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1.
Nat Commun ; 12(1): 3254, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059668

RESUMO

The capability to design microbiomes with predictable functions would enable new technologies for applications in health, agriculture, and bioprocessing. Towards this goal, we develop a model-guided approach to design synthetic human gut microbiomes for production of the health-relevant metabolite butyrate. Our data-driven model quantifies microbial interactions impacting growth and butyrate production separately, providing key insights into ecological mechanisms driving butyrate production. We use our model to explore a vast community design space using a design-test-learn cycle to identify high butyrate-producing communities. Our model can accurately predict community assembly and butyrate production across a wide range of species richness. Guided by the model, we identify constraints on butyrate production by high species richness and key molecular factors driving butyrate production, including hydrogen sulfide, environmental pH, and resource competition. In sum, our model-guided approach provides a flexible and generalizable framework for understanding and accurately predicting community assembly and metabolic functions.


Assuntos
Bactérias/metabolismo , Técnicas Bacteriológicas/métodos , Butiratos/metabolismo , Microbioma Gastrointestinal/fisiologia , Anaerobiose , Bactérias/genética , Bactérias/isolamento & purificação , Biologia Computacional , DNA Bacteriano/isolamento & purificação , Genoma Bacteriano , Humanos , Sulfeto de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Microbiologia Industrial/métodos , Engenharia Metabólica , Análise de Sequência de DNA
2.
J Med Microbiol ; 70(5)2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34048334

RESUMO

Introduction. Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) are the most common pathogens from the genus Staphylococcus causing biofilm-associated infections. Generally, biofilm-associated infections represent a clinical challenge. Bacteria in biofilms are difficult to eradicate due to their resistance and serve as a reservoir for recurring persistent infections.Gap Statement. A variety of protocols for in vitro drug activity testing against staphylococcal biofilms have been introduced. However, there are often fundamental differences. All these differences in methodical approaches can then be reflected in the form of discrepancies between results.Aim. In this study, we aimed to develop optimal conditions for staphylococcal biofilm formation on pegs. The impact of peg surface modification was also studied.Methodology. The impact of tryptic soy broth alone or supplemented with foetal bovine serum (FBS) or human plasma (HP), together with the impact of the inoculum density of bacterial suspensions and the shaking versus the static mode of cultivation, on total biofilm biomass production in SA and SE reference strains was studied. The surface of pegs was modified with FBS, HP, or poly-l-lysine (PLL). The impact on total biofilm biomass was evaluated using the crystal violet staining method and statistical data analysis.Results. Tryptic soy broth supplemented with HP together with the shaking mode led to crucial potentiation of biofilm formation on pegs in SA strains. The SE strain did not produce biofilm biomass under the same conditions on pegs. Preconditioning of peg surfaces with FBS and HP led to a statistically significant increase in biofilm biomass formation in the SE strain.Conclusion. Optimal cultivation conditions for robust staphylococcal biofilm formation in vitro might differ among different bacterial strains and methodical approaches. The shaking mode and supplementation of cultivation medium with HP was beneficial for biofilm formation on pegs for SA (ATCC 29213) and methicillin-resistant SA (ATCC 43300). Peg conditioning with HP and PLL had no impact on biofilm formation in either of these strains. Peg coating with FBS showed an adverse effect on the biofilm formation of these strains. By contrast, there was a statistically significant increase in biofilm biomass production on pegs coated with FBS and HP for SE (ATCC 35983).


Assuntos
Técnicas Bacteriológicas/instrumentação , Biofilmes/crescimento & desenvolvimento , Staphylococcus/fisiologia , Animais , Técnicas Bacteriológicas/métodos , Biofilmes/classificação , Biofilmes/efeitos dos fármacos , Biomassa , Meios de Cultura/química , Meios de Cultura/farmacologia , Matriz Extracelular de Substâncias Poliméricas/classificação , Matriz Extracelular de Substâncias Poliméricas/efeitos dos fármacos , Humanos , Especificidade da Espécie , Staphylococcus/classificação , Staphylococcus/efeitos dos fármacos
3.
Food Chem ; 358: 129907, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33930712

RESUMO

Rapid detection of pathogenic bacteria particularly in food samples demands efficient separation and enrichment strategies. Here, hydrophilic temperature-responsive boronate affinity magnetic nanocomposites were established for selective enrichment of bacteria. The thermo-responsive polymer brushes were developed by surface-initiated atom transfer radical polymerization of N-isopropylacrylamide (NIPAm) and allyl glycidyl ether (AGE), followed by a reaction of epoxy groups, and incorporation of fluorophenylboronic acid. The physical and chemical characteristics of the magnetic nanocomposites were analyzed systematically. After optimization, S. aureus and Salmonella spp. showed high binding capacities of 32.14 × 106 CFU/mg and 50.98 × 106 CFU/mg in 0.01 M PBS (pH 7.4) without bacteria death. Bacterial bindings can be controlled by altering temperature and the application of competing monosaccharides. The nanocomposite was then utilized to enrich S. aureus and Salmonella spp. from the spiked tap water, 25% milk, and turbot extraction samples followed by multiplex polymerase chain reaction (mPCR), which resulted in high bacteria enrichment, and demonstrated great potential in separation of bacteria from food samples.


