Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 9.477
Filtrar
1.
J Appl Oral Sci ; 27: e20180256, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31365706

RESUMO

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.


Assuntos
DNA Ribossômico/isolamento & purificação , Cavidade Pulpar/microbiologia , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Streptococcus/isolamento & purificação , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Ribossômico/genética , Humanos , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Ribossômico/genética , Reprodutibilidade dos Testes , Tratamento do Canal Radicular/métodos , Streptococcus/genética
2.
BMC Infect Dis ; 19(1): 598, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31288744

RESUMO

BACKGROUND: In Japan and other countries, the number of patients with syphilis is increasing year by year. Recently, the cases of the pulmonary involvement in patients with secondary syphilis have been reported. However, it is still undetermined how to obtain a desirable specimen for a diagnosis of the pulmonary involvement, and how to treat it if not cured. CASE PRESENTATION: A 34-year-old man presented with cough and swelling of the right inguinal nodes. A physical examination revealed erythematous papular rash over the palms, soles and abdomen. A 4 cm mass in the right lower lobe of the lung was detected on computed tomography. He was diagnosed as having secondary syphilis, because he was tested positive for the rapid plasma reagin and Treponema pallidum hemagglutination assay. Amoxycillin and probenecid were orally administered for 2 weeks. Subsequently, rash and serological markers were improved, however, the lung mass remained unchanged in size. Transbronchial biopsy (TBB) confirmed the pulmonary involvement of syphilis using polymerase chain reaction techniques (tpp47- and polA-PCR). Furthermore, following surgical resection revealed the lung mass to be an abscess. CONCLUSIONS: To our knowledge, this is the first surgically treated case of a lung abscess caused by syphilis, which was diagnosed by PCR techniques in TBB. This report could propose a useful diagnostic method for the pulmonary involvement of syphilis.


Assuntos
Reação em Cadeia da Polimerase/métodos , Sífilis/diagnóstico , Adulto , Brônquios/microbiologia , Brônquios/patologia , Proteína C-Reativa/análise , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Humanos , Masculino , Sífilis/microbiologia , Tomografia Computadorizada por Raios X , Treponema pallidum/genética , Treponema pallidum/isolamento & purificação
3.
BMC Infect Dis ; 19(1): 618, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299893

RESUMO

BACKGROUND: The increased transmission of multidrug-resistant (MDR) tuberculosis (TB) poses a challenge to tuberculosis prevention and control in Sri Lanka. Isoniazid (INH) is a key element of the first line anti tuberculosis treatment regimen. Resistance to INH may lead to development of MDR TB. Therefore, early detection of INH resistance is important to curb spread of resistance. Due to the limited availability of rapid molecular methods for detection of drug resistance in Sri Lanka, this study was aimed at developing a simple and rapid gold nanoparticle (AuNP) based lateral flow strip for the simultaneous detection of the most common INH resistance mutation (katG S315 T, 78.6%) and Mycobacterium tuberculosis (MTb). METHODS: Lateral flow strip was designed on an inert plastic backing layer containing a sample pad, nitrocellulose membrane and an absorption pad. Biotin labeled 4 capture probes which separately conjugated with streptavidin were immobilized on the nitrocellulose. The test sample was prepared by multiplex PCR using primers to amplify codon 315 region of the katG gene and MTb specific IS6110 region. The two detection probes complementary to the 5' end of each amplified fragment was conjugated with gold nanoparticles (20 nm) and coupled with the above amplified PCR products were applied on the sample pad. The hybridization of the amplified target regions to the respective capture probes takes place when the sample moves towards the absorption pad. Positive hybridization is indicated by red colour lines. RESULTS: The three immobilized capture probes on the strip (for the detection of TB, katG wild type and mutation) were 100 and 96.6% specific and 100 and 92.1% sensitive respectively. CONCLUSION: The AuNP based lateral flow assay was capable of differentiating the specific mutation and the wild type along with MTb identification within 3 h.


Assuntos
Doenças Transmissíveis/diagnóstico , Nanotecnologia/métodos , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Antituberculosos/uso terapêutico , Proteínas de Bactérias/genética , Catalase/genética , Doenças Transmissíveis/tratamento farmacológico , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Ouro/química , Humanos , Isoniazida/uso terapêutico , Limite de Detecção , Nanopartículas Metálicas/química , Reação em Cadeia da Polimerase Multiplex , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Sri Lanka , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico
4.
World J Microbiol Biotechnol ; 35(8): 115, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332542

