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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.
Article in English | WPRIM | ID: wpr-887737

ABSTRACT

Objective@#To evaluate multidrug resistant loop-mediated isothermal amplification (MDR-LAMP) assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the @*Methods@#MDR-LAMP assay was evaluated using 100 @*Results@#The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MDR-LAMP were 85.5%, 93.6%, 96.7%, and 74.4% for the detection of resistance to isoniazid and rifampicin, respectively, and 80.5%, 92.3%, 98.6%, and 41.4% for the detection of @*Conclusion@#MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of


Subject(s)
Antitubercular Agents , Bacterial Proteins/genetics , Catalase/genetics , DNA, Bacterial/analysis , DNA-Directed RNA Polymerases/genetics , Drug Resistance, Multiple, Bacterial/genetics , Isoniazid , Molecular Diagnostic Techniques/methods , Mutation , Mycobacterium tuberculosis/isolation & purification , Nucleic Acid Amplification Techniques/methods , Oxidoreductases/genetics , Phenotype , Rifampin , Whole Genome Sequencing
4.
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
5.
Rev. Assoc. Med. Bras. (1992) ; 66(11): 1509-1514, Nov. 2020. tab, graf
Article in English | SES-SP, LILACS, SES-SP | ID: biblio-1143632

ABSTRACT

SUMMARY INTRODUCTION: Nearly 73% of the Chilean population is infected with Helicobacter pylori (H. pylori), a factor predisposing for gastric cancer. Recent studies have demonstrated the presence of this pathogen within yeasts, suggesting that this fact can directly influence the failure of a treatment, transmission, and reinfection. AIM: To detect the presence of H. pylori inside oral yeasts isolated from students of the University of Concepción (Chile). METHODS: 72 samples, obtained from the oral cavity using cotton swabs were incubated in YPD broth for 48h at 37°C and posteriorly seeded in Sabouraud Dextrose agar plus chloramphenicol at the same temperature and for the same time. Yeasts isolated were observed microscopically (wet mounting and Gram-stained) and identified using microbiological techniques. Intracellular H. pylori detection was performed by the amplification of 16S rDNA by PCR. RESULTS: Oral yeasts were detected in 24 samples (33.3%), being C. albicans (79.2%) the most frequent species, followed by C. dubliniensis (12.4%), C. krusei (4.2%), and C. tropicalis (4.2%). When analyzed by PCR, 15 of the 24 oral yeasts 62.5 % were positive for H. pylori 16S rDNA. From the 15 individuals positive for yeast harboring H. pylori, 81% of them reported stomach discomfort, and the presence of the bacteria was diagnosed at some moment in 20% of them. CONCLUSION: The intracellular presence of the H. pylori in oral yeasts suggests an endosymbiotic relationship of these microorganisms, which could favor H. pylori transmission and reinfection in the gastrointestinal tract.


RESUMO INTRODUÇÃO: Quase 73% da população chilena estão infectadas pelo Helicobacter pylori (H. pylori), fator predisponente ao câncer gástrico. Estudos recentes demonstraram a presença desse patógeno em leveduras, sugerindo que esse fato pode influenciar diretamente a falha de um tratamento, transmissão e reinfecção. OBJETIVO: Detectar a presença de H. pylori em leveduras orais isoladas de estudantes da Universidade de Concepción (Chile). MÉTODOS: 72 amostras, obtidas da cavidade oral utilizando cotonetes, foram incubadas em caldo YPD por 48h a 37°C e posteriormente sementes em ágar Sabouraud Dextrose mais cloranfenicol na mesma temperatura e ao mesmo tempo. Leveduras isoladas foram observadas microscopicamente (montagem úmida e corada por Gram) e identificadas utilizando técnicas microbiológicas. A detecção intracelular de H. pylori foi realizada pela amplificação do 16S rDNA por PCR. RESULTADOS: Leveduras orais foram detectadas em 24 amostras (33,3%), sendo C. albicans (79,2%), a espécie mais frequente seguida por C. dubliniensis (12,4%), C. krusei (4,2%) e C. tropicalis (4,2 %) Quando analisadas por PCR, 15 das 24 leveduras orais 62,5% foram positivas para o H. pylori 16S rDNA. Dos 15 indivíduos positivos para leveduras que abrigam H. pylori, 81% deles relataram desconforto estomacal e a presença da bactéria foi diagnosticada em algum momento em 20% deles. CONCLUSÃO: A presença intracelular do H. pylori em leveduras orais sugere uma relação endossimbiótica desses microrganismos, o que poderia favorecer a transmissão e a reinfecção do H. pylori no trato gastrointestinal.


