Detection of enterotoxin and toxic shock syndrome toxin 1 genes in Staphylococcus, with emphasis on coagulase-negative staphylococci

The detection of staphylococcal enterotoxins is decisive for the confirmation of an outbreak and for the determination of the enterotoxigenicity of strains. Since the recognition of their antigenicity, a large number of serological methods for the detection of enterotoxins in food and culture media have been proposed. Since immunological methods require detectable amounts of toxin, molecular biology techniques represent important tools in the microbiology laboratory. In the present study, polymerase chain reaction (PCR) was used to identify genes responsible for the production of enterotoxins and toxic shock syndrome toxin 1 (TSST-1) in S. aureus and coagulase-negative staphylococci (CNS) isolated from patients and the results were compared with those obtained by the reverse passive latex agglutination (RPLA) assay. PCR detection of toxin genes revealed a higher percentage of toxigenic S. aureus strains (46.7%) than the RPLA method (38.3%). Analysis of the toxigenic profile of CNS strains showed that 26.7% of the isolates produced some type of toxin, and one or more toxin-specific genes were detected in 40% of the isolates. These results suggests the need for further studies in order to better characterize the pathogenic potential of CNS and indicate that attention should be paid to the toxigenic capacity of this group of microorganisms.

Optimal clone identifier for genomic shotgun libraries: OC Identifier tool

In DNA microarray experiments, the gene fragments that are spotted on the slides are usually obtained by the synthesis of specific oligonucleotides that are able to amplify genes through PCR. Shotgun library sequences are an alternative to synthesis of primers for the study of each gene in the genome. The possibility of putting thousands of gene sequences into a single slide allows the use of shotgun clones in order to proceed with microarray analysis without a completely sequenced genome. We developed an OC Identifier tool (optimal clone identifier for genomic shotgun libraries) for the identification of unique genes in shotgun libraries based on a partially sequenced genome; this allows simultaneous use of clones in projects such as transcriptome and phylogeny studies, using comparative genomic hybridization and genome assembly. The OC Identifier tool allows comparative genome analysis, biological databases, query language in relational databases, and provides bioinformatics tools to identify clones that contain unique genes as alternatives to primer synthesis. The OC Identifier allows analysis of clones during the sequencing phase, making it possible to select genes of interest for construction of a DNA microarray. ©FUNPEC-RP.

Determinação da resistência de Staphylococcus aureus: Um desafio?

The aim of this study was to identify the resistance profile of Staphylococcus aureus strains, in relation to induced clindamycin resistance, and to detect oxacillin resistance by the routine phenotypic methods. The strains were isolated from nasal or lingual swabs taken from healthy adult carriers with no medical history of hospitalization or antibiotic treatment. Eighteen strains were distinguished by the different patterns generated by pulsed field gel electrophoresis (PFGE). Four (22.2%) of these showed sensitivity to clindamycin by the conventional antibacterial susceptibility test, but demonstrated inducible resistance to it by the D-test. One strain (5.6%) was characterized as borderline oxacillin-resistant S. aureus (BORSA), and another (5.6%) as CA MRSA (community-associated methicillin-resistant Staphylococcus aureus). Both of these strains were shown to be cefoxitin susceptible by the disk diffusion test. The polymerase chain reaction (PCR) failed to detect the mecA gene in this last strain and it was thus classified as BORSA. These results show the importance of incorporating the D-test into the routine lab tests for S. aureus inducible clindamycin resistance and also of including the cefoxitin resistance test among the phenotypic methods for MRSA characterization.

