Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 14.069
Filtrar
1.
Food Microbiol ; 109: 104127, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36309437

RESUMO

Salmonella spp. is one of the leading causes of foodborne outbreaks worldwide. Salmonella spp. has been associated with a variety of food sources, particularly egg products. They can enter a viable but nonculturable (VBNC) state in response to harsh stress. VBNC cells still retain membrane integrity and metabolic activity, which may pose health risks. However, the formation mechanism and resuscitation ability of VBNC cells are not well understood. In this work, Salmonella spp. cocktails, including Salmonella enterica serovar Newport and Salmonella enterica serovar Enteritidis, in liquid egg products was induced into a VBNC state by mild heat treatment, a commonly used method to inhibit the growth of pathogenic in liquid egg industry. Mild heat induced VBNC cells were found to resuscitate in liquid egg yolk (LEY) and liquid whole egg (LWE), but they failed to recover in liquid egg white (LEW). In addition, a certain number of cells remained as VBNC state after in vitro digestion. The membrane vesicle (MV) protein encoding gene pagC, two-component system encoding genes phoP/Q and sigma factor encoding gene rpoS were highly expressed in VBNC cells compared with the culturable counterparts. The results of this study can contribute to a better understanding of the health risks associated with Salmonella spp. in VBNC state and provide a theoretical basis for formation mechanism of VBNC state.


Assuntos
Temperatura Alta , Salmonella enterica , Viabilidade Microbiana , Salmonella enteritidis/genética , Fator sigma
2.
J Med Microbiol ; 71(10)2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36201343

RESUMO

Healthcare-associated infections (HCAIs) are a major challenge and the near patient surface is important in harbouring causes such as methicillin-resistant Staphylococcus aureus (MRSA) and Clostridioides difficile. Current approaches to decontamination are sub-optimal and many studies have demonstrated that microbial causes of HCAIs may persist with onward transmission. This may be due to the capacity of these microbes to survive in biofilms on surfaces. New technologies to enhance hospital decontamination may have a role in addressing this challenge. We have reviewed current technologies such as UV light and hydrogen peroxide and also assessed the potential use of cold atmospheric pressure plasma (CAPP) in surface decontamination. The antimicrobial mechanisms of CAPP are not fully understood but the production of reactive oxygen and other species is believed to be important. CAPP systems have been shown to partially or completely remove a variety of biofilms including those caused by Candida albicans, and multi-drug-resistant bacteria such as MRSA. There are some studies that suggest promise for CAPP in the challenge of surface decontamination in the healthcare setting. However, further work is required to define better the mechanism of action. We need to know what surfaces are most amenable to treatment, how microbial components and the maturity of biofilms may affect successful treatment, and how would CAPP be used in the clinical setting.


Assuntos
Infecção Hospitalar , Staphylococcus aureus Resistente à Meticilina , Gases em Plasma , Bactérias , Biofilmes , Infecção Hospitalar/microbiologia , Descontaminação , Hospitais , Humanos , Peróxido de Hidrogênio/farmacologia , Viabilidade Microbiana , Oxigênio , Gases em Plasma/farmacologia
3.
PLoS Pathog ; 18(10): e1010908, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36260637

RESUMO

Extra-intestinal Pathogenic Escherichia coli (ExPEC) is defined as an extra-intestinal foodborne pathogen, and several dominant sequence types (STs) ExPEC isolates are highly virulent, with zoonotic potential. Bacteria extracellular vesicles (EVs) carry specific subsets of molecular cargo, which affect various biological processes in bacteria and host. The mechanisms of EVs formation in ExPEC remains to be elucidated. Here, the purified EVs of ExPEC strains of different STs were isolated with ultracentrifugation processes. A comparative analysis of the strain proteomes showed that cytoplasmic proteins accounted for a relatively high proportion of the proteins among ExPEC EVs. The proportion of cytoplasm-carrying vesicles in ExPEC EVs was calculated with a simple green fluorescent protein (GFP) expression method. The RecA/LexA-dependent SOS response is a critical mediator of generation of cytoplasm-carrying EVs. The SOS response activates the expression of prophage-associated endolysins, Epel1, Epel2.1, and Epel2.2, which triggered cell lysis, increasing the production of ExPEC cytoplasm-carrying EVs. The repressor LexA controlled directly the expression of these endolysins by binding to the SOS boxes in the endolysin promoter regions. Reducing bacterial viability stimulated the production of ExPEC EVs, especially cytoplasm-carrying EVs. The imbalance in cell division caused by exposure to H2O2, the deletion of ftsK genes, or t6A synthesis defects activated the RecA/LexA-dependent SOS response, inducing the expression of endolysins, and thus increasing the proportion of cytoplasm-carrying EVs in the total ExPEC EVs. Antibiotics, which decreased bacterial viability, also increase the production of ExPEC cytoplasm-carrying EVs through the SOS response. Changes in the proportion of cytoplasm-carrying EVs affected the total DNA content of ExPEC EVs. When macrophages are exposed to a higher proportion of cytoplasm-carrying vesicles, ExPEC EVs were more cytotoxic to macrophages, accompanied with more-severe mitochondrial disruption and a higher level of induced intrinsic apoptosis. In summary, we offered comprehensive insight into the proteome analysis of ExPEC EVs. This study demonstrated the novel formation mechanisms of E. coli cytoplasm-carrying EVs.


Assuntos
Proteínas de Escherichia coli , Vesículas Extracelulares , Escherichia coli Extraintestinal Patogênica , Viabilidade Microbiana , Citoplasma/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Vesículas Extracelulares/metabolismo , Escherichia coli Extraintestinal Patogênica/genética , Peróxido de Hidrogênio/metabolismo , Proteínas de Membrana/metabolismo
4.
PLoS Genet ; 18(10): e1010456, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36279294

RESUMO

Thymidine starvation causes rapid cell death. This enigmatic process known as thymineless death (TLD) is the underlying killing mechanism of diverse antimicrobial and antineoplastic drugs. Despite decades of investigation, we still lack a mechanistic understanding of the causal sequence of events that culminate in TLD. Here, we used a diverse set of unbiased approaches to systematically determine the genetic and regulatory underpinnings of TLD in Escherichia coli. In addition to discovering novel genes in previously implicated pathways, our studies revealed a critical and previously unknown role for intracellular acidification in TLD. We observed that a decrease in cytoplasmic pH is a robust early event in TLD across different genetic backgrounds. Furthermore, we show that acidification is a causal event in the death process, as chemical and genetic perturbations that increase intracellular pH substantially reduce killing. We also observe a decrease in intracellular pH in response to exposure to the antibiotic gentamicin, suggesting that intracellular acidification may be a common mechanistic step in the bactericidal effects of other antibiotics.


Assuntos
Escherichia coli , Timina , Escherichia coli/metabolismo , DNA Bacteriano/genética , Viabilidade Microbiana , Timina/metabolismo , Recombinação Genética , Concentração de Íons de Hidrogênio
5.
Ultrason Sonochem ; 90: 106166, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36215891

RESUMO

Although both ultraviolet (UV) radiation and ultrasound (US) treatment have their capabilities in microbial inactivation, applying any one method alone may require a high dose for complete inactivation, which may affect the sensory and nutritional properties of pineapple juice. Hence, this study was intended to analyse and optimise the effect of combined US and UV treatments on microbial inactivation without affecting the selected quality parameters of pineapple juice. US treatment (33 kHz) was done at three different time intervals, viz. 10 min, 20 min and 30 min., after which, juice samples were subjected to UV treatment for 10 min at three UV dosage levels, viz. 1 J/cm2, 1.3 J/cm2, and 1.6 J/cm2. The samples were evaluated for total colour difference, pH, total soluble solids (TSS), titrable acidity (TA), and ascorbic acid content; total bacterial count and total yeast count; and the standardization of process parameters was done using Response Surface Methodology and Artificial Neural Network. The results showed that the individual, as well as combined treatments, did not significantly impact the physicochemical properties while retaining the quality characteristics. It was observed that combined treatment resulted in 5 log cycle reduction in bacterial and yeast populations while the individual treatment failed. From the optimization studies, it was found that combined US and UV treatments with 22.95 min and1.577 J/cm2 ensured a microbiologically safe product while retaining organoleptic quality close to that of fresh juice.


Assuntos
Ananas , Malus , Malus/química , Manipulação de Alimentos/métodos , Saccharomyces cerevisiae , Sucos de Frutas e Vegetais , Viabilidade Microbiana/efeitos da radiação , Ananas/química
6.
Food Res Int ; 160: 111699, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36076451

RESUMO

A unique double-layered vehicle was fabricated for the first time based on a millifluidic/direct gelation to encapsulate probiotics. Free probiotic bacteria are usually very sensitive to severe gastrointestinal conditions and maintaining their survival when passing through the digestive tract is essential. The effects of alginate concentration (20-30 g/L), flow rates of alginate (0.8-1.2 mL/min), and W/O emulsion (0.5-0.7 mL/min) on encapsulation efficiency (EE), size, and sphericity of core-shell millicapsules were optimized for encapsulation of Bifidobacterium animalis subsp. lactis and Lactobacillus plantarum. The optimized calcium-alginate millicapsule was spherical (0.97 ± 0.01 SF), with an average diameter of 4.49 ± 0.19 mm, and encapsulation efficiency of 98.17 ± 0.5 %. Two strains were encapsulated separately in W/O emulsion as a core of the millicapsule. After coating with chitosan, the encapsulation yield of the bacteria, survival rates under simulated gastrointestinal (GI) conditions, and viability during storage were determined. Survival efficiency of B. animalis subsp. lactis and L. plantarum after millifluidic encapsulation were found to be 92.33 and 90.81 %, respectively. Cell viability of encapsulated probiotics after passing through the GI system was improved (7.5 log CFU mL-1 for both strains). Although the viability of the encapsulated probiotics stored at -18 °C for five months significantly decreased (p<0.05), the number of live cells was approximately in accordance with the standard definition of long-term probiotic survival (6 log CFU/g). This work provides a pathway for the construction of an innovative delivery system with high efficiency and protective effects for probiotics.


Assuntos
Lactobacillus plantarum , Probióticos , Alginatos/química , Emulsões , Viabilidade Microbiana , Probióticos/química
7.
Food Res Int ; 160: 111723, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36076461

RESUMO

Probiotics are living microorganisms that can produce health benefits to the host only when they are ingested in sufficient quantities and reach the intestines active state. However, the external environment that probiotics face for a long time before administration and the low pH environment in the stomach after administration can greatly reduce their activity. In this work, we proposed a simple microfluidic encapsulation strategy to efficiently prepare the probiotics-loaded nanocellulose/alginate delivery system, which can improve the storage stability and gastrointestinal survival rate of probiotics. The microcapsules were found to be monodisperse, and the average particle size was<500 µm by observing the microstructure and macroscopic morphology. The kelp nanocellulose was cross-linked in the microcapsule and formed a dense surface with alginate. Through the simulated gastrointestinal digestion experiment, it was found that the survival of probiotics in microcapsules containing 0.5 % and 1.5 % kelp nanocellulose decreased by 1.77 log CFU/g and 1.65 log CFU/g respectively, which was significantly lower than that of nanocellulose-free microcapsules (3.70 log CFU/g). And all the treated groups could release probiotics above 7 log CFU/g after digesting intestinal juice for 6 h. Furthermore, through the storage experiment, it was found that the microcapsules with 1.5 % kelp nanocellulose could still release 8.07 log CFU/g probiotics after four weeks. The results provide a new strategy for probiotics processing and extensive high-value utilization of marine natural products.


Assuntos
Kelp , Probióticos , Alginatos/química , Cápsulas/química , Composição de Medicamentos/métodos , Dispositivos Lab-On-A-Chip , Viabilidade Microbiana , Probióticos/química
8.
Front Cell Infect Microbiol ; 12: 977944, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36093179

RESUMO

Two-component regulatory systems (TCRS) are ubiquitous signal transduction mechanisms evolved by bacteria for sensing and adapting to the constant changes that occur in their environment. Typically consisting of two types of proteins, a membrane sensor kinase and an effector cytosolic response regulator, the TCRS modulate via transcriptional regulation a plethora of key physiological processes, thereby becoming essential for bacterial viability and/or pathogenicity and making them attractive targets for novel antibacterial drugs. Some members of the phylum Campylobacterota (formerly Epsilonproteobacteria), including Helicobacter pylori and Campylobacter jejuni, have been classified by WHO as "high priority pathogens" for research and development of new antimicrobials due to the rapid emergence and dissemination of resistance mechanisms against first-line antibiotics and the alarming increase of multidrug-resistant strains worldwide. Notably, these clinically relevant pathogens express a variety of TCRS and orphan response regulators, sometimes unique among its phylum, that control transcription, translation, energy metabolism and redox homeostasis, as well as the expression of relevant enzymes and virulence factors. In the present mini-review, we describe the signalling mechanisms and functional diversity of TCRS in H. pylori and C. jejuni, and provide an overview of the most recent findings in the use of these microbial molecules as potential novel therapeutic targets for the development of new antibiotics.


Assuntos
Campylobacter jejuni , Helicobacter pylori , Antibacterianos/farmacologia , Campylobacter jejuni/genética , Helicobacter pylori/genética , Viabilidade Microbiana , Virulência
9.
Nature ; 609(7929): 1029-1037, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36104562

RESUMO

Advancing the spontaneous bottom-up construction of artificial cells with high organizational complexity and diverse functionality remains an unresolved issue at the interface between living and non-living matter1-4. Here, to address this challenge, we developed a living material assembly process based on the capture and on-site processing of spatially segregated bacterial colonies within individual coacervate microdroplets for the endogenous construction of membrane-bounded, molecularly crowded, and compositionally, structurally and morphologically complex synthetic cells. The bacteriogenic protocells inherit diverse biological components, exhibit multifunctional cytomimetic properties and can be endogenously remodelled to include a spatially partitioned DNA-histone nucleus-like condensate, membranized water vacuoles and a three-dimensional network of F-actin proto-cytoskeletal filaments. The ensemble is biochemically energized by ATP production derived from implanted live Escherichia coli cells to produce a cellular bionic system with amoeba-like external morphology and integrated life-like properties. Our results demonstrate a bacteriogenic strategy for the bottom-up construction of functional protoliving microdevices and provide opportunities for the fabrication of new synthetic cell modules and augmented living/synthetic cell constructs with potential applications in engineered synthetic biology and biotechnology.


Assuntos
Células Artificiais , Escherichia coli , Viabilidade Microbiana , Biologia Sintética , Citoesqueleto de Actina/química , Actinas/química , Trifosfato de Adenosina/metabolismo , Células Artificiais/química , Biotecnologia , Escherichia coli/citologia , Histonas/química , Vacúolos/química , Água/química
10.
Viruses ; 14(9)2022 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-36146663

RESUMO

Respiratory pathogens can be spread though the transmission of aerosolised expiratory secretions in the form of droplets or particulates. Understanding the fundamental aerosol parameters that govern how such pathogens survive whilst airborne is essential to understanding and developing methods of restricting their dissemination. Pathogen viability measurements made using Controlled Electrodynamic Levitation and Extraction of Bioaerosol onto Substrate (CELEBS) in tandem with a comparative kinetics electrodynamic balance (CKEDB) measurements allow for a direct comparison between viral viability and evaporation kinetics of the aerosol with a time resolution of seconds. Here, we report the airborne survival of mouse hepatitis virus (MHV) and determine a comparable loss of infectivity in the aerosol phase to our previous observations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through the addition of clinically relevant concentrations of mucin to the bioaerosol, there is a transient mitigation of the loss of viral infectivity at 40% RH. Increased concentrations of mucin promoted heterogenous phase change during aerosol evaporation, characterised as the formation of inclusions within the host droplet. This research demonstrates the role of mucus in the aerosol phase and its influence on short-term airborne viral stability.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Camundongos , Viabilidade Microbiana , Mucinas , Aerossóis e Gotículas Respiratórios
11.
Food Res Int ; 160: 111720, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36076413

RESUMO

As a novel microbe inactivation strategy, cold atmospheric plasma (CAP) technology has attracted great attractions in the past two decades. This study demonstrated that the CAP treatment was a robust inactivation approach for P. aeruginosa. Air and nitrogen-CAP achieved 100 % inactivation efficiency in 5 and 9 min, respectively. Furthermore, the inactivation mechanisms were explained by measuring several physicochemical indexes, including the characteristics of bacterial suspension, cell membrane integrity, cell viability, and the concentration of intracellular substances. The possible inactivation mechanisms might be that the RONS generated by air and nitrogen attacked the cell envelope, resulted in the leakage of intracellular substances; meanwhile, RONS also destroyed the intracellular anti-oxidative system, accelerated the oxidative stress and disrupted the intracellular redox homeostasis, subsequently the death of the cells. Moreover, the inactivation application in chicken breasts proved CAP could be a novel sanitizing process in practical industries.


Assuntos
Gases em Plasma , Animais , Galinhas , Viabilidade Microbiana , Nitrogênio , Gases em Plasma/farmacologia , Pseudomonas aeruginosa
12.
Anal Chem ; 94(40): 13921-13926, 2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36166663

RESUMO

Assessing bacterial viability is crucial in public health, food safety, environmental microbiology, and other relevant fields. The classical agar plate counting method and the popular dye-based assays have shown their strengths, but they also have limitations including high time consumption, relatively complex sample preparations, and cytotoxicity. In this work, we present a new bacterial viability assay based on optical tweezers utilizing a power sweeping strategy. By monitoring and analyzing bacterial nanomotion in optical traps under different trapping laser powers, the slope of the proportionality between the quantified extent of motion and the trapping laser power was defined as the mobility restriction coefficient (MRC) to quantify bacterial viability. We first established a firm correlation between the viability and MRC by measuring alive and dead Escherichia coli and Photobacterium phosphoreum. Then the capability of real-time long-term characterization of the assay was validated by measuring the viability of individual P. phosphoreum while regulating the viability with an inactivation light. Notably, a 'spinning-induced stabilization' mechanism was proposed to explain the surprising increase of apparent bacterial mobility after inactivation. Overall, the assay was proved to be a reliable label-free bacterial viability assay at a single-cell level, which holds potential in antibiotic susceptibility testing, drug screening, and rapid diagnostics.


Assuntos
Escherichia coli , Pinças Ópticas , Ágar , Antibacterianos , Viabilidade Microbiana
13.
Sci Rep ; 12(1): 13726, 2022 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-35962051

RESUMO

Pseudomonas aeruginosa is a Gram-negative bacterium responsible for numerous human infections. Previously, novel antibiotic tolerant variants known as phoenix colonies as well as variants similar to viable but non-culturable (VBNC) colonies were identified in response to high concentrations of aminoglycosides. In this study, the mechanisms behind phoenix colony and VBNC-like colony emergence were further explored using both whole genome sequencing and RNA sequencing. Phoenix colonies were found to have a single nucleotide polymorphism (SNP) in the PA4673 gene, which is predicted to encode a GTP-binding protein. No SNPs were identified within VBNC-like colonies compared to the founder population. RNA sequencing did not detect change in expression of PA4673 but revealed multiple differentially expressed genes that may play a role in phoenix colony emergence. One of these differentially expressed genes, PA3626, encodes for a tRNA pseudouridine synthase which when knocked out led to a complete lack of phoenix colonies. Although not immediately clear whether the identified genes in this study may have interactions which have not yet been recognized, they may contribute to the understanding of how phoenix colonies are able to emerge and survive in the presence of antibiotic exposure.


Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Antibacterianos/farmacologia , Genômica , Humanos , Viabilidade Microbiana/genética
14.
Int J Food Microbiol ; 380: 109871, 2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-35985079

RESUMO

A novel method is proposed for fitting microbial inactivation models to data on liquid media: the Most Probable Curve (MPC) method. It is a multilevel model that makes a separation between the "true" microbial concentration according to the model, the "actual" concentration in the media considering chance, and the actual counts on the plate. It is based on the assumptions that stress resistance is homogeneous within a microbial population, and that there is no aggregation of microbial cells. Under these assumptions, the number of colonies in/on a plate follows a Poisson distribution with expected value depending on the proposed kinetic model, the number of dilutions and the plated volume. The novel method is compared against (non)linear regression based on a normal likelihood distribution (traditional method), Poisson regression and gamma-Poisson regression using data on the inactivation of Listeria monocytogenes. The conclusion is that the traditional method has limitations when the data includes plates with low (or zero) cell counts, which can be mitigated using more complex (discrete) likelihoods. However, Poisson regression uses an unrealistic likelihood function, making it unsuitable for survivor curves with several log-reductions. Gamma-Poisson regression uses a more realistic likelihood function, even though it is based mostly on empirical hypotheses. We conclude that the MPC method can be used reliably, especially when the data includes plates with low or zero counts. Furthermore, it generates a more realistic description of uncertainty, integrating the contribution of the plating error and reducing the uncertainty of the primary model parameters. Consequently, although it increases modelling complexity, the MPC method can be of great interest in predictive microbiology, especially in studies focused on variability analysis.


Assuntos
Microbiologia de Alimentos , Listeria monocytogenes , Viabilidade Microbiana , Distribuição de Poisson , Incerteza
15.
Nature ; 608(7922): 390-396, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35922513

RESUMO

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1-3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a ß-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.


Assuntos
Antibacterianos , Bactérias , Membrana Celular , Depsipeptídeos , Viabilidade Microbiana , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/citologia , Bactérias/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Difosfatos/química , Farmacorresistência Bacteriana/efeitos dos fármacos , Humanos , Lipídeos/química , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Pirrolidinas/química , Açúcares/química
16.
Arch Microbiol ; 204(9): 557, 2022 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-35972563

RESUMO

Stool is the most commonly used sample for gut microbiota analysis in humans and animals. Cryopreservation of stool at - 80 °C is a feasible and simple method in clinics and researches, especially in large-scale cohort studies. However, the viability of bacteria in stool after freezing has yet well-demonstrated quantitatively and compositionally. This study determined the viable microbiota of samples under cryopreservation at - 80 °C, relative to fresh samples and that stored at ambient. Stool samples were collected from three healthy adults. Propidium monoazide treatment combined with quantitative PCR and 16S rRNA gene sequencing was performed to target viable microbiota. After freezing, the number of viable bacteria decreased, though inter-individual difference existed. Notably, the alpha diversity of viable microbiota after freezing did not change significantly, while its composition changed. Freezing significantly reduced the viable bacteria in Gram-negative genera of Bacteroidetes and Firmicutes, and proportionally increased Gram-positive bacteria in genera of Actinobacteria and Firmicutes, including Bifidobacterium, Collinsella and Blautia, implying that the cell envelope structure associated with the bacterial sensitivity to freezing. On the contrary, the room temperature storage not only decreased the number of viable bacteria, but also decreased the microbial alpha diversity, and remarkably enriched facultative anaerobes of Escherichia-Shigella, Enterococcus and Lactococcus, some of which are opportunistic pathogens. Our findings suggested that changes in viable microbiota in stool samples caused by cryopreservation should be paid enough attention for downstream utilization.


Assuntos
Microbioma Gastrointestinal , Animais , Bactérias/genética , Criopreservação , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Humanos , Viabilidade Microbiana , RNA Ribossômico 16S/genética
17.
Braz J Microbiol ; 53(4): 2107-2119, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35962856

RESUMO

The reference material (RM) is a technical requirement for the quality assurance of analytical results and proficiency tests or interlaboratory comparisons. Microbiological RMs are most available in the dehydrated form, mainly by freeze-drying, and maintaining bacterial survival after preparation is a challenge. Thus, obtaining the most resistant cells is essential. Considering that bacteria present cross-response to dehydration after being submitted to an array of stress conditions, this study aimed to evaluate the influence of growth conditions on enterobacteria for the production of mixed microbiological RMs by freeze-drying in skim milk powder. Salmonella enterica serovar Enteritidis, Cronobacter sakazakii, Escherichia coli, and Citrobacter freundii were grown in a minimal medium with 0.5 M NaCl and 0 to 5.0 mM of manganese sulfate (MnSO4) until stationary phase. Salmonella Enteritidis presented an increased resistance to dehydration in the presence of Mn, while C. sakazakii was the most resistant to freeze-drying and further storage for 90 days. Mixed microbiological RMs were produced by freeze-drying and containing Salmonella Enteritidis and coliforms in skim milk powder with 100 mM of trehalose and the Salmonella survival rate was 91.2 to 93.6%. The mixed RM was stable after 30 days at -20 °C, and Salmonella and coliforms were detected by different methods being, the Rambach Agar the best for the bacterial differentiation. The results showed that the culture conditions applied in this study resulted in bacterial cells being more resistant to dehydration, freeze-drying, and stabilization for the production of mixed microbiological RMs more stable and homogeneous.


Assuntos
Desidratação , Salmonella , Humanos , Viabilidade Microbiana , Pós , Liofilização/métodos , Bactérias , Microbiologia de Alimentos
18.
Proc Natl Acad Sci U S A ; 119(35): e2201204119, 2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-35994658

RESUMO

Bacteria utilize two-component system (TCS) signal transduction pathways to sense and adapt to changing environments. In a typical TCS, a stimulus induces a sensor histidine kinase (SHK) to phosphorylate a response regulator (RR), which then dimerizes and activates a transcriptional response. Here, we demonstrate that oligomerization-dependent depolarization of excitation light by fused mNeonGreen fluorescent protein probes enables real-time monitoring of RR dimerization dynamics in live bacteria. Using inducible promoters to independently express SHKs and RRs, we detect RR dimerization within seconds of stimulus addition in several model pathways. We go on to combine experiments with mathematical modeling to reveal that TCS phosphosignaling accelerates with SHK expression but decelerates with RR expression and SHK phosphatase activity. We further observe pulsatile activation of the SHK NarX in response to addition and depletion of the extracellular electron acceptor nitrate when the corresponding TCS is expressed from both inducible systems and the native chromosomal operon. Finally, we combine our method with polarized light microscopy to enable single-cell measurements of RR dimerization under changing stimulus conditions. Direct in vivo characterization of RR oligomerization dynamics should enable insights into the regulation of bacterial physiology.


Assuntos
Bactérias , Proteínas de Bactérias , Histidina Quinase , Viabilidade Microbiana , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/efeitos da radiação , Elétrons , Histidina Quinase/genética , Histidina Quinase/metabolismo , Microscopia de Polarização , Nitratos , Óperon/genética , Fosforilação , Regiões Promotoras Genéticas , Multimerização Proteica/efeitos dos fármacos , Análise de Célula Única , Fatores de Tempo
19.
J Microbiol Methods ; 199: 106537, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35798134

RESUMO

Marine-derived Bacillus velezensis B-9987 is an important biocontrol bacterium with a broad-spectrum antibacterial effect. The traditional plate counting method is widely used for quantitative detection of viable bacteria and spores but has some disadvantages such as being laborious and time-consuming (at least 24-48 h). This study aimed to develop a new PMA-qPCR method for rapid and accurate detection of viable bacteria and spores of B-9987. The specific primers were designed for qPCR amplification based on the conserved region of the bmmA gene (encoding a malonyl CoA-ACP transacylase) of B-9987. According to the characteristic that propidium monoazide (PMA) dye can distinguish viable and dead bacteria, the optimal PMA concentration of 10 µg/ml and optimal exposure time of 10 min were achieved under PMA treatment conditions. The B-9987 spores' genomic DNA was successfully extracted after the spore coat was removed and spore germination was induced. The quantification limits of the PMA-qPCR method were determined for viable B-9987 bacteria, spores in pure culture, and spores in marine Bacillus wettable powder (marine Bacillus WP) and were 1.5 × 103 CFU/ml, 6.5 × 102 CFU/ml, and 103 CFU/ml, respectively. Compared with the qPCR method, the PMA-qPCR method could sensitively detect viable bacteria in the viable/dead bacterial mixture. In this study, the developed PMA-qPCR method was found to have excellent sensitivity and specificity in the context of a pure culture of B-9987 strain, which could accurately and rapidly detect viable B-9987 bacteria within 3-4 h and viable B-9987 spores in marine Bacillus WP within 4-6 h.


Assuntos
Azidas , Bacillus , Bacillus/genética , Bactérias/genética , Viabilidade Microbiana , Propídio/análogos & derivados , Reação em Cadeia da Polimerase em Tempo Real/métodos , Esporos
20.
Food Res Int ; 158: 111477, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35840198

RESUMO

In this article, the thermal inactivation of two Salmonella strains (Salmonella Enteritidis CECT4300 and Salmonella Senftenberg CECT4565) was studied under both isothermal and dynamic conditions. We observed large differences between these two strains, with S. Senftenberg being much more resistant than S. Enteritidis. Under isothermal conditions, S. Senftenberg had non-linear survivor curves, whereas the response of S. Enteritidis was log-linear. Therefore, weibullian inactivation models were used to describe the response of S. Senftenberg, with the Mafart model being the more suitable one. For S. Enteritidis, the Bigelow (log-linear) inactivation model was successful at describing the isothermal response. Under dynamic conditions, a combination of the Peleg and Mafart models (secondary model of Mafart; t* of Peleg) fitted to the isothermal data could predict the response of S. Senftenberg to the dynamic treatments tested (heating rates between 0.5 and 10 °C/min). This was not the case for S. Enteritidis, where the model predictions based on isothermal data underestimated the microbial concentrations. Therefore, a dynamic model that considers stress acclimation to one of the dynamic profiles was fitted, using the remaining profiles as validation. In light of this, besides its quantitative impact, variability between strains of bacterial species can also cause qualitative differences in microbial inactivation. This is demonstrated by S. Enteritidis being able to develop stress acclimation where S. Senftenbenberg could not. This has important implications for the development of microbial inactivation models to support process design, as every industrial treatment is dynamic. Consequently, it is crucial to consider different model hypotheses, and how they affect the model predictions both under isothermal and dynamic conditions.


Assuntos
Microbiologia de Alimentos , Salmonella enteritidis , Aclimatação , Viabilidade Microbiana
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...