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1.
Braz. j. biol ; 81(3): 692-700, July-Sept. 2021. graf
Article in English | LILACS | ID: biblio-1153403

ABSTRACT

Abstract Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.


Resumo A contaminação bacteriana dos componentes sanguíneos é um grande desafio na medicina transfusional, principalmente nos concentrados de plaquetas (PCs) devido às condições de armazenamento que favorecem a proliferação bacteriana. Neste estudo, desenvolvemos um protocolo de PCR em tempo real rápido, sensível e específico para a triagem bacteriana de PCs. Um método baseado em PCR em tempo real, controlado internamente, foi otimizado e validado com um Master Mix Universal PCR 16S (IBMP / Fiocruz), que detecta uma região conservada do gene 16S rRNA bacteriano. O background de DNA não específico foi completamente eliminado tratando a PCR Master Mix com monoazida de etídio (EMA). O limite de detecção inferior observado foi de 10 cópias equivalentes do genoma com um valor de Ct 34 ± 1,07, a curva de calibração foi gerada com diluições seriada de 10 vezes do DNA de E. coli. O tempo de processamento, incluindo a purificação microbiana do DNA, foi de aproximadamente 4 horas. O método desenvolvido mostrou alta sensibilidade sem amplificação inespecífica e menor tempo de detecção do que os métodos microbiológicos tradicionais, demonstrando ser um meio eficiente de triagem de PCs pré-transfusionais.


Subject(s)
Blood Platelets , Escherichia coli , Bacteria/genetics , DNA, Bacterial/genetics , RNA, Ribosomal, 16S , Real-Time Polymerase Chain Reaction
2.
J. appl. oral sci ; 29: e20200787, 2021. tab, graf
Article in English | LILACS | ID: biblio-1250191

ABSTRACT

Abstract Objective: To define the subgingival microbial profile associated with Stage II generalized periodontitis using next-generation sequencing and to determine the relative abundance of novel periodontal pathogens and bacterial complexes. Methodology: Subgingival biofilm samples were collected from 80 subjects diagnosed with Stage II generalized periodontitis. Bacterial DNA was extracted, and 16S rRNA-based bacterial profiling via next-generation sequencing was carried out. The bacterial composition and diversity of microbial communities based on the age and sex of the patients were analyzed. The bacterial species were organized into groups: bacterial complexes (red, orange, purple, yellow, and green), novel periodontal pathogens, periodontal health-related species, and unclassified periodontal species. The results were analyzed and statistically evaluated. Results: The highest number of bacteria belonged to the phylum Bacteroidetes and Firmicutes. In terms of relative abundance, the orange complex represented 18.99%, novel bacterial species (Fretibacterium spp. and Saccharibacteria spp.) comprised 17.34%, periodontal health-related species accounted for 16.75% and unclassified periodontal species represented (Leptotrichia spp. and Selenomonas spp.) 15.61%. Novel periodontal pathogens had outweighed the periodontal disease-related red complex (5.3%). The one-sample z-test performed was statistically significant at p<0.05. The Beta diversity based on the unweighted UniFrac distance at the species level demonstrated a total variance of 15.77% based on age and 39.19% on sex, which was not statistically significant. Conclusion: The bacterial species corresponding to the disease-related orange complex and novel periodontal pathogens are predominant in Stage II generalized periodontitis.


Subject(s)
Humans , Adult , Periodontitis , Dental Plaque , Bacteria/genetics , DNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Prevalence , High-Throughput Nucleotide Sequencing
3.
Braz. j. biol ; 80(4): 872-880, Oct.-Dec. 2020. tab, graf
Article in English | LILACS | ID: biblio-1142543

ABSTRACT

Abstract Studies on the bacterial diversity associated with wild plants are rare, especially on those that grow in association with bromeliads. In the present study, we isolated and identified epiphytic and endophytic bacteria from the roots of the bromeliads Dyckia excelsa, Dyckia leptostachya and Deuterocohnia meziana occurring in the "cangas" in the Pantanal from Mato Grosso do Sul State, Brazil. The epiphytic bacteria were isolated from washed roots, while the endophytic bacteria were isolated from surface disinfested roots. Bacterial representatives corresponding to each BOX-PCR fingerprint, as well as those that did not result in amplicons, were selected for 16S rDNA gene sequence analysis. The BOX-PCR data showed intrageneric and intraspecific diversity and could discriminate strains and identify their phenotypic characteristics. The 16S rDNA gene sequence and phylogeny analysis showed a higher occurrence of strains belonging to the genus Bacillus than Mycobacterium and Brevibacterium, which were found in lower numbers. Species from the Bacillus genus are well known for their sporulation capacity and longer survival in arid locations, such as the "cangas". This study clearly showed that the bromeliad species represent a vast reservoir of bacterial community diversity, and the cultivable strains represent a new source for biotechnological prospecting.