Genetic polymorphism of Streptococcus mutans strains associated with incomplete caries removal

Aim: Despite the antibacterial properties of dental materials, the survival of residual bacteria under restorations has been demonstrated after incomplete caries removal. The aim of this study was to evaluate the genetic polymorphism of Streptococcus mutans strains isolated from deep dentinal lesions before and three months after incomplete caries removal. Methods: Samples of carious dentin were collected from 33 primary and/or permanent molars before and after indirect pulp treatment and processed for microbiological isolation of mutans streptococci (MS). After three months of the dental treatment, positive cultures for MS were detected in only ten of these teeth. DNA of MS isolates were obtained and subjected to polymerase chain reaction (PCR) for identification of S mutans. The arbitrary primed-PCR method (primer OPA-13) was used to detect the genetic polymorphism of S. mutans strains. Results: Identical or highly related S. mutans genotypes were observed in each tooth, regardless of the collect. Considering each tooth separately, a maximum of nine genotypic patterns were found in each tooth from all the collects. In addition, at least one genotypic pattern was repeated in the three collects. Genetic diversity was observed among the S. mutans isolates, obtained from different teeth after three months of the dental treatment. Conclusions: The persistence of identical genotypic patterns and the genetic similarity among the isolates, from the same tooth in distinct collects, showed the resistance of some S. mutans strains after incomplete caries removal treatment.

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

SNaPshot minisequencing reaction is in increasing use because of its fast detection of many polymorphisms in a single assay. In this work we described a highly sensitive single nucleotide polymorphisms (SNPs) typing method with detection of 42 mitochondrial DNA (mtDNA) SNPs in a single PCR and SNaPshot multiplex reaction in order to allow haplogroup classification in Latin American admixture population. We validated the panel typing 160 Brazilian individuals. DNA was extracted from blood spotted on filter paper using Chelex protocol. Forty SNPs were selected targeting haplogroup-specific mutations in Europeans, Africans and Asians (only precursors of Native Americans haplogroups A2, B2, C1, and D1) and two non-coding SNPs were chosen to increase the power of discrimination between individuals (SNPs positions 16,519 and 16,362). It was done using a modified version of a previously published multiplex SNaPshot minisequencing reaction established to resolve European haplogroups, adding SNPs targeting Africans (L0, L1, L2, L3, and L*) and Asians (A, B, C, and D) haplogroups based on SNPs described at PhyloTree.org build 2. PCR primers were designed using PerlPrimer software and checked with the Autodimer program. Thirty-three primer-pairs were used to amplify 42 SNPs. Using this panel, we were able to successfully classify 160 individuals into their correct haplogroups. Complete SNP profiles were obtained from 10. pg of total DNA. We conclude that it is possible to build and genotype more than 40 mtDNA SNPs in a single multiplex PCR and SNaPshot reaction, with sensitivity and reliability, resolving haplogroup classification in admixture populations. © 2011.

Exequibility of differential gene expression analysis by DDRT-PCR in murine bone marrow cells

Model of study: Experimental study. Introduction: Recently, stem cell research has generated great interest due to its applicability in regenerative medicine. Bone marrow is considered the most important source of adult stem cells and the establishment of new methods towards gene expression analysis regarding stem cells has become necessary. Thus Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT-PCR) may be an accessible tool to investigate small differences in the gene expression of different stem cells in distinct situations. Aim: In the present study, we investigated the exequibility of DDRT-PCR to identify differences in global gene expression of mice bone marrow cells under two conditions. Methods: First, bone marrow cells were isolated fresh and a part was cultivated during one week without medium replacement. Afterwards, both bone marrow cells (fresh and cultivated) were submitted to gene expression analyses by DDRT-PCR. Results: Initially, it was possible to observe in one week-cultured bone marrow cells, changes in morphology (oval cells to fibroblastic-like cells) and protein profile, which was seen through differences in band distribution in SDS-Page gels. Finally through gene expression analysis, we detected three bands (1300, 1000 and 225 bp) exclusively expressed in the fresh bone marrow group and two bands (400 and 300 bp) expressed specifically in the cultivated bone marrow cell group. Conclusions: In summary, the DDRT-PCR method was proved efficient towards the identification of small differences in gene expression of bone marrow cells in two defined conditions. Thus, we expect that DDRT-PCR can be fast and efficiently designed to analyze differential gene expression in several stem cell types under distinct conditions.