Assuntos
Bactérias/isolamento & purificação , Microbiologia de Alimentos/instrumentação , Microbiologia de Alimentos/métodos , Nanocompostos/química , Acrilamidas/química , Animais , Bactérias/metabolismo , Técnicas Bacteriológicas/instrumentação , Técnicas Bacteriológicas/métodos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Fenômenos Magnéticos , Leite/microbiologia , Polimerização , Polímeros/química , Salmonella/isolamento & purificação , Salmonella/metabolismo , Staphylococcus aureus/metabolismo , Temperatura , Microbiologia da Água
4.
Int J Mol Sci ; 22(7)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805483

RESUMO

Using two different types of impedance biochips (PS5 and BS5) with ring top electrodes, a distinct change of measured impedance has been detected after adding 1-5 µL (with dead or live Gram-positive Lysinibacillus sphaericus JG-A12 cells to 20 µL DI water inside the ring top electrode. We relate observed change of measured impedance to change of membrane potential of L. sphaericus JG-A12 cells. In contrast to impedance measurements, optical density (OD) measurements cannot be used to distinguish between dead and live cells. Dead L. sphaericus JG-A12 cells have been obtained by adding 0.02 mg/mL of the antibiotics tetracycline and 0.1 mg/mL chloramphenicol to a batch with OD0.5 and by incubation for 24 h, 30 °C, 120 rpm in the dark. For impedance measurements, we have used batches with a cell density of 25.5 × 108 cells/mL (OD8.5) and 270.0 × 108 cells/mL (OD90.0). The impedance biochip PS5 can be used to detect the more resistive and less capacitive live L. sphaericus JG-A12 cells. Also, the impedance biochip BS5 can be used to detect the less resistive and more capacitive dead L. sphaericus JG-A12 cells. An outlook on the application of the impedance biochips for high-throughput drug screening, e.g., against multi-drug-resistant Gram-positive bacteria, is given.


Assuntos
Técnicas Bacteriológicas/instrumentação , Técnicas Bacteriológicas/métodos , Espectroscopia Dielétrica/métodos , Viabilidade Microbiana , Bacillaceae , Espectroscopia Dielétrica/instrumentação , Eletrodos , Dispositivos Lab-On-A-Chip , Microscopia/métodos , Microscopia de Força Atômica , Silício
5.
Methods Mol Biol ; 2297: 125-140, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33656676

RESUMO

Photosynthesis is the most important chemical reaction on the earth, and about 60% of the CO2 is fixed by algae through photosynthesis. Photosynthetic organisms including algae experience half of the entire life in the dark due to diel cycles, and dark metabolism is critical and necessary for photosynthetic organisms to restart photosynthesis when receiving light again. Briefly, dark metabolism provides necessary materials and energy for restoring photosynthesis, reoxidizes NADH to form NAD+, rationally stores photosynthates, and maintains correct redox balance. Chlamydomonas reinhardtii grows under both autotrophic and heterotrophic conditions, making it an ideal organism to study photosynthesis, dark metabolism, and light dark transitions as well. In addition, it provides a good model to identify key molecular components and elucidate the molecular regulatory mechanisms of heterotrophic, which provides new clues to understand how photosynthetic organisms restart photosynthesis from the dark. Chlamydomonas mutants with dark growth deficiency or slower growth phenotypes are likely caused by the inefficient uptake and transport of acetate, the damaged proteins of mitochondrial electron transport chain, the malfunctioned mitochondrion, the redox state alteration in the dark or failed communication between mitochondrion and other organelles, the imbalanced redox or the disrupted distribution of the photosynthetic products. Here we summarize the methods and strategies for analyzing these mutants in Chlamydomonas reinhardtii.


Assuntos
Técnicas Bacteriológicas/métodos , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Meios de Cultura , Escuridão , Fluorescência , Processos Heterotróficos , Luz , Proteínas Mitocondriais/metabolismo , Oxirredução , Oxigênio/metabolismo , Consumo de Oxigênio , Fenótipo , Fotossíntese
6.
Cochrane Database Syst Rev ; 3: CD013694, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33755189

