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1.
Sci Rep ; 14(1): 5460, 2024 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-38443572

RESUMO

Autistic Children often struggle with social interaction and communication, studies have found that many of them prefer to interact with objects than people. However, there is a lack of research exploring the specific characteristics and factors involved in interactions within families with autistic children where objects are the center of the interaction. This paper describes the process and findings of a diary study exploring how young autistic children interact with their families through objects in natural scenarios. A one-week diary study was conducted with six families with young autistic children. Diary videos were recorded onsite and coded later according to a social interaction behavior scheme with corresponding diary entries. Qualitative data analysis was conducted to reveal possible patterns. Results revealed ongoing difficulties in establishing and maintaining family interaction and identified influential factors of object-centered family interaction. The most prevalent pattern observed was parents taking the lead in interactions, followed by the child's confirmation response. Remarkably, daily necessities emerged as potential physical mediums for enhancing family interactions, opening avenues for exploring tangible designs in human-computer interaction. These findings offer valuable implications for future research and the development of innovative designs that promote enriching interactions for autistic children and their families.


Assuntos
Transtorno Autístico , Criança , Humanos , Comunicação , Meios de Cultura , Pais , Exame Físico
2.
PeerJ ; 12: e16995, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38426145

RESUMO

Background: Hermetia illucens (HI), commonly known as the black soldier fly, has been recognized for its prowess in resource utilization and environmental protection because of its ability to transform organic waste into animal feed for livestock, poultry, and aquaculture. However, the potential of the black soldier fly's high protein content for more than cheap feedstock is still largely unexplored. Methods: This study innovatively explores the potential of H. illucens larvae (HIL) protein as a peptone substitute for microbial culture media. Four commercial proteases (alkaline protease, trypsin, trypsase, and papain) were explored to hydrolyze the defatted HIL, and the experimental conditions were optimized via response surface methodology experimental design. The hydrolysate of the defatted HIL was subsequently vacuum freeze-dried and deployed as a growth medium for three bacterial strains (Staphylococcus aureus, Bacillus subtilis, and Escherichia coli) to determine the growth kinetics between the HIL peptone and commercial peptone. Results: The optimal conditions were 1.70% w/w complex enzyme (alkaline protease: trypsin at 1:1 ratio) at pH 7.0 and 54 °C for a duration of 4 h. Under these conditions, the hydrolysis of defatted HIL yielded 19.25% ±0.49%. A growth kinetic analysis showed no significant difference in growth parameters (µmax, Xmax, and λ) between the HIL peptone and commercial peptone, demonstrating that the HIL hydrolysate could serve as an effective, low-cost alternative to commercial peptone. This study introduces an innovative approach to HIL protein resource utilization, broadening its application beyond its current use in animal feed.


Assuntos
Dípteros , Peptonas , Animais , Tripsina , Hidrólise , Cinética , Larva , Meios de Cultura
3.
J Vis Exp ; (205)2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38497647

RESUMO

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized method for measuring bacterial colonization in the rhizosphere is necessary to improve reproducibility. We first cultured sterile A.thaliana in hydroponic conditions and then inoculated the bacterial cells in the rhizosphere at a final concentration of OD600 of 0.01. At 2 days post-inoculation, the root tissue was harvested and washed three times in sterile water to remove the uncolonized bacterial cells. The roots were then weighed, and the bacterial cells colonized on the root were collected by vortex. The cell suspension was diluted in a gradient with a phosphate-buffered saline (PBS) buffer, followed by plating onto a Luria-Bertani (LB) agar medium. The plates were incubated at 37 °C for 10 h, and then, the single colonies on LB plates were counted and normalized to indicate the bacterial cells colonized on roots. This method is used to detect bacterial colonization in the rhizosphere in mono-interaction conditions, with good reproducibility.


Assuntos
Arabidopsis , Hidroponia , Reprodutibilidade dos Testes , Meios de Cultura , Interações Microbianas
4.
Appl Microbiol Biotechnol ; 108(1): 262, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38483568

