Germination Of Bacterial Spores Research Articles

Occurrence of spore-forming bacterial in brazilian dairy desserts

Dairy desserts are commercially sold refrigerated products that are widely consumed due to their nutritional characteristics, practicality, and sensory appeal. Their deterioration is related to changes in texture, odor, and the production of off-flavors caused by spore-forming bacteria and other contaminating bacteria that produce heat-stable spoilage enzymes. This study aimed to determine the presence of spore-forming bacteria in three types of dairy desserts, which were subjected to thermal shock to induce the germination of bacterial spores. The ability of vegetative cells to grow under different conditions, as well as the synthesis of proteolytic enzymes and their multiplication rates, were evaluated. The results indicated a higher occurrence of mesophilic aerobic bacteria with higher proteolytic and lipolytic activity, and a higher growth rate. Twenty isolates showed statistically significant differences (P<0.05), exhibiting superior capacity for synthesis of enzymes and growth rate. These isolates were genetically identified as B. subtilis, B. cereus, B. thuringiensis, B. tequilensis and B. parabrevis. These results reinforce the necessity for control measures against spore-forming aerobic mesophilic or thermoduric bacteria to ensure quality. Therefore, production losses, reduced shelf life, damage to brand reputation, and sales losses continue to be challenge for the Brazilian dairy industry.

Morphotextural, microbiological, and volatile characterization of flatbread containing cricket (Acheta domesticus) powder and buckwheat (Fagopyrum esculentum) flour

The growing awareness of the consumers on the advantages of a proper nutrition is deeply modifying their demands. Hence, the exploitation of innovative ingredients to enrich the nutritional values of staple foods is continuously explored by research institutions and food industries. This paper represents a feasibility study on the use of nonconventional ingredients, including house cricket (Acheta domesticus) powder and buckwheat (Fagopyrum esculentum) flour, for the production of novel flatbread formulations. Experimental flatbread prototypes were evaluated by analyzing microbiological, physico-chemical, textural, colorimetric, and volatile parameters. Microbiological viable counts revealed low levels of bacterial spores in the formulations comprising cricket powder. Water activity results showed adequate values, inhibiting the growth of spoilage and pathogenic bacteria, and preventing the germination of bacterial spores. The addition of cricket powder, influenced textural properties of flatbread samples, characterized by lower hardness values respect to those not containing insects seems likely due to a high content of dietary fiber (chitin from insects). As for the color analysis, flatbread samples added with cricket powder evidenced darker tones respect to those not containing insects, thus resulting visibly comparable with whole grain products. Worthy to mention that the addition of buckwheat flours did not cause hardness reduction or color variation of experimental prototypes. The volatile component analysis highlighted numerous compounds associated with enzymatic activities and nonconventional ingredients. Overall, the results collected demonstrated that cricket powder and buckwheat flour possess a great potential to produce innovative flatbreads.

The underlying mechanism of bacterial spore germination: An update review.

Bacterial spores are highly resilient and universally present on earth and can irreversibly enter the food chain to cause food spoilage or foodborne illness once revived to resume vegetative growth. Traditionally, extensive thermal processing has been employed to efficiently kill spores; however, the relatively high thermal load adversely affects food quality attributes. In recent years, the germination-inactivation strategy has been developed to mildly kill spores based on the circumstance that germination can decrease spore-resilient properties. However, the failure to induce all spores to geminate, mainly owing to the heterogeneous germination behavior of spores, hampers the success of applying this strategy in the food industry. Undoubtedly, elucidating the detailed germination pathway and underlying mechanism can fill the gap in our understanding of germination heterogeneity, thereby facilitating the development of full-scale germination regimes to mildly kill spores. In this review, we comprehensively discuss the mechanisms of spore germination of Bacillus and Clostridium species, and update the molecular basis of the early germination events, for example, the activation of germination receptors, ion release, Ca-DPA release, and molecular events, combined with the latest research evidence. Moreover, high hydrostatic pressure (HHP), an advanced non-thermal food processing technology, can also trigger spore germination, providing a basis for the application of a germination-inactivation strategy in HHP processing. Here, we also summarize the diverse germination behaviors and mechanisms of spores of Bacillus and Clostridium species under HHP, with the aim of facilitating HHP as a mild processing technology with possible applications in food sterilization. Practical Application: This work provides fundamental basis for developing efficient killing strategies of bacterial spores in food industry.