RESUMO

Antibiotic and arsenic (As) contaminations are worldwide public health problems. Previously, the bacterial ABC-type efflux protein MacAB reportedly conferred resistance to macrolide-type antibiotics but not to other metal(loid)s. In this study, the roles of MacAB for the co-resistance of different antibiotics and several metal(loid)s were analyzed in Agrobacterium tumefaciens 5A, a strain resistant to arsenite [As(III)] and several types of antibiotics. The macA and macB genes were cotranscribed, and macB was deleted in A. tumefaciens 5A and heterologously expressed in Escherichia coli AW3110 and E. coli S17-1. Compared to the wild-type strain 5A, the macB deletion strain reduced bacterial resistance levels to several macrolide-type and penicillin-type antibiotics but not to cephalosporin-type antibiotics. In addition, the macB deletion strain showed lower resistance to As(III) but not to arsenate [As(V)], antimonite [Sb(III)] and cadmium chloride [Cd(II)]. The mutant strain 5A-ΔmacB cells accumulated more As(III) than the cells of the wild-type. Furthermore, heterologous expression of MacAB in E. coli S17-1 showed that MacAB was essential for resistance to macrolide, several penicillin-type antibiotics and As(III) but not to As(V). Heterologous expression of MacAB in E. coli AW3110 reduced the cellular accumulation of As(III) but not of As(V), indicating that MacAB is responsible for the efflux of As(III). These results demonstrated that, in addition to macrolide-type antibiotics, MacAB also conferred resistance to penicillin-type antibiotics and As(III) by extruding them out of cells. This finding contributes to a better understanding of the bacterial resistance mechanisms of antibiotics and metal(loid)s.


Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , DNA Bacteriano/isolamento & purificação , Farmacorresistência Bacteriana Múltipla/genética , Macrolídeos/farmacologia , Agrobacterium tumefaciens/metabolismo , Arsenitos/farmacologia , Proteínas de Bactérias/metabolismo , Cefalosporinas/farmacologia , DNA Bacteriano/genética , Eritromicina/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Penicilinas/farmacologia
5.
World J Microbiol Biotechnol ; 35(8): 117, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332532

RESUMO

Iron- and sulfur-oxidizing bacteria inhabiting rice rhizoplane play a significant role on arsenic biogeochemistry in flooded rice paddies, influencing arsenic translocation to rice grains. In the present study, the selective pressure of arsenic species on these microbial populations was evaluated. Rice roots from continuously flooded plants were incubated in iron sulfide (FeS) gradient tubes and exposed to either arsenate or arsenite. The biomass developed in the visible iron-oxidation band of the enrichments was analyzed by Scanning Electron Microscopy and Energy-Dispersive Spectroscopy (SEM-EDS) and the bacterial communities were characterized by 16S rRNA gene sequencing. Different Proteobacteria communities were selected depending on exposure to arsenate and arsenite. Arsenate addition favored the versatile iron-oxidizers Dechloromonas and Azospira, associated to putative iron (hydr)oxide crystals. Arsenite exposure decreased the diversity in the enrichments, with the development of the sulfur-oxidizer Thiobacillus thioparus, likely growing on sulfide released by FeS. Whereas sulfur-oxidizers were observed in all treatments, iron-oxidizers disappeared when exposed to arsenite. These results reveal a strong impact of different inorganic arsenics on rhizospheric iron-oxidizers as well as a crucial role of sulfur-oxidizing bacteria in establishing rice rhizosphere communities under arsenic pressure.


Assuntos
Arsênico/metabolismo , DNA Bacteriano/isolamento & purificação , Oryza/efeitos dos fármacos , Oryza/microbiologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/microbiologia , Arseniatos/metabolismo , Arsenitos/metabolismo , DNA Bacteriano/genética , Ferro/metabolismo , Oxirredução , Proteobactérias/efeitos dos fármacos , Proteobactérias/isolamento & purificação , Proteobactérias/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação , Solo/química , Microbiologia do Solo , Poluentes do Solo/metabolismo , Enxofre/metabolismo
6.
World J Microbiol Biotechnol ; 35(8): 123, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31346774