RESUMO

The increasing demand for rare earth elements (REEs) has spurred interest in the development of recovery methods from aqueous waste streams. Acidophilic microalgae have gained attention for REE biosorption as they can withstand high concentrations of transition metals and do not require added organic carbon to grow, potentially allowing simultaneous sorption and self-replication of the sorbent. Here, we assessed the potential of Galdieria sulphuraria for REE biosorption under acidic, nutrient-replete conditions from solutions containing ≤ 15 ppm REEs. Sorption at pH 1.5-2.5 (the growth optimum of G. sulphuraria) was poor but improved up to 24-fold at pH 5.0 in phosphate-free conditions. Metabolic activity had a negative impact on REE sorption, additionally challenging the feasibility of REE biosorption under ideal growth conditions for acidophiles. We further examined the possibility of REE biosorption in the presence of phosphate for biomass growth at elevated pH (pH ≥ 2.5) by assessing aqueous La concentrations in various culture media. Three days after adding La into the media, dissolved La concentrations were up to three orders of magnitude higher than solubility predictions due to supersaturation, though LaPO4 precipitation occurred under all conditions when seed was added. We concluded that biosorption should occur separately from biomass growth to avoid REE phosphate precipitation. Furthermore, we demonstrated the importance of proper control experiments in biosorption studies to assess potential interactions between REEs and matrix ions such as phosphates. KEY POINTS: • REE biosorption with G. sulphuraria increases significantly when raising pH to 5 • Phosphate for biosorbent growth has to be supplied separately from biosorption • Biosorption studies have to assess potential matrix effects on REE behavior.


Assuntos
Metais Terras Raras , Microalgas , Microalgas/metabolismo , Fosfatos , Metais Terras Raras/metabolismo , Meios de Cultura , Concentração de Íons de Hidrogênio
5.
Bioresour Technol ; 398: 130511, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38437963

RESUMO

The effect of thiamine (TA), ascorbic acid (AA), citric acid, and gallic acid (GA) on bacterial cellulose (BC) production by Komagataeibacter sucrofermentans, in synthetic (Hestrin and Schramm, HS) and natural substrates (industrial raisins finishing side stream extract, FSSE; orange juice, OJ; green tea extract, GTE), was investigated. The Response Surface Methodology was found reliable for BC yield prediction and optimization. Higher yields were achieved in the FSSE substrates, especially those supplemented with AA, TA, and GA (up to 19.4 g BC/L). The yield in the non-fortified substrates was 1.1-5.4 and 11.6-15.7 g/L, in HS and FSSE, respectively. The best yield in the natural non-fortified substrate FSSE-OJ-GTE (50-20-30 %), was 5.9 g/L. The porosity, crystallinity, and antioxidant properties of the produced BC films were affected by both the substrate and the drying method (freeze- or oven-drying). The natural substrates and the process wastewaters can be further exploited towards added value and sustainability. Take Home Message Sentence: Raisin and citrus side-streams can be efficiently combined for bacterial cellulose production, enhanced by other vitamin- and phenolic-rich substrates such as green tea.


Assuntos
Acetobacteraceae , Celulose , Vitaminas , Celulose/química , Rios , Vitamina A , Vitamina K , Compostos Orgânicos , Meios de Cultura , Chá , Extratos Vegetais
6.
Food Res Int ; 182: 114064, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38519157

RESUMO

Bacillus subtilis spores are important food spoilage agents and are occasionally involved in food poisoning. In foods that are not processed with intense heat, such bacterial spores are controlled by a combination of different hurdles, such as refrigeration, acidification, and low water activity (aw), which inhibit or delay germination and/or growth. Sporulation temperature has long been regarded as a relevant factor for the assessment of germination in chemically defined media, but little is known about its impact on food preservation environments. In this study, we compared germination dynamics of B. subtilis spores produced at optimal temperature (37 °C) with others incubated at suboptimal (20 °C) and supraoptimal (43 °C) temperatures in a variety of nutrients (rich-growth medium, L-alanine, L-valine, and AGFK) under optimal conditions as well as under food-related stresses (low aw, pH, and temperature). Spores produced at 20 °C had a lower germination rate and efficiency than those incubated at 37 °C in all the nutrients, while those sporulated at 43 °C displayed a higher germination rate and/or efficiency in response to rich-growth medium and mostly to L-alanine and AGFK under optimal environmental conditions. However, differences in germination induced by changes in sporulation temperature decreased when spores were activated by heat, mainly due to the greater benefit of heat for spores produced at 20 °C and 37 °C than at 43 °C, especially in AGFK. Non-heat-activated spores produced at 43 °C still displayed superior germination fitness under certain stresses that had considerably impaired the germination of the other two populations, such as reduced temperature and aw. Moreover, they presented lower temperature and pH boundaries for the inhibition of germination in rich-growth medium, while requiring a higher NaCl concentration threshold compared to spores obtained at optimal and suboptimal temperature. Sporulation temperature is therefore a relevant source of variability in spore germination that should be taken into account for the accurate prediction of spore behaviour under variable food preservation conditions with the aim of improving food safety and stability.


Assuntos
Bacillus subtilis , Esporos Bacterianos , Temperatura , Temperatura Alta , Meios de Cultura , Alanina
7.
Food Res Int ; 182: 114138, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38519170

RESUMO

Selecting the primary cells in an optimal state for cultured meat production is a crucial challenge in commercializing cultured meat. We investigated the metabolomic changes in culture media according to passage numbers for indirectly assessing the state of primary cells. Pig skeletal muscle stem cells (PSCs) harvested from the biceps femoris muscles of 7-d-old crossbred pigs (Landrace × Yorkshire × Duroc, LYD) were used for cell characterization. Fresh media (FM) and spent media (SM) of PSCs during passages 1 to 3 in vitro culture were prepared for metabolomics analysis. SM was collected on the third day of proliferation for each passage of PSCs. Cell characterization analysis revealed that the proliferation rate was highest at passage 2; however, a significant loss of expression of myogenic marker genes was observed at passage 3. Based on metabolomic profiles of culture media, FM and SM groups (SM1, SM2, and SM3) were clearly separated by partial least squares-discriminant analysis. A total of seven differentially abundant metabolites (DAMs) were identified from FM and SM for each passage, based on the following criteria: P < 0.05, fold change > 1.5 or < 0.66, and a variable importance in projection score > 1.5. All seven DAMs and their interconnected metabolites might be primarily used as substrates for energy production and most of them were relatively abundant in SM3. Among the seven DAMs, the three potential biomarkers (γ-glutamyl-L-leucine, cytosine, and ketoleucine), which showed significant changes exclusively in SM3, each had an area under the curve value of 1. Therefore, monitoring the levels of these key metabolites in culture media could serve as a quality control measure for cultured meat production by enabling the indirect detection of suboptimal PSCs based on their proliferation ability.


Assuntos
Técnicas de Cultura de Células , Suínos , Animais , Meios de Cultura/química , Biomarcadores , Músculos
8.
J Agric Food Chem ; 72(11): 6064-6076, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38465450

RESUMO

The process of producing cell-cultured meat involves utilizing a significant amount of culture medium, including fetal bovine serum (FBS), which represents a considerable portion of production expense while also raising environmental and safety concerns. This study demonstrated that supplementation with Auxenochlorella pyrenoidosa protein extract (APE) under low-serum conditions substantially increased Carassius auratus muscle (CAM) cell proliferation and heightened the expression of Myf5 compared to the absence of APE. An integrated intracellular metabolomics and proteomics analysis revealed a total of 13 and 67 differentially expressed metabolites and proteins, respectively, after supplementation with APE in the medium containing 5%FBS, modulating specific metabolism and signaling pathways, which explained the application of APE for passage cell culture under low-serum conditions. Further analysis revealed that the bioactive factors in the APE were protein components. Moreover, CAM cells cultured in reconstructed serum-free media containing APE, l-ascorbic acid, insulin, transferrin, selenium, and ethanolamine exhibited significantly accelerated growth in a scale-up culture. These findings suggest a promising alternative to FBS for fish muscle cell culture that can help reduce production costs and environmental impact in the production of cultured meat.


Assuntos
Hominidae , Soroalbumina Bovina , Animais , Células Cultivadas , Meios de Cultura , Técnicas de Cultura de Células , Músculos
9.
Sci Rep ; 14(1): 5720, 2024 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-38459251

RESUMO

Severe Fusarium wilt and crown root symptoms were observed in almond orchards in Portugal. The present study elucidates the etiology of the disease through molecular, phenotypic, and pathogenic characterization. Three Fusarium isolates from Portugal were tested and 12 Fusarium isolates from almond from Spain were included for comparative purposes. Their identity was inferred by phylogenetic analysis combining tef1 and rpb2 sequences. The Portuguese isolates were identified as Fusarium oxysporum sensu stricto (s.s.), and the Spanish isolates as Fusarium nirenbergiae, F. oxysporum (s.s.), Fusarium proliferatum, Fusarium redolens (s.s.), Fusarium sambucinum (s.s.), and Fusarium sp. Fungal colonies and conidia were characterized on potato dextrose agar (PDA) and on Synthetischer Nährstoffarmer agar, respectively. The colonies had a variable morphology and their color ranged from white to pale violet. Typical Fusarium micro- and macroconidia were characterized. Temperature effect on mycelial growth was evaluated on PDA from 5 to 35 °C, with optimal growth temperature ranging between 16.8 and 26.4 °C. The pathogenicity of F. oxysporum was demonstrated by inoculating almond plants ('Lauranne') grafted on GF-677 or Rootpac 20 rootstocks. A significant reduction in plant growth, wilting, and xylem discoloration was observed, with Rootpac 20 being more susceptible than GF-677. Infections were also reproduced using naturally infested soils. Almond plants ('Lauranne') were inoculated with isolates of all Fusarium species, with F. redolens from Spain and F. oxysporum from Portugal being the most aggressive.


Assuntos
Fusarium , Prunus dulcis , Fusarium/genética , Virulência , Ágar , Filogenia , Meios de Cultura
10.
Sci Rep ; 14(1): 5606, 2024 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-38453984

RESUMO

Fetal bovine serum (FBS) plays a pivotal role in animal cell culture. Due to ethical and scientific issues, searching for an alternative, comprising the three R's (Refinement, Reduction and Replacement) gained global attention. In this context, we have identified the heat inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a potential alternative for FBS. Briefly, we formulated HI-CF (f-HICF) containing serum free medium which can aid the growth, attachment, and proliferation of adherent cells, similar to FBS. In this study, we investigated the biochemical characterization, sterility, stability, formulation, and functional analysis of HI-CF as a supplement in culturing animal cells. Notably, vitamins, micronutrients, proteins, lipids, and trace elements are identified and compared with FBS for effective normalization of the serum free media. HI-CF is tested to be devoid of endotoxin and mycoplasma contamination thus can qualify the cell culture grade. The f-HICF serum free media was prepared, optimised, and tested with A549, HeLa, 3T3, Vero and C2C12 cell lines. Our results conclude that f-HICF is a potential alternative to FBS, in accordance with ethical concern; compliance with 3R's; lack of unintended antibody interactions; presence of macro and micronutrients; simple extraction; cost-effectiveness and availability.


Assuntos
Oligoquetos , Soroalbumina Bovina , Humanos , Animais , Meios de Cultura Livres de Soro , Meios de Cultura/química , Temperatura Alta , Técnicas de Cultura de Células/métodos , Células HeLa , Vitaminas , Células Cultivadas
11.
J Vis Exp ; (204)2024 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-38465948

RESUMO

This article presents a rapid yet robust protocol for isolating Campylobacter spp. from raw meats, specifically focusing on Campylobacter jejuni and Campylobacter coli. The protocol builds upon established methods, ensuring compatibility with the prevailing techniques employed by regulatory bodies such as the Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) in the USA, as well as the International Organization for Standardization (ISO) in Europe. Central to this protocol is collecting a rinsate, which is concentrated and resuspended in Bolton Broth media containing horse blood. This medium has been proven to facilitate the recovery of stressed Campylobacter cells and reduce the required enrichment duration by 50%. The enriched samples are then transferred onto nitrocellulose membranes on brucella plates. To improve the sensitivity and specificity of the method, 0.45 µm and 0.65 µm pore-size filter membranes were evaluated. Data revealed a 29-fold increase in cell recovery with the 0.65 µm pore-size filter compared to the 0.45 µm pore-size without impacting specificity. The highly motile characteristics of Campylobacter allow cells to actively move through the membrane filters towards the agar medium, which enables effective isolation of pure Campylobacter colonies. The protocol incorporates multiplex quantitative real-time polymerase chain reaction (mqPCR) assay to identify the isolates at the species level. This molecular technique offers a reliable and efficient means of species identification. Investigations conducted over the past twelve years involving retail meats have demonstrated the ability of this method to enhance recovery of Campylobacter from naturally contaminated meat samples compared to current reference methods. Furthermore, this protocol boasts reduced preparation and processing time. As a result, it presents a promising alternative for the efficient recovery of Campylobacter from meat. Moreover, this procedure can be seamlessly integrated with DNA-based methods, facilitating rapid screening of positive samples alongside comprehensive whole-genome sequencing analysis.


Assuntos
Campylobacter jejuni , Campylobacter , Animais , Cavalos , Galinhas , Microbiologia de Alimentos , Carne , Campylobacter/genética , Meios de Cultura
12.
Sci Rep ; 14(1): 7081, 2024 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-38528099

RESUMO

In this article, we focused on the impact of precisely chemically modified FLI maturation medium enriched with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF1), and polyvinyl alcohol (PVA) and its potential to improve the efficiency of in vitro production of porcine embryos. We hypothesized that enhancing the composition of the maturation medium could result in an elevated production of embryos in vitro and can affect EGA. FLI medium resulted in a significantly higher rate of oocyte blastocyst maturation and formation compared to the control DMEM medium. In addition, immunocytochemical labelling confirmed the detection of UBF in 4-cell FLI parthenogenic embryos, suggesting similarities with natural embryo development. Through RNAseq analysis, upregulated genes present in 4-cell FLI embryos were found to play key roles in important biological processes such as cell proliferation, cell differentiation, and transcriptional regulation. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos.


Assuntos
Fertilização In Vitro , Fator 2 de Crescimento de Fibroblastos , Animais , Suínos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator Inibidor de Leucemia/farmacologia , Proteômica , Blastocisto , Oócitos , Meios de Cultura/farmacologia
13.
Int J Food Microbiol ; 414: 110616, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38325257

RESUMO

Escherichia albertii is an emerging enteropathogen. Although E. albertii-specific detection and isolation methods have been developed, their efficiency on food samples have not yet been systematically studied. To establish a series of effective methods for detecting E. albertii in food, an interlaboratory study was conducted in 11 laboratories using enrichment with modified E. coli broth supplemented with cefixime and tellurite (CT-mEC), real-time PCR assay, and plating on four kinds of selective agars. This study focused on the detection efficiency of an E. albertii-specific real-time PCR assay (EA-rtPCR) and plating on deoxycholate hydrogen sulfide lactose agar (DHL), MacConkey agar (MAC), DHL supplemented with rhamnose and xylose (RX-DHL), and MAC supplemented with rhamnose and xylose (RX-MAC). Chicken and bean sprout samples were inoculated with E. albertii either at 17.7 CFU/25 g (low inoculation level) or 88.5 CFU/25 g (high inoculation level), and uninoculated samples were used as controls. The sensitivity of EA-rtPCR was 1.000 for chicken and bean sprout samples inoculated with E. albertii at low and high inoculation levels. The Ct values of bean sprout samples were higher than those of the chicken samples. Analysis of microbial distribution by 16S rRNA gene amplicon sequencing in enriched cultures of bean sprout samples showed that approximately >96 % of the population comprised unidentified genus of family Enterobacteriaceae and genus Acinetobacter in samples which E. albertii was not isolated. The sensitivity of the plating methods for chicken and bean sprout samples inoculated with a high inoculation level of E. albertii was 1.000 and 0.848-0.970, respectively. The sensitivity of the plating methods for chicken and bean sprout samples inoculated with a low inoculation level of E. albertii was 0.939-1.000 and 0.515-0.727, respectively. The E. albertii-positive rate in all colonies isolated in this study was 89-90 % in RX-DHL and RX-MAC, and 64 and 44 % in DHL and MAC, respectively. Therefore, the sensitivity of RX-supplemented agar was higher than that of the agars without these sugars. Using a combination of enrichment in CT-mEC and E. albertii isolation on selective agars supplemented with RX, E. albertii at an inoculation level of over 17.5 CFU/25 g of food was detected with a sensitivity of 1.000 and 0.667-0.727 in chicken and bean sprouts, respectively. Therefore, screening for E. albertii-specific genes using EA-rtPCR followed by isolation with RX-DHL or RX-MAC is an efficient method for E. albertii detection in food.


Assuntos
Escherichia coli , Escherichia , Xilose , Ágar , Reação em Cadeia da Polimerase em Tempo Real , RNA Ribossômico 16S , Ramnose , Meios de Cultura , Carne , Microbiologia de Alimentos , Lactose
14.
Clin Lab ; 70(2)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38345975

RESUMO

BACKGROUND: Bloodstream infections (BSI) represent a common cause of sepsis and mortality in children. Blood culture (BC) is the gold standard for diagnosis of BSI. The low sensitivity of BC in the pediatric population is usually due to the small volume of blood used for inoculation and to the antibiotics used before sampling. Here, we explore the ways to effectively reduce antibiotic activity to maximize the chances of pathogen recovery, and to enhance the growth of microorganisms in lower blood volume and bacterial counts. METHODS: The recovery of common pathogens causing blood stream infections was analyzed after exposure to cefo-perazone/sulbactam, vancomycin, and caspofung by using resin-containing or not BacT/Alert PF Plus and BD FX400 peds plus pediatric bottles. The microbial growth in the resin-containing bottles was assessed using 0.5 colony-forming units (CFU) bacterial inoculum to mimic the bacteremia/fungemia condition. The usefulness of a diagnosis to confirm or exclude BSI was evaluated by lower than recommended blood culture sampling (102 CFU/mL, 0.3 mL). RESULTS: Staphylococcus aureus (S. aureus), and Candida glabrata (C. glabrata) were recovered from 100% of two types of resin-containing bottles in the presence of a sufficient antibiotic dose, while Escherichia coli (E. coli) was not restored to 100% in BD FX400 peds plus pediatric bottles. The shorter TTD for S. aureus, C. glabrata, and E. coli were observed in antibiotic-containing BacT/Alert PF Plus bottles. Both the PF Plus and BD resin test bottles showed consistently good TTD performances to Gram-negative, Gram-positive, and yeast species in low inoculum levels, with the exception of S. aureus. The lower volume of blood inoculated into culture bottles hardly affected the growth of most bacteria, but optimized PF Plus resin-bottles accelerated the detection of infectious agents, especially S. aureus, Streptococcus pneumoniae, and C. glabrata. CONCLUSIONS: It is possible to enhance recovery from antibiotic-containing pediatric bottles and shorten TTD for the identification of pathogens by using the BacT/Alert blood culture system combination with new resin-containing media.


