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1.
Anal Chem ; 93(3): 1443-1450, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33369381

RESUMO

A prophage comprises a bacteriophage genome that has integrated into a host bacterium's DNA, which generally permits the cell to grow and divide normally. However, the prophage can be induced by various stresses, or induction can occur spontaneously. After prophage induction, viral replication and production of endolysins begin until the cell lyses and phage particles are released. However, the heterogeneity of prophage induction and lysis of individual cells in a population and the dynamics of a cell undergoing lysis by prophage induction have not been fully characterized. Here, we used Raman tweezers and live-cell phase-contrast microscopy to characterize the Raman spectral and cell length changes that occur during the lysis of individual Bacillus subtilis cells from spores that carry PBSX prophage during spores' germination, outgrowth, and then vegetative growth. Major findings of this work are as follows: (i) After addition of xylose to trigger prophage induction, the intensities of Raman spectral bands associated with nucleic acids of single cells in induced cultures gradually fell to zero, in contrast to the much smaller changes in protein band intensities and no changes in nucleic acid bands in uninduced cultures; (ii) the nucleic acid band intensities from an individual induced cell exhibited a rapid decrease, following a long lag period; (iii) after the addition of nutrient-rich medium with xylose, single spores underwent a long period (228 ± 41.4 min) for germination, outgrowth, and vegetative growth, followed by a short period of cell burst in 1.5 ± 0.8 min at a cell length of 8.2 ± 5.5 µm; (iv) the latent time (Tlatent) between the addition of xylose and the start of cell burst was heterogeneous in cell populations; however, the period (ΔTburst) from the latent time to the completion of cell lysis was quite small; (v) in a poor medium with l-alanine alone, addition of xylose caused prophage induction following spore germination but with longer Tlatent and ΔTburst times and without cell elongation; (vi) spontaneous prophage induction and lysis of individual cells from spores in a minimal nutrient medium were observed without xylose addition, and cell length prior to cell lysis was ∼4.1 µm, but spontaneous prophage induction was not observed in a rich medium; (vii) in a rich medium, addition of xylose at a time well after spore germination and outgrowth significantly shortened the average Tlatent time. The results of this study provide new insights into the heterogeneity and dynamics of lysis of individual B. subtilis cells derived from spores upon prophage induction.


Assuntos
Bacillus subtilis/citologia , Análise de Célula Única , Esporos Bacterianos/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Microscopia de Contraste de Fase , Pinças Ópticas , Análise Espectral Raman , Esporos Bacterianos/química , Esporos Bacterianos/metabolismo
2.
Int J Food Microbiol ; 337: 108915, 2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33152569

RESUMO

Spore-forming bacteria are implicated in cases of food spoilage or food poisoning. In their sporulated form, they are resistant to physical and chemical treatments applied in the food industry and can persist throughout the food chain. The sporulation leads to an increase in the concentration of resistant forms in final products or food processing equipment. In order to identify sporulation environments in the food industry, it is necessary to be able to predict bacterial sporulation according to environmental factors. As sporulation occurs after bacterial growth, a kinetic model of growth-sporulation was used to describe the evolution of vegetative cells and spores through time. The effects of temperature, pH and water activity on the growth and the sporulation abilities of Bacillus subtilis BSB1 were modelled. The values of the growth boundaries were used as inputs to predict these effects. The good description of the sporulation kinetics by growth parameters suggests that the impact of the studied environmental factors is the same on both physiological process. Suboptimal conditions for growth delay the appearance of the first spores, and spores appear more synchronously in suboptimal conditions for growth. The developed model was also applicable to describe the growth and sporulation curves in changing temperature and pH conditions over time.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Microbiologia de Alimentos , Esporos Bacterianos/crescimento & desenvolvimento , Temperatura , Água/química , Manipulação de Alimentos , Concentração de Íons de Hidrogênio , Cinética
3.
Proc Natl Acad Sci U S A ; 117(25): 14444-14452, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513721

RESUMO

Chemical-induced spores of the Gram-negative bacterium Myxococcus xanthus are peptidoglycan (PG)-deficient. It is unclear how these spherical spores germinate into rod-shaped, walled cells without preexisting PG templates. We found that germinating spores first synthesize PG randomly on spherical surfaces. MglB, a GTPase-activating protein, forms a cluster that responds to the status of PG growth and stabilizes at one future cell pole. Following MglB, the Ras family GTPase MglA localizes to the second pole. MglA directs molecular motors to transport the bacterial actin homolog MreB and the Rod PG synthesis complexes away from poles. The Rod system establishes rod shape de novo by elongating PG at nonpolar regions. Thus, similar to eukaryotic cells, the interactions between GTPase, cytoskeletons, and molecular motors initiate spontaneous polarization in bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Myxococcus xanthus/citologia , Peptidoglicano/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Polaridade Celular , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Microscopia Eletrônica , Morfogênese , Myxococcus xanthus/crescimento & desenvolvimento , Myxococcus xanthus/metabolismo , Myxococcus xanthus/ultraestrutura , Peptidoglicano/genética , Esporos Bacterianos/metabolismo , Esporos Bacterianos/ultraestrutura
4.
Can J Microbiol ; 66(11): 664-669, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32497447