Bacterial spore germination receptors are nutrient-gated ion channels.

Bacterial spores resist antibiotics and sterilization and can remain metabolically inactive for decades, but they can rapidly germinate and resume growth in response to nutrients. Broadly conserved receptors embedded in the spore membrane detect nutrients, but how spores transduce these signals remains unclear. Here, we found that these receptors form oligomeric membrane channels. Mutations predicted to widen the channel initiated germination in the absence of nutrients, whereas those that narrow it prevented ion release and germination in response to nutrients. Expressing receptors with widened channels during vegetative growth caused loss of membrane potential and cell death, whereas the addition of germinants to cells expressing wild-type receptors triggered membrane depolarization. Therefore, germinant receptors act as nutrient-gated ion channels such that ion release initiates exit from dormancy.

Identification and characterization of new proteins crucial for bacterial spore resistance and germination.

2Duf, named after the presence of a transmembrane (TM) Duf421 domain and a small Duf1657 domain in its sequence, is likely located in the inner membrane (IM) of spores in some Bacillus species carrying a transposon with an operon termed spoVA 2mob. These spores are known for their extreme resistance to wet heat, and 2Duf is believed to be the primary contributor to this trait. In this study, we found that the absence of YetF or YdfS, both Duf421 domain-containing proteins and found only in wild-type (wt) B. subtilis spores with YetF more abundant, leads to decreased resistance to wet heat and agents that can damage spore core components. The IM phospholipid compositions and core water and calcium-dipicolinic acid levels of YetF-deficient spores are similar to those of wt spores, but the deficiency could be restored by ectopic insertion of yetF, and overexpression of YetF increased wt spore resistance to wet heat. In addition, yetF and ydfS spores have decreased germination rates as individuals and populations with germinant receptor-dependent germinants and increased sensitivity to wet heat during germination, potentially due to damage to IM proteins. These data are consistent with a model in which YetF, YdfS and their homologs modify IM structure to reduce IM permeability and stabilize IM proteins against wet heat damage. Multiple yetF homologs are also present in other spore forming Bacilli and Clostridia, and even some asporogenous Firmicutes, but fewer in asporogenous species. The crystal structure of a YetF tetramer lacking the TM helices has been reported and features two distinct globular subdomains in each monomer. Sequence alignment and structure prediction suggest this fold is likely shared by other Duf421-containing proteins, including 2Duf. We have also identified naturally occurring 2duf homologs in some Bacilli and Clostridia species and in wt Bacillus cereus spores, but not in wt B. subtilis. Notably, the genomic organization around the 2duf gene in most of these species is similar to that in spoVA 2mob, suggesting that one of these species was the source of the genes on this operon in the extremely wet heat resistant spore formers.

Monitoring bacterial spore metabolic activity using heavy water-induced Raman peak evolution.

Endospore-forming bacteria are associated with food spoilage, food poisoning, and infection in hospitals. Therefore, methods to monitor spore metabolic activity and verify sterilization are of great interest. However, current methods for tracking metabolic activity are time-consuming and resource intensive. This work investigates isotope labeling and Raman microscopy as a low-cost rapid alternative. Specifically, we monitor the Raman spectrum of enterotoxic B. cereus spores undergoing germination and cell division in D2O-infused broth. During germination and cell division, water is metabolized and deuterium from the broth is incorporated into proteins and lipids, resulting in the appearance of a Raman peak related to C-D bonds at 2190 cm-1. We find that a significant C-D peak appears after 2 h of incubation at 37 °C. Further, we found that the peak appearance coincides with the observed first cell division indicating little metabolic activity during germination. Lastly, the germination and cell growth rate of spores were not affected by adding 30% heavy water to the broth. This shows the potential for real-time monitoring of metabolic activity from a bacterial spore to a dividing cell. In conclusion, our work proposes tracking the evolution of the C-D Raman peak in spores incubated with D2O-infused broth as an effective and time-, and cost-efficient method to monitor the outgrowth of a spore population, simultaneously allowing us to track for how long the bacteria have grown and divided.