RESUMO

BACKGROUND: Tuberculosis is a leading cause of infectious disease-related death and is one of the top 10 causes of death worldwide. The World Health Organization (WHO) recommends the use of specific rapid molecular tests, including Xpert MTB/RIF or Xpert Ultra, as initial diagnostic tests for the detection of tuberculosis and rifampicin resistance in people with signs and symptoms of tuberculosis. However, the WHO estimates that nearly one-third of all active tuberculosis cases go undiagnosed and unreported. We were interested in whether a single test, Xpert MTB/RIF or Xpert Ultra, could be useful as a screening test to close this diagnostic gap and improve tuberculosis case detection. OBJECTIVES: To estimate the accuracy of Xpert MTB/RIF and Xpert Ultra for screening for pulmonary tuberculosis in adults, irrespective of signs or symptoms of pulmonary tuberculosis in high-risk groups and in the general population. Screening "irrespective of signs or symptoms" refers to screening of people who have not been assessed for the presence of tuberculosis symptoms (e.g. cough). To estimate the accuracy of Xpert MTB/RIF and Xpert Ultra for detecting rifampicin resistance in adults screened for tuberculosis, irrespective of signs and symptoms of pulmonary tuberculosis in high-risk groups and in the general population. SEARCH METHODS: We searched 12 databases including the Cochrane Infectious Diseases Group Specialized Register, MEDLINE and Embase, on 19 March 2020 without language restrictions. We also reviewed reference lists of included articles and related Cochrane Reviews, and contacted researchers in the field to identify additional studies. SELECTION CRITERIA: Cross-sectional and cohort studies in which adults (15 years and older) in high-risk groups (e.g. people living with HIV, household contacts of people with tuberculosis) or in the general population were screened for pulmonary tuberculosis using Xpert MTB/RIF or Xpert Ultra. For tuberculosis detection, the reference standard was culture. For rifampicin resistance detection, the reference standards were culture-based drug susceptibility testing and line probe assays. DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data using a standardized form and assessed risk of bias and applicability using QUADAS-2. We used a bivariate random-effects model to estimate pooled sensitivity and specificity with 95% credible intervals (CrIs) separately for tuberculosis detection and rifampicin resistance detection. We estimated all models using a Bayesian approach. For tuberculosis detection, we first estimated screening accuracy in distinct high-risk groups, including people living with HIV, household contacts, people residing in prisons, and miners, and then in several high-risk groups combined. MAIN RESULTS: We included a total of 21 studies: 18 studies (13,114 participants) evaluated Xpert MTB/RIF as a screening test for pulmonary tuberculosis and one study (571 participants) evaluated both Xpert MTB/RIF and Xpert Ultra. Three studies (159 participants) evaluated Xpert MTB/RIF for rifampicin resistance. Fifteen studies (75%) were conducted in high tuberculosis burden and 16 (80%) in high TB/HIV-burden countries. We judged most studies to have low risk of bias in all four QUADAS-2 domains and low concern for applicability. Xpert MTB/RIF and Xpert Ultra as screening tests for pulmonary tuberculosis In people living with HIV (12 studies), Xpert MTB/RIF pooled sensitivity and specificity (95% CrI) were 61.8% (53.6 to 69.9) (602 participants; moderate-certainty evidence) and 98.8% (98.0 to 99.4) (4173 participants; high-certainty evidence). Of 1000 people where 50 have tuberculosis on culture, 40 would be Xpert MTB/RIF-positive; of these, 9 (22%) would not have tuberculosis (false-positives); and 960 would be Xpert MTB/RIF-negative; of these, 19 (2%) would have tuberculosis (false-negatives). In people living with HIV (1 study), Xpert Ultra sensitivity and specificity (95% CI) were 69% (57 to 80) (68 participants; very low-certainty evidence) and 98% (97 to 99) (503 participants; moderate-certainty evidence). Of 1000 people where 50 have tuberculosis on culture, 53 would be Xpert Ultra-positive; of these, 19 (36%) would not have tuberculosis (false-positives); and 947 would be Xpert Ultra-negative; of these, 16 (2%) would have tuberculosis (false-negatives). In non-hospitalized people in high-risk groups (5 studies), Xpert MTB/RIF pooled sensitivity and specificity were 69.4% (47.7 to 86.2) (337 participants, low-certainty evidence) and 98.8% (97.2 to 99.5) (8619 participants, moderate-certainty evidence). Of 1000 people where 10 have tuberculosis on culture, 19 would be Xpert MTB/RIF-positive; of these, 12 (63%) would not have tuberculosis (false-positives); and 981 would be Xpert MTB/RIF-negative; of these, 3 (0%) would have tuberculosis (false-negatives). We did not identify any studies using Xpert MTB/RIF or Xpert Ultra for screening in the general population. Xpert MTB/RIF as a screening test for rifampicin resistance Xpert MTB/RIF sensitivity was 81% and 100% (2 studies, 20 participants; very low-certainty evidence), and specificity was 94% to 100%, (3 studies, 139 participants; moderate-certainty evidence). AUTHORS' CONCLUSIONS: Of the high-risks groups evaluated, Xpert MTB/RIF applied as a screening test was accurate for tuberculosis in high tuberculosis burden settings. Sensitivity and specificity were similar in people living with HIV and non-hospitalized people in high-risk groups. In people living with HIV, Xpert Ultra sensitivity was slightly higher than that of Xpert MTB/RIF and specificity similar. As there was only one study of Xpert Ultra in this analysis, results should be interpreted with caution. There were no studies that evaluated the tests in people with diabetes mellitus and other groups considered at high-risk for tuberculosis, or in the general population.


Assuntos
Antibióticos Antituberculose/farmacologia , Farmacorresistência Bacteriana , Mycobacterium tuberculosis/efeitos dos fármacos , Reação em Cadeia da Polimerase/métodos , Rifampina/farmacologia , Tuberculose Pulmonar/diagnóstico , Adulto , Técnicas Bacteriológicas/métodos , Teorema de Bayes , Viés , Estudos de Coortes , Estudos Transversais , Reações Falso-Negativas , Reações Falso-Positivas , Infecções por HIV/complicações , Humanos , Mycobacterium tuberculosis/isolamento & purificação , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose Pulmonar/complicações , Tuberculose Pulmonar/tratamento farmacológico
7.
J Vet Diagn Invest ; 33(2): 375-378, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33525989

RESUMO

Johne's disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.