Resumo Estudos sobre a diversidade bacteriana associada a plantas silvestres são raros, especialmente naqueles que crescem em associação com bromélias. No presente estudo, isolamos e identificamos bactérias epífitas e endofíticas das raízes das bromélias Dyckia excelsa, D. leptostachya e Deuterocohnia meziana ocorrentes nas "cangas" no Pantanal do Mato Grosso do Sul, Brasil. As bactérias epifíticas foram isoladas de raízes lavadas, enquanto as bactérias endofíticas foram isoladas de raízes desinfestadas na superfície. Representantes bacterianos correspondentes a cada perfil do BOX-PCR, bem como aqueles que não resultaram em amplificações, foram selecionados para o sequenciamento do gene 16S rDNA. Os dados da BOX-PCR mostraram diversidade intragênica e intraespecífica e puderam discriminar cepas e identificar suas características fenotípicas. A seqüência do gene 16S rDNA e a análise filogenética mostraram uma maior ocorrência de cepas pertencentes ao gênero Bacillus do que as bactérias Mycobacterium e Brevibacterium, encontradas em menor número. Espécies do gênero Bacillus são bem conhecidas por sua capacidade de esporulação e maior sobrevida em locais áridos, como as "cangas". Este estudo mostrou claramente que as espécies de bromélias representam um vasto reservatório de diversidade de comunidades bacterianas, e as linhagens cultiváveis podem representar uma nova fonte para a prospecção biotecnológica.


Subject(s)
Bacteria/genetics , Biodiversity , Phylogeny , Brazil , DNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Plant Roots
4.
Braz. j. otorhinolaryngol. (Impr.) ; 86(2): 217-221, March-Apr. 2020. tab, graf
Article in English | LILACS | ID: biblio-1132581

ABSTRACT

Abstract Introduction: It is proposed that Helicobacter pylori can be responsible for the development of otitis media with effusion. Objective: The aim of this study is to investigate the prevalence of H. pylori in the adenoid tissue and fluid of the middle ear in patients who suffer from adenoid hyperplasia and otitis media with effusion in comparison with those who suffer from adenoid hyperplasia without otitis media with effusion. Methods: This is a case-control study that was carried out in 50 children of age 2-7 years old who were admitted with adenoid hyperplasia. Patients were divided into case and control groups. The study group included 25 patients with adenoid hyperplasia and otitis media with effusion and the control group included 25 patients with adenoid hyperplasia without otitis media with effusion. The patients in both groups underwent surgical adenoidectomy. For the case group we carried out myringotomy and placement of tympanostomy tube, and fluid samples were collected under sterile conditions. The samples were sent to the laboratory for polymerase chain reactions. Results: In the case group H. pylori was found to be positive in 18 samples of the middle ear fluid (70%) and in 1 polymerase chain reaction adenoid tissue sample (4%). In the control group H. pylori was positive in 3 samples of adenoid tissues (12%). There was no gender difference. Conclusion: H. pylori is one of the important bacteria that plays a role in the pathogenesis of otitis media with effusion. Whether adenoid tissue may be a reservoir for H. Pylori is unclear.


Resumo Introdução: Propõe-se que o Helicobacter pylori possa ser responsável pelo desenvolvimento de otite média com efusão. Objetivo: Investigar a prevalência de H. pylori no tecido adenoideano e no fluido da orelha média em pacientes com hiperplasia de adenoide e otite média com efusão em comparação àqueles com hiperplasia de adenoide sem otite média com efusão. Método: Este é um estudo de caso-controle feito em 50 crianças de 2 a 7 anos, com sinais e sintomas de hiperplasia de adenoide. Os pacientes foram divididos em grupo de estudo e grupo controle. O grupo de estudo incluiu 25 pacientes com hiperplasia de adenoide e otite média com efusão e o grupo controle incluiu 25 pacientes com hiperplasia de adenoide sem otite média com efusão. Os pacientes dos dois grupos foram submetidos a adenoidectomia e, no grupo de estudo, realizou-se também miringotomia com colocação de tubo de ventilação e amostras de fluidos foram coletadas sob condições estéreis. As amostras foram enviadas para o laboratório, para investigação por reação de polimerase em cadeia. Resultados: No grupo de estudo, houve positividade para H. pylori em 18 amostras do fluido de orelha média (70%) e uma amostra de tecido adenoideano foi positiva na reação de polimerase em cadeia (4%). No grupo controle, houve positividade para H. pylori em 3 amostras de tecido adenoideano (12%). Não houve diferença entre os gêneros. Conclusão: H. pylori é uma das bactérias importantes que desempenham um papel na patogênese da otite médica com efusão. Se o tecido adenoideano pode ou não representar um reservatório para H. pylori ainda necessita ser esclarecido.