RESUMO

Constituents of the seed microbiota and initial changes in the microbiota in fermentations are important in fermentation progression. To identify the origin of indigo-reducing bacteria and understand the initial changes in the microbiota that occur concomitantly with the initiation of indigo reduction during indigo fermentation, we analysed the initial changes in the microbiota. The proportions of the reported indigo-reducing taxa Alkalibacterium, Amphibacillus and Polygonibacillus increased to 24.0% on the 5th day, to 15.2% on the 7th day and to 42.8% at 4.5 months, and the relative abundances of these taxa were 0.048%, 0.14% and 0.02%, respectively, in sukumo (composted Japanese indigo plant material used for fermentation). In the early phase of the microbiota transition, two substantial changes were observed. The first change may be attributed to the substantial environmental changes caused by the introduction of heated wood ash extract (pH ≥ 10.5, temperature ≥ 60 °C). This change increased the proportions of Alkalibacterium and the family Bacillaceae. The second change in microbiota might be initiated by the consumption of oxygen by aerobic microorganisms until the 5th day followed by an increase in the abundance of the obligate anaerobe Anaerobranca and the aerotolerant Amphibacillus and a decrease in the abundance of Bacillaceae. This experiment demonstrated that the 0.048% Alkalibacterium in the original material was augmented to 23.6% of the microbiological community within 5 days. This means that using the appropriate material and performing appropriate pretreatment and adjustment of fermentation conditions are important to increase the abundance of the taxa that reduce indigo.


Assuntos
DNA Bacteriano/isolamento & purificação , Fermentação , Índigo Carmim/metabolismo , Microbiota , Bacillaceae/classificação , Bacillaceae/isolamento & purificação , Bacillaceae/metabolismo , DNA Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Concentração de Íons de Hidrogênio , RNA Ribossômico 16S , Análise de Sequência de DNA
7.
World J Microbiol Biotechnol ; 35(8): 122, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31346836

RESUMO

To promote enzymatic unhairing for leather production, a new unhairing enzyme is developed. The Keratinase (kerT) gene, which is amplified from B. amyloliquefaciens TCCC11319 by PCR, is expressed in B. subtilis WB600. The recombinant KerT reduces the collagenolytic protease content as well as improving the keratinase content effectively. Therefore, the improved keratinase leads to the obviously unhairing effect, whereas the low collagenolytic protease ensures the integrity of collagen fibers in hide. It represents, the leather grain surface isn't destroyed thereby the value of finished leather can be maintained. In addition, by analyzing the properties of KerT, tits activity isn't inhibited with Na+, K+ and Ca2+ which are commonly used in leather production. The freeze-dried fermentation broth can be used directly as unhairing enzyme without addition of traditional sulfide chemicals. By evaluating the properties of unhaired hide, the results show that the collagen degradation ability of this new unhairing enzyme is slightly and it does not cause any adverse effects on the leather quality. Besides, this unhairing enzyme doesn't further degrade collagen in the time range of 8 h to 24 h, thus it is safely and easy-control in actual production. In conclusion, the enzymatic unhairing method with recombinant KerT has the potential to be more sustainable and efficient alternative than current sulphur-lime method, and it does not require the further purification thereby saving the cost.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , DNA Bacteriano/isolamento & purificação , Peptídeo Hidrolases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Fragmentação do DNA , DNA Bacteriano/genética , Fermentação , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
8.
Anal Chim Acta ; 1075: 144-151, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31196420

RESUMO

Salmonella is a widely distributed, extremely harmful bacteria, the presence of which requires confirmation via an on-site visual biosensor. In this study, we constructed a label-free, cascade amplification visualization biosensor for the sensitive and rapid detection of Salmonella enterica subsp. enterica serovar typhimurium based on the RDTG principle (recombinase polymerase amplification (RPA), duplex-specific enzyme (DSN) cleavage, terminal deoxynucleotidyl transferase (TdT) extension and G-quadruplexes output). Following DNA extraction of Salmonella spp., the first step in the construction involved the recognition and amplification of nucleic acids, carried out by RPA, to achieve the first signal amplification within 10 min. This RPA product was then specifically cleaved by DSN to produce a large number of small double-stranded DNA (dsDNA) products with 3'-OH within 15 min to achieve the second signal amplification. Thereafter, TdT was employed to empower these small 3'-OH dsDNA products to extend and produce a large number of long G-rich single-stranded DNAs (ssDNAs) within 20 min, thus realizing the third signal increase. These long G-rich ssDNA products displayed a color change that could be directly observed through the naked eye by adding H2O2/3,3',5,5'-tetramethylbenzidine (TMB). The RDTG biosensor for the detection of Salmonella spp. has several advantages, including a low limit of 6 cfu/mL. It is an isothermal-free instrument, simple to operate, with a rapid detection time of less than 1.5 h. Furthermore, it can be visually characterized and quantified by a microplate reader to detect Salmonella spp., in food and environmental samples, and it has broad application prospects.