Characterization of Putative Sporulation and Germination Genes in Clostridium perfringens Food-Poisoning Strain SM101.

Bacterial sporulation and spore germination are two intriguing processes that involve the expression of many genes coherently. Phylogenetic analyses revealed gene conservation among spore-forming Firmicutes, especially in Bacilli and Clostridia. In this study, by homology search, we found Bacillus subtilis sporulation gene homologs of bkdR, ylmC, ylxY, ylzA, ytaF, ytxC, yyaC1, and yyaC2 in Clostridium perfringenes food-poisoning Type F strain SM101. The β-glucuronidase reporter assay revealed that promoters of six out of eight tested genes (i.e., bkdR, ylmC, ytaF, ytxC, yyaC1, and yyaC2) were expressed only during sporulation, but not vegetative growth, suggesting that these genes are sporulation-specific. Gene knock-out studies demonstrated that C. perfringens ΔbkdR, ΔylmC, ΔytxC, and ΔyyaC1 mutant strains produced a significantly lower number of spores compared to the wild-type strain. When the spores of these six mutant strains were examined for their germination abilities in presence of known germinants, an almost wild-type level germination was observed with spores of ΔytaF or ΔyyaC1 mutants; and a slightly lower level with spores of ΔbkdR or ΔylmC mutants. In contrast, almost no germination was observed with spores of ΔytxC or ΔyyaC2 mutants. Consistent with germination defects, ΔytxC or ΔyyaC2 spores were also defective in spore outgrowth and colony formation. The germination, outgrowth, and colony formation defects of ΔytxC or ΔyyaC2 spores were restored when ΔytxC or ΔyyaC2 mutant was complemented with wild-type ytxC or yyaC2, respectively. Collectively, our current study identified new sporulation and germination genes in C. perfringens.

Reactive oxygen species generated by infrared laser light in optical tweezers inhibits the germination of bacterial spores.

Bacterial spores are highly resistant to heat, radiation and various disinfection chemicals. The impact of these on the biophysical and physicochemical properties of spores can be studied on the single-cell level using optical tweezers. However, the effect of the trapping laser on spores' germination rate is not fully understood. In this work, we assess the impact of 1064 nm laser light on the germination of Bacillus thuringiensis spores. The results show that the germination rate of spores after laser exposure follows a sigmoid dose-response relationship, with only 15% of spores germinating after 20 J of laser light. Under anaerobic growth conditions, the percentage of germinating spores at 20 J increased to 65%. The results thereby indicate that molecular oxygen is a major contributor to the germination-inhibiting effect observed. Thus, our study highlights the risk for optical trapping of spores and ways to mitigate it.

Reviving the view: evidence that macromolecule synthesis fuels bacterial spore germination.

The Gram positive bacterium Bacillus subtilis and its relatives are capable of forming a durable dormant long-lasting spore. Although spores can remain dormant for years, they possess the remarkable capacity to rapidly resume life and convert into actively growing cells. This cellular transition initiates with a most enigmatic irreversible event, termed germination, lasting only for a few minutes. Germination is typified by a morphological conversion that culminates in loss of spore resilient properties. Yet, the molecular events occurring during this brief critical phase are largely unknown. The current widely accepted view considers germination to occur without the need for any macromolecule synthesis; however, accumulating data from our laboratory and others, highlighted here, provide evidence that both transcription and translation occur during germination and are required for its execution. We further underline numerous overlooked studies, conducted mainly during the 1960s-1970s,reinforcing this notion. We propose to revisit the fascinating process of spore germination and redefine it as a pathway involving macromolecule synthesis. We expect our perspective to shed new light on the awakening process of a variety of spore-forming environmental, commensal, and pathogenic bacteria and possibly be applicable to additional organisms displaying a quiescent life form.

Antibacterial and Sporicidal Activity Evaluation of Theaflavin-3,3'-digallate.

Theaflavin-3,3′-digallate (TFDG), a polyphenol derived from the leaves of Camellia sinensis, is known to have many health benefits. In this study, the antibacterial effect of TFDG against nine bacteria and the sporicidal activities on spore-forming Bacillus spp. have been investigated. Microplate assay, colony-forming unit, BacTiter-GloTM, and Live/Dead Assays showed that 250 µg/mL TFDG was able to inhibit bacterial growth up to 99.97%, while 625 µg/mL TFDG was able to inhibit up to 99.92% of the spores from germinating after a one-hour treatment. Binding analysis revealed the favorable binding affinity of two germination-associated proteins, GPR and Lgt (GerF), to TFDG, ranging from −7.6 to −10.3 kcal/mol. Semi-quantitative RT-PCR showed that TFDG treatment lowered the expression of gpr, ranging from 0.20 to 0.39 compared to the control in both Bacillus spp. The results suggest that TFDG not only inhibits the growth of vegetative cells but also prevents the germination of bacterial spores. This report indicates that TFDG is a promising broad-spectrum antibacterial and anti-spore agent against Gram-positive, Gram-negative, acid-fast bacteria, and endospores. The potential anti-germination mechanism has also been elucidated.

Combination of multi-focus Raman spectroscopy and compressive sensing for parallel monitoring of single-cell dynamics

To overcome the low efficiency of conventional confocal Raman spectroscopy, many efforts have been devoted to parallelizing the Raman excitation and acquisition, in which the scattering from multiple foci is projected onto different locations on a spectrometer’s CCD, along either its vertical, horizontal dimension, or even both. While the latter projection scheme relieves the limitation on the row numbers of the CCD, the spectra of multiple foci are recorded in one spectral channel, resulting in spectral overlapping. Here, we developed a method under a compressive sensing framework to demultiplex the superimposed spectra of multiple cells during their dynamic processes. Unlike the previous methods which ignore the information connection between the spectra of the cells recorded at different time, the proposed method utilizes a prior that a cell’s spectra acquired at different time have the same sparsity structure in their principal components. Rather than independently demultiplexing the mixed spectra at the individual time intervals, the method demultiplexes the whole spectral sequence acquired continuously during the dynamic process. By penalizing the sparsity combined from all time intervals, the collaborative optimization of the inversion problem gave more accurate recovery results. The performances of the method were substantiated by a 1D Raman tweezers array, which monitored the germination of multiple bacterial spores. The method can be extended to the monitoring of many living cells randomly scattering on a coverslip, and has a potential to improve the throughput by a few orders.

Effect of a Monascus sp. Red Yeast Rice Extract on Germination of Bacterial Spores.

The pink-red color of traditional sausages (cured meat) is the result of nitrite addition and the formation of nitrosomyoglobin. However, the pleasant color of processed meat products is a side effect of nitrite addition while the main anticipated goal is to suppress the germination of clostridial spores. The fungus Monascus is known as a producer of oligoketide pigments, which are used in Asian countries, especially in China, for coloring foods, including meat products. Although, different biological activities of Monascus pigments have been tested and confirmed in many studies, their effect on germination of bacterial spores has never been investigated. This study is focused on testing the activity of red yeast rice (RYR) extract, containing monascin, rubropunctatin, rubropunctamine complexes and monascuspiloin as the main pigments, on germination of Clostridium and Bacillus spores. It was found that addition of nitrite alone, at the permitted concentration, had no effect on spore germination. However, the combined effects of nitrite with NaCl, tested after addition of pickling salt, was efficient in inhibiting the germination of C. beijerinckii spores but had no effect on B. subtilis spores. In contrast, total suppression of C. beijerinckii spore germination was reached after addition of RYR extract to the medium at a concentration of 2% v/v. For B. subtilis, total inhibition of spore germination was observed only after addition of 4% v/v RYR extract to the medium containing 1.3% w/w NaCl.