Subject(s)
Humans , Male , Female , Child, Preschool , Child , Otitis Media with Effusion/microbiology , DNA, Bacterial/genetics , Helicobacter pylori/genetics , Helicobacter Infections/diagnosis , Case-Control Studies , Polymerase Chain Reaction , Helicobacter pylori/isolation & purification
5.
Rev. Soc. Bras. Med. Trop ; 53: e20190333, 2020. graf
Article in English | LILACS | ID: biblio-1092187

ABSTRACT

Abstract INTRODUCTION: Phylogenetic analysis of the 16S ribosomal gene initial region is used to identify Leptospira isolates at the species level from clinical samples. Unfortunately, this method cannot differentiate between some intermediates and saprophytic species. METHODS: We used comparative genomic analysis between 35 Leptospira species to find new molecular targets for Leptospira species identification. RESULTS: We proposed the use of the rpoC gene, encoding the DNA-directed RNA polymerase β-subunit, for identifying 35 Leptospira species. CONCLUSIONS: The rpoC gene can be a molecular target to identify the main species of the Leptospira genus directly from clinical samples.


Subject(s)
Humans , Animals , DNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Leptospira/genetics , Phylogeny , DNA-Directed RNA Polymerases , Leptospira/classification
6.
Mem. Inst. Oswaldo Cruz ; 115: e190407, 2020. tab
Article in English | LILACS | ID: biblio-1101275

ABSTRACT

BACKGROUND Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. OBJECTIVES To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. METHODS Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. FINDINGS The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. MAIN CONCLUSIONS qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.


Subject(s)
Humans , Genetic Markers/genetics , Drug Resistance, Bacterial/genetics , Isoniazid/pharmacology , Mutation/genetics , Mycobacterium tuberculosis/genetics , Antitubercular Agents/pharmacology , DNA, Bacterial/genetics , Microbial Sensitivity Tests , Sensitivity and Specificity , Real-Time Polymerase Chain Reaction , Genotype , Mycobacterium tuberculosis/drug effects
7.
Mem. Inst. Oswaldo Cruz ; 115: e200370, 2020. tab, graf
Article in English | SES-SP, LILACS, SES-SP | ID: biblio-1135225

ABSTRACT

BACKGROUND Bacillus anthracis is the aetiologic agent of anthrax, a re-emerging, septicaemic, haemorrhagic and lethal disease that affects humans, domestic ruminants and wildlife. Plasmids pXO1 and pXO2 are attributes that confer pathogenicity to B. anthracis strains. This bacterium was used as biological weapon in the World Wars and in the biological attack in the United States of America at 2001. B. anthracis is classified as a Tier 1 bioterrorism agent by the Centers for Diseases Control and Prevention. Anthrax is recognised as a re-emerging disease. Several studies concerning the dynamics of B. anthracis cycle in soil revealed that nonpathogenic B. anthracis strains due to lack of pXO2 plasmid are commonly found in some types of soil. OBJECTIVES This study aimed isolation and identification of B. anthracis spores in soil samples of the state of Rio de Janeiro, Brazil. METHODS Phenotypic and genotypic approaches were used to identify isolates including MALDI-TOF/MS, motility test, susceptibility to gamma phage and penicillin, survey for pag and cap genes as surrogates of pXO1 and pXO2 plasmids, respectively, and sequencing of 16SrRNA-encoding gene. Physicochemical analysis of the soil samples were carried out to describe soil characteristics. FINDINGS We observed the presence of one B. anthracis pXO1+ and pXO2- isolated from clay loam soil; one B. anthracis-like strain pXO1+ and pXO2-isolated from loamy sand; and 10 Bacillus spp. strains sensitive to phage-gamma that need better characterisation to define which their species were recovered from loamy sand. MAIN CONCLUSIONS This work showed promising results and it was the first study to report results from an active surveillance for B. anthracis in Brazil.


Subject(s)
Humans , Plasmids/analysis , Soil Microbiology , Spores, Bacterial , Bacillus anthracis/isolation & purification , DNA, Bacterial/genetics , Polymerase Chain Reaction/methods , Virulence Factors/genetics , Plasmids/genetics , Soil , Bacillus anthracis/genetics , Bacillus anthracis/pathogenicity , Bacterial Toxins , Virulence , Brazil , DNA, Bacterial/analysis , Sequence Analysis, DNA , Antigens, Bacterial
8.
Mem. Inst. Oswaldo Cruz ; 115: e200184, 2020. tab, graf
Article in English | SES-SP, LILACS, SES-SP | ID: biblio-1135263