Assuntos
Técnicas Biossensoriais/métodos , DNA Bacteriano/análise , Salmonella/isolamento & purificação , Técnicas de Tipagem Bacteriana/métodos , Benzidinas/química , DNA Nucleotidilexotransferase/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Quadruplex G , Peróxido de Hidrogênio/química , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases/química , Salmonella/genética , Sensibilidade e Especificidade , Iogurte/microbiologia
9.
BMC Infect Dis ; 19(1): 542, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221109

RESUMO

BACKGROUND: Tuberculosis rapid molecular assays, including GeneXpert MTB/RIF® and Loopamp MTBC Detection Kit®, are highly sensitive and specific. Such performance does not automatically translate in improved disease control and highly depends on their use, local epidemiology and the diagnostic algorithms they're implemented within. We evaluate the performance of both assays and assess their impact on additional cases notification when implemented within WHO recommended tuberculosis diagnostic algorithms in Madagascar. METHODS: Five hundred forty eight presumptive pulmonary tuberculosis patients were prospectively recruited between November 2013 and December 2014 in Antananarivo, Madagascar, a high TB incidence sub-Saharan African urban setting. Both molecular assays were evaluated as first line or add-on testing following negative smear microscopy. Based on locally defined assay performance characteristics we measure the impact of both assays and WHO-recommended diagnostic algorithms on additional tuberculosis case notifications. RESULTS: High sensitivity and specificity was confirmed for both GeneXpert MTB/RIF® (86.6% (95% CI 81.1-90.7%) and 97.4% (95% CI 94.9-98.8%)) and Loopamp MTBC Detection Kit® (84.6% (95% CI 78.9-89.0%) and 98.4% (95% CI 96.2-99.4%)). Implementation of GeneXpert MTB/RIF® and Loopamp MTBC Detection Kit® increased tuberculosis diagnostic algorithms sensitivity from 73.6% (95% CI 67.1-79.3%) up to 88.1% (95% CI 82.8-91.9%). This increase was highest when molecular assays were used as add-on testing following negative smear microscopy. As add-on testing, GeneXpert MTB/RIF® and Loopamp MTBC Detection Kit® respectively improved case detection by 23.8 and 21.2% (p < 0.05). CONCLUSION: Including GeneXpert MTB/RIF® or Loopamp MTBC Detection Kit® molecular assays for TB detection on sputum samples from presumptive TB cases can significantly increase case notification in TB diagnostic centers. The TB case detection rate is further increased when those tests are use as second-line follow-on testing following negative smear microscopy results. A country wide scale-up and digital integration of molecular-based TB diagnosis assays shows promises for TB control in Madagascar.


Assuntos
Mycobacterium tuberculosis/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Tuberculose Pulmonar/diagnóstico , Adulto , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Feminino , Humanos , Madagáscar , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose Pulmonar/microbiologia
10.
BMC Infect Dis ; 19(1): 553, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234780

RESUMO

BACKGROUND: Kazakhstan remains a high-burden TB prevalence country with a concomitent high-burden of multi-drug resistant tuberculosis. For this reason, we performed an in depth genetic diversity and population structure characterization of Mycobacterium tuberculosis complex (MTC) genetic diversity in Kazakhstan with both patient and community benefit. METHODS: A convenience sample of 700 MTC DNA cultures extracts from 630 tuberculosis patients recruited from 12 out of 14 regions in Kazakhstan, between 2010 and 2015, was independently studied by high-throughput hybridization-based methods, TB-SPRINT (59-Plex, n = 700), TB-SNPID (50-Plex, n = 543). DNA from 391 clinical isolates was successfully typed by two methods. To resolve the population structure of drug-resistant clades in more detail two complementary assays were run on the L2 isolates: an IS6110-NTF insertion site typing assay and a SigE SNP polymorphism assay. RESULTS: Strains belonged to L2/Beijing and L4/Euro-American sublineages; L2/Beijing prevalence totaled almost 80%. 50% of all samples were resistant to RIF and to INH., Subtyping showed that: (1) all L2/Beijing were "modern" Beijing and (2) most of these belonged to the previously described 94-32 sublineage (Central Asian/Russian), (3) at least two populations of the Central Asian/Russian sublineages are circulating in Kazakhstan, with different evolutionary dynamics. CONCLUSIONS: For the first time, the global genetic diversity and population structure of M. tuberculosis genotypes circulating in Kazakhstan was obtained and compared to previous local studies. Results suggest a region-specific spread of a very limited number of L2/Beijing clonal complexes in Kazakhstan many strongly associated with an MDR phenotype.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Adulto , Idoso , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Evolução Molecular , Variação Genética , Genótipo , Humanos , Cazaquistão/epidemiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico/métodos , Fenótipo , Prevalência , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adulto Jovem
11.
BMC Infect Dis ; 19(1): 561, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31248368