Subject(s)
Humans , Helicobacter pylori/genetics , Helicobacter Infections , Students , Universities , DNA, Bacterial , Chile/epidemiology
6.
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
7.
Braz. oral res. (Online) ; 34: e042, 2020. tab, graf
Article in English | LILACS, BBO | ID: biblio-1132668

ABSTRACT

Abstract A few investigations of caries biofilms have identified Scardovia spp.; however, little is known about its involvement in caries pathogenesis. The purpose of this study was to assess the gene expression profile of Scardovia spp. in root caries, and compare it with other microorganisms. Clinical samples from active root caries lesions were collected. Microbial mRNA was isolated and cDNA sequenced. The function and composition of the Scardovia were investigated using two methods: a) de novo assembly of the read data and mapping to contigs, and b) reads mapping to reference genomes. Pearson correlation was performed (p < 0.05). Proportion of Scardovia inopinata and Scardovia wiggsiae sequences ranged from 0-6% in the root caries metatranscriptome. There was a positive correlation between the transcriptome of Lactobacillus spp. and Scardovia spp. (r = 0.70; p = 0.03), as well as with other Bifidobacteriaceae (r = 0.91; p = 0.0006). Genes that code for fructose 6-phosphate phosphoketolase (the key enzyme for "Bifid shunt"), as well as ABC transporters and glycosyl-hydrolases were highly expressed. In conclusion, "Bifid shunt" and starch metabolism are involved in carbohydrate metabolism of S. inopinata and S. wiggsiae in root caries. There is a positive correlation between the metabolism abundance of Lactobacillus spp., Bifidobacteriaceae members, and Scardovia in root caries.


Subject(s)
Humans , Male , Female , Adult , Aged , Aged, 80 and over , Gene Expression , Actinobacteria/genetics , Root Caries/microbiology , Reference Values , DNA, Bacterial , Chromosome Mapping , Actinobacteria/isolation & purification , Sequence Analysis, DNA , Statistics, Nonparametric , Biofilms , Gene Expression Profiling , Transcriptome , Middle Aged
8.
Braz. oral res. (Online) ; 34: e093, 2020. tab, graf
Article in English | LILACS, BBO | ID: biblio-1132652

ABSTRACT

Abstract Information about bacterial diversity, such as the number of each species in the root canals of primary teeth, contributes to improving our effective management of infections of endodontic origin in primary teeth. This study made a qualitative and quantitative assessment of the bacteria in the root canals of primary teeth with necrotic pulp, using the fluorescence in situ hybridization (FISH) technique. Thirty-one primary teeth with pulp necrosis from 31 children were evaluated using the FISH technique, to detect the presence and density of Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Enterococcus faecalis, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Streptococcus, Streptococcus mutans, Streptococcus sobrinus, Tannerella forsythia and Treponema denticola. Descriptive measures explained the data related to density, and Student's t-test assessed the differences among the densities of each bacterium, according to signs and symptoms. The bacterial density was paired and correlated. All bacteria tested were detected and identified in all the samples. The average number of bacterial individuals from each species ranged from 1.9 x 108 cells/mL (S. mutans) to 3.1 x 108 cells/mL (F. nucleatum) (p > 0.05). The sum of the mean counts of each bacterium represented almost 80% of the entire microbial community. Patients with pain had significantly more T. denticola, and those with edema showed a greater density of Streptococcus and P. nigrescens (p < 0.05). This study revealed that all 12 bacteria evaluated were found in all primary teeth with pulp necrosis. There was no predominance among the species studied; all species had a similar number of individuals.