RESUMO

Bacillus subtilis and Bacillus velezensis are used in organic agriculture as an alternative to chemical pesticides to fight against phytopathogen organisms. These Gram-positive soil-dwelling bacteria are able to resist harsh conditions and survive by differentiating into endospores. Few studies have examined how bacterial populations change on plants over time, and whether they remain active or enter a dormant state. Nonetheless, these characteristics are strikingly important to determine the usage of B. subtilis and B. velezensis and their efficacy in environmental conditions. Here, we investigated the population dynamics of B. subtilis NCIB3610 and B. velezensis QST713 when applied as spores on different ornamental plants. We report that on all the plants studied (Echinacea purpurea 'Salsa red', Echinacea purpurea 'Fatal attraction', and Lavandula angustifolia 'Hidecote blue'), spores rapidly germinated and colonized the rhizoplane, maintaining a relatively low proportion of spores in the population over time, whereas the bacterial population on the leaves rapidly declined. Bacteria in the surrounding soil did not germinate and persisted as spores. Taken together, these results suggest that only cells found at the rhizosphere remain metabolically active to allow the formation of a lasting relationship with the plant, making possible beneficial effects from the inoculated bacteria.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Bacillus/crescimento & desenvolvimento , Plantas/microbiologia , Contagem de Colônia Microbiana , Folhas de Planta/microbiologia , Rizosfera , Esporos Bacterianos/crescimento & desenvolvimento
5.
Int J Food Microbiol ; 325: 108629, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32325344

RESUMO

High pressure processing is a mild preservation process that inactivates pathogenic and spoilage micro-organisms in food products, but preserves the fresh characteristics of a product. Compared to untreated product, an enhanced shelf life is obtained during refrigerated storage. Knowledge on the use of high pressure pasteurisation aimed for ambient storage is limited. The aim of this research was to investigate if a combination of high pressure and moderate heat could be used to produce a shelf-stable high-acid fruit product. Ascospores of the heat resistant fungi Talaromyces macrosporus and Aspergillus fischeri were added to fresh strawberry puree that served as a model system. The effect of the processing steps and storage at ambient temperature for 2 weeks was studied on viability of the ascospores. A preheating step at 69 °C/2 min resulted in full or partial activation of A. fischeri and T. macrosporus spores, respectively. The pressure build-up by the process without any holding time resulted in additional activation of spores. A combination of moderate heat (maximum 85-90 °C) and high pressure (500-700 MPa) for holding times up to 13 min inactivated these highly resistant spores much faster than a heat treatment alone. At Tmax = 85 °C and 600 MPa the spores of T. macrosporus and A. fischeri were inactivated by 5.0 and 5.5 log10 after 13 and 7 min, respectively. At Tmax = 85 °C the heat treatment alone did not reduce the viability of these spores up to 60 min of treatment. At Tmax = 90 °C the holding time of the combined pressure-heat treatment could be reduced to obtain the same degree of inactivation of the heat resistant fungi. In addition, treated and untreated ascospores in strawberry puree were stored for 14 days at room temperature to evaluate delayed outgrowth of spores. Untreated ascospores of A. fischeri were activated by storage in the puree. However, at conditions combining high pressure ≥ 600 MPa with Tmax ≥ 85 °C for 13 min, heat resistant fungi were successfully inactivated. This research showed that a combination of moderate heat and pressure can drastically improve the effectiveness to inactivate heat-resistant ascospores in a high-acid fruit product compared to a heat treatment, potentially resulting in a better product quality.


Assuntos
Aspergillus/crescimento & desenvolvimento , Pasteurização/métodos , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Fúngicos/crescimento & desenvolvimento , Talaromyces/crescimento & desenvolvimento , Microbiologia de Alimentos , Fragaria/microbiologia , Frutas/microbiologia , Temperatura Alta
6.
Nucleic Acids Res ; 48(9): 4902-4914, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32313947

RESUMO

Reversible lysine acetylation plays regulatory roles in diverse biological processes, including cell metabolism, gene transcription, cell apoptosis and ageing. Here, we show that lysine acetylation is involved in the regulation of chromosome segregation, a pivotal step during cell division in Streptomyces coelicolor. Specifically, deacetylation increases the DNA-binding affinity of the chromosome segregation protein ParB to the centromere-like sequence parS. Both biochemical and genetic experiments suggest that the deacetylation process is mainly modulated by a sirtuin-like deacetylase ScCobB1. The Lys-183 residue in the helix-turn-helix region of ParB is the major deacetylation site responsible for the regulation of ParB-parS binding. In-frame deletion of SccobB1 represses formation of ParB segregation complexes and leads to generation of abnormal spores. Taken together, these observations provide direct evidence that deacetylation participates in the regulation of chromosome segregation by targeting ParB in S. coelicolor.


Assuntos
Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Streptomyces coelicolor/genética , Acetilação , Proteínas de Bactérias/química , Centrômero/metabolismo , Proteínas de Ligação a DNA/química , Deleção de Genes , Lisina/metabolismo , Ligação Proteica , Sirtuínas/genética , Sirtuínas/metabolismo , Esporos Bacterianos/genética , Esporos Bacterianos/crescimento & desenvolvimento , Streptomyces coelicolor/enzimologia , Streptomyces coelicolor/metabolismo , Streptomyces coelicolor/fisiologia
7.
J Food Sci ; 85(5): 1536-1541, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32249415

RESUMO

The aim of this study was to investigate the influence of surface hydrophobicity and roughness of carrier materials on the inactivation of bacterial spores with gaseous hydrogen peroxide whereas condensate formation is prevented. Spores of Bacillus subtilis and Bacillus atrophaeus were applied either as single spore culture or as a mixed spore population to simulate natural contamination with microorganisms of different characteristics. Inactivation with gaseous hydrogen peroxide was carried out at 5200 ppm hydrogen peroxide without condensate formation. The inactivation results of B. subtilis and B. atrophaeus spores on carrier materials with varying surface hydrophobicity differed significantly. However, inactivation of the mixed spore populations resulted in similar resistance compared to the single spore batches. The results of this study indicate that surface hydrophobicity most probably has an impact on the inactivation with gaseous hydrogen peroxide whereas surface roughness only plays a minor role.


Assuntos
Bacillus subtilis/efeitos dos fármacos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Esporos Bacterianos/química , Bacillus/efeitos dos fármacos , Bacillus/crescimento & desenvolvimento , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/fisiologia , Gases/química , Gases/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Solo/química , Microbiologia do Solo , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/crescimento & desenvolvimento , Propriedades de Superfície , Volatilização
8.
Food Microbiol ; 89: 103413, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32138984

RESUMO

Clostridium perfringens is a strictly anaerobic pathogen that requires absence of oxygen for its growth in laboratory experiments, which is usually attained by using an anaerobic chamber or anaerobic jars. However, it has been demonstrated that C. perfringens may survive for short periods of times due to its adaptive response to O2. Therefore, the objective of this study was to explore the application of Oxyrase (OX) and sodium thioglycolate (ST) as oxygen scavengers, used alone or in combination, for observation of the growth of C. perfringens under aerobic incubation. The growth of C. perfringens from spores in Schaedler Anaerobe Agar containing different levels and combinations of OX and ST was observed at temperatures between 20 and 50 °C. The kinetic parameters, including lag time, specific growth rate, and maximum cell concentrations in the stationary phase, were determined. The results indicated that ST at concentrations of 0.025 and 0.05% (w/w), although allowing eventual growth of C. perfringens, prolonged its lag times, while OX at 1.5% only allowed growth at a lower growth rate in comparison to anaerobic incubation. OX at 3% enhanced the growth of C. perfringens at temperatures between 30 and 50 °C, while higher levels of OX were needed in the medium to support the growth of C. perfringens during storage at 25 °C (>6% OX) and 20 °C (>9% OX), due to the effect of temperature on enzyme activity. No significant difference was found in the kinetic parameters of C. perfringens incubated aerobically with OX and the control (without OX or ST) in an anaerobic chamber. Therefore, OX at appropriate concentrations may allow the observation of the growth of C. perfringens under aerobic incubation conditions without the need of an anaerobic device.


Assuntos
Clostridium perfringens/crescimento & desenvolvimento , Inocuidade dos Alimentos , Oxigenases/farmacologia , Esporos Bacterianos/crescimento & desenvolvimento , Tioglicolatos/farmacologia , Clostridium perfringens/efeitos dos fármacos , Esporos Bacterianos/efeitos dos fármacos , Temperatura
9.
Lett Appl Microbiol ; 70(6): 407-412, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32133659

RESUMO

Inactivation of Bacillales and Clostridiales spores is of interest, since some cause food spoilage and human diseases. A recent publication (mSphere 3: e00597-1, 2018) reported that glycerol monolaurate (GML) in a non-aqueous gel (GMLg) effectively killed spores of Bacillus subtilis, Bacillus cereus and Clostridioides difficile, and Bacillus anthracis spores to a lesser extent. We now show that (i) the B. subtilis spores prepared as in the prior work were impure; (ii) if spore viability was measured by diluting spores 1/10 in GMLg, serially diluting incubations 10-fold and spotting aliquots on recovery plates, there was no colony formation from the 1/10 to 1/1000 dilutions due to GMLg carryover, although thorough ethanol washes of incubated spores eliminated this problem and (iii) GMLg did not kill highly purified spores of B. subtilis, B. cereus, Bacillus megaterium and C. difficile in 3-20 h in the conditions used in the recent publication. GMLg also gave no killing of crude B. subtilis spores prepared as in the recent publication in 5 h but gave ~1·5 log killing at 24 h. Thus, GMLg does not appear to be an effective sporicide, although the gel likely inhibits spore germination and could kill spores somewhat upon long incubations. SIGNIFICANCE AND IMPACT OF THE STUDY: Given potential deleterious effects of spores of Bacillales and Clostridiales, there is an ongoing interest in new ways of spore killing. A recent paper (mSphere 3: e00597-1, 2018) reported that glycerol monolaurate (GML) in a non-aqueous gel (GMLg) effectively killed spores of many species. We now find that (i) the Bacillus subtilis spores prepared as in the previous report were impure and (ii) GMLg gave no killing of purified spores of Bacillales and Clostridiales species in ≤5 h under the published conditions. Thus, GMLg is not an effective sporicide, though may prevent spore germination or kill germinated spores.


Assuntos
Antibacterianos/farmacologia , Bacillales/efeitos dos fármacos , Clostridiales/efeitos dos fármacos , Lauratos/farmacologia , Monoglicerídeos/farmacologia , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/crescimento & desenvolvimento , Bacillales/crescimento & desenvolvimento , Bacillus cereus/efeitos dos fármacos , Bacillus megaterium/efeitos dos fármacos , Bacillus subtilis/efeitos dos fármacos , Clostridiales/crescimento & desenvolvimento , Microbiologia de Alimentos , Géis/farmacologia
10.
Food Microbiol ; 87: 103353, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31948638

RESUMO

Bacillus subtilis spore inactivation mechanisms under low energy electron beam (LEEB) and high energy electron beam (HEEB) treatment were investigated using seven mutants lacking specific DNA repair mechanisms. The results showed that most of the DNA repair-deficient mutants, including ΔrecA, ΔKu ΔligD, Δexo Δnfo, ΔuvrAB and ΔsbcDC, had reduced resistances towards electron beam (EB) treatments at all investigated energy levels (80 keV, 200 keV and 10 MeV) compared to their wild type. This result suggested DNA damage was induced during EB treatments. The mutant lacking recA showed the lowest resistance, followed by the mutant lacking Ku and ligD. These findings indicated that recA, Ku and ligD and their associated DNA repair mechanisms, namely, homologous recombination and non-homologous end joining, play important roles in spore survival under EB treatment. Furthermore, exoA, nfo, uvrAB, splB, polY1 and polY2, which are involved in nucleotide damage repair/removal, showed different levels of effects on spore resistance under EB treatment. Finally, the results suggested that HEEB and LEEB inactivate B. subtilis spores through similar mechanisms. This research will provide a better understanding of how EB technologies inactivate B. subtilis spores and will contribute to the application of these technologies as a non-thermal, gentle spore control approach.


Assuntos
Bacillus subtilis/genética , Reparo do DNA , Esporos Bacterianos/efeitos da radiação , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/efeitos da radiação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dano ao DNA/efeitos da radiação , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Elétrons , Viabilidade Microbiana/efeitos da radiação , Mutação , Esporos Bacterianos/genética , Esporos Bacterianos/crescimento & desenvolvimento
11.
Syst Appl Microbiol ; 43(2): 126055, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31992497

RESUMO

Fifteen enterobacterial strains were isolated from fresh produce. The 16S rRNA gene sequences indicated that these belong to Serratia, with twelve strains showing 99.57%-99.93% and three strains showing 99.86-100% 16S rRNA gene sequence similarity with Serratia marcescens and Serratia nematodiphila as nearest neighbors, respectively. Further comparative multi locus sequence analyses, as well as phylogenomic comparisons, revealed that 6 of the 15 strains were well-separated from their nearest neighbors and formed two clearly distinct taxa. Strains S2, S9, S10 and S15T were urease-positive, while strains S3T and S13 were urease-negative. Average nucleotide identity and digital DNA-DNA hybridization comparisons of representative strains S3T and S15T with type strains of S. marcescens, S. nematodiphila and S. ureilytica indicated that these shared less than 96% and 70% homology, respectively. Major fatty acids of strains S3T and S15T included C16:0, C16:1 ω7c/C16:1 ω6c, C17:0 Cyclo and C18:1 ω6c /C18:1 ω7c. The mol% G+C of genomic DNA of strain S15T was 59.49% and of strain S3T was 59.04. These results support the description of two novel species, Serratia nevei and Serratia bockelmannii, with strains S15T (=LMG 31536T =DSM 110085T) and S3T (=LMG 31535T =DSM 110152T) as type strains, respectively. Although Serratia marcescens subsp. sakuensis was previously described to form spores, spores could not be determined in this study. As spore formation was the only differential characteristic of this subspecies, S. marcescens subsp. sakuensis is a later heterotypic synonym of Serratia marcescens.


Assuntos
Microbiologia de Alimentos , Filogenia , Serratia/classificação , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Ácidos Graxos/análise , Genoma Bacteriano/genética , Genótipo , Alemanha , Hibridização de Ácido Nucleico , Fenótipo , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Serratia/química , Serratia/genética , Serratia/isolamento & purificação , Esporos Bacterianos/citologia , Esporos Bacterianos/crescimento & desenvolvimento
12.
PLoS One ; 15(1): e0228270, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978128

RESUMO

Bacillus anthracis is the causative agent of anthrax in animals and humans. The organism lies in a dormant state in the soil until introduced into an animal via, ingestion, cutaneous inoculation or inhalation. Once in the host, spores germinate into rapidly growing vegetative cells elaborating toxins. When animals die of anthrax, vegetative bacteria sporulate upon nutrient limitation in the carcass or soil while in the presence of air. After release into the soil environment, spores form a localized infectious zone (LIZ) at and around the carcass. Laboratory strains of B. anthracis produce fewer proteins associated with growth and sporulation compared to wild strains isolated from recent zoonotic disease events. We verified wild strains grow more rapidly than lab strains demonstrating a greater responsiveness to nutrient availability. Sporulation was significantly more rapid in these wild strains compared to lab strains, indicating wild strains are able to sporulate faster due to nutrient limitation while laboratory strains have a decrease in the speed at which they utilize nutrients and an increase in time to sporulation. These findings have implications for disease control at the LIZ as well as on the infectious cycle of this dangerous zoonotic pathogen.


Assuntos
Bacillus anthracis/fisiologia , Esporos Bacterianos/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Laboratórios , Microbiologia do Solo , Especificidade da Espécie
13.
Appl Environ Microbiol ; 86(6)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31900309

RESUMO

Changes with time of a population of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 dormant spores into germinated spores and vegetative cells were followed by flow cytometry, at pH ranges of 4.7 to 7.4 and temperatures of 10°C to 37°C for B. weihenstephanensis and 18°C to 59°C for B. licheniformis Incubation conditions lower than optimal temperatures or pH led to lower proportions of dormant spores able to germinate and extended time of germination, a lower proportion of germinated spores able to outgrow, an extension of their times of outgrowth, and an increase of the heterogeneity of spore outgrowth time. A model based on the strain growth limits was proposed to quantify the impact of incubation temperature and pH on the passage through each physiological stage. The heat treatment temperature or time acted independently on spore recovery. Indeed, a treatment at 85°C for 12 min or at 95°C for 2 min did not have the same impact on spore germination and outgrowth kinetics of B. weihenstephanensis despite the fact that they both led to a 10-fold reduction of the population. Moreover, acidic sporulation pH increased the time of outgrowth 1.2-fold and lowered the proportion of spores able to germinate and outgrow 1.4-fold. Interestingly, we showed by proteomic analysis that some proteins involved in germination and outgrowth were detected at a lower abundance in spores produced at pH 5.5 than in those produced at pH 7.0, maybe at the origin of germination and outgrowth behavior of spores produced at suboptimal pH.IMPORTANCE Sporulation and incubation conditions have an impact on the numbers of spores able to recover after exposure to sublethal heat treatment. Using flow cytometry, we were able to follow at a single-cell level the changes in the physiological states of heat-stressed spores of Bacillus spp. and to discriminate between dormant spores, germinated spores, and outgrowing vegetative cells. We developed original mathematical models that describe (i) the changes with time of the proportion of cells in their different states during germination and outgrowth and (ii) the influence of temperature and pH on the kinetics of spore recovery using the growth limits of the tested strains as model parameters. We think that these models better predict spore recovery after a sublethal heat treatment, a common situation in food processing and a concern for food preservation and safety.


Assuntos
Bacillus licheniformis/crescimento & desenvolvimento , Bacillus/crescimento & desenvolvimento , Esporos Bacterianos/crescimento & desenvolvimento , Temperatura Alta , Modelos Teóricos
14.
Food Microbiol ; 85: 103285, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31500704

RESUMO

The objective of this study was to determine the kinetic parameters and apply Markov Chain Monte Carlo (MCMC) simulation to predict the growth of Clostridium perfringens from spores in cooked ground chicken meat during dynamic cooling. Inoculated samples were exposed to various cooling conditions to observe dynamic growth. A combination of 4 cooling profiles was used in one-step inverse analysis with the Baranyi model as the primary model and the cardinal parameters model as the secondary model. Six kinetic parameters of the Baranyi model and the cardinal parameters model, including Q0, Ymax, µopt, Tmin, Topt, and Tmax, were estimated. The estimated Tmin, Topt, and Tmax were 14.8, 42.9, and 50.5 °C, respectively, with a µopt of 5.25 h-1 and maximum cell density of 8.4 log CFU/g. Correlation analysis showed that both Q0 and Ymax are weakly correlated to other parameters, while the remaining parameters are mostly mildly to strongly correlated with each other. Although it may be difficult to estimate highly correlated parameters using a single temperature profile, one-step analysis with multiple different temperature profiles helped estimate them successfully. The estimated parameters were used as the prior information to construct the posterior distribution for Bayesian analysis. MCMC simulation was used to predict the bacterial growth using different dynamic temperature profiles for validation of the accuracy of the predictive models. The MCMC simulation results showed that the Bayesian analysis produced more accurate predictions of bacterial growth during cooling than the deterministic method. With Bayesian analysis, the root-mean-square-error (RMSE) of prediction was only 0.1 log CFU/g with all residual errors within ±0.25 log CFU/g. Therefore, Bayesian analysis is recommended for predicting the growth of C. perfringens in cooked meat during cooling.


Assuntos
Clostridium perfringens/crescimento & desenvolvimento , Culinária , Manipulação de Alimentos , Produtos da Carne/microbiologia , Temperatura , Animais , Teorema de Bayes , Galinhas , Contagem de Colônia Microbiana , Simulação por Computador , Cinética , Cadeias de Markov , Modelos Biológicos , Método de Monte Carlo , Esporos Bacterianos/crescimento & desenvolvimento
15.
Food Microbiol ; 86: 103325, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31703860

RESUMO

Clostridium perfringens SM101 genome encodes three serine proteases (CspA, CspB, and CspC), and genetic evidence indicates that CspB is required for processing of pro-SleC into active SleC, an enzyme essential for degradation of the peptidoglycan cortex during spore germination. In this study, the expression of cspA and cspC, as well as the germination and colony formation by spores of cspAC and cspC mutants of strain SM101, were assessed. We demonstrated that 1) the cspA and cspC genes were expressed as a bicistronic operon only during sporulation in the mother cell compartment of SM101; 2) both cspAC and cspC mutant spores were unable to germinate significantly with either KCl, l-glutamine, brain heart infusion (BHI) broth, or a 1:1 chelate of Ca2+ and dipicolinic acid (DPA); 3) consistent with germination results, both cspAC and cspC mutant spores were defective in normal DPA release; 4) the colony formation by cspAC and cspC mutant spores was ~106-fold lower than that of wild-type spores, although decoated mutant spores yielded wild-type level colony formation on plates containing lysozyme; 5) no processing of inactive pro-SleC into active SleC was observed in cspAC and cspC mutant spores during germination; and finally, 6) the defects in germination, DPA release, colony formation and SleC processing in cspAC and cspC mutant spores were complemented by the wild-type cspA-cspC operon. Collectively, these results indicate that both CspA and CspC are essential for C. perfringens spore germination through activating SleC and inducing cortex hydrolysis.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Clostridium perfringens/enzimologia , Esporos Bacterianos/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Clostridium perfringens/efeitos dos fármacos , Clostridium perfringens/genética , Clostridium perfringens/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hidrólise , Óperon , Ácidos Picolínicos/farmacologia , Processamento de Proteína Pós-Traducional , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/enzimologia , Esporos Bacterianos/genética
16.
Environ Microbiol ; 22(1): 170-182, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31713316