RESUMO

BACKGROUND: In a context of increasing use of Nucleic Acid Amplification Test, diagnoses of Neisseria gonorrhoeae and Chlamydia trachomatis infections among men increased in Europe and USA since 2007. We aimed to describe trends in the incidence of male urethritis in France between 2007 and 2017. METHODS: We analysed male urethritis clinical cases reported by the French GPs' Sentinelles network. RESULTS: GPs reported 1944 cases of male urethritis during the study period. The estimated annual incidence rates in men aged 15 years and older remained stable between 226 cases per 100,000 seen in 2007 and 196 in 2017 (P value = 0.9). A third-generation cephalosporin with macrolide or tetracycline was prescribed in 17.5% of cases in 2009 (27/154) and 32.4% in 2017 (47/145) (P value = 0.0327). CONCLUSIONS: The incidence rates for adult male urethritis diagnosed in primary care have remained stable since 2007 in France in contrast with the increasing trend of Neisseria gonorrhoeae and Chlamydia trachomatis infections based on microbiological surveillance. Using stable clinical definition for male urethritis seems essential to follow correctly epidemiological dynamic.


Assuntos
Chlamydia trachomatis/genética , Neisseria gonorrhoeae/genética , Trichomonas vaginalis/genética , Uretrite/diagnóstico , Adolescente , Adulto , Idoso , Antibacterianos/uso terapêutico , Cefalosporinas/uso terapêutico , Chlamydia trachomatis/isolamento & purificação , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , França/epidemiologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Neisseria gonorrhoeae/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , Trichomonas vaginalis/isolamento & purificação , Uretrite/tratamento farmacológico , Uretrite/epidemiologia , Uretrite/microbiologia , Adulto Jovem
12.
BMC Infect Dis ; 19(1): 563, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31248383

RESUMO

BACKGROUND: Rapid and accurate diagnosis of childhood tuberculosis (TB) is challenging because children are often unable to produce the sputum sample required for conventional tests. Stool is an alternative sample type that is easy to collect from children, and studies investigating the use of stool for molecular detection of Mycobacterium tuberculosis (Mtb) have led to promising results. Our objective was to evaluate stool as an alternative specimen to sputum for Mtb detection in children. We did so using the TruTip workstation (Akonni Biosystems), a novel automated lysis and extraction platform. METHODS: We tested stool samples from 259 children aged 0-14 years old, in Lima, Peru who presented with TB symptoms. Following extraction with TruTip, we detected the presence of Mtb DNA by IS6110 real-time PCR. We calculated assay sensitivity in two groups: (1) children with culture confirmed TB (N = 22); and (2) children with clinically-diagnosed unconfirmed TB (N = 84). We calculated specificity among children in whom TB was ruled out (N = 153). Among children who were diagnosed with TB, we examined factors associated with a positive stool test. RESULTS: Assay sensitivity was 59% (95% confidence interval [CI]: 39-80%) and 1.2% (95% CI: 0.0-6.5%) in children with culture-confirmed and clinically-diagnosed unconfirmed TB, respectively, and specificity was 97% (95% CI: 93-99%). The assay detected Mtb in stool of 7/7 children with smear-positive TB (100% sensitivity; 95% CI: 59-100%), and in 6/15 of children with smear-negative, culture-confirmed TB (40% sensitivity; 95% CI: 16-68%). Older age, smear positivity, culture positivity, ability to produce sputum and cavitary disease were associated with a positive stool result. CONCLUSION: Testing of stool samples with the TruTip workstation and IS6110 amplification yielded sensitivity and specificity estimates comparable to other tests such as Xpert. Future work should include detection of resistance using the TruTip closed amplification system and assay optimization to improve sensitivity in children with low bacillary loads.


Assuntos
Fezes/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Tuberculose/diagnóstico , Adolescente , Criança , Pré-Escolar , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Peru , Sensibilidade e Especificidade , Tuberculose/microbiologia
13.
World J Microbiol Biotechnol ; 35(7): 102, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31236715