Case report: Disseminated Mycobacterium tuberculosis infection in a dog

An uncommon disseminated Mycobacterium tuberculosis infection is described in a 12-year-old female dog presenting with fever, dyspnea, cough, weight loss, lymphadenopathy, melena, epistaxis, and emesis. The dog had a history of close contact with its owner, who died of pulmonary tuberculosis. Radiographic examination revealed diffuse radio-opaque images in both lung lobes, diffuse visible masses in abdominal organs, and hilar and mesenteric lymphadenopathy. Bronchial washing samples and feces were negative for acid-fast organisms. Polymerase chain reaction (PCR)-based species identification of bronchial washing samples, feces, and urine revealed M. tuberculosis using PCR-restriction enzyme pattern analysis-PRA. Because of public health concerns, which were worsened by the physical condition of the dog, euthanasia of the animal was recommended. Rough and tough colonies suggestive of M. tuberculosis were observed after microbiological culture of lung, liver, spleen, heart, and lymph node fragments in Löwenstein-Jensen and Stonebrink media. The PRA analysis enabled diagnosis of M. tuberculosis strains isolated from organs. Copyright © 2013 by The American Society of Tropical Medicine and Hygiene.

An Integrative Genomic and Transcriptomic Analysis Reveals Potential Targets Associated with Cell Proliferation in Uterine Leiomyomas

Background: Uterine Leiomyomas (ULs) are the most common benign tumours affecting women of reproductive age. ULs represent a major problem in public health, as they are the main indication for hysterectomy. Approximately 40-50% of ULs have non-random cytogenetic abnormalities, and half of ULs may have copy number alterations (CNAs). Gene expression microarrays studies have demonstrated that cell proliferation genes act in response to growth factors and steroids. However, only a few genes mapping to CNAs regions were found to be associated with ULs. Methodology: We applied an integrative analysis using genomic and transcriptomic data to identify the pathways and molecular markers associated with ULs. Fifty-one fresh frozen specimens were evaluated by array CGH (JISTIC) and gene expression microarrays (SAM). The CONEXIC algorithm was applied to integrate the data. Principal Findings: The integrated analysis identified the top 30 significant genes (P<0.01), which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell proliferation, including FGFR1 and IGFBP5. Transcriptional and protein analyses showed that FGFR1 (P = 0.006 and P<0.01, respectively) and IGFBP5 (P = 0.0002 and P = 0.006, respectively) were up-regulated in the tumours when compared with the adjacent normal myometrium. Conclusions: The integrative genomic and transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs. © 2013 Cirilo et al.

Occurrence of mycobacteria in bovine milk samples from both individual and collective bulk tanks at farms and informal markets in the southeast region of Sao Paulo, Brazil

Background: Mycobacterium spp. is one of the most important species of zoonotic pathogens that can be transmitted from cattle to humans. The presence of these opportunistic, pathogenic bacteria in bovine milk has emerged as a public-health concern, especially among individuals who consume raw milk and related dairy products. To address this concern, the Brazilian control and eradication program focusing on bovine tuberculosis, was established in 2001. However, bovine tuberculosis continues to afflict approximately 1,3 percent of the cattle in Brazil. In the present study, 300 samples of milk from bovine herds, obtained from both individual and collective bulk tanks and informal points of sale, were cultured on Löwenstein-Jensen and Stonebrink media. Polymerase chain reaction (PCR)-based tests and restriction-enzyme pattern analysis were then performed on the colonies exhibiting phenotypes suggestive of Mycobacterium spp., which were characterized as acid-fast bacilli.Results: Of the 300 bovine milk samples that were processed, 24 were positively identified as Mycobacterium spp.Molecular identification detected 15 unique mycobacterial species: Mycobacterium bovis, M. gordonae, M. fortuitum, M. intracellulare, M. flavescens, M. duvalii, M. haemophilum, M. immunogenum, M. lentiflavum, M. mucogenicum, M. novocastrense, M. parafortuitum, M. smegmatis, M. terrae and M. vaccae. The isolation of bacteria from the various locations occurred in the following proportions: 9 percent of the individual bulk-tank samples, 7 percent of the collective bulk-tank samples and 8 percent of the informal-trade samples. No statistically significant difference was observed between the presence of Mycobacterium spp. in the three types of samples collected, the milk production profiles, the presence of veterinary assistance and the reported concerns about bovine tuberculosis prevention in the herds.Conclusion: The microbiological cultures associated with PCR-based identification tests are possible tools for the investigation of the presence of Mycobacterium spp. in milk samples. Using these methods, we found that the Brazilian population may be regularly exposed to mycobacteria by consuming raw bovine milk and related dairy products. These evidences reinforces the need to optimize quality programs of dairy products, to intensify the sanitary inspection of these products and the necessity of further studies on the presence of Mycobacterium spp. in milk and milk-based products. © 2013 Franco et al.; licensee BioMed Central Ltd.

