scholarly journals Analysis of Germination Capacity and Germinant Receptor (Sub)clusters of Genome-Sequenced Bacillus cereus Environmental Isolates and Model Strains

2016 ◽  
Vol 83 (4) ◽  
Author(s):  
Alicja K. Warda ◽  
Yinghua Xiao ◽  
Jos Boekhorst ◽  
Marjon H. J. Wells-Bennik ◽  
Masja N. Nierop Groot ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Spore Germination ◽  
Brain Heart Infusion ◽  
Whole Genome Sequence ◽  
Single Spore ◽  
Content Type ◽  
Germination Response ◽  
Natural Isolates ◽  
Heat Activation ◽  
Different Strains

ABSTRACT Spore germination of 17 Bacillus cereus food isolates and reference strains was evaluated using flow cytometry analysis in combination with fluorescent staining at a single-spore level. This approach allowed for rapid collection of germination data under more than 20 conditions, including heat activation of spores, germination in complex media (brain heart infusion [BHI] and tryptone soy broth [TSB]), and exposure to saturating concentrations of single amino acids and the combination of alanine and inosine. Whole-genome sequence comparison revealed a total of 11 clusters of operons encoding germinant receptors (GRs): GerK, GerI, and GerL were present in all strains, whereas GerR, GerS, GerG, GerQ, GerX, GerF, GerW, and GerZ (sub)clusters showed a more diverse presence/absence in different strains. The spores of tested strains displayed high diversity with regard to their sensitivity and responsiveness to selected germinants and heat activation. The two laboratory strains, B. cereus ATCC 14579 and ATCC 10987, and 11 food isolates showed a good germination response under a range of conditions, whereas four other strains (B. cereus B4085, B4086, B4116, and B4153) belonging to phylogenetic group IIIA showed a very weak germination response even in BHI and TSB media. Germination responses could not be linked to specific (combinations of) GRs, but it was noted that the four group IIIA strains contained pseudogenes or variants of subunit C in their gerL cluster. Additionally, two of those strains (B4086 and B4153) carried pseudogenes in the gerK and gerR I (sub)clusters that possibly affected the functionality of these GRs. IMPORTANCE Germination of bacterial spores is a critical step before vegetative growth can resume. Food products may contain nutrient germinants that trigger germination and outgrowth of Bacillus species spores, possibly leading to food spoilage or foodborne illness. Prediction of spore germination behavior is, however, very challenging, especially for spores of natural isolates that tend to show more diverse germination responses than laboratory strains. The approach used has provided information on the genetic diversity in GRs and corresponding subclusters encoded by B. cereus strains, as well as their germination behavior and possible associations with GRs, and it provides a basis for further extension of knowledge on the role of GRs in B. cereus (group member) ecology and transmission to the host.

scholarly journals The CasKR Two-Component System Is Required for the Growth of Mesophilic and Psychrotolerant Bacillus cereus Strains at Low Temperatures

2014 ◽  
Vol 80 (8) ◽  
pp. 2493-2503 ◽  
Author(s):  
Sara Esther Diomandé ◽  
Stéphanie Chamot ◽  
Vera Antolinos ◽  
Florian Vasai ◽  
Marie-Hélène Guinebretière ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Food Poisoning ◽  
Two Component System ◽  
Diverse Range ◽  
Component System ◽  
Content Type ◽  
Two Component ◽  
Temperature Ranges ◽  
Different Strains

ABSTRACTThe different strains ofBacillus cereuscan grow at temperatures covering a very diverse range. SomeB. cereusstrains can grow in chilled food and consequently cause food poisoning. We have identified a new sensor/regulator mechanism involved in low-temperatureB. cereusgrowth. Construction of a mutant of this two-component system enabled us to show that this system, called CasKR, is required for growth at the minimal temperature (Tmin). CasKR was also involved in optimal cold growth aboveTminand in cell survival belowTmin. Microscopic observation showed that CasKR plays a key role in cell shape during cold growth. Introducing thecasKRgenes in a ΔcasKRmutant restored its ability to grow atTmin. Although it was first identified in the ATCC 14579 model strain, this mechanism has been conserved in most strains of theB. cereusgroup. We show that the role of CasKR in cold growth is similar in otherB. cereus sensu latostrains with different growth temperature ranges, including psychrotolerant strains.