Subject(s)
Humans , Child , Tooth, Deciduous , Dental Pulp Necrosis , Dental Pulp Cavity , DNA, Bacterial , In Situ Hybridization, Fluorescence , Porphyromonas gingivalis , Prevotella intermedia
9.
Rev. bras. parasitol. vet ; 29(1): e022419, 2020. tab, graf
Article in English | LILACS | ID: biblio-1092691

ABSTRACT

Abstract This study aimed to evaluate the occurrence of diseases transmitted by Amblyomma ovale in 61 dogs monitored for three years through collections of ticks and blood, interviews, telemetry and camera traps in three areas of Serra do Mar State Park, Brazil. Blood samples were used to investigate infection by Rangelia vitalii by real-time TaqMan PCR and Rickettsia parkeri by IIFA. The collected ticks were submitted to conventional PCR to investigate the presence of R. parkeri . These data were compared with the monitoring results and interviews with the owners. Dogs considered as companion presented a risk of infection by R. parkeri strain Mata Atlantica 5.4 times higher than those not considered as companion (p = 0.009). Dogs that had at least one A. ovale collected during the campaigns had a 10 times higher risk of infection by R. parkeri strain Mata Atlantica than those who did not (p = 0.009). One dog positive for R. vitalii by real-time TaqMan PCR was parasitized by A. ovale frequently during monitoring. Sequenced ompaA - positive DNA samples had 100% identity of R. parkeri strain Mata Atlantica clone As106. From the findings, it is urgent to control domestic dogs around rainforests to reduce zoonoses transmission.


Resumo A ocorrência de doenças transmitidas por Amblyomma ovale em 61 cães monitorados por três anos através de coletas de carrapatos, sangue, entrevistas, telemetria e armadilhas fotográficas foi avaliada em três áreas do Parque Estadual da Serra do Mar - SP. Amostras de sangue foram utilizadas para investigação de Rangelia vitalii através de PCR TaqMan em tempo real e Rickettsia parkeri através da RIFI. Carrapatos coletados foram submetidos à PCR convencional para investigação de R. parkeri . Estes dados foram comparados considerando os resultados do monitoramento e entrevistas. Cães de companhia apresentaram risco de infecção pela R. parkeri cepa Mata Atlântica 5,4 vezes maior que os não considerados como de companhia (p = 0,009). Cães que tiveram pelo menos um A. ovale coletado apresentaram risco de infecção por R. parkeri cepa Mata Atlântica 10 vezes maior do que aqueles que não tiveram (p = 0,009). Um cão positivo para R. vitalii através de PCR TaqMan em tempo real foi parasitado por A. ovale durante o monitoramento. Amostras positivas para o gene ompaA possuíam 100% de identidade do clone As106 de R. parkeri cepa de Mata Atlântica. Assim, é urgente o controle de cães na Mata Atlântica para redução dos riscos de zoonoses.


Subject(s)
Animals , Dogs , Rickettsia/isolation & purification , Rickettsia Infections/veterinary , Ixodidae/microbiology , Dog Diseases/epidemiology , Rickettsia/classification , Rickettsia/genetics , Rickettsia Infections/diagnosis , Rickettsia Infections/epidemiology , Telemetry , Brazil/epidemiology , DNA, Bacterial/analysis , Polymerase Chain Reaction , Sequence Analysis, DNA , Fluorescent Antibody Technique, Indirect , Dog Diseases/diagnosis , Dog Diseases/microbiology , Rainforest
10.
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
11.
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
12.
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
13.
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
14.
Rev. Soc. Bras. Med. Trop ; 53: e20200064, 2020. tab, graf
Article in English | ColecionaSUS, LILACS, ColecionaSUS, SES-SP | ID: biblio-1136871

ABSTRACT

Abstract INTRODUCTION: Carbapenem-resistant Klebsiella pneumoniae infection lacks treatment options and is associated with prolonged hospital stays and high mortality rates. The production of carbapenemases is one of the most important factors responsible for this multi-resistance phenomenon. METHODS: In the present study, we analyzed the presence of genes encoding carbapenemases in K. pneumoniae isolates circulating in one of the public hospitals in the city of Aracaju, Sergipe, Brazil. We also determined the best combination of drugs that display in vitro antimicrobial synergy. First, 147 carbapenem-resistant K. pneumoniae isolates were validated for the presence of blaKPC, bla GES, bla NDM, bla SPM, bla IMP, bla VIM, and bla OXA-48 genes using multiplex polymerase chain reaction. Thereafter, using two isolates (97 and 102), the role of double and triple combinational drug therapy as a treatment option was analyzed. RESULTS: Seventy-four (50.3%) isolates were positive for bla NDM, eight (5.4%) for bla KPC, and one (1.2%) for both bla NDM and bla KPC. In the synergy tests, double combinations were better than triple combinations. Polymyxin B and amikacin for isolate 97 and polymyxin B coupled with meropenem for isolate 102 showed the best response. CONCLUSIONS: Clinicians in normal practice use multiple drugs to treat infections caused by multi-resistant microorganism; however, in most cases, the benefit of the combinations is unknown. In vitro synergistic tests, such as those described herein, are important as they might help select an appropriate multi-drug antibiotic therapy and a correct dosage, ultimately reducing toxicities and the development of antibiotic resistance.