RESUMO

Preparation of curd vary worldwide due to which its taste, texture and impact on human health also differ. In Assam, curd prepared from raw milk (RMC) is preferred over curd prepared from boiled milk (BMC), a tradition believed to have originated from the Mongoloid customs. Microbial diversity of raw milk (RM), boiled milk (BM), RMC and BMC collected from three farms were investigated by culture dependent and independent techniques. Additionally, metabolite profiles of RMC and BMC were studied by gas chromatography and mass spectroscopy. A total of 59 bacterial isolates were identified from the four different dairy products. In RM, lactic acid bacteria such as Lactococcus, Enterococcus, Lactobacillus and Leuconostoc were obtained along with the environmental bacteria like Bacillus, Staphylococcus, Acetobacter, Chryseobacterium, Streptococcus, Acinetobacter, Kocuria, Klebsiella and Macrococcus. Additionally, Prevotella, Oscillospira, Phascolarctobacterium and Akkermansia were also detected in BM by culture independent technique. In RMC and BMC, Lactococcus, Leuconostoc and Lactobacillus were prevalent. RM and RMC shared Enterococcus, Lactococcus, Streptococcus and Acinetobacter as common bacterial genera. However, no bacterial genus was common in BM and BMC. The correlation analysis revealed that Lactobacillus was negatively correlated to other bacterial genera. Oligotyping analysis revealed that Lactobacillus brevis and L.fermentum were abundant in RMC and BMC, respectively. In metabolomic study, ascorbic acid, dodecanoic acid and hexadecanoic acid were found to be significantly higher in RMC. Presence of different types of probiotics in these curds samples opens a new avenue to understand their effects on human health.


Assuntos
Bactérias/isolamento & purificação , Biodiversidade , Fermentação , Leite/microbiologia , Animais , Bactérias/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Enterococcus/isolamento & purificação , Enterococcus/metabolismo , Manipulação de Alimentos , Microbiologia de Alimentos , Sequenciamento de Nucleotídeos em Larga Escala , Lactobacillales , Lactobacillus/isolamento & purificação , Lactobacillus/metabolismo , Lactococcus/isolamento & purificação , Lactococcus/metabolismo , Leuconostoc/isolamento & purificação , Leuconostoc/metabolismo , Metagenômica , Análise Multivariada , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação
14.
World J Microbiol Biotechnol ; 35(7): 104, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31236765

RESUMO

Endophytic bacterial diversity in plants presents the level of interaction between culturable and non-culturable endophytic bacteria, thereby providing an appropriate insight into the endophytic environment. This study was conducted to determine the trend of culturable and non-culturable endophytic bacteria at two different sites encompassing four consecutive growth stages. For culturable endophytic bacteria, isolation was carried out using the dilution plate technique, and the obtained colonies were compared using PCR-restriction fragment length polymorphism (RFLP). Different RFLP-types were identified to their nearest neighbour using 16S rRNA sequencing. The non-culturable endophytic bacterial diversity was obtained by next generation sequencing. Results suggested a similar trend among the culturable and non-culturable bacteria for observed operational taxonomic units and diversity indices. It is noticeable that the endophytic bacteria inhabiting in stage 1 disappeared, and instead, different endophytic bacteria appeared. Moreover, the temporal persistence of certain culturable and non-culturable bacteria was also observed. In conclusion, the endophytic bacterial diversity in cucumber initially increased with the plant growth and then decreased at a later stage. Furthermore, it was suggested that plants regulate the number and diversity of endophytes throughout the lifecycle of plants.


Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Biodiversidade , Cucumis sativus/microbiologia , Endófitos/classificação , Endófitos/isolamento & purificação , Microbiota , Bactérias/genética , Cucumis sativus/crescimento & desenvolvimento , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Endófitos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Microbiota/genética , Filogenia , Raízes de Plantas/microbiologia , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética
15.
Parasit Vectors ; 12(1): 290, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31174587

RESUMO

BACKGROUND: Head louse, Pediculus humanus capitis, is an obligatory blood-sucking ectoparasite, distributed worldwide. Phylogenetically, it occurs in five divergent mitochondrial clades (A-E); each exhibiting a particular geographical distribution. Recent studies suggest that, as in the case of body louse, head louse could be a disease vector. We aimed to study the genetic diversity of head lice collected in the Democratic Republic of the Congo (DR Congo) and to screen for louse-borne pathogens in these lice. METHODS: A total of 181 head lice were collected from 27 individuals at the Monkole Hospital Center located in Kinshasa. All head lice were genotyped and screened for the presence of louse-borne bacteria using molecular methods. We searched for Bartonella quintana, Borrelia recurrentis, Rickettsia prowazekii, Anaplasma spp., Yersinia pestis, Coxiella burnetii and Acinetobacter spp. RESULTS: Among these head lice, 67.4% (122/181) belonged to clade A and 24.3% (44/181) belonged to clade D. Additionally, for the first time in this area, we found clade E in 8.3% (15/181) of tested lice, from two infested individuals. Dual infestation with clades A and D was observed for 44.4% individuals. Thirty-three of the 181 head lice were infected only by different bacterial species of the genus Acinetobacter. Overall, 16 out of 27 individuals were infested (59.3%). Six Acinetobacter species were detected including Acinetobacter baumannii (8.3%), Acinetobacter johnsonii (1.7%), Acinetobacter soli (1.7%), Acinetobacter pittii (1.7%), Acinetobacter guillouiae (1.1%), as well as a new potential species named "Candidatus Acinetobacter pediculi". CONCLUSIONS: To our knowledge, this study reports for the first time, the presence of clade E head lice in DR Congo. This study is also the first to report the presence of Acinetobacter species DNAs in human head lice in DR Congo.