Assuntos
Bacteriemia , Sepse , Infecções Estafilocócicas , Criança , Humanos , Antibacterianos/farmacologia , Escherichia coli , Meios de Cultura , Staphylococcus aureus , Bacteriemia/diagnóstico , Bacteriemia/microbiologia , Bactérias , Técnicas Bacteriológicas
15.
Clin Lab ; 70(2)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38345982

RESUMO

BACKGROUND: Urinary tract infections (UTIs) are the most common infections in adults, and urine culture is the parameter that uses the most time and cost in microbiology laboratories. For this reason, the selection of fast and cost-effective methods in the evaluation of urine samples is one of the priority issues of microbiology laboratories. The aim of this study was to investigate the compatibility and cost-effectiveness of routinely used Blood Agar (BA), Eosin Methylene Blue (EMB) medium, and CHROMagar Orientation Medium (CO Medium) in the identification of microorganisms in urine samples. METHODS: Consecutive urine samples (n: 700) sent to our laboratory were simultaneously inoculated onto BA/EMB media and CO medium. Urine samples were evaluated after 18 - 24 hours of incubation at 37℃ and the compatibility of the two methods was compared. The use of 104 Gram stains, 198 biochemical tests, and 9 identification kits was required with BA/EMB agar. RESULTS: When 104 colonies with single growth were evaluated, presumptive identification with CO medium was found to be 100% compatible with the VITEK 2 system. The most isolated 62 Escherichia coli (E. coli) colonies gave dark pink-red color and were found to be fully compatible with the VITEK 2 system. Compatibility of BA and EMB medium evaluations with VITEK 2 system; E. coli (n: 62), KES group (n: 26), Pseudomonas spp. (n: 6) and Proteus spp. For (n: 2), it was determined as 69.3%, 57.69%, 100%, and 100%, respectively. According to the results of our study, when BA/EMB and CO Medium methods were compared, 182 Euro (€) savings were achieved in 700 urine cultures with CO Medium. It was estimated that the amount of savings could be 15,600 € per year. CONCLUSIONS: CHROMagar Orientation Medium method can be used routinely with its advantages such as being cost-effective, reducing the workload, and not requiring additional operations. CHROMagar Orientation Medium can also be considered as an easily accessible method and opportunity that does not require infrastructure and trained personnel, especially for laboratories with low test capacity and having problems with the supply of com-mercial kits and automated systems.


Assuntos
Escherichia coli , Infecções Urinárias , Humanos , Ágar , Meios de Cultura , Infecções Urinárias/microbiologia , Azul de Metileno
16.
BMC Biotechnol ; 24(1): 9, 2024 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-38331794

RESUMO

BACKGROUND: The production of Pleurotus ostreatus mycelium as a promising object for use in food and other industries is hampered by a lack of information about the strain-specificity of this fungus mycelium growth and its acquisition of various biological activities. Therefore, this research aimed to investigate mycelial growth of different P. ostreatus strains on varies solid and liquid media as well as to evaluate strains antagonistic, antibacterial, antiradical scavenging activities, and total phenolic content. RESULTS: Potato Dextrose Agar medium was suitable for all strains except P. ostreatus strain 2460. The best growth rate of P. ostreatus 2462 strain on solid culture media was 15.0 ± 0.8 mm/day, and mycelia best growth on liquid culture media-36.5 ± 0.2 g/l. P. ostreatus strains 551 and 1685 were more susceptible to positive effect of plant growth regulators Ivin, Methyur and Kamethur. Using of nutrient media based on combination of natural waste (amaranth flour cake and wheat germ, wheat bran, broken vermicelli and crumbs) has been increased the yield of P. ostreatus strains mycelium by 2.2-2.9 times compared to the control. All used P. ostreatus strains displayed strong antagonistic activity in co-cultivation with Aspergillus niger, Candida albicans, Issatchenkia orientalis, Fusarium poae, Microdochium nivale in dual-culture assay. P. ostreatus 2462 EtOAc mycelial extract good inhibited growth of Escherichia coli (17.0 ± 0.9 mm) while P. ostreatus 2460 suppressed Staphylococcus aureus growth (21.5 ± 0.5 mm) by agar well diffusion method. The highest radical scavenging effect displayed both mycelial extracts (EtOH and EtOAc) of P. ostreatus 1685 (61 and 56%) by DPPH assay as well as high phenolic content (7.17 and 6.73 mg GAE/g) by the Folin-Ciocalteu's method. The maximal total phenol content (7.52 mg GAE/g) demonstrated of P. ostreatus 2461 EtOH extract. CONCLUSIONS: It is found that the growth, antibacterial, antiradical scavenging activity as well as total phenolic content were dependent on studied P. ostreatus strains in contrast to antagonistic activity. The proposed culture mediums of natural waste could be an alternative to commercial mediums for the production mycelial biomass of P. ostreatus strains.


Assuntos
Pleurotus , Ágar/análise , Ágar/farmacologia , Antibacterianos/farmacologia , Meios de Cultura/química , Extratos Vegetais/farmacologia , Micélio
17.
Photodiagnosis Photodyn Ther ; 45: 103996, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38336150

RESUMO

BACKGROUND: This study aimed to assess the effect of antibacterial photodynamic therapy (aPDT) with chitosan nanoparticles on Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) in the culture medium. MATERIALS AND METHODS: In this in vitro, experimental study, chitosan nanoparticles (CHNPs) containing indocyanine green (ICG) were first synthesized and characterized. A. actinomycetemcomitans was cultured on trypticase soy agar. The culture media containing A. actinomycetemcomitans were randomly subjected to the following six decontamination protocols: negative control subjected to sterile phosphate buffered saline (PBS) for 5 min, positive control exposed to 0.2 % chlorhexidine (CHX) for 5 min, exposure to 0.25 mg/mL ICG in the dark at 37 °C for 5 min, aPDT with 0.25 mg/mL ICG and diode laser (808 nm, 250 mW, 14.94 J/cm2, 30 s, 1 mm distance, 8 mm tip diameter), exposure to CHNPs containing 0.25 mg/mL ICG in the dark at 37 °C for 5 min, and aPDT with CHNPs containing 0.25 mg/mL ICG and diode laser. The number of colonies was counted, and analyzed by one-way ANOVA and Tamhane test (alpha=0.050). RESULTS: Antimicrobial PDT with CHNPs, and CHX groups comparably showed the highest decontamination efficacy (P = 0.000). CONCLUSION: The results showed optimal efficacy of aPDT with CHNPs containing 0.25 mg/mL ICG and 808 nm diode laser for reduction of A. actinomycetemcomitans colony count. Thus, aPDT appears to be as effective as CHX, but with fewer adverse effects.


Assuntos
Quitosana , Nanopartículas , Fotoquimioterapia , Aggregatibacter actinomycetemcomitans , Quitosana/farmacologia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Antibacterianos/farmacologia , Clorexidina , Meios de Cultura , Verde de Indocianina/farmacologia
18.
Mar Drugs ; 22(2)2024 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-38393037

RESUMO

Co-cultivation, coupled with the OSMAC approach, is considered an efficient method for expanding microbial chemical diversity through the activation of cryptic biosynthetic gene clusters (BGCs). As part of our project aiming to discover new fungal metabolites for crop protection, we previously reported five polyketides, the macrolides dendrodolides E (1) and N (2), the azaphilones spiciferinone (3) and 8α-hydroxy-spiciferinone (4), and the bis-naphtho-γ-pyrone cephalochromin (5) from the solid Potato Dextrose Agar (PDA) co-culture of two marine sediment-derived fungi, Plenodomus influorescens and Pyrenochaeta nobilis. However, some of the purified metabolites could not be tested due to their minute quantities. Here we cultivated these fungi (both axenic and co-cultures) in liquid regime using three different media, Potato Dextrose Broth (PDB), Sabouraud Dextrose Broth (SDB), and Czapek-Dox Broth (CDB), with or without shaking. The aim was to determine the most ideal co-cultivation conditions to enhance the titers of the previously isolated compounds and to produce extracts with stronger anti-phytopathogenic activity as a basis for future upscaled fermentation. Comparative metabolomics by UPLC-MS/MS-based molecular networking and manual dereplication was employed for chemical profiling and compound annotations. Liquid co-cultivation in PDB under shaking led to the strongest activity against the phytopathogen Phytophthora infestans. Except for compound 1, all target compounds were detected in the co-culture in PDB. Compounds 2 and 5 were produced in lower titers, whereas the azaphilones (3 and 4) were overexpressed in PDB compared to PDA. Notably, liquid PDB co-cultures contained meroterpenoids and depside clusters that were absent in the solid PDA co-cultures. This study demonstrates the importance of culture regime in BGC regulation and chemical diversity of fungal strains in co-culture studies.


Assuntos
Metaboloma , Espectrometria de Massas em Tandem , Técnicas de Cocultura , Cromatografia Líquida , Meios de Cultura , Glucose
19.
Lett Appl Microbiol ; 77(3)2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38364315

RESUMO

The objective of this study is to validate the US Food and Drug Administration (FDA) rea-time polymerase chain reaction (qPCR) assay, the Neogen Amplified Nucleic Single Temperature Reaction (ANSR) assay, and the Vitek ImmunoDiagnostic Assay System (VIDAS) SLM procedure against the FDA cultural procedure for Salmonella detection in green chile pepper. Green chile was artificially contaminated with Salmonella according to the FDA guidelines (FDA. Guidelines for the Validation of Microbiological Methods for the FDA Foods Program, 3rd Edition. 2019. www.fda.gov/media/83812/download?attachment (17 March 2024, date last accessed)) at a fractional recovery level (where 50%-25% tests positive and at a level +1 log greater for each organism tested). Enriched samples were tested directly by the ANSR Salmonella test and by qPCR, and were subcultured into Rappaport-Vassiliadis and tetrathionate brilliant green broth for cultural detection and qPCR. For the VIDAS-SLM assay, the selective enrichments were further cultured in M broth before testing. Presumptive salmonellae were confirmed with biochemical tests, serology, and qPCR. All three rapid assays were compared favorably with the FDA-BAM (Bacteriological Analytical Manual) method. No significant differences at P < .05 were found between the procedures using McNemar's χ2 test. The three procedures were found to be rapid and reliable alternatives to cultural detection of Salmonella enterica in green chile.


Assuntos
Microbiologia de Alimentos , Salmonella enterica , Meios de Cultura , Salmonella enterica/genética , Chile , Técnicas Bacteriológicas/métodos , Salmonella
20.
Metab Eng ; 82: 201-215, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38364997

RESUMO

Chemically defined media for cultivation of Saccharomyces cerevisiae strains are commonly supplemented with a mixture of multiple Class-B vitamins, whose omission leads to strongly reduced growth rates. Fast growth without vitamin supplementation is interesting for industrial applications, as it reduces costs and complexity of medium preparation and may decrease susceptibility to contamination by auxotrophic microbes. In this study, suboptimal growth rates of S. cerevisiae CEN.PK113-7D in the absence of pantothenic acid, para-aminobenzoic acid (pABA), pyridoxine, inositol and/or biotin were corrected by single or combined overexpression of ScFMS1, ScABZ1/ScABZ2, ScSNZ1/ScSNO1, ScINO1 and Cyberlindnera fabianii BIO1, respectively. Several strategies were explored to improve growth of S. cerevisiae CEN.PK113-7D in thiamine-free medium. Overexpression of ScTHI4 and/or ScTHI5 enabled thiamine-independent growth at 83% of the maximum specific growth rate of the reference strain in vitamin-supplemented medium. Combined overexpression of seven native S. cerevisiae genes and CfBIO1 enabled a maximum specific growth rate of 0.33 ± 0.01 h-1 in vitamin-free synthetic medium. This growth rate was only 17 % lower than that of a congenic reference strain in vitamin-supplemented medium. Physiological parameters of the engineered vitamin-independent strain in aerobic glucose-limited chemostat cultures (dilution rate 0.10 h-1) grown on vitamin-free synthetic medium were similar to those of similar cultures of the parental strain grown on vitamin-supplemented medium. Transcriptome analysis revealed only few differences in gene expression between these cultures, which primarily involved genes with roles in Class-B vitamin metabolism. These results pave the way for development of fast-growing vitamin-independent industrial strains of S. cerevisiae.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Vitaminas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biotina/metabolismo , Tiamina , Meios de Cultura
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