Assuntos
Doenças dos Bovinos/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/diagnóstico , Reação em Cadeia da Polimerase/veterinária , Animais , Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/veterinária , Bovinos , Fezes/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos
8.
Appl Environ Microbiol ; 87(9)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33608294

RESUMO

Depressurization and sample processing delays may impact the outcome of shipboard microbial incubations of samples collected from the deep sea. To address this knowledge gap, we developed a remotely operated vehicle (ROV)-powered incubator instrument to carry out and compare results from in situ and shipboard RNA stable isotope probing (RNA-SIP) experiments to identify the key chemolithoautotrophic microbes and metabolisms in diffuse, low-temperature venting fluids from Axial Seamount. All the incubations showed microbial uptake of labeled bicarbonate primarily by thermophilic autotrophic Epsilonbacteraeota that oxidized hydrogen coupled with nitrate reduction. However, the in situ seafloor incubations showed higher abundances of transcripts annotated for aerobic processes, suggesting that oxygen was lost from the hydrothermal fluid samples prior to shipboard analysis. Furthermore, transcripts for thermal stress proteins such as heat shock chaperones and proteases were significantly more abundant in the shipboard incubations, suggesting that depressurization induced thermal stress in the metabolically active microbes in these incubations. Together, the results indicate that while the autotrophic microbial communities in the shipboard and seafloor experiments behaved similarly, there were distinct differences that provide new insight into the activities of natural microbial assemblages under nearly native conditions in the ocean.IMPORTANCE Diverse microbial communities drive biogeochemical cycles in Earth's ocean, yet studying these organisms and processes is often limited by technological capabilities, especially in the deep ocean. In this study, we used a novel marine microbial incubator instrument capable of in situ experimentation to investigate microbial primary producers at deep-sea hydrothermal vents. We carried out identical stable isotope probing experiments coupled to RNA sequencing both on the seafloor and on the ship to examine thermophilic, microbial autotrophs in venting fluids from an active submarine volcano. Our results indicate that microbial communities were significantly impacted by the effects of depressurization and sample processing delays, with shipboard microbial communities being more stressed than seafloor incubations. Differences in metabolism were also apparent and are likely linked to the chemistry of the fluid at the beginning of the experiment. Microbial experimentation in the natural habitat provides new insights into understanding microbial activities in the ocean.


Assuntos
Técnicas Bacteriológicas/métodos , Fontes Hidrotermais/microbiologia , Microbiota/genética , Processos Autotróficos , Bactérias/genética , Sequência de Bases , Metagenoma , Pressão , RNA Ribossômico 16S/genética , Água do Mar , Navios , Fatores de Tempo
9.
Rev. Pesqui. (Univ. Fed. Estado Rio J., Online) ; 13: 17-26, jan.-dez. 2021. tab
Artigo em Inglês, Português | LILACS, BDENF - Enfermagem | ID: biblio-1145877

RESUMO

Objetivo: verificar a demanda de hemoculturas, aspirados traqueais e uroculturas realizadas no HU-UNIVASF/ EBSERH e a prevalência dos microrganismos identificados no período de janeiro a junho de 2016. Métodos: estudo retrospectivo documental com abordagem quantitativa. Resultados: o setor de microbiologia realizou 488 hemoculturas, 427 uroculturas e 197 aspirados traqueais. A positividade de hemoculturas mostrou-se entre 10,9 à 25,7%, e o percentual de contaminações variou de 6,8 à 14,0%. Os microrganismos mais prevalência nas hemoculturas foram Staphylococcus epidermidis (23,7%), Staphylococcus aureus (19,3%) e Klebisiella pneumoniae (9,6%). Nas uroculturas foram Klebisiella pneumoniae (23,1%), Candida sp. (13,5%) e Escherichia coli (12,5%). Nos aspirados traqueais foram Acinetobacter baumannii (29,2%), Pseudomonas aeruginosa (26,6%) e Staphylococcus aureus (16,2%). Conclusão: a cultura mais solicitada foi hemocultura. A bactéria mais prevalente nas hemoculturas foi Staphylococcus epidermidis, nos aspirados traqueais Acinetobacter baumannii e nas uroculturas Klebisiella pneumoniae


Objective: the study's purpose has been to verify the demand for blood cultures, tracheal aspirates and urine cultures performed at a University Hospital from the Universidade Federal do Vale do São Francisco (HU-UNIVASF/EBSERH), as well as the predominance of microorganisms identified over the period from January to June 2016. Methods: it is a retrospective documentary study with a quantitative approach. Results: the microbiology sector carried out 488 blood cultures, 427 urine cultures and 197 tracheal aspirates. The positivity of blood cultures was between 10.9 and 25.7%, and the percentage of contaminations ranged from 6.8 to 14.0%. The most prevalent microorganisms in blood cultures were Staphylococcus epidermidis (23.7%), Staphylococcus aureus (19.3%) and Klebsiella pneumoniae (9.6%). In urine cultures were Klebsiella pneumoniae (23.1%), Candida sp. (13.5%) and Escherichia coli (12.5%). In tracheal aspirates were Acinetobacter baumannii (29.2%), Pseudomonas aeruginosa (26.6%) and Staphylococcus aureus (16.2%). Conclusion: the most requested culture was blood culture. The most prevalent bacterium in blood cultures was Staphylococcus epidermidis, in tracheal aspirates was Acinetobacter baumannii, and in urine cultures was Klebsiella pneumoniae


Objetivo: el propósito del trabajo es verificar la demanda de hemocultivos, aspirados traqueales y urocultivos realizados en el Hospital Universitário de la Universidade Federal do Vale do São Francisco (HU-UNIVASF/ EBSERH) y la prevalencia de los microorganismos identificados en el período de enero a junio de 2016. Métodos: este trabajo es un estudio retrospectivo documental con abordaje cuantitativo. Resultados: el sector de microbiología realizó 488 hemocultivos, 427 urocultivos y 197 aspirados traqueales. La positividad de hemocultivos se mostró entre el 10,9 al 25,7%, y el porcentaje de contaminaciones varía de 6,8 a 14,0%. Los microorganismos más prevalentes en los hemocultivos fueron Staphylococcus epidermidis (23,7%), Staphylococcus aureus (19,3%) y Klebsiella pneumoniae (9,6%). En los urocultivos fueron Klebisiella pneumoniae (23,1%), Candida sp. (13,5%) y Escherichia coli (12,5%). En los aspirados traqueales fueron Acinetobacter baumannii (29,2%), Pseudomonas aeruginosa (26,6%) y Staphylococcus aureus (16,2%). Conclusión: la cultura más solicitada fue hemocultivo. La bacteria más prevalente en los hemocultivos fue Staphylococcus epidermidis, en los aspirados traqueales, Acinetobacter baumannii y en los urocultivos, Klebisiella pneumoniae


Assuntos
Urina/microbiologia , Infecção Hospitalar/microbiologia , Infecção Hospitalar/epidemiologia , Técnicas Bacteriológicas/métodos , Hemocultura , Staphylococcus aureus , Staphylococcus epidermidis , Prevalência , Acinetobacter baumannii , Escherichia coli , Hospitais Universitários , Klebsiella pneumoniae
10.
Int J Food Microbiol ; 337: 108949, 2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33220648

RESUMO

The 2014 listeriosis outbreak caused by caramel-coated apples was linked to apples cross-contaminated within an apple packing facility. This outbreak has increased the focus on effective cleaning and sanitation methods that must be validated and monitored during apple packing. Thus, rapid and reliable testing methods are necessary for assessing cleanliness in the apple packing industry. The objectives of this study were to assess the prevalence of common indicator organisms [Aerobic plate count (APC), Enterobacteriaceae, coliforms, Escherichia coli, and Listeria spp.] on food contact surfaces (zone 1) in apple packinghouses and to evaluate the utility and accuracy of currently used rapid tests (ATP and glucose/lactose residue swabs). Food contact surfaces were sampled over a 100 cm2 area in five commercial apple packinghouses to evaluate populations of indicator organisms APC, Enterobacteriaceae, coliforms, E. coli (n = 741), and rapid test readings (n = 659). Petrifilm plates were used for the quantification of APC, Enterobacteriaceae, and coliform/E. coli. Rapid tests [ATP swabs (UltraSnap) and glucose/lactose residue swabs (SpotCheck Plus)] were processed on-site. A larger area (0.93 m2) was sampled for the detection of Listeria spp. (n = 747), following a modified protocol of the FDA's Bacteriological Analytical Manual method, and confirmed with PCR and gel electrophoresis via the iap gene. No significant association was found between either rapid test and populations of APC, Enterobacteriaceae, coliforms, E. coli, and Listeria spp. detection. However, recovery of APC (log CFU/100 cm2) was higher with a failed glucose/lactose residue swab surface hygiene result (3.1) than a passed result (2.9) (p = 0.03). Populations of APC, Enterobacteriaceae, and coliforms were significantly different at each unit operation during the packing process (p ≤ 0.05). This study concluded that ATP and glucose/lactose residue rapid tests were poorly suited for determining microbial load since they were not related to populations of any common indicator organisms or the detection of Listeria spp. These findings emphasize the need to utilize a rapid test, which can be a good indicator of residual matter on a surface, along with traditional microbiological methods to assess cleaning and sanitation practices in apple packinghouses.


Assuntos
Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Manipulação de Alimentos/estatística & dados numéricos , Microbiologia de Alimentos , Malus/microbiologia , Bactérias/classificação , Contagem de Colônia Microbiana , Biomarcadores Ambientais , Microbiologia de Alimentos/métodos , Microbiologia de Alimentos/estatística & dados numéricos , Higiene , Prevalência
11.
Diagn Microbiol Infect Dis ; 99(1): 115183, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33069002

RESUMO

The FilmArray® Pneumonia Plus (FA-PP) panel can provide rapid identifications and semiquantitative results for many pathogens. We performed a prospective single-center study in 43 critically ill patients with coronavirus disease 2019 (COVID-19) in which we performed 96 FA-PP tests and cultures of blind bronchoalveolar lavage (BBAL). FA-PP detected 1 or more pathogens in 32% (31/96 of samples), whereas culture methods detected at least 1 pathogen in 35% (34/96 of samples). The most prevalent bacteria detected were Pseudomonas aeruginosa (n = 14) and Staphylococcus aureus (n = 11) on both FA-PP and culture. The FA-PP results from BBAL in critically ill patients with COVID-19 were consistent with bacterial culture findings for bacteria present in the FA-PP panel, showing sensitivity, specificity, and positive and negative predictive value of 95%, 99%, 82%, and 100%, respectively. Median turnaround time for FA-PP was 5.5 h, which was significantly shorter than for standard culture (26 h) and antimicrobial susceptibility testing results (57 h).


Assuntos
Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , COVID-19/complicações , Reação em Cadeia da Polimerase Multiplex/métodos , Pneumonia Bacteriana/diagnóstico , Idoso , Bactérias/classificação , Bactérias/genética , Líquido da Lavagem Broncoalveolar/microbiologia , Estado Terminal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pneumonia Bacteriana/microbiologia , SARS-CoV-2 , Sensibilidade e Especificidade , Fatores de Tempo
12.
Methods Mol Biol ; 2220: 107-113, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975769

RESUMO

High-throughput biochemical screening techniques are an important tool in phenotypic analysis of bacteria. New methods, simultaneously measuring many phenotype responses, increase the output of such investigations and allow a more complete overview of the bacterial phenotype, facilitating large-scale correlation to related genotypes. This chapter describes the application of OmniLog phenotype microarray analysis, a high-throughput assay for the phenotypic characterization of bacterial strains across a variety of different traits such as nutrient utilization and antimicrobial sensitivity, to Listeria species.


Assuntos
Listeria monocytogenes/metabolismo , Antibacterianos/farmacologia , Técnicas Bacteriológicas/métodos , Humanos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genética , Listeria monocytogenes/crescimento & desenvolvimento , Listeriose/tratamento farmacológico , Listeriose/microbiologia , Testes de Sensibilidade Microbiana/métodos , Nutrientes/metabolismo , Fenótipo
13.
J Agric Food Chem ; 69(1): 135-145, 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33371673

RESUMO

Composite cryogels containing boronic acid ligands are synthesized for effective separation and isolation of bacteria. The large and interconnected pores in cryogels enable fast binding and release of microbial cells. To control bacterial binding, an alkyne-tagged boronic acid ligand is conjugated to azide-functionalized cryogel via the Cu(I)-catalyzed azide-alkyne cycloaddition reaction. The boronic acid-functionalized cryogel binds Gram-positive and Gram-negative bacteria through reversible boronate ester bonds, which can be controlled by pH and simple monosaccharides. To increase the capacity of affinity separation, a new approach is used to couple the alkyne-tagged phenylboronic acid to cryogel via an intermediate polymer layer that provides multiple immobilization sites. The morphology and chemical composition of the composite cryogel are characterized systematically. The capability of the composite cryogel for the separation of Gram-positive and Gram-negative bacteria is investigated. The binding capacities of the composite cryogel for Escherichia coli and Staphylococcus epidermidis are 2.15 × 109 and 3.36 × 109 cfu/g, respectively. The bacterial binding of the composite cryogel can be controlled by adjusting pH. The results suggest that the composite cryogel may be used as affinity medium for rapid separation and isolation of bacteria from complex samples.


Assuntos
Técnicas Bacteriológicas/métodos , Ácidos Borônicos/química , Criogéis/química , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/isolamento & purificação , Técnicas Bacteriológicas/instrumentação , Bactérias Gram-Negativas/química , Bactérias Gram-Positivas/química
14.
Methods Mol Biol ; 2182: 33-38, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32894484

RESUMO

Developed by 3M Company, 3M ™ Molecular Detection Assays-3M MDS-enable detection of Salmonella from advanced isothermal DNA amplification and bioluminescence detection technology. It can be used for a wide variety of products, including poultry, eggs, pet foods, and environmental samples, and results are obtained within about 24 h. In this chapter, all steps of the 3M MDS™ method for detection of Salmonella are described and detailed.


Assuntos
Técnicas Bacteriológicas/métodos , Microbiologia de Alimentos/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Salmonella/genética , Animais , Ovos/microbiologia , Contaminação de Alimentos/análise , Aves Domésticas/microbiologia
15.
Medicine (Baltimore) ; 99(50): e23410, 2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33327265

RESUMO

Carbapenemase-producing organisms (CPO) have been identified as an urgent healthcare threat. Various methods have been used for the detection of CPO using rectal swabs. Recently, an on-demand polymerase chain reaction (PCR) assay, namely, the Xpert Carba-R assay, that requires less than an hour of turnaround time, had been developed for CPO detection in clinical samples. This study focused on the use of this assay to determine the intestinal colonization rate of CPO in patients admitted to emergency rooms (ERs).A retrospective review of medical records was conducted at a tertiary hospital between July 2017 and June 2018. CPO screening using rectal swabs was performed for patients transferred from other hospitals or for those who tested positive in CPO culture tests in the previous three months. The Xpert Carba-R assay and culture tests were used as the CPO screening methods, and the results of both tests were compared.Medical records of 705 patients admitted to our hospital during the study period were reviewed. Of these, 31 (4.4%) showed positive results for CPO using the Xpert Carba-R assay, and these patients were then transferred from the ERs to isolation rooms. Fifteen of the Xpert Carba-R assay-positive patients were also positive for the culture test; hence, early detection enabled the rapid isolation of CPO-infected patients and prevented the spread of the CPO.The Xpert Carba-R assay is a rapid test to identify and guide infection control programs to contain the spread of the rectal colonization of CPO within a hospital.


Assuntos
Técnicas Bacteriológicas/métodos , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Infecções por Enterobacteriaceae/diagnóstico , Enterococos Resistentes à Vancomicina/isolamento & purificação , Serviço Hospitalar de Emergência , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Isolamento de Pacientes , Prevalência , Reto/microbiologia , República da Coreia/epidemiologia , Estudos Retrospectivos , Sensibilidade e Especificidade
16.
Int J Food Microbiol ; 334: 108850, 2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-32919261

RESUMO

The complex and highly diverse microbial environment of drinking water, consisting mainly of bacteria at different metabolic states, is still underexplored. The aim of this work was to characterize the bacterial communities in tap water and bottled mineral water, the two predominant sources of drinking water in modern societies. A total of 11 tap water samples from a range of locations and distribution networks and 10 brands of bottled natural mineral water were analysed using two approaches: a) heterotrophic plate counts by matrix-assisted laser desorption/ionization time of flight mass-spectrometry (MALDI-TOF MS) for the culturable heterotrophic communities, and b) Illumina amplicon sequencing for total bacteria including non-culturable bacteria. Culturable heterotrophic bacteria were isolated in WPCA (ISO) agar at 22 ± 2 °C for 72 h and 2046 isolates were identified using MALDI-TOF MS. The Bruker Daltonics Library and a previously customized library (Drinking Water Library) were used as reference databases. For the total bacteria fraction, DNA was extracted from 6 L of water and submitted to Illumina 16S rRNA sequencing of the v4 region. Significant differences were observed between mineral and tap water, with a general dominance of Alphaproteobacteria (mainly the genus Blastomonas) in tap water and Gammaproteobacteria in mineral water with Acidovorax being the dominant genus in 3 out of 7 mineral water brands. The bacterial communities in the different brands of mineral water were highly diverse and characteristic of each one. Moreover, the season in which the water was bottled also affected the species distribution, with some of them identified in only one season. Among the culturable bacteria, the most abundant phylum was Proteobacteria (around 85% of the isolates), followed by Actinobacteria, Firmicutes and Bacteroidetes. Proteobacteria was also the most abundant phylum detected with Illumina sequencing (>99% of the reads). The two methods gave distinct results at the different taxonomic levels and could therefore have a complimentary application in the study of microbiota in mineral water environments. MALDI-TOF MS is a promising method for the rapid identification of heterotrophic bacteria in routine water analysis in the bottling industry. SIGNIFICANCE AND IMPACT OF THE STUDY: The complementarity of MALDI-TOF MS and NGS in the assessment of bacterial community diversity has been demonstrated in water intended for human consumption. The two methods are suitable for routine use in the water industry for water quality management.


Assuntos
Técnicas Bacteriológicas , Água Potável/microbiologia , Microbiota , Águas Minerais/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/metabolismo , Técnicas Bacteriológicas/métodos , Meios de Cultura/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Ribossômico 16S/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
PLoS One ; 15(8): e0237655, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32810167

RESUMO

BACKGROUND: Several tests are available for plague confirmation but bacteriological culture with Yersinia pestis strain isolation remains the gold standard according to the World Health Organization. However, this is a time consuming procedure; requiring specific devices and well-qualified staff. In addition, strain isolation is challenging if antibiotics have been administered prior to sampling. Here, we developed a loop-mediated isothermal amplification (LAMP) technique, a rapid, simple, sensitive and specific technique that would be able to detect Y. pestis in human biological samples. METHODS: LAMP primers were designed to target the caf1 gene which is specific to Y. pestis. The detection limit was determined by testing 10-fold serial dilution of Y. pestis DNA. Cross-reactivity was tested using DNA extracts from 14 pathogens and 47 residual samples from patients suffering from non-plague diseases. Specificity and sensitivity of the LAMP caf1 were assessed on DNA extracts of 160 human biological samples. Then, the performance of the LAMP caf1 assay was compared to conventional PCR and bacteriological culture. RESULTS: The detection limit of the developed Y. pestis LAMP assay was 3.79 pg/µl, similar to conventional PCR. The result could be read out within 45 min and as early as 35 minutes in presence of loop primer, using a simple water bath at 63°C. This is superior to culture with respect to time (requires up to 10 days) and simplicity of equipment compared to PCR. Furthermore, no cross-reactivity was found when tested on DNA extracts from other pathogens and human biological samples from patients with non-plague diseases. Compared to the gold standard, LAMP sensitivity and specificity were 97.9% (95% CI: 89.1%-99.9%) and 94.6% (95% CI: 88.6%-97.9%), respectively. CONCLUSION: LAMP detected Y. pestis effectively with high sensitivity and specificity in human plague biological samples. It can potentially be used in the field during outbreaks in resource limited countries such as Madagascar.


Assuntos
Técnicas Bacteriológicas/métodos , DNA Bacteriano/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Peste/diagnóstico , Yersinia pestis/isolamento & purificação , Técnicas Bacteriológicas/economia , Estudos de Viabilidade , Humanos , Limite de Detecção , Madagáscar , Técnicas de Amplificação de Ácido Nucleico/economia , Peste/microbiologia , Fatores de Tempo , Yersinia pestis/genética
18.
PLoS One ; 15(8): e0236700, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32750088

RESUMO

Mycobacterial culture remains the gold standard for the diagnosis of tuberculosis. However, an appropriate digestion and decontamination method (DDM) is essential for the effective recovery of tubercle bacilli in culture. Therefore, the current study was designed to compare the performance of papain-cetylpyridinium chloride [papain-CPC] and pepsin-cetylpyridinium chloride [pepsin-CPC] DDMs against N-acetyl L-Cysteine-sodium hydroxide (NALC-NaOH) DDM for recovery of mycobacteria from clinically suspected pulmonary tuberculosis cases. To evaluate papain-CPC, pepsin-CPC and NALC-NaOH DDMs, sputum samples (N = 1381) were cultured on Löwenstein-Jensen medium and the results were compared. The papain-CPC DDM showed sensitivity, specificity, positive predictive value, and negative predictive value of 100%, 93.27%, 71.7%, and 100%, respectively as compared to NALC-NaOH DDM. Similarly, pepsin-CPC DDM demonstrated sensitivity, specificity, positive predictive value and negative predictive value of 98.94%, 94.7%, 76.11%, and 99.81%, respectively. In summary, both papain-CPC and pepsin-CPC DDMs are highly sensitive and specific techniques for recovery of mycobacteria as compared to NALC-NaOH DDM. However, when the overall performances of all DDMs compared, papain-CPC DDM isolated increased number of mycobacterial isolates with comparatively higher numbers of colonies on LJ media than both pepsin-CPC and NALC-NaOH DDMs, indicating its potential to replace the NALC-NaOH DDM for recovery of mycobacteria from sputum samples.


Assuntos
Técnicas Bacteriológicas/métodos , Meios de Cultura/química , Mycobacterium tuberculosis/isolamento & purificação , Escarro/microbiologia , Tuberculose Pulmonar/microbiologia , Acetilcisteína/química , Cetilpiridínio/química , Humanos , Papaína/química , Pepsina A/química , Sensibilidade e Especificidade , Manejo de Espécimes
19.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 908-919, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567274

RESUMO

A multiplex PCR method was developed to detect the main pathogens of Qinghai Tibetan sheep endometritis. First, the genomes of five standard bacterial strains were extracted and specific primers were selected; the multiplex PCR method was established by using the genome of the standard strain as a template. The samples were collected by sterile cotton swab from Tibetan sheep uterus, and then placed in LB medium and numbered. After 48 h, the genomes of cultured bacteria were extracted and detected by single PCR method, then the positive samples were recorded. The positive samples detected by single PCR were selected for multiplex PCR detection and recorded again. The coincidence rate between these two methods was calculated to measure the accuracy of multiplex PCR. In order to identify the species of the pathogen, 30 positive samples verified by single and multiplex PCR were randomly selected for bacterial isolation and identification. In the 600 samples, the infected ratio of Streptococcus agalactiae (GBS) was 47.33%, Escherichia coli 34.83%, Staphylococcus aureus 6.5%, Salmonella and Trueperella pyogenes were negatively detected. Among the positive samples detected by multiplex PCR, the positive ratio of GBS was 45.50%, E. coli 33.50%, S. aureus 6.5%. Comparison of two detection results, Multiplex PCR detection coincidence rate is more than 95%. The isolated pathogens were identified as E. coli, GBS and S. aureus, which was consistent with the results of two methods. The multiplex PCR method was successfully established and the main pathogens of endometritis in Qinghai Tibetan sheep were GBS, E. coli and S. aureus.


Assuntos
Bactérias , Técnicas Bacteriológicas , Endometrite , Reação em Cadeia da Polimerase Multiplex , Doenças dos Ovinos , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Endometrite/microbiologia , Endometrite/veterinária , Feminino , Reação em Cadeia da Polimerase Multiplex/normas , Reação em Cadeia da Polimerase/veterinária , Sensibilidade e Especificidade , Ovinos , Doenças dos Ovinos/microbiologia , Tibet
20.
J Med Microbiol ; 69(6): 806-811, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32490793

RESUMO

Introduction. Bloodstream infections (BSI) are growing in incidence and present a serious health threat. Most patients wait up to 48 h before microbiological cultures can confirm a diagnosis. Low numbers of circulating bacteria in patients with BSI mean we need to develop new methods and optimize current methods to facilitate efficient recovery of bacteria from the bloodstream. This will allow detection of positive blood cultures in a more clinically useful timeframe. Many bacterial blood recovery methods are available and usually include a combination of techniques such as centrifugation, filtration, serum separation or lysis treatment. Here, we evaluate nine different bacteria recovery methods performed directly from blood culture.Aim. We sought to identify a bacterial recovery method that would allow for a cost-effective and efficient recovery of common BSI pathogens directly from blood culture.Methods. Simulated E. coli ATCC 25922 blood culture was used as a model system to evaluate nine different bacteria recovery methods. Each method was assessed on recovery yield, cost, hands-on time, risk of contamination and ease of use. The highest scoring recovery method was further evaluated using simulated blood cultures spiked with seven of the most frequently occurring bloodstream pathogens. The recovery yield was calculated based on c.f.u. count before and after each recovery method. Independent t-tests were performed to determine if the recovery methods evaluated were significantly different based on c.f.u. ml-1 log recovery.Results. All nine methods evaluated successfully recovered E. coli ATCC 25922 from simulated blood cultures although the bacterial yield differed significantly. The MALDI-TOF intact cell method offered the poorest recovery with a mean loss of 2.94±0.37 log c.f.u. ml-1. In contrast, a method developed by Bio-Rad achieved the greatest bacterial yield with a mean bacteria loss of 0.27±0.013 log c.f.u. ml-1. Overall, a low-speed serum-separation method was demonstrated to be the most efficient method in terms of time, cost and recovery efficiency and successfully recovered seven of the most frequent BSI pathogens with a mean bacteria loss of 0.717±0.18 log c.f.u. ml-1.Conclusion. The efficiency of bacterial recovery can vary significantly between different methods and thereby can have a critical impact on downstream analysis. The low-speed serum-separation method offered a simple and effective means of recovering common BSI pathogens from blood culture and will be further investigated for use in the rapid detection of bacteraemia and susceptibility testing in clinical practice.


Assuntos
Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Hemocultura/métodos , Patógenos Transmitidos pelo Sangue/isolamento & purificação , Bacteriemia/diagnóstico , Escherichia coli/isolamento & purificação , Humanos
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