Assuntos
Bactérias/genética , Variação Genética , Pediculus/genética , Acinetobacter/genética , Acinetobacter/patogenicidade , Animais , Bactérias/isolamento & purificação , Bactérias/patogenicidade , Bartonella quintana/genética , Bartonella quintana/patogenicidade , Borrelia/genética , Borrelia/patogenicidade , Coxiella burnetii/genética , Coxiella burnetii/patogenicidade , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , República Democrática do Congo , Vetores de Doenças , Genótipo , Humanos , Infestações por Piolhos/microbiologia , Pediculus/microbiologia , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Yersinia pestis/genética , Yersinia pestis/patogenicidade
16.
Meat Sci ; 155: 20-26, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31059938

RESUMO

Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of paratuberculosis, is considered to be a potential zoonotic pathogen and meat is one of the sources of MAP exposure for humans. MAP has been shown to be relatively resistant to different food processing methods, but there is a lack of information about the effects of ripening and fermentation processes on MAP survival in meat. Our results demonstrate that a short ripening process during teewurst production did not reduce MAP counts and viable mycobacteria were detected even during 4 weeks of storage. Although no viable MAP was recovered during the dry fermented sausage production process, there was no reduction in MAP count detected by real time PCR during production and storage of both sausages. Although the impact of foodborne exposure to viable MAP and/or mycobacterial components has not yet been clearly determined, the consumption of raw fermented meat products may be considered as a possible route of MAP transmission to humans.


Assuntos
Produtos da Carne/microbiologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Animais , Bovinos , DNA Bacteriano/isolamento & purificação , Fermentação , Manipulação de Alimentos/métodos , Armazenamento de Alimentos , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/crescimento & desenvolvimento , Paratuberculose/microbiologia , Reação em Cadeia da Polimerase em Tempo Real , Suínos
17.
J Appl Microbiol ; 127(2): 429-444, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31102430

RESUMO

AIMS: This study evaluated methods to sample and extract nucleic acids from Pacific oysters to accurately determine the microbiome associated with different tissues. METHODS AND RESULTS: Samples were collected from haemolymph, gill, gut and adductor muscle, using swabs and homogenates of solid tissues. Nucleic acids were extracted from fresh and frozen samples using three different commercial kits. The bacterial DNA yield varied between methods (P < 0·05) and each tissue harboured a unique microbiota, except for gill and muscle. Higher bacterial DNA yields were obtained by swabbing compared to tissue homogenates and from fresh tissues compared to frozen tissues, without impacting the bacterial community composition estimated by 16S rRNA gene (V1-V3 region) sequencing. Despite the higher bacterial DNA yields with QIAamp® DNA Microbiome Kit, the E.Z.N.A.® Mollusc DNA Kit identified twice as many operational taxonomic units (OTUs) and eliminated PCR inhibition from gut tissues. CONCLUSIONS: Sampling and nucleic acid purification substantially affected the quantity and diversity of bacteria identified in Pacific oyster microbiome studies and a fit-for-purpose strategy is recommended. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurate identification of Pacific oyster microbial diversity is instrumental for understanding the polymicrobial aetiology of Pacific oyster mortality diseases which greatly impact oyster production.


Assuntos
Bactérias/isolamento & purificação , Crassostrea/microbiologia , DNA Bacteriano/isolamento & purificação , Microbiota/genética , Animais , Bactérias/genética , DNA Ribossômico/genética , Trato Gastrointestinal/microbiologia , Brânquias/microbiologia , Hemolinfa/microbiologia , Músculos/microbiologia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética
18.
Microb Pathog ; 132: 166-177, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31054870

RESUMO

The macrophage innate immune response is outlined through recognition of the components of Mycobacterium tuberculosis. DNA of M. tuberculosis (MtbDNA) is recognized by macrophages, but the implications of this recognition are poorly characterized. Stimulation of murine macrophages with MtbDNA induces autophagy, a process that promotes elimination of intracellular pathogens. However, it remains unknown whether this or other phenomena also occur in human cells. In this work, we studied the innate response profiles of human macrophages after stimulation with DNA from virulent M. tuberculosis H37Rv. Human monocyte-derived macrophages were polarized into M1 and M2 phenotypes and stimulated with MtbDNA. The plasma membrane markers of the phenotype, production of TNF-α, and induction of autophagy were evaluated. Our results indicate that MtbDNA induced phenotypical changes, the significant production of TNF-α, and autophagy confirmed by the augmented expression of immunity related GTPase M (IRGM) and autophagy related ATG16L1 genes in M1 macrophages, whereas M2 macrophages exhibited limited responses. In addition, MtbDNA activation was TLR-9-dependent. Although TLR-9 expression was similar between M1 and M2 macrophages, only M1 macrophages were fully responsive to MtbDNA. In conclusion, MtbDNA recognition enhanced the antimicrobial mechanisms of M1 macrophages.