scholarly journals Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA2mob Operon in Foodborne Strains of Bacillus subtilis

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Antonina O. Krawczyk ◽  
Anne de Jong ◽  
Jimmy Omony ◽  
Siger Holsappel ◽  
Marjon H. J. Wells-Bennik ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Heat Resistance ◽  
Spore Germination ◽  
Food Industry ◽  
Heat Treatments ◽  
Phenotypic Traits ◽  
Content Type ◽  
Heat Resistant ◽  
Heat Activation ◽  
High Level

ABSTRACT Spore heat resistance, germination, and outgrowth are problematic bacterial properties compromising food safety and quality. Large interstrain variation in these properties makes prediction and control of spore behavior challenging. High-level heat resistance and slow germination of spores of some natural Bacillus subtilis isolates, encountered in foods, have been attributed to the occurrence of the spoVA 2mob operon carried on the Tn1546 transposon. In this study, we further investigate the correlation between the presence of this operon in high-level-heat-resistant spores and their germination efficiencies before and after exposure to various sublethal heat treatments (heat activation, or HA), which are known to significantly improve spore responses to nutrient germinants. We show that high-level-heat-resistant spores harboring spoVA 2mob required higher HA temperatures for efficient germination than spores lacking spoVA 2mob. The optimal spore HA requirements additionally depended on the nutrients used to trigger germination, l-alanine (l-Ala), or a mixture of l-asparagine, d-glucose, d-fructose, and K+ (AGFK). The distinct HA requirements of these two spore germination pathways are likely related to differences in properties of specific germinant receptors. Moreover, spores that germinated inefficiently in AGFK contained specific changes in sequences of the GerB and GerK germinant receptors, which are involved in this germination response. In contrast, no relation was found between transcription levels of main germination genes and spore germination phenotypes. The findings presented in this study have great implications for practices in the food industry, where heat treatments are commonly used to inactivate pathogenic and spoilage microbes, including bacterial spore formers. IMPORTANCE This study describes a strong variation in spore germination capacities and requirements for a heat activation treatment, i.e., an exposure to sublethal heat that increases spore responsiveness to nutrient germination triggers, among 17 strains of B. subtilis, including 9 isolates from spoiled food products. Spores of industrial foodborne isolates exhibited, on average, less efficient and slower germination responses and required more severe heat activation than spores from other sources. High heat activation requirements and inefficient, slow germination correlated with elevated resistance of spores to heat and with specific genetic features, indicating a common genetic basis of these three phenotypic traits. Clearly, interstrain variation and numerous factors that shape spore germination behavior challenge standardization of methods to recover highly heat-resistant spores from the environment and have an impact on the efficacy of preservation techniques used by the food industry to control spores.

scholarly journals Molecular Signatures Related to the Virulence of Bacillus cereus Sensu Lato, a Leading Cause of Devastating Endophthalmitis

mSystems ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Jian Yuan ◽  
Yu-Yu Li ◽  
Yi Xu ◽  
Bian-Jin Sun ◽  
Jiao Shao ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Association Studies ◽  
Whole Genome Sequence ◽  
Data Sets ◽  
Molecular Signatures ◽  
Sequencing Data ◽  
Content Type ◽  
Genome Wide ◽  
Medical University ◽  
Bacillus Toyonensis