Quantifying bacterial spore germination by single-cell impedance cytometry for assessment of host microbiota susceptibility to Clostridioides difficile infection

The germination of ingested spores is often a necessary first step required for enabling bacterial outgrowth and host colonization, as in the case of Clostridioides difficile (C. difficile) infection. Spore germination rate in the colon depends on microbiota composition and its level of disruption by antibiotic treatment since secretions by commensal bacteria modulate primary to secondary bile salt levels to control germination. Assessment of C. difficile spore germination typically requires measurement of colony-forming units, which is labor intensive and takes at least 24 h to perform but is regularly required due to the high recurrence rates of nosocomial antibiotic-associated diarrhea. We present a rapid method to assess spore germination by using high throughput single-cell impedance cytometry (>300 events/s) to quantify live bacterial cells, by gating for their characteristic electrophysiology versus spores, so that germination can be assessed after just 4 h of culture at a detection limit of ~100 live cells per 50 μL sample. To detect the phenotype of germinated C. difficile bacteria, we utilize its characteristically higher net conductivity versus that of spore aggregates and non-viable C. difficile forms, which causes a distinctive high-frequency (10 MHz) impedance phase dispersion within moderately conductive media (0.8 S/m). In this manner, we can detect significant differences in spore germination rates within just 4 h, with increasing primary bile salt levels in vitro and using ex vivo microbiota samples from an antibiotic-treated mouse model to assess susceptibility to C. difficile infection. We envision a rapid diagnostic tool for assessing host microbiota susceptibility to bacterial colonization after key antibiotic treatments.

Flow Cytometry Combined With Single Cell Sorting to Study Heterogeneous Germination of Bacillus Spores Under High Pressure.

Isostatic high pressure (HP) of 150 MPa can trigger the germination of bacterial spores, making them lose their extreme resistance to stress factors, and increasing their susceptibility to milder inactivation strategies. However, germination response of spores within a population is very heterogeneous, and tools are needed to study this heterogeneity. Here, classical methods were combined with more recent and powerful techniques such as flow cytometry (FCM) and fluorescence activated cell sorting (FACS) to investigate spore germination behavior under HP. Bacillus subtilis spores were treated with HP at 150 MPa and 37°C, stained with SYTO16 and PI, and analyzed via FCM. Four sub-populations were detected. These sub-populations were for the first time isolated on single cell level using FACS and characterized in terms of their heat resistance (80°C, 10 min) and cultivability in a nutrient-rich environment. The four isolated sub-populations were found to include (1) heat-resistant and mostly cultivable superdormant spores, i.e., spores that remained dormant after this specific HP treatment, (2) heat-sensitive and cultivable germinated spores, (3) heat-sensitive and partially-cultivable germinated spores, and (4) membrane-compromised cells with barely detectable cultivability. Of particular interest was the physiological state of the third sub-population, which was previously referred to as “unknown”. Moreover, the kinetic transitions between different physiological states were characterized. After less than 10 min of HP treatment, the majority of spores germinated and ended up in a sublethally damaged stage. HP treatment at 150 MPa and 37°C did not cause inactivation of all geminated spores, suggesting that subsequent inactivation strategies such as mild heat inactivation or other inactivation techniques are necessary to control spores in food. This study validated FCM as a powerful technique to investigate the heterogeneous behavior of spores under HP, and provided a pipeline using FACS for isolation of different sub-populations and subsequent characterization to understand their physiological states.

Placeholder Strategy with Upconversion Nanoparticles-Eriochrome Black T Conjugate for a Colorimetric Assay of an Anthrax Biomarker.

The timely warning of the germination of bacterial spores and their prevention are highly important to minimize their potential detrimental effects and for disease control. Thus, a sensitive and selective assay of biomarkers is most desirable. In this work, a nanoprobe is constructed by conjugating lanthanide upconversion nanoparticles (UCNPs) with sodium tripolyphosphate (TPP) and eriochrome black T (EBT). The nanoprobe, UCNPs-TPP/EBT, serves as a platform for the detection of the anthrax biomarker, dipicolinic acid (DPA). In principle, DPA displaces EBT from the UCNPs-TPP/EBT nanoconjugate, resulting in a color change from magenta to blue because of the release of free EBT into the aqueous solution. The binding sites on UCNPs are partly preblocked with TPP as the placeholder molecule, leaving a desired number of binding sites for EBT conjugation. On the basis of this dye displacement reaction, a novel colorimetric assay protocol for DPA is developed, deriving a linear calibration range from 2 to 200 μM with a detection limit of 0.9 μM, which is well below the infectious dose of the spores (60 μM). The assay platform exhibits excellent anti-interference capability when treating a real biological sample matrix. The present method is validated by the analysis of DPA in human serum, and its practical application is further demonstrated by monitoring the DPA release upon spore germination.