Assuntos
Autofagia , DNA Bacteriano/isolamento & purificação , Macrófagos/metabolismo , Mycobacterium tuberculosis/genética , Fator de Necrose Tumoral alfa/metabolismo , DNA Bacteriano/genética , Humanos , Imunidade Inata , Monócitos , Mycobacterium tuberculosis/metabolismo , Fenótipo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo
19.
Microb Pathog ; 132: 150-155, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31059757

RESUMO

Tuberculosis is an airborne infectious disease caused by Mycobacterium tuberculosis which threatens the globe. Aminoglycosides {Amikacin (AK) & Kanamycin (KM)} are WHO recommended second-line anti-TB drugs used against the treatment of drug-resistant tuberculosis. Aminoglycosides target the steps of protein translation machinery of M.tuberculosis. Several mechanisms have been put forward to elucidate the phenomena of aminoglycosides resistance but our knowledge is still insufficient. The aim of the study was to understand the involvement of Mycobacterium tuberculosis universal stress protein (Rv2005c) in aminoglycosides resistance and virulence. To establish the relationship of universal stress protein Rv2005c with AK & KM resistance, Rv2005c was cloned, expressed in E.coli BL21 using pQE2 expression vector and antimicrobial drug susceptibility testing (DST) was carried out. STRING-10 was also used to predict the interacting protein partners of Rv2005c. DST showed that the minimum inhibitory concentration of induced recombinant cells (Rv2005c) were five and four folds shifted with AK and KM E-strips, respectively. STRING-10 showed the interacting protein partners of Rv2005c. Overexpression of Rv2005c leads to shifting in MIC which might be signifying its involvement in the survival/resistance of Mycobacteria by inhibiting/modulating the effects of AK and KM released from the E-strips. Interactome also suggests that Rv2005c and its interacting protein partners are cumulatively involved in M.tuberculosis resistance, stresses, and latency.


Assuntos
Aminoglicosídeos/farmacologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , DNA Bacteriano/isolamento & purificação , Farmacorresistência Bacteriana Múltipla/genética , Amicacina/farmacologia , Antígenos de Bactérias/metabolismo , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Clonagem Molecular , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Canamicina/farmacologia , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Mapas de Interação de Proteínas , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico
20.
Future Microbiol ; 14: 653-660, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31137965

RESUMO

Aim: This study aims to characterize circulating strains to predict their relationship with sexually transmitted microorganisms, Chlamydia trachomatis, HIV, HCV, Treponema pallidum, HPV, Mycoplasmas, in an Italian multiethnic area, which has revealed a recent increase of Neisseria gonorrhoeae first-line antibiotic resistance. Materials & methods: We performed N. gonorrhoeae multiantigen sequence typing and the N. gonorrhoeae sequence typing for antimicrobial resistance. Results: We identified mutations in genes conferring resistance to cephalosporins, macrolides, fluoroquinolones through por and tbpB loci, and we reported new combinations of already known alleles. N. gonorrhoeae resistance to ciprofloxacin was associated with C. trachomatis coinfection. Conclusion: This study's data proved the utility of a routine N. gonorrhoeae molecular characterization to monitor the evolution of antibiotic resistance and to detect the most effective clinical treatment.


Assuntos
Antibacterianos/farmacologia , Chlamydia trachomatis/patogenicidade , Coinfecção , Farmacorresistência Bacteriana/genética , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/patogenicidade , Adulto , Alelos , Antibacterianos/uso terapêutico , Cefalosporinas/farmacologia , Ciprofloxacino/farmacologia , Coinfecção/epidemiologia , DNA Bacteriano/isolamento & purificação , Farmacorresistência Bacteriana/efeitos dos fármacos , Feminino , Fluoroquinolonas/farmacologia , Genes Bacterianos/genética , Genótipo , Humanos , Itália/epidemiologia , Macrolídeos/farmacologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Mutação , Neisseria gonorrhoeae/efeitos dos fármacos , Projetos Piloto , Adulto Jovem
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA