated in the presence of CuS. The differential expression of the seven genes was confirmed by real-time PCR. An atomic absorption analysis of the covellite samples showed that the quantity of copper(II) ions in solution changed from zero to approximately 1.11 g/l after 24 h, and the pH changed from 1.8 to 4.0, suggesting that the copper ions in solution and the pH alteration may be responsible, at least in part, for the up-regulation of the transporter genes in the presence of covellite. An in silico protein-protein interaction analysis of the proteins encoded by the seven transporter protein genes, as well as of proteins encoded by genes of the same functional category that are adjacent to the seven genes identified by RAP-PCR, showed that besides the correlated function, the transporter proteins may act in different steps of the bacterial response to covellite.
The Journal of Microbiology, 2009
Plasmid pRJ6 is the first known bacteriocinogenic mobilizable (Mob) plasmid of Staphylococcus aur... more Plasmid pRJ6 is the first known bacteriocinogenic mobilizable (Mob) plasmid of Staphylococcus aureus. Its Mob region is composed of four mob genes (mobCDAB) arranged as an operon, a genetic organization uncommon among S. aureus Mob plasmids. oriT (pRJ6) was detected in a region of 431 bp, positioned immediately upstream of mobC. This region, when cloned into pCN37, was able to confer mobilization to the re-combinant plasmid only in the presence of pRJ6. The entire Mob region, including oriT (pRJ6), is much more similar to Mob regions from several coagulase-negative staphylococci plasmids, although some remarkable similarities with S. aureus Mob plasmids can also be noted. These similarities include the presence within oriT (pRJ6) of the three mcb (MobC binding sites), firstly described in pC221 and pC223, an identical nick site also found in these same plasmids, and a nearly identical sra(pC223) site (sequence recognized by MobA). pRJ6 was successfully transferred to S. epidermidis by conjugation in the presence of the conjugative plasmid pGOl. Altogether these findings suggest that pRJ6 might have been originally a coagulase-negative staphylococci plasmid that had been transferred successfully to S. aureus.
Scandinavian Journal of Infectious Diseases, 2007
Fever and a petechial rash are strongly associated with meningococcal disease in the city of Rio ... more Fever and a petechial rash are strongly associated with meningococcal disease in the city of Rio de Janeiro. Early antibiotic therapy is indicated and, consequently, a reduction of confirmed cases by culture, Gram stain, and latex agglutination test is expected. We evaluated a multiplex PCR assay to identify Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae in biological samples from cases of non-culture proven meningitis with a petechial rash at presentation. To detect DNA in cerebrospinal fluid (n = 71) or blood (n = 5), a PCR screen was performed, based on the crgA, ply and bexA targets, respectively. Of the total, 70 CSF and 3 blood samples (96%) were positive by PCR for the presence of N. meningitidis DNA. Another PCR assay predicted in 82% of these samples N. meningitidis serogroups A (2%), B (60%), C (7%), X (3%), Y (2%), 29E (2%) or W135 (24%). In non-culture proven meningitis, PCR was found to be a valuable adjunct for the demonstration of meningococcal aetiology.
Research in Microbiology, 2007
The inhibitory activity of seven bacteriocins produced by Staphylococcus aureus (aureocins A70, A... more The inhibitory activity of seven bacteriocins produced by Staphylococcus aureus (aureocins A70, A53, and 215FN) and Staphylococcus epidermidis (Pep5, epidermin, epilancin K7 and epicidin 280) was tested against strains of both S. aureus (165 strains) and Streptococcus agalactiae (74 strains) isolated from udders of cows suffering from bovine mastitis. Most strains of the two species were inhibited by epidermin (>85%), aureocin A53 (>67%) and by a combination of aureocins A70 and A53 (>91%), co-expressed in the genetic background of strain A70, the native producer of aureocin A70. Synergy between aureocins A70 and A53 was also demonstrated, which broadened the spectrum of strains inhibited. The remaining staphylococcins inhibited either none of, or a lower percentage (<48%) of, the mastitis-causing pathogens tested. Our results therefore show that the use of epidermin and/or a combination of aureocins A53 and A70 may represent a new non-antibiotic alternative for successfully inhibiting both mastitic staphylococci and streptococci.
Memórias do Instituto Oswaldo Cruz, 2007
This study analyzed the involvement of tetA and tetE genes in the tetracycline resistance of 16 s... more This study analyzed the involvement of tetA and tetE genes in the tetracycline resistance of 16 strains of genus Aeromonas, isolated from clinical and food sources. Polymerase chain reactions revealed that 37.5% of the samples were positive for tetA, and also 37.5% were tetE positive. One isolate was positive for both genes. Only the isolate A. caviae 5.2 had its resistance associated to the presence of a plasmid, pSS2. The molecular characterization of pSS2 involved the construction of its restriction map and the determination of its size. The digestion of pSS2 with HindIII originated two fragments (A and B) that were cloned separately into the pUC18 vector. The tetA gene was shown to be located on the HindIII-A fragment by PCR. After transforming a tetracycline-sensitive strain with pSS2, the transformants expressed the resistance phenotype and harbored a plasmid whose size was identical to that of pSS2. The results confirmed the association between pSS2 and the tetracycline resistance phenotype, and suggest a feasible dissemination of tetA and tetE among strains of Aeromonas. This study suggests the spreading tetA and tetE genes in Aeromonas in Brazil and describes a resistance plasmid that probably contributes to the dissemination of the resistance.
Memórias do Instituto Oswaldo Cruz, 2006
Diagnosis of bacterial meningitis has long been based on classical methods of Gram stain, serolog... more Diagnosis of bacterial meningitis has long been based on classical methods of Gram stain, serological tests, and culture of cerebrospinal fluid (CSF). The performance of these methods, especially culture and direct smear, is thwarted by failure to detect bacteria following administration of antimicrobial agents and reluctance to performance lumbar punctures at admission. Indeed, patients with meningitis frequently receive antibiotics orally or by injection before the diagnosis is suspected or established. Thus an alternative method has become necessary to help clinicians and epidemiologists to management and control of bacterial meningitis. We evaluate the application of a polymerase chain reaction-based (PCR) assay for amplification of pneumolysin gene (ply) to diagnosis of Streptococcus pneumoniae meningitis. The PCR assay sensitivity for CSF was 96% (95% confidence interval, CI, 90-99%) compared to a sensitivity of 59% for culture (95% CI 49-69%), 66% for Gram stain (95% CI 56-74%), and 78% for latex agglutination test (95% CI 69-86%); PCR specificity was 100% (95% CI 83-100%). PCR results were available within 4 h of the start of the assay. This molecular approach proved to be reliable and useful to identify this bacterium compared with other classical laboratory methods for identification of bacterial meningitis pathogens.
Current Microbiology, 2008
Acidithiobacillus ferrooxidans is a gram-negative bacterium that obtains energy from the oxidatio... more Acidithiobacillus ferrooxidans is a gram-negative bacterium that obtains energy from the oxidation of ferrous iron or reduced sulfur compounds. In this bacterium, the proteins encoded by the rus operon are involved in electron transfer from Fe(II) to O(2), and the first two proteins in this pathway also participate in the electron transfer pathway from Fe(II) to NAD(P). In this work we analyzed the expression, by real-time PCR, of the eight genes from the rus operon when A. ferrooxidans LR was grown in the presence of iron (control) and then kept in contact with chalcopyrite (CuFeS(2)) and covellite (CuS). A small decrease in rus operon gene expression was observed in the presence of chalcopyrite, while in the presence of covellite the expression of these genes showed a remarkable decrease. These results can be explained by the absence of ferrous iron in covellite. To explain the expression difference observed between the gene cyc1 and the gene rus, we investigated the information content presented at the Translation Initiation Site (TIS) of both genes. cyc1 showed a highly information content (8.4 bits) that can maximize translation, and rus showed a less favorable context (5.5 bits). Our hypothesis is that the energetic metabolism in A. ferrooxidans may be controlled at the transcriptional and posttranscriptional level by different mechanisms.
BMC Genomics, 2011
Background Small molecule ligands often have multiple effects on the transcriptional program of a... more Background Small molecule ligands often have multiple effects on the transcriptional program of a cell: they trigger a receptor specific response and additional, indirect responses ("side effects"). Distinguishing those responses is important for understanding side effects of drugs and for elucidating molecular mechanisms of toxic chemicals. Results We explored this problem by exposing cells to the environmental contaminant benzo-[a]-pyrene (B[a]P). B[a]P exposure activates the aryl hydrocarbon receptor (Ahr) and causes toxic stress resulting in transcriptional changes that are not regulated through Ahr. We sought to distinguish these two types of responses based on a time course of expression changes measured after B[a]P exposure. Using Random Forest machine learning we classified 81 primary Ahr responders and 1,308 genes regulated as side effects. Subsequent weighted clustering gave further insight into the connection between expression pattern, mode of regulation, and b...
BMC Genomics, 2011
Background Small molecule ligands often have multiple effects on the transcriptional program of a... more Background Small molecule ligands often have multiple effects on the transcriptional program of a cell: they trigger a receptor specific response and additional, indirect responses ("side effects"). Distinguishing those responses is important for understanding side effects of drugs and for elucidating molecular mechanisms of toxic chemicals. Results We explored this problem by exposing cells to the environmental contaminant benzo-[a]-pyrene (B[a]P). B[a]P exposure activates the aryl hydrocarbon receptor (Ahr) and causes toxic stress resulting in transcriptional changes that are not regulated through Ahr. We sought to distinguish these two types of responses based on a time course of expression changes measured after B[a]P exposure. Using Random Forest machine learning we classified 81 primary Ahr responders and 1,308 genes regulated as side effects. Subsequent weighted clustering gave further insight into the connection between expression pattern, mode of regulation, and b...
Chemical Research in Toxicology, 2014
Cell-based models are important for deriving mechanistic information about stress response pathwa... more Cell-based models are important for deriving mechanistic information about stress response pathways that have evolved to protect cells from toxic insult, such as exposure to environmental pollutants. One determinant of the stress response is the amount of chemical entering the cell and the cell's ability to detoxify and remove the chemical. If the stress response is overwhelmed, an adverse outcome will ensue. It was the goal of our study to quantify uptake and elimination rates of benzo(a)pyrene (BaP), a ubiquitous environmental pollutant, in a murine liver cell line. We evaluated the kinetic behavior in the context of BaP uptake, biotransformation, DNA adduct formation and repair along with the transcriptional and cell proliferation response. A low (50 nM) and a high (5 μM) BaP concentration were chosen in order to differentiate the role of exposure concentration in the time-resolved interaction of BaP with cells. While rates of uptake and the initial transcriptional response were similar for both BaP concentrations, cells exposed to 50 nM BaP completely recovered from exposure within 24 h, whereas cells exposed to 5 μM BaP did not. Biotransformation proceeded faster on 50 nM BaP, and the few DNA adducts formed were completely repaired after transient cell cycle arrest. In contrast, DNA adducts greatly accumulated in cells exposed to 5 μM BaP, despite significant biotransformation; complete cell cycle arrest and toxicity evolved. On the basis of the kinetic rate constants and cellular response, we conclude that at least short-term, pulsed exposures to 50 nM BaP, which we consider environmentally relevant, can be handled by cells without adverse outcome. Further studies are needed to determine the ability of cells to recover from repeated exposure. Our study emphasizes the importance of quantifying chemical uptake and fate in cell models to differentiate a stress response from an adverse outcome for better risk assessment.
Benzo(a)pyrene (BaP) is a ubiquitous pollutant derived by incomplete combustion of organic matter... more Benzo(a)pyrene (BaP) is a ubiquitous pollutant derived by incomplete combustion of organic matter. BaP is classified by the International Agency of Cancer (IARC) as carcinogenic to humans and is controlled in drinking water in the United States and Europe.
Analytical and Bioanalytical Chemistry, Apr 1, 2010
The emergence of analytical tools for highthroughput screening of biomolecules has revolutionized... more The emergence of analytical tools for highthroughput screening of biomolecules has revolutionized the way in which toxicologists explore the impact of chemicals or other stressors on organisms. One of the most developed and routinely applied high-throughput analysis approaches is transcriptomics, also often referred to as gene expression profiling. The transcriptome represents all RNA molecules, including the messenger RNA (mRNA), which constitutes the building blocks for translating DNA into amino acids to form proteins. The entirety of mRNA is a mirror of the genes that are actively expressed in a cell or an organism at a given time. This in turn allows one to deduce how organisms respond to changes in the external environment. In this article we explore how transcriptomics is currently applied in ecotoxicology and highlight challenges and trends.
Cell-based models are important for deriving mechanistic information about stress response pathwa... more Cell-based models are important for deriving mechanistic information about stress response pathways that have evolved to protect cells from toxic insult, such as exposure to environmental pollutants. One determinant of the stress response is the amount of chemical entering the cell and the cell's ability to detoxify and remove the chemical. If the stress response is overwhelmed, an adverse outcome will ensue. It was the goal of our study to quantify uptake and elimination rates of benzo(a)pyrene (BaP), a ubiquitous environmental pollutant, in a murine liver cell line. We evaluated the kinetic behavior in the context of BaP uptake, biotransformation, DNA adduct formation and repair along with the transcriptional and cell proliferation response. A low (50 nM) and a high (5 μM) BaP concentration were chosen in order to differentiate the role of exposure concentration in the time-resolved interaction of BaP with cells. While rates of uptake and the initial transcriptional response were similar for both BaP concentrations, cells exposed to 50 nM BaP completely recovered from exposure within 24 h, whereas cells exposed to 5 μM BaP did not. Biotransformation proceeded faster on 50 nM BaP, and the few DNA adducts formed were completely repaired after transient cell cycle arrest. In contrast, DNA adducts greatly accumulated in cells exposed to 5 μM BaP, despite significant biotransformation; complete cell cycle arrest and toxicity evolved. On the basis of the kinetic rate constants and cellular response, we conclude that at least short-term, pulsed exposures to 50 nM BaP, which we consider environmentally relevant, can be handled by cells without adverse outcome. Further studies are needed to determine the ability of cells to recover from repeated exposure. Our study emphasizes the importance of quantifying chemical uptake and fate in cell models to differentiate a stress response from an adverse outcome for better risk assessment.
Background: Small molecule ligands often have multiple effects on the transcriptional program of ... more Background: Small molecule ligands often have multiple effects on the transcriptional program of a cell: they trigger a receptor specific response and additional, indirect responses ("side effects"). Distinguishing those responses is important for understanding side effects of drugs and for elucidating molecular mechanisms of toxic chemicals.