Subject(s)
Humans , Bacterial Proteins/genetics , beta-Lactamases/genetics , Klebsiella Infections/microbiology , Klebsiella pneumoniae/drug effects , Anti-Bacterial Agents/pharmacology , Brazil , DNA, Bacterial , Microbial Sensitivity Tests , Polymerase Chain Reaction , Prevalence , Drug Resistance, Multiple, Bacterial/genetics , Klebsiella pneumoniae/isolation & purification , Klebsiella pneumoniae/genetics
15.
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
16.
Chinese Journal of Biotechnology ; (12): 2695-2706, 2020.
Article in Chinese | WPRIM | ID: wpr-878522

ABSTRACT

The marine genus Marinobacterium was first identified in 1997, and a total of 18 species have been characterized so far, 10 of which have published whole-genome sequencing data. This article summarizes the characteristics of Marinobacterium genus and analyzes the genome sequencing data related to the carbon source utilization, polyhydroxyalkanoate metabolism, and aromatic compounds degradation. The Marinobacterium species possess the complete glycolysis pathway and tricarboxylic acid cycle, yet lack genes involved in xylose utilization. All strains of the Marinobacterium genus contain the genes encoding for the typeⅠand type Ⅲ polyhydroxyalkanoate synthases, suggesting that the genus may have ability of polyhydroxyalkanoate accumulation. The Marinobacterium species contain the degradation pathways of aromatic compounds. Benzene, phenol and benzoic acid can be degraded into catechol via different enzymes, subsequently catechol is converted to 3-ketoadipate through the ortho-cleavage pathway. Alternatively, catechol can be degraded into pyruvate and acetyl-CoA. The analysis of genome sequencing data of the Marinobacterium genus provides in-depth understanding of the metabolic characteristics, indicating that the genus may have certain applications in the synthesis of polyhydroxyalkanoate and the removal of marine aromatic compounds.


Subject(s)
Alteromonadaceae , DNA, Bacterial , Phylogeny , RNA, Ribosomal, 16S , Sequence Analysis, DNA
17.
Chinese Journal of Biotechnology ; (12): 2556-2565, 2020.
Article in Chinese | WPRIM | ID: wpr-878511

ABSTRACT

The important role of intestinal microorganisms in human health has been widely confirmed. At present, most of the studies on intestinal microorganisms are based on amplification of the V3-V4 region of bacterial 16S rRNA gene, and little attention has been paid to archaea. In this study, a primer set which can amplify 16S rRNA gene of both bacteria and archaea at the same time was used. By comparing the community changes before and after probiotics intake, it showed that this primer set is suitable for analyzing the changes of human intestinal bacteria and archaea communities. The fecal samples of volunteers were collected, and the amplification and high-throughput sequencing were carried out by using bacterial primer set (B primer) and bacterial and archaeal universal primer (AB primer); several commonly used rRNA databases were used to determine the amplification ability of the primer set to bacteria and archaea. The results showed that AB primer could display the bacterial community amplified by B primer, and could obtain the sequence of common methanogenic archaea in intestinal tract. AB primer set can analyze the bacteria and archaea in the intestinal tract at the same time by only one amplification and sequencing, which can show the structure of intestinal microbial community more comprehensively, which is suitable for the research of intestinal microorganisms.


Subject(s)
Archaea/genetics , Bacteria/genetics , DNA Primers , DNA, Bacterial , Humans , Microbiota/genetics , Phylogeny , RNA, Ribosomal, 16S/genetics
18.
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
19.
Article in Chinese | WPRIM | ID: wpr-828944