ABSTRACT

BACKGROUND Carrion's disease (CD) is a neglected biphasic illness caused by Bartonella bacilliformis, a Gram-negative bacteria found in the Andean valleys. The spread of resistant strains underlines the need for novel antimicrobials against B. bacilliformis and related bacterial pathogens. OBJECTIVE The main aim of this study was to integrate genomic-scale data to shortlist a set of proteins that could serve as attractive targets for new antimicrobial discovery to combat B. bacilliformis. METHODS We performed a multidimensional genomic scale analysis of potential and relevant targets which includes structural druggability, metabolic analysis and essentiality criteria to select proteins with attractive features for drug discovery. FINDINGS We shortlisted seventeen relevant proteins to develop new drugs against the causative agent of Carrion's disease. Particularly, the protein products of fabI, folA, aroA, trmFO, uppP and murE genes, meet an important number of desirable features that make them attractive targets for new drug development. This data compendium is freely available as a web server (http://target.sbg.qb.fcen.uba.ar/). MAIN CONCLUSION This work represents an effort to reduce the costs in the first phases of B. bacilliformis drug discovery.


Subject(s)
Humans , Bartonella Infections/drug therapy , Bartonella bacilliformis/drug effects , Anti-Bacterial Agents/therapeutic use , DNA, Bacterial/isolation & purification , DNA, Bacterial/genetics , Polymerase Chain Reaction , Genomics , Bartonella bacilliformis/isolation & purification , Bartonella bacilliformis/genetics
9.
Rev. Soc. Bras. Med. Trop ; 53: e20190526, 2020. tab, graf
Article in English | ColecionaSUS, LILACS, ColecionaSUS, SES-SP | ID: biblio-1136834

ABSTRACT

Abstract INTRODUCTION: This study investigated the genetic environment of bla KPC-2 in Klebsiella pnemoniae multi-drug resistant clinical isolates. METHODS: Four carbapenemase gene isolates resistant to carbapenems, collected from infected patients from two hospitals in Brazil, were investigated using polymerase chain reaction and plasmid DNA sequencing. RESULTS: The bla KPC-2 gene was located between ISKpn6 and a resolvase tnpR in the non-Tn4401 element (NTEKPC-IId). It was detected on a plasmid belonging to the IncQ1 group. CONCLUSIONS To our knowledge, this is the first report of the presence of the bla KPC-2 gene in the NTEKPC-IId element carried by plasmid IncQ1 from infections in Brazil.


Subject(s)
Humans , beta-Lactamases/genetics , Klebsiella Infections/microbiology , Klebsiella pneumoniae/genetics , Anti-Bacterial Agents/pharmacology , Plasmids/genetics , DNA, Bacterial/genetics , Microbial Sensitivity Tests , Polymerase Chain Reaction , Drug Resistance, Multiple, Bacterial , Klebsiella pneumoniae/isolation & purification , Klebsiella pneumoniae/enzymology
10.
Chinese Journal of Biotechnology ; (12): 2541-2547, 2020.
Article in Chinese | WPRIM | ID: wpr-878509

ABSTRACT

Metagenomic sequencing provides a powerful tool for microbial research. However, traditional experimental DNA extraction process will inevitably mix with environmental microorganisms which float in the air. It is still unclear whether the mixed environmental microbial DNA will heavily affect the metagenomic results of samples with extremely low microbial content. In this study, we first collected environmental bacteria in the laboratory and quantified the mixed environmental microbial DNA content during DNA extraction based on a qPCR-based quantification assay. We then extracted DNA from pure water in order to determine the mixed microbial taxons during extraction under open environment. At last, we extracted total DNA from a skin sample in a Biosafety cabinet or under open laboratory environment, to assess the impact of the mixed environmental microorganisms on the metagenomic results. Our results showed that DNA extraction under open laboratory environment in Beijing region resulted in 28.9 pg contaminant, which may accout for 30% of total DNA amount from skin samples. Metagenomic analysis revealed that the main incorporated environmental taxons were Cutibacterium acnes and Escherichia coli. Tens of environmental bacteria were foisted in the skin DNA samples, which largely decreased the relative abundance of dominant species and thus deteriorated the result accuracy. Therefore, analyzing microbial composition of samples with extremely low DNA content should better performed under aseptic environment.


Subject(s)
DNA , DNA, Bacterial/genetics , Laboratories , Metagenomics , RNA, Ribosomal, 16S , Sequence Analysis, DNA
11.
Rev. bras. parasitol. vet ; 28(3): 451-457, July-Sept. 2019. tab, graf
Article in English | LILACS | ID: biblio-1042527

ABSTRACT

Abstract The msp4 gene of A. marginale is unicodon, stable and mostly homogeneous, being considered as a useful marker for phylogeographic characterization of this bacterium. The objective of this work was to analyze the phylogeography of A. marginale based on the msp4 gene in beef cattle from the Brazilian Pantanal, compared to those found in other regions worldwide. The blood samples investigated were collected from 400 animals (200 cows and 200 calves) reared in five extensive breeding farms in this region. The results indicated that of the evaluated samples, 56.75% (227/400) were positive for A. marginale based on the msp1β gene by quantitatitve PCR (qPCR), while 8.37% (19/227) were positive for the msp4 gene in the conventional PCR. In the Network distance analysis, 14 sequences from the Brazilian Pantanal were grouped into a single group with those from Thailand, India, Spain, Colombia, Parana (Brazil), Mexico, Portugal, Argentina, China, Venezuela, Australia, Italy and Minas Gerais (Brazil). Among 68 sequences from Brazil and the world, 15 genotypes were present while genotype number one (#1) was the most distributed worldwide. Both Splitstree and network analyses showed that the A. marginale msp4 sequences detected in beef cattle from the Brazilian Pantanal showed low polymorphism, with the formation of one genogroup phylogenetically related to those found in ruminants from South and Central America, Europe, and Asia.


Resumo O gene msp4 de A. marginale é unicodon, estável e pouco heterogêneo, sendo considerado como um marcador útil para caracterização filogeográfica desta bactéria. Este trabalho teve como objetivo analisar a filogeografia de A. marginale com base no gene msp4 em bovinos de corte do Pantanal Brasileiro, comparativamente a outra regiões do mundo. Alíquotas de sangue foram colhidas de 400 bovinos (200 vacas e 200 bezerros) em cinco propriedades de cria e recria extensiva. Como resultado, 56,75% (227/400) mostraram-se positivas para A. marginale pela qPCR para o gene msp1β e destas, 8,37% (19/227) amostras foram positivas na PCR convencional para o gene msp4. Na análise de distância Network, 14 sequências do Pantanal brasileiro foram agrupadas em um único grupo com as da Thailândia, Índia, Espanha, Colômbia, Paraná (Brasil), México, Portugal, Argentina, China, Venezuela, Austrália, Italia e Minas Gerais (Brasil). Dentre 68 sequências do Brasil e do mundo, constatou-se a presença de 15 genótipos, sendo o genótipo número um (#1) o mais distribuído. As sequências msp4 de A. marginale detectadas em bovinos de corte no Pantanal brasileiro apresentaram baixo polimorfismo com formação de dois genogrupos filogeneticamente relacionados àqueles encontrados em ruminantes de países das América do Sul e Central, Europa e Ásia.


Subject(s)
Animals , Male , Female , Bacterial Proteins/genetics , Cattle/microbiology , Anaplasma marginale/genetics , Phylogeography/methods , Membrane Proteins/genetics , Asia , Americas , Brazil , DNA, Bacterial/genetics , Molecular Sequence Data , Polymerase Chain Reaction , Amino Acid Sequence , Anaplasma marginale/isolation & purification , Europe , Genotype
12.
Rev. bras. parasitol. vet ; 28(2): 306-309, Apr.-June 2019. tab
Article in English | LILACS | ID: biblio-1042510

ABSTRACT

Abstract Mycoplasma suis is a bacterium that causes hemoplasmosis in pigs. This agent is capable of adhering to the surface of porcine erythrocytes, inducing structural changes on these cells. In Brazil, there are few reports about the disease, its causal agent, and the economic impact of this pathogen on pig production systems and farm sanitation. The present study aimed to investigate the occurrence of M. suis in extensive swine farms located in the counties of Itapecuru Mirim, Santa Rita and Rosario, State of Maranhão, northeast Brazil. For such purpose, 64 blood samples of pigs from these facilities were tested for M. suis using a 16S rRNA gene-based quantitative real-time PCR (qPCR); 82.3%, 65.2% and 25% of blood samples of swine from farms in the cities of Itapecuru Mirim, Santa Rita and Rosario were positive for M. suis by qPCR, respectively. This study shows, for the first time, that M. suis circulates in pig populations from the state of Maranhão, Northeast Brazil.


Resumo Mycoplasma suis é uma bactéria que causa a hemoplasmose em suínos. Este agente é capaz de se aderir à superfície dos eritrócitos de suínos, ocasionando deformações estruturais nestas células. No Brasil, poucos são os relatos acerca do parasita, da infecção e de seus impactos econômicos nas esferas produtiva e sanitária. O objetivo deste estudo foi investigar, por meio da PCR em tempo real quantitativa (qPCR) baseada no gene 16S rRNA, a ocorrência de M. suis em 64 amostras de sangue de suínos de criações extensivas dos municípios de Itapecuru Mirim, Santa Rita e Rosário, localizados no estado do Maranhão. Foram obtidos um percentual de 82,3%, 65,2% e 25% de amostras positivas na qPCR para M. suis nos municípios de Itapecuru Mirim, Santa Rita e Rosário, respectivamente. Este estudo mostra que M. suis circula entre os suínos de criações extensivas no estado do Maranhão.


Subject(s)
Animals , Male , Female , Mycoplasma/genetics , Mycoplasma Infections/microbiology , Mycoplasma Infections/veterinary , Swine , Swine Diseases/diagnosis , Swine Diseases/microbiology , Brazil , DNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Real-Time Polymerase Chain Reaction , Mycoplasma/classification , Mycoplasma Infections/diagnosis
13.
J. oral res. (Impresa) ; 8(1): 30-36, feb. 28, 2019. ilus, tab
Article in English | LILACS | ID: biblio-1145264

ABSTRACT

Objectives: To study the prevalence of methicillin resistant Staphylococcus aureus (MRSA) in saliva samples of pre-surgical oral squamous cell carcinoma (OSCC) patients along with their resistance pattern to other antibiotics. Methods: Saliva samples of OSCC patients were collected and processed for isolation of MRSA. Staphylococcus aureus isolates were primarily identified using standard microbiological methods like biochemical assays, specialized media and latex agglutination test. Confirmation of MRSA strains was done by growing the isolates on MRSA agar and by using PCR to amplify two MRSA specific genes. All the isolated Staphylococcus aureus strains were subjected to antibiotic sensitivity tests. Results: A total of 17 Staphylococcus aureus strains were isolated from 50 saliva samples of pre-surgical OSCC patients of which 13 were confirmed to be MRSA. These MRSA strains were also found to be mostly resistant to other commonly used antibiotics. Univariate analysis revealed that most patients with MRSA infections had a prior history of hospitalization and surgery. Also, it was confirmed that patients with other comorbidities and infections were more prone to having MRSA present in the saliva. Conclusion: The majority of Staphylococcus aureus isolates from the saliva of OSCC patients were MRSA, and were resistant to several other commonly used antibiotics.


Objetivos: Estudiar la prevalencia de Staphylococcus aureus resistente a la meticilina (MRSA) en muestras de saliva prequirúrgicas de pacientes con carcinoma oral de células escamosas (COCE) junto con su patrón de resistencia a otros antibióticos. Métodos: Se recolectaron muestras de saliva de pacientes con COCE y se procesaron para el aislamiento de SARM. Los aislamientos de Staphylococcus aureus se identificaron principalmente mediante métodos microbiológicos estándar, como los análisis bioquímicos, los medios especializados y la prueba de aglutinación con látex. La confirmación de las cepas de SARM se realizó cultivando los aislados en agar SARM y utilizando PCR para amplificar dos genes específicos de SARM. Todas las cepas aisladas de Staphylococcus aureus se sometieron a pruebas de sensibilidad a los antibióticos. Resultados: Se aislaron un total de 17 cepas de Staphylococcus aureus a partir de 50 muestras de saliva de pacientes prequirúrgicos con COCE, de los cuales solo se confirmó que 13 eran SARM. También se encontró que estas cepas de SARM son resistentes a otros antibióticos de uso común. El análisis univariado reveló que la mayoría de los pacientes con infecciones por SARM tenían antecedentes previos de hospitalización y cirugía. Además, se confirmó que los pacientes con otras comorbilidades e infecciones eran más propensos a las infecciones por SARM. Conclusión: la mayoría de los aislamientos de Staphylococcus aureusde la saliva de los pacientes con OSCC fueron MRSA y fueron resistentes a varios otros antibióticos de uso común.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Aged, 80 and over , Young Adult , Staphylococcus/isolation & purification , Drug Resistance, Microbial , Methicillin-Resistant Staphylococcus aureus/genetics , Squamous Cell Carcinoma of Head and Neck , Mouth/microbiology , Saliva , DNA, Bacterial/genetics , Prevalence , Anti-Bacterial Agents
14.
J. appl. oral sci ; 27: e20180256, 2019. tab
Article in English | LILACS, BBO | ID: biblio-1012514

ABSTRACT

Abstract Objective The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). Results Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.


Subject(s)
Humans , Streptococcus/isolation & purification , DNA, Ribosomal/isolation & purification , RNA, Ribosomal/isolation & purification , Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Dental Pulp Cavity/microbiology , Root Canal Therapy/methods , Streptococcus/genetics , DNA, Bacterial/isolation & purification , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , RNA, Bacterial/isolation & purification , RNA, Bacterial/genetics , RNA, Ribosomal/genetics , Reproducibility of Results
15.
Biol. Res ; 52: 5, 2019. tab, graf
Article in English | LILACS | ID: biblio-1011408

ABSTRACT

BACKGROUND: A moderately thermophilic, slightly halophilic, aerobic, Gram-stain negative, bacterial strain, SLM16, was isolated from a mixed of seawater-sand-sediment sample collected from a coastal fumarole located in Whalers Bay, Deception Island, Antarctica. The aim was to screen for thermophilic microorganisms able to degrade primary amines and search for amine transaminase activity for potential industrial application. RESULTS: Identification and partial characterization of the microorganism SLM16 were carried out by means of morphological, physiological and biochemical tests along with molecular methods. Cells of strain SLM16 were non-motile irregular rods of 1.5-2.5 µm long and 0.3-0.45 µm wide. Growth occurred in the presence of 0.5-5.5% NaCl within temperature range of 35-55 °C and pH range of 5.5-9.5, respectively. The DNA G+C composition, estimated from ftsY gene, was 66% mol. Phylogenetic analysis using de 16S rRNA gene sequence showed that strain SLM16 belongs to the marine bacterial genus Albidovulum. CONCLUSION: Strain SLM16 is a moderate thermophilic Gram negative microorganisms which belongs to the marine bacterial genus Albidovulum and is closely related to Albidovulum inexpectatum species based on phylogenetic analysis. Additionally, amine-transaminase activity towards the arylaliphatic amine α-methylbenzylamine was detected.


Subject(s)
Seawater/microbiology , DNA, Bacterial/genetics , Rhodobacteraceae/isolation & purification , Rhodobacteraceae/enzymology , Transaminases/metabolism , Phylogeny , RNA, Ribosomal, 16S/genetics , Bacterial Typing Techniques , Sequence Analysis, DNA , Rhodobacteraceae/classification , Antarctic Regions
16.
Rev. Soc. Bras. Med. Trop ; 52: e20190243, 2019. tab
Article in English | LILACS | ID: biblio-1020442

ABSTRACT

Abstract INTRODUCTION In recent decades, the prevalence of carbapenem-resistant Acinetobacter isolates has increased, and the production of oxacillinase (OXA)-type carbapenemases is the main mechanism underlying resistance. We evaluated OXA production from 114 Acinetobacter isolates collected between March and December 2013 from different clinical specimens of patients in two hospitals (Hospital 1 [n = 61] and Hospital 2 [n = 53]) located in Niterói, Rio de Janeiro, Brazil. We also evaluated the genetic diversity of OXA-producing isolates. METHODS All the isolates were identified through the automated system Vitek II and matrix-assisted laser desorption ionization-time of flight mass spectrometry MALDI-TOF MS as belonging to the A. baumannii-A. calcoaceticuscomplex. Antimicrobial susceptibility profiles were verified through agar diffusion tests. The presence of OXA-encoding genes was confirmed by PCR. The genetic diversity of isolates positive for carbapenemase production was analyzed through pulsed-field gel electrophoresis. RESULTS There was a high rate of resistance to carbapenems in the isolates (imipenem: 96%; meropenem: 92%) from both hospitals. Moreover, a high percentage (95.6%) of OXA-23-positive isolates was observed for both hospitals, indicating that this was the main mechanism of carbapenem-resistance among the studied population. In addition, most isolates (96.5%) were positive for bla OXA-51. A high genetic diversity and a few major genotypes were found among the OXA-23-positive isolates analyzed. Only intra-hospital dissemination was observed. CONCLUSIONS The elevated dissemination of bla OXA-23-like observed among Acinetobacter isolates from both the studied hospitals highlights the need for continuous epidemiological surveillance in these institutions.


Subject(s)
Humans , Acinetobacter/enzymology , beta-Lactamases/drug effects , Acinetobacter Infections/microbiology , Acinetobacter/drug effects , Acinetobacter/genetics , beta-Lactamases/biosynthesis , Brazil , DNA, Bacterial/genetics , Microbial Sensitivity Tests , Polymerase Chain Reaction , Bacterial Typing Techniques , Electrophoresis, Gel, Pulsed-Field , Hospitals, General , Anti-Bacterial Agents/pharmacology
17.
Braz. j. microbiol ; 49(4): 816-822, Oct.-Dec. 2018. tab, graf
Article in English | LILACS | ID: biblio-974289

ABSTRACT

ABSTRACT Fifty seven soil-borne actinomycete strains were assessed for the antibiotic production. Two of the most active isolates, designed as Streptomyces ST-13 and DK-15 exhibited a broad range of antimicrobial activity and therefore they were selected for HPLC fractionation against the most suppressed bacteria Staphylococcus aureus (ST-13) and Chromobacterium violaceum (DK-15). LC/MS analysis of extracts showed the presence of polyketides factumycin (DK15) and tetrangomycin (ST13). The taxonomic position of the antibiotic-producing actinomycetes was determined using a polyphasic approach. Phenotypic characterization and 16S rRNA gene sequence analysis of the isolates matched those described for members of the genus Streptomyces. DK-15 strain exhibited the highest 16S rRNA gene sequence similarity to Streptomyces globosus DSM-40815 (T) and Streptomyces toxytricini DSM-40178 (T) and ST-13 strain to Streptomyces ederensis DSM-40741 (T) and Streptomyces phaeochromogenes DSM-40073 (T). For the proper identification, MALDI-TOF/MS profile of whole-cell proteins led to the identification of S. globosus DK-15 (accession number: KX527570) and S. ederensis ST13 (accession number: KX527568). To our knowledge, there is no report about the production of these antibiotics by S.globosus and S. ederensis, thus isolates DK15 and ST13 identified as S. globosus DK-15 and S.ederensis ST-13 can be considered as new sources of these unique antibacterial metabolites.