Detection of post-F1 fish hybrids in broodstock using molecular markers: Approaches for genetic management in aquaculture

Herein, we have developed molecular markers for nuclear genes to use in multiplex-PCR and PCR-RFLP, with the goal of characterising hybrid lines derived from crosses between pintado Pseudoplatystoma corruscans and cachara P. reticulatum. These markers, together with others described previously, were used to perform molecular identification analyses as genetic subsidies for Brazilian aquaculture. These analyses were performed due to the problems of high mortality in the offspring reported by the aquaculturist. From a total of 16 broodstock samples, 13 were genetically identified as hybrids; surprisingly, nine of these hybrids were found to be post-F1 lineages. These data show that the fertility of these animals can seriously affect the cultivated stocks, thus causing financial damage in this aquaculture system. The establishment of PCR-RFLP and multiplex-PCR as molecular techniques allows for both the correct management of these animals and the routine monitoring of production and trade of fish hybrids in aquaculture. Consequently, such tools will enable a sustainable development in the aquaculture industry. © 2012 Blackwell Publishing Ltd.

Enterotoxin genes in coagulase-negative and coagulase-positive staphylococci isolated from bovine milk

The objective of this study was to isolate and identify the main staphylococcal species causing bovine mastitis in 10 Brazilian dairy herds and study their capability to produce enterotoxins. Herds were selected based on size and use of milking technology, and farms were visited once during the study. All mammary glands of all lactating cows were screened using the California Mastitis Test (CMT) and a strip cup. A single aseptic milk sample (20. mL) was collected from all CMT-positive quarters. Identification of Staphylococcus spp. was performed using conventional microbiology, and PCR was used to determine the presence of enterotoxin-encoding genes (sea, seb, sec, and sed). Of the 1,318 CMT-positive milk samples, Staphylococcus spp. were isolated from 263 (19.9%). Of these isolates, 135 (51%) were coagulase-positive staphylococci (CPS) and 128 (49%) were coagulase-negative staphylococci (CNS). Eighteen different species of CNS were isolated, among which S. warneri, S. epidermidis and S. hyicus were the most frequent. The distribution of Staphylococcus species was different among herds: S. epidermidis was found in 8 herds, S. warneri was found in 7 herds, and S. hyicus in 6 herds. Some of the CNS species (S. saprophyticus ssp. saprophyticus, S. auricularis, S. capitis, and S. chromogenes) were isolated in only one of the farms. Genes related to production of enterotoxins were found in 66% (n = 85) of all CNS and in 35% of the CPS isolates. For both CNS and CPS isolates, the most frequently identified enterotoxin genes were sea, seb, and sec; the prevalence of sea differed between CPS (9.5%) and CNS (35.1%) isolates. Staphylococcus warneri isolates showed a greater percentage of sea than seb, sec, or sed, whereas S. hyicus isolates showed a greater percentage of sea than sec. Over 60% of CNS belonged to 3 major species, which carried 62.2 to 81.3% of the enterotoxin genes. The high prevalence highlights the potential for food poisoning caused by these species. For possible high-risk situations for food poisoning, such as milk produced with total bacterial counts greater than regulatory levels and stored under inappropriate temperatures, monitoring contamination with CNS could be important to protect human health. Because the prevalence of CNS intramammary infections in dairy herds is usually high, and these species can be found in great numbers in bulk milk, identification of risk factors for production of staphylococcal enterotoxins should be considered in future studies. © 2013 American Dairy Science Association.