RESUMO

Bacterial spores are commonly isolated from a variety of different environments, including extreme habitats. Although it is well established that such ubiquitous distribution reflects the spore resistance properties, it is not clear whether the growing conditions affect the spore structure and function. We used Bacillus subtilis spores of similar age but produced at 25, 37, or 42°C to compare their surface structures and functional properties. Spores produced at the 25°C were more hydrophobic while those produced at 42°C contained more dipicolinic acid, and were more resistant to heat or lysozyme treatments. Electron microscopy analysis showed that while 25°C spores had a coat with a compact outer coat, not tightly attached to the inner coat, 42°C spores had a granular, not compact outer coat, reminiscent of the coat produced at 37°C by mutant spores lacking the protein CotG. Indeed, CotH and a series of CotH-dependent coat proteins including CotG were more abundantly extracted from the coat of 25 or 37°C than 42°C spores. Our data indicated that CotH is a heat-labile protein with a major regulatory role on coat formation when sporulation occurs at low temperatures, suggesting that B. subtilis builds structurally and functionally different spores in response to the external conditions.


Assuntos
Bacillus subtilis/fisiologia , Esporos Bacterianos/crescimento & desenvolvimento , Temperatura , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/metabolismo , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Muramidase , Ácidos Picolínicos/análise , Esporos Bacterianos/química , Esporos Bacterianos/metabolismo , Esporos Bacterianos/ultraestrutura
17.
Appl Environ Microbiol ; 86(4)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31811041

RESUMO

Clostridioides difficile is a pathogenic bacterium that infects the human colon to cause diarrheal disease. Growth of the bacterium is known to be dependent on certain bile acids, oxygen levels, and nutrient availability in the intestine, but how the environmental pH can influence C. difficile is mostly unknown. Previous studies indicated that C. difficile modulates the intestinal pH, and prospective cohort studies have found a strong association between a more alkaline fecal pH and C. difficile infection. Based on these data, we hypothesized that C. difficile physiology can be affected by various pH conditions. In this study, we investigated the impact of a range of pH conditions on C. difficile to assess potential effects on growth, sporulation, motility, and toxin production in the strains 630Δerm and R20291. We observed pH-dependent differences in sporulation rate, spore morphology, and viability. Sporulation frequency was lowest under acidic conditions, and differences in cell morphology were apparent at low pH. In alkaline environments, C. difficile sporulation was greater for strain 630Δerm, whereas R20291 produced relatively high levels of spores in a broad range of pH conditions. Rapid changes in pH during exponential growth impacted sporulation similarly among the strains. Furthermore, we observed an increase in C. difficile motility with increases in pH, and strain-dependent differences in toxin production under acidic conditions. The data demonstrate that pH is an important parameter that affects C. difficile physiology and may reveal relevant insights into the growth and dissemination of this pathogen.IMPORTANCE Clostridioides difficile is an anaerobic bacterium that causes gastrointestinal disease. C. difficile forms dormant spores which can survive harsh environmental conditions, allowing their spread to new hosts. In this study, we determine how intestinally relevant pH conditions impact C. difficile physiology in the two divergent strains, 630Δerm and R20291. Our data demonstrate that low pH conditions reduce C. difficile growth, sporulation, and motility. However, toxin production and spore morphology were differentially impacted in the two strains at low pH. In addition, we observed that alkaline environments reduce C. difficile growth, but increase cell motility. When pH was adjusted rapidly during growth, we observed similar impacts on both strains. This study provides new insights into the phenotypic diversity of C. difficile grown under diverse pH conditions present in the intestinal tract, and demonstrates similarities and differences in the pH responses of different C. difficile isolates.


Assuntos
Toxinas Bacterianas/biossíntese , Esporos Bacterianos/crescimento & desenvolvimento , /crescimento & desenvolvimento , Concentração de Íons de Hidrogênio
18.
Nat Chem Biol ; 16(2): 126-133, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31792444

RESUMO

Materials can be made multifunctional by embedding them with living cells that perform sensing, synthesis, energy production, and physical movement. A challenge is that the conditions needed for living cells are not conducive to materials processing and require continuous water and nutrients. Here, we present a three dimensional (3D) printer that can mix material and cell streams to build 3D objects. Bacillus subtilis spores were printed within the material and germinated on its exterior surface, including spontaneously in new cracks. The material was resilient to extreme stresses, including desiccation, solvents, osmolarity, pH, ultraviolet light, and γ-radiation. Genetic engineering enabled the bacteria to respond to stimuli or produce chemicals on demand. As a demonstration, we printed custom-shaped hydrogels containing bacteria that can sense or kill Staphylococcus aureus, a causative agent of infections. This work demonstrates materials endued with living functions that can be used in applications that require storage or exposure to environmental stresses.