Biomarkers of bacterial spore germination

Exposure to specific germinant can induce germination in dormant bacterial spores converting them into vegetative cells which are metabolically active and fragile. This phenomenon of conversion of spores from one phase to another could be a keynote potential strategy for development of different type of techniques ranging from spore detection to their eradication and spore-based biosensing. To extend this biphasic spore germination-based approach in order to facilitate development of detection systems, mechanistic details of markers that signals the process of germination initiation in bacterial spores are required. These markers possess a high level of specificity for differentiation in germinating and dormant spores. In this line, present review underscores the structural properties, sporulation and germination in bacterial spores, and covers a detailed description on various biomarkers namely absorbance (600 nm), dipicolinic acid (DPA), refractility of spores, nucleic acid, ATP, spore’s heat resistance, and enzymes which could be valuable in perceiving germination in bacterial spores. Assaying germination using these markers can further explore the efficient use of spores in development of detection devices for food and environment safety.

Incorporating germination-induction into decontamination strategies for bacterial spores.

This article reviews implementation of germination-induction as part of a decontamination strategy for the cleanup of bacterial spores. To our knowledge this is the first time that germination-induction studies have been reviewed in this context. This article will provide a resource which summarizes the mechanisms of germination in Clostridia and Bacillus species, challenges and successes in germination-induction, and potential areas where this strategy may be implemented.

Mycelium-mediated transfer of water and nutrients stimulates bacterial activity in dry and oligotrophic environments

Fungal–bacterial interactions are highly diverse and contribute to many ecosystem processes. Their emergence under common environmental stress scenarios however, remains elusive. Here we use a synthetic microbial ecosystem based on the germination of Bacillus subtilis spores to examine whether fungal and fungal-like (oomycete) mycelia reduce bacterial water and nutrient stress in an otherwise dry and nutrient-poor microhabitat. We find that the presence of mycelia enables the germination and subsequent growth of bacterial spores near the hyphae. Using a combination of time of flight- and nanoscale secondary ion mass spectrometry (ToF- and nanoSIMS) coupled with stable isotope labelling, we link spore germination to hyphal transfer of water, carbon and nitrogen. Our study provides direct experimental evidence for the stimulation of bacterial activity by mycelial supply of scarce resources in dry and nutrient-free environments. We propose that mycelia may stimulate bacterial activity and thus contribute to sustaining ecosystem functioning in stressed habitats.

A Quasi-chemical Model for Bacterial Spore Germination Kinetics by High Pressure

High pressure processing (HPP) is an emerging non-thermal technology that is growing exponentially in use worldwide for the pasteurization of commercial foodstuffs. At combinations of elevated pressures and temperatures, HPP inactivates bacterial spores, but HPP has not yet been implemented commercially for food sterilization. Studies of the mechanisms of bacterial spore inactivation by HPP using primarily spores of Bacillus species have shown that spore germination precedes inactivation, with the release of dipicolinic acid from the spore core as the rate-determining step. Investigations probing spore resistance to and germination by HPP using Bacillus subtilis, a number of selected B. subtilis mutants, Bacillus amyloliquefaciens, and Clostridium difficile spores have compiled a wealth of detailed mechanistic information, while also accumulating abundant germination kinetics data that has not previously been analyzed by predictive models. Presently, we devise a “quasi-chemical” model for bacterial spore germination dynamics by HPP. This quasi-chemical germination model (QCGM) hypothesizes a three-step mechanism and derives a set of ordinary differential equations to model the observed germination dynamics. The results with this model are viewed in the context of historical studies of spore activation, germination, and inactivation, with an eye toward potentially integrating differential equation models for germination and inactivation into a single, comprehensive model for spore dynamics by HPP. With the increasing use of high hydrostatic pressure to investigate mechanisms of bacterial spore resistance and physiology, the QCGM results help promote the efficient control of bacterial spores, whether for the inactivation of Clostridium botulinum spores in low-acid foods or aerosolized Bacillus anthracis spores on textiles used in protective clothing, tents, or shelters.

Bacterial Spore Germination

Bacterial Spore Germination