ABSTRACT Bacillus endophthalmitis is a devastating eye infection that causes rapid blindness through extracellular tissue-destructive exotoxins. Despite its importance, knowledge of the phylogenetic relationships and population structure of intraocular Bacillus spp. is lacking. In this study, we sequenced the whole genomes of eight Bacillus intraocular pathogens independently isolated from 8/52 patients with posttraumatic Bacillus endophthalmitis infections in the Eye Hospital of Wenzhou Medical University between January 2010 and December 2018. Phylogenetic analysis revealed that the pathogenic intraocular isolates belonged to Bacillus cereus, Bacillus thuringiensis and Bacillus toyonensis. To determine the virulence of the ocular isolates, three representative strains were injected into mouse models, and severe endophthalmitis leading to blindness was observed. Through incorporating publicly available genomes for Bacillus spp., we found that the intraocular pathogens could be isolated independently but displayed a similar genetic context. In addition, our data provide genome-wide support for intraocular and gastrointestinal sources of Bacillus spp. belonging to different lineages. Importantly, we identified five molecular signatures of virulence and motility genes associated with intraocular infection, namely, plcA-2, InhA-3, InhA-4, hblA-5, and fliD using pangenome-wide association studies. The characterization of overrepresented genes in the intraocular isolates holds value to predict bacterial evolution and for the design of future intervention strategies in patients with endophthalmitis. IMPORTANCE In this study, we provided a detailed and comprehensive clinicopathological and pathogenic report of Bacillus endophthalmitis over the 8 years of the study period. We first reported the whole-genome sequence of Bacillus spp. causing devastating endophthalmitis and found that Bacillus toyonensis is able to cause endophthalmitis. Finally, we revealed significant endophthalmitis-associated virulence genes involved in hemolysis, immunity inhibition, and pathogenesis. Overall, as more sequencing data sets become available, these data will facilitate comparative research and will reveal the emergence of pathogenic “ocular bacteria.”

scholarly journals The Effects of Heat Activation on Bacillus Spore Germination, with Nutrients or under High Pressure, with or without Various Germination Proteins

2015 ◽  
Vol 81 (8) ◽  
pp. 2927-2938 ◽  
Author(s):  
Stephanie Luu ◽  
Jose Cruz-Mora ◽  
Barbara Setlow ◽  
Florence E. Feeherry ◽  
Christopher J. Doona ◽  
...  
Keyword(s):  
High Pressure ◽  
Spore Germination ◽  
Protein Conformation ◽  
Bacillus Amyloliquefaciens ◽  
Clostridium Botulinum ◽  
Nutrient Concentrations ◽  
Content Type ◽  
Bacillus Spore ◽  
Heat Activation ◽  
Activation Times

ABSTRACTNutrient germination of spores ofBacillusspecies occurs through germinant receptors (GRs) in spores' inner membrane (IM) in a process stimulated by sublethal heat activation.Bacillus subtilisspores maximum germination rates via different GRs required different 75°C heat activation times: 15 min forl-valine germination via the GerA GR and 4 h for germination with thel-asparagine–glucose–fructose–K+mixture via the GerB and GerK GRs, with GerK requiring the most heat activation. In some cases, optimal heat activation decreased nutrient concentrations for half-maximal germination rates. Germination of spores via various GRs by high pressure (HP) of 150 MPa exhibited heat activation requirements similar to those of nutrient germination, and the loss of the GerD protein, required for optimal GR function, did not eliminate heat activation requirements for maximal germination rates. These results are consistent with heat activation acting primarily on GRs. However, (i) heat activation had no effects on GR or GerD protein conformation, as probed by biotinylation by an external reagent; (ii) spores prepared at low and high temperatures that affect spores' IM properties exhibited large differences in heat activation requirements for nutrient germination; and (iii) spore germination by 550 MPa of HP was also affected by heat activation, but the effects were relatively GR independent. The last results are consistent with heat activation affecting spores' IM and only indirectly affecting GRs. The 150- and 550-MPa HP germinations ofBacillus amyloliquefaciensspores, a potential surrogate forClostridium botulinumspores in HP treatments of foods, were also stimulated by heat activation.

scholarly journals The Bacillus cereus GerN and GerT Protein Homologs Have Distinct Roles in Spore Germination and Outgrowth, Respectively

2008 ◽  
Vol 190 (18) ◽  
pp. 6148-6152 ◽  
Author(s):  
Adam Senior ◽  
Anne Moir
Keyword(s):  
Amino Acid ◽  
Bacillus Cereus ◽  
Significant Role ◽  
Spore Germination ◽  
Amino Acid Identity ◽  
Wild Type ◽  
Acid Identity ◽  
Germination Response ◽  
Alkaline Conditions ◽  
Spore Outgrowth

ABSTRACT The GerT protein of Bacillus cereus shares 74% amino acid identity with its homolog GerN. The latter is a Na+/H+-K+ antiporter that is required for normal spore germination in inosine. The germination properties of single and double mutants of B. cereus ATCC 10876 reveal that unlike GerN, which is required for all germination responses that involve the GerI germinant receptor, the GerT protein does not have a significant role in germination, although it is required for the residual GerI-mediated inosine germination response of a gerN mutant. In contrast, GerT has a significant role in outgrowth; gerT mutant spores do not outgrow efficiently under alkaline conditions and outgrow more slowly than the wild type in the presence of high NaCl concentrations. The GerT protein in B. cereus therefore contributes to the success of spore outgrowth from the germinated state during alkaline or Na+ stress.

scholarly journals The Cooperative and Interdependent Roles of GerA, GerK, and Ynd in Germination of Bacillus licheniformis Spores

2016 ◽  
Vol 82 (14) ◽  
pp. 4279-4287 ◽  
Author(s):  
Kristina Borch-Pedersen ◽  
Toril Lindbäck ◽  
Elisabeth H. Madslien ◽  
Shani W. Kidd ◽  
Kristin O'Sullivan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Bacillus Licheniformis ◽  
Spore Germination ◽  
Food Poisoning ◽  
Food Spoilage ◽  
Important Species ◽  
Content Type ◽  
Germination Response ◽  
Food Borne ◽  
Safe Food

ABSTRACTWhen nutrients are scarce,Bacillusspecies form metabolically dormant and extremely resistant spores that enable survival over long periods of time under conditions not permitting growth. The presence of specific nutrients triggers spore germination through interaction with germinant receptors located in the spore's inner membrane.Bacillus licheniformisis a biotechnologically important species, but it is also associated with food spoilage and food-borne disease. TheB. licheniformisATCC 14580/DSM13 genome exhibits threegerAfamily operons (gerA,gerK, andynd) encoding germinant receptors. We show that spores ofB. licheniformisgerminate efficiently in response to a range of different singlel-amino acid germinants, in addition to a weak germination response seen withd-glucose. Mutational analyses revealed that the GerA and Ynd germination receptors function cooperatively in triggering an efficient germination response with singlel-amino acid germinants, whereas the GerK germination receptor is essential for germination withd-glucose. Mutant spores expressing only GerA and GerK or only Ynd and GerK show reduced or severely impaired germination responses, respectively, with singlel-amino acid germinants. Neither GerA nor Ynd could function alone in stimulating spore germination. Together, these results functionally characterize the germination receptor operons present inB. licheniformis. We demonstrate the overlapping germinant recognition patterns of the GerA and Ynd germination receptors and the cooperative functionalities between GerA, Ynd, and GerK in inducing germination.IMPORTANCETo ensure safe food production and durable foods, there is an obvious need for more knowledge on spore-forming bacteria. It is the process of spore germination that ultimately leads to food spoilage and food poisoning.Bacillus licheniformisis a biotechnologically important species that is also associated with food spoilage and food-borne disease. Despite its importance, the mechanisms of spore germination are poorly characterized in this species. This study provides novel knowledge on germination ofB. licheniformisspores. We characterize the germinant recognition profiles of the three germinant receptors present inB. licheniformisspores and demonstrate that the GerA germinant receptor cooperates with the Ynd and GerK germinant receptors to enable an effective germination response tol-amino acids. We also demonstrate that GerK is required for germination in response to the single germinant glucose. This study demonstrates the complex interactions between germinant receptors necessary for efficient germination ofB. licheniformisspores.

scholarly journals In vitro antivirulence activity of baicalin against Clostridioides difficile

2020 ◽  
Vol 69 (4) ◽  
pp. 631-639
Author(s):  
Abraham Joseph Pellissery ◽  
Poonam Gopika Vinayamohan ◽  
Kumar Venkitanarayanan
Keyword(s):  
Spore Germination ◽  
Type Species ◽  
Brain Heart Infusion ◽  
Toxin Production ◽  
Strictly Anaerobic ◽  
Content Type ◽  
Link Type ◽  
Clostridioides Difficile ◽  
Spore Outgrowth

Introduction. Clostridioides difficile is an enteric pathogen that causes a serious toxin-mediated colitis in humans. Bacterial exotoxins and sporulation are critical virulence components that contribute to pathogenesis, and disease transmission and relapse, respectively. Therefore, reducing toxin production and sporulation could significantly minimize C. difficile pathogenicity and disease outcome in affected individuals. Aim. This study investigated the efficacy of a natural flavone glycoside, baicalin, in reducing toxin synthesis, sporulation and spore germination in C. difficile in vitro. Methodology. Hypervirulent C. difficile isolates BAA 1870 or 1803 were cultured in brain heart infusion broth with or without the subinhibitory concentration (SIC) of baicalin, and incubated at 37 °C for 24 h under strictly anaerobic conditions. The supernatant was harvested after 24 h for determining C. difficile toxin production by ELISA. In addition, a similar experiment was performed wherein samples were harvested for assessing total viable counts, and heat-resistant spore counts at 72 h of incubation. Furthermore, C. difficile spore germination and spore outgrowth kinetics, with or without baicalin treatment, was measured in a plate reader by recording optical density at 600 nm. Finally, the effect of baicalin on C. difficile toxin, sporulation and virulence-associated genes was investigated using real-time quantitative PCR. Results. The SIC of baicalin significantly reduced toxin synthesis, sporulation and spore outgrowth when compared to control. In addition, C. difficile genes critical for pathogenesis were significantly down-regulated in the presence of baicalin. Conclusion. Our results suggest that baicalin could potentially be used to control C. difficile , and warrant future studies in vivo.

scholarly journals Heat activation and inactivation of bacterial spores. Is there an overlap?

2021 ◽  
Author(s):  
Juan Wen ◽  
Jan P.P.M. Smelt ◽  
Norbert O.E. Vischer ◽  
Arend D Vos ◽  
Peter Setlow ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Spore Germination ◽  
Bacterial Spores ◽  
Heat Inactivation ◽  
Bacillus Species ◽  
Dependent Manner ◽  
Single Spore ◽  
Heat Damage ◽  
Germination Process ◽  
Heat Activation

Heat activation at a sublethal temperature is widely applied to promote Bacillus species spore germination. This treatment also has potential to be employed in food processing to eliminate undesired bacterial spores by enhancing their germination, and then inactivating the less heat resistant germinated spores at a milder temperature. However, incorrect heat treatment could also generate heat damage in spores, and lead to more heterogeneous spore germination. Here, the heat activation and heat damage profile of Bacillus subtilis spores was determined by testing spore germination and outgrowth at both population and single spore levels. The heat treatments used were 40-80 degrees Celcius, and for 0-300 min. The results were as follows. 1) Heat activation at 40-70 degrees Celcius promoted L-valine and L-asparagine-glucose-fructose-potassium (AGFK) induced germination in a time dependent manner. 2) The optimal heat activation temperatures for AGFK and L-valine germination via the GerB plus GerK or GerA germinant receptors were 65 and 50-65 degrees Celcius, respectively. 3) Heat inactivation of dormant spores appeared at 70 degrees Celcius, and the heat damage of molecules essential for germination and growth began at 70 and 65 degrees Celcius, respectively. 4) Heat treatment at 75 degrees Celcius resulted in both activation of germination and damage to the germination apparatus, and 80 degrees Celcius treatment caused more pronounced heat damage. 5) For the spores that should withstand adverse environmental temperatures in nature, heat activation seems functional for a subsequent optimal germination process, while heat damage affected both germination and outgrowth.

scholarly journals Heat activation and inactivation of bacterial spores. Is there an overlap?