Assuntos
Bacillus subtilis , Impressão Tridimensional , Esporos Bacterianos , Ferimentos e Lesões/microbiologia , Antibacterianos/metabolismo , Bacillus subtilis/genética , Fenômenos Fisiológicos Bacterianos , Desenho de Equipamento , Escherichia coli , Concentração de Íons de Hidrogênio , Teste de Materiais , Microrganismos Geneticamente Modificados , Impressão Tridimensional/instrumentação , Percepção de Quorum , Sefarose/química , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/fisiologia , Staphylococcus aureus , Estresse Fisiológico , Temperatura , Ácido Vanílico/análise
19.
Environ Microbiol ; 22(1): 456-465, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31742859

RESUMO

Thermophilic endospores are widespread in cold marine sediments where the temperature is too low to support growth and activity of thermophiles in situ. These endospores are likely expelled from warm subsurface environments and subsequently dispersed by ocean currents. The endospore upper temperature limit for survival is 140°C, which can be tolerated in repeated short exposures, potentially enabling transit through hot crustal fluids. Longer-term thermal tolerance of endospores, and how long they could persist in an environment hotter than their maximum growth temperature, is less understood. To test whether thermophilic endospores can survive prolonged exposure to high temperatures, sediments were incubated at 80-90°C for 6, 12 or 463 days. Sediments were then cooled by 10-40°C, mimicking the cooling in subsurface oil reservoirs subjected to seawater injection. Cooling the sediments induced sulfate reduction, coinciding with an enrichment of endospore-forming Clostridia. Different Desulfofundulus, Desulfohalotomaculum, Desulfallas, Desulfotomaculum and Desulfofarcimen demonstrated different thermal tolerances, with some Desulfofundulus strains surviving for >1 year at 80°C. In an oil reservoir context, heat-resistant endospore-forming sulfate-reducing bacteria have a survival advantage if they are introduced to, or are resident in, an oil reservoir normally too hot for germination and growth, explaining observations of reservoir souring following cold seawater injection.


Assuntos
Clostridiaceae/metabolismo , Sedimentos Geológicos/microbiologia , Peptococcaceae/metabolismo , Água do Mar/microbiologia , Sulfatos/metabolismo , Archaea , Clostridiaceae/classificação , Clostridiaceae/genética , Temperatura Baixa , Temperatura Alta , Oxirredução , Peptococcaceae/classificação , Peptococcaceae/genética , Filogenia , Esporos Bacterianos/genética , Esporos Bacterianos/crescimento & desenvolvimento
20.
J Appl Microbiol ; 128(1): 124-137, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31573710

RESUMO

AIMS: To add a spore germination step in order to reduce decontamination temperature and time requirements compared to the current hot, humid air decontamination parameters, which are 75-80°C, ≥72 h, 70-90% RH, down to ≤60°C and ≤24 h total decontamination time. METHODS AND RESULTS: Bacillus anthracis spore germination with l-alanine+inosine+calcium dipicolinate (CaDPA) was quantified at 0-40°C, several time points and spore concentrations of 5-9 log10 per ml. Germination efficiency at 0-40°C was >99% at <8 log10 spores per ml. The temperature optimum was 20°C. Germination efficiency was significantly higher but slower at 0°C compared to ≥30°C at ≥8 log10 spores per ml. A single germinant application followed by 60°C, 1-h treatment consistently inactivated >2 log10 (>99%) of spores. However, a repeat application of germinant was needed to achieve the objective of ≥6 log10 spore inactivation out of a 7 log10 challenge (≥99·9999%) for ≤24 h total decontamination time for nylon and aircraft performance coating. CONCLUSIONS: l-alanine+inosine+CaDPA stimulated germination across wide temperature and spore concentration ranges. SIGNIFICANCE AND IMPACT OF THE STUDY: Germination expands the scope of spore decontamination to include materials from any industry sector that can be sprayed with an aqueous germinant solution.


Assuntos
Bacillus anthracis/fisiologia , Descontaminação/métodos , Esporos Bacterianos/fisiologia , Alanina/farmacologia , Bacillus anthracis/efeitos dos fármacos , Bacillus anthracis/crescimento & desenvolvimento , Temperatura Alta , Inosina/farmacologia , Ácidos Picolínicos/farmacologia , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/crescimento & desenvolvimento , Fatores de Tempo
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