Fetal-Maternal Interactions in the Synepitheliochorial Placenta Using the eGFP Cloned Cattle Model

Background: To investigate mechanisms of fetal-maternal cell interactions in the bovine placenta, we developed a model of transgenic enhanced Green Fluorescent Protein (t-eGFP) expressing bovine embryos produced by nuclear transfer (NT) to assess the distribution of fetal-derived products in the bovine placenta. In addition, we searched for male specific DNA in the blood of females carrying in vitro produced male embryos. Our hypothesis is that the bovine placenta is more permeable to fetal-derived products than described elsewhere. Methodology/Principal Findings: Samples of placentomes, chorion, endometrium, maternal peripheral blood leukocytes and blood plasma were collected during early gestation and processed for nested-PCR for eGFP and testis-specific Y-encoded protein (TSPY), western blotting and immunohistochemistry for eGFP detection, as well as transmission electron microscopy to verify the level of interaction between maternal and fetal cells. TSPY and eGFP DNA were present in the blood of cows carrying male pregnancies at day 60 of pregnancy. Protein and mRNA of eGFP were observed in the trophoblast and uterine tissues. In the placentomes, the protein expression was weak in the syncytial regions, but intense in neighboring cells on both sides of the fetal-maternal interface. Ultrastructurally, our samples from t-eGFP expressing NT pregnancies showed to be normal, such as the presence of interdigitating structures between fetal and maternal cells. In addition, channels-like structures were present in the trophoblast cells. Conclusions/Significance: Data suggested that there is a delivery of fetal contents to the maternal system on both systemic and local levels that involved nuclear acids and proteins. It not clear the mechanisms involved in the transfer of fetal-derived molecules to the maternal system. This delivery may occur through nonclassical protein secretion; throughout transtrophoblastic-like channels and/or by apoptotic processes previously described. In conclusion, the bovine synepitheliochorial placenta displays an intimate fetal-maternal interaction, similar to other placental types for instance human and mouse. © 2013 Pereira et al.

Early endosome antigen 1 (EEA1) decreases in macrophages infected with Paracoccidioides brasiliensis

Paracoccidioidomycosis (PCM) is a chronic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis, endemic in Latin America. P. brasiliensis has been observed in epithelial cells in vivo and in vitro, as well as within the macrophages. The identification of the mechanism by which it survives within the host cell is fertile ground for the discovery of its pathogenesis since this organism has the ability to induce its own endocytosis in epithelial cells and most likely in macrophages. The study of the expression of endocytic proteins pathway and co-localization of microorganisms enable detection of the mechanism by which microorganisms survive within the host cell. The aim of this study was to evaluate the expression of the endocytic protein EEA1 (early endosome antigen 1) in macrophages infected with P. brasiliensis. For detection of EEA1, three different techniques were employed: immunofluorescence, real-time polymerase chain reaction (PCR) and immunoblotting. In the present study, decreased expression of EEA1 as well as the rearrangement of the actin was observed when the fungus was internalized, confirming that the input mechanism of the fungus in macrophages occurs through phagocytosis. © 2013 ISHAM.

Detecção molecular quantitativa de cianobactérias hepatotóxicas através de PCR competitiva

Pós-graduação em Ciências Biológicas (Biologia Vegetal) - IBRC

Inquérito sorológico, molecular e fatores de risco para leptospirose em mamíferos cativos, papel dos animais sinantrópicos presentes no local e aspectos de saúde pública

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