2022 ◽  
Author(s):  
Juan Wen ◽  
Jan P. P. M. Smelt ◽  
Norbert O.E. Vischer ◽  
Arend L. de Vos ◽  
Peter Setlow ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Spore Germination ◽  
Bacterial Spores ◽  
Heat Inactivation ◽  
Dependent Manner ◽  
Single Spore ◽  
Heat Damage ◽  
Heat Resistant ◽  
New Information ◽  
Heat Activation

Heat activation at a sublethal temperature is widely applied to promote Bacillus species spore germination. This treatment also has potential to be employed in food processing to eliminate undesired bacterial spores by enhancing their germination, and then inactivating the less heat resistant germinated spores at a milder temperature. However, incorrect heat treatment could also generate heat damage in spores, and lead to more heterogeneous spore germination. Here, the heat activation and heat damage profile of Bacillus subtilis spores was determined by testing spore germination and outgrowth at both population and single spore levels. The heat treatments used were 40-80°C, and for 0-300 min. The results were as follows. 1) Heat activation at 40-70°C promoted L-valine and L-asparagine-glucose-fructose-potassium (AGFK) induced germination in a time dependent manner. 2) The optimal heat activation temperatures for AGFK and L-valine germination via the GerB plus GerK or GerA germinant receptors were 65 and 50-65°C, respectively. 3) Heat inactivation of dormant spores appeared at 70°C, and the heat damage of molecules essential for germination and growth began at 70 and 65°C, respectively. 4) Heat treatment at 75°C resulted in both activation of germination and damage to the germination apparatus, and 80°C treatment caused more pronounced heat damage. 5) For the spores that should withstand adverse environmental temperatures in nature, heat activation seems functional for a subsequent optimal germination process, while heat damage affected both germination and outgrowth. Importance Bacterial spores are thermal resistant structures that can thus survive preservation strategies and revive through the process of spore germination. The more heat resistant spores are the more heterogeneous they germinate upon adding germinants. Upon germination spores can cause food spoilage and cause food intoxication. Here we provide new information on both heat activation and inactivation regimes and their effects on the (heterogeneity of) spore germination.

scholarly journals Genomic Changes Associated with the Loss of Nocardia brasiliensis Virulence in Mice after 200In VitroPassages

2016 ◽  
Vol 84 (9) ◽  
pp. 2595-2606 ◽  
Author(s):  
Carolina Gonzalez-Carrillo ◽  
Cassandra Millan-Sauceda ◽  
Hector Gerardo Lozano-Garza ◽  
Rocio Ortiz-Lopez ◽  
Ramiro Elizondo-Gonzalez ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Genome Sequence ◽  
Brain Heart Infusion ◽  
Open Reading Frames ◽  
Whole Genome Sequence ◽  
Bacterial Cells ◽  
Content Type ◽  
Nocardia Brasiliensis ◽  
Genomic Changes ◽  
Rich Media

Nocardiaspecies, particularlyNocardia brasiliensis, are etiologic agents of mycetoma, a chronic subcutaneous infection. Until now, little has been known about the pathogenic mechanisms involved in nocardial infection. Traditionally, subculture in rich media has been a simple way to induce attenuation. In this work, we report the changes in virulence toward mice and in genomic constitution ofN. brasiliensisproduced after 200 continuous subcultures in brain heart infusion (BHI) medium (P-200 strain). The ability of theN. brasiliensisP-200 strain to produce experimental infection was tested using BALB/c mice. P-200 was also used to immunize mice to determine whether it could induce resistance against a challenge with a nonsubcultured isolate (P-0). Comparative proteomic analysis betweenN. brasiliensisP-0 and P-200 was performed by two-dimensional (2-D) electrophoresis, and the genome sequence was obtained through Roche 454 sequence analysis. Virulence in BALB/c mice was completely lost, and BALB/c mice immunized with P-200 bacterial cells were resistant to mycetoma production by the nonsubcultured strain. Whole-genome sequence analysis revealed that P-200 lost a total of 262,913 bp distributed in 19 deleted regions, involving a total of 213 open reading frames (ORFs). The deleted genes included those encoding bacterial virulence factors, e.g., catalase, nitrate reductase enzymes, and a group of mammalian cell entry (MCE) family proteins, which may explain the loss of virulence of the isolate. Thus, completely attenuatedN. brasiliensiswas obtained after 200 passages in BHI medium, and putativeNocardiavirulence genes were identified for the first time.

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