ABSTRACT

OBJECTIVE@#To investigate changes in intestinal flora in patients with primary Sj?gren syndrome (pSS) and explore the relationship between pSS disease activity and intestinal flora structure.@*METHODS@#Fecal samples were collected from 18 female pSS patients, including 9 patients with active disease (group A) and 9 with disease inactivity or low activity (group B), with 10 healthy subjects as the control group. The total bacterial DNA was extracted from the fecal samples for PCR amplification, and Illumina Hiseq 2500 high-throughput sequencing was performed for the v3-v4 region of 16Sr DNA gene to obtain the biological information of the intestinal flora. The intergroup OTU analysis, structural diversity analysis, significant difference analysis and LEFSE analysis were performed with information mining of the literature think tanks.@*RESULTS@#The dilution curves generated based on the OTUshannon index for analysis of sample complexity showed that the measured data were relatively complete and could reflect the diversity of the microorganisms in the subjects. Analysis of the Alpha diversity index showed that the Shannon index differed significantly between group A and group B, and the Simpson index differed significantly between group A and group B and between group A and the control group ( < 0.05). Sequence analysis the 3 groups all consisted mainly of 4 phylum (, , , showed that the intestinal flora in and ) and 4 genera (, , , and ), all showing no significant differences among the 3 groups ( > 0.05) with the exception of genus, which differed significantly among the 3 groups ( < 0.05). The 16S v3-v4 region in the genus , , , , , , , , , , -, and differed significantly among the 3 groups ( < 0.05). The high-dimensional biometrics and genomic characteristics of the intestinal microorganisms differed significantly among the 3 groups ( < 0.05). According to the size of LDA SCORE (effect size), the core flora in group A included the genera , , -, , -, , , , and , as compared with the genera , , , , , -, , - and in the control group.@*CONCLUSIONS@#Patients with pSS have significant changes in the diversity of intestinal flora, especially in some specific bacteria in genus and in 16S v3-v4 region of the bacteria. The differences in the core bacteria in the intestinal flora of pSS patients suggest the role of flora structure changes in the pathogenesis of pSS.


Subject(s)
Bacteria , DNA, Bacterial , Feces , Female , Gastrointestinal Microbiome , Humans , RNA, Ribosomal, 16S , Sjogren's Syndrome
20.
Article in Chinese | WPRIM | ID: wpr-828525

ABSTRACT

OBJECTIVE@#To investigate changes in intestinal flora in patients with primary Sj?gren syndrome (pSS) and explore the relationship between pSS disease activity and intestinal flora structure.@*METHODS@#Fecal samples were collected from 18 female pSS patients, including 9 patients with active disease (group A) and 9 with disease inactivity or low activity (group B), with 10 healthy subjects as the control group. The total bacterial DNA was extracted from the fecal samples for PCR amplification, and Illumina Hiseq 2500 high-throughput sequencing was performed for the v3-v4 region of 16Sr DNA gene to obtain the biological information of the intestinal flora. The intergroup OTU analysis, structural diversity analysis, significant difference analysis and LEFSE analysis were performed with information mining of the literature think tanks.@*RESULTS@#The dilution curves generated based on the OTUshannon index for analysis of sample complexity showed that the measured data were relatively complete and could reflect the diversity of the microorganisms in the subjects. Analysis of the Alpha diversity index showed that the Shannon index differed significantly between group A and group B, and the Simpson index differed significantly between group A and group B and between group A and the control group ( < 0.05). Sequence analysis the 3 groups all consisted mainly of 4 phylum (, , , showed that the intestinal flora in and ) and 4 genera (, , , and ), all showing no significant differences among the 3 groups ( > 0.05) with the exception of genus, which differed significantly among the 3 groups ( < 0.05). The 16S v3-v4 region in the genus , , , , , , , , , , -, and differed significantly among the 3 groups ( < 0.05). The high-dimensional biometrics and genomic characteristics of the intestinal microorganisms differed significantly among the 3 groups ( < 0.05). According to the size of LDA SCORE (effect size), the core flora in group A included the genera , , -, , -, , , , and , as compared with the genera , , , , , -, , - and in the control group.@*CONCLUSIONS@#Patients with pSS have significant changes in the diversity of intestinal flora, especially in some specific bacteria in genus and in 16S v3-v4 region of the bacteria. The differences in the core bacteria in the intestinal flora of pSS patients suggest the role of flora structure changes in the pathogenesis of pSS.


Subject(s)
Bacteria , Classification , Genetics , Biodiversity , DNA, Bacterial , Genetics , Feces , Microbiology , Gastrointestinal Microbiome , Humans , RNA, Ribosomal, 16S , Genetics , Sjogren's Syndrome , Microbiology
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