Subject(s)
Streptomyces/isolation & purification , Streptomyces/metabolism , Anti-Bacterial Agents/biosynthesis , Phylogeny , Pyridones/metabolism , Soil Microbiology , Streptomyces/classification , Streptomyces/genetics , Benz(a)Anthracenes/metabolism , DNA, Bacterial/genetics , Bacterial Typing Techniques
18.
Braz. j. microbiol ; 49(4): 900-908, Oct.-Dec. 2018. tab, graf
Article in English | LILACS | ID: biblio-974290

ABSTRACT

ABSTRACT Matrix Assisted Laser Desorption/Ionization and Time of Flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the identification of bacteria through the detection and analysis of their proteins or fragments derived from ribosomes. Slight sequence variations in conserved ribosomal proteins distinguish microorganisms at the subspecies and strain levels. Characterization of Leptospira spp. by 16S RNA sequencing is costly and time-consuming, and recent studies have shown that closely related species (e.g., Leptospira interrogans and Leptospira kirschneri) may not be discriminated using this technology. Herein, we report an in-house Leptospira reference spectra database using Leptospira reference strains that were validated with a collection of well-identified Brazilian isolates kept in the Bacterial Zoonosis Laboratory at the Veterinary Preventive Medicine and Animal Health Department at Sao Paulo University. In addition, L. interrogans and L. kirschneri were differentiated using an in-depth mass spectrometry analysis with ClinProTools™ software. In conclusion, our in-house reference spectra database has the necessary accuracy to differentiate pathogenic and non-pathogenic species and to distinguish L. interrogans and L. kirschneri.


Subject(s)
Humans , Bacterial Typing Techniques/methods , Tandem Mass Spectrometry/methods , Leptospira/isolation & purification , Leptospirosis/microbiology , Brazil , DNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Leptospira/classification , Leptospira/genetics , Leptospira/chemistry
19.
Braz. j. microbiol ; 49(4): 742-748, Oct.-Dec. 2018. graf
Article in English | LILACS | ID: biblio-974296

ABSTRACT

ABSTRACT We examined microbial communities from enriched fine and retorted shale particles using sequencing of V4 variable region of 16S rRNA. High number of microbial genera was found in both enriched shale by-products that were dominate by Actinobacteria, Firmicutes and Proteobacteria, showing differences due to microbial colonization after the pyrolysis process.


Subject(s)
Bacteria/isolation & purification , Waste Products/analysis , Geologic Sediments/microbiology , Microbiota , Phylogeny , Bacteria/classification , Bacteria/genetics , Brazil , DNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Geologic Sediments/chemistry , Biodiversity
20.
Braz. j. microbiol ; 49(4): 695-702, Oct.-Dec. 2018. tab, graf
Article in English | LILACS | ID: biblio-974298

ABSTRACT

ABSTRACT Antarctica harbors a great diversity of microorganisms, including bacteria, archaea, microalgae and yeasts. The Pseudomonas genus is one of the most diverse and successful bacterial groups described to date, but only eight species isolated from Antarctica have been characterized. Here, we present three potentially novel species isolated on King George Island. The most abundant isolates from four different environments, were genotypically and phenotypically characterized. Multilocus sequence analysis and 16S rRNA gene analysis of a sequence concatenate for six genes (16S, aroE, glnS, gyrB, ileS and rpoD), determined one of the isolates to be a new Pseudomonas mandelii strain, while the other three are good candidates for new Pseudomonas species. Additionally, genotype analyses showed the three candidates to be part of a new subgroup within the Pseudomonas fluorescens complex, together with the Antarctic species Pseudomonas antarctica and Pseudomonas extremaustralis. We propose terming this new subgroup P. antarctica. Likewise, phenotypic analyses using API 20 NE and BIOLOG® corroborated the genotyping results, confirming that all presented isolates form part of the P. fluorescens complex. Pseudomonas genus research on the Antarctic continent is in its infancy. To understand these microorganisms' role in this extreme environment, the characterization and description of new species is vital.


Subject(s)
Phylogeny , Pseudomonas/isolation & purification , Pseudomonas/classification , Phenotype , Pseudomonas/genetics , Soil Microbiology , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , RNA, Ribosomal, 16S/genetics , Multilocus Sequence Typing , Islands , Genotype , Antarctic Regions
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