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Adrian Ponce - One of the best experts on this subject based on the ideXlab platform.
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Detection and Viability Assessment of Endospore-Forming Pathogens
Principles of Bacterial Detection: Biosensors Recognition Receptors and Microsystems, 2020Co-Authors: Adrian Ponce, Stephanie A Connon, Pun To YungAbstract:In this chapter, we explore technology developments for the rapid detection, identification, and viability assessment of endospore-forming pathogens with a focus on Bacillus anthracis. First, we introduce various toxin-producing species and their role as bioinsecticides, probiotics, and bioweapons. We also review the role of Endospores as biological indicators (i.e., dosimeters) for evaluating sterilization regimens, such as autoclaving and wastewater remediation. Monitoring the effectiveness of cleaning and sterilization regimens to maintain good hygiene is required in several major industries, including health care, food, and pharmaceutical industries. In the next section, we review recent developments in DNA-, immuno-, and dipicolinic acid assays, and their applications for detection and monitoring of Bacillus anthracis and other endospore-forming pathogens. Finally, we review viability assays capable of rapid validation of endospore inactivation after sterilization, including assays based on ATP synthesis during stage II germination, and DPA release during stage I germination.
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quantitative and fast sterility assurance testing of surfaces by enumeration of germinable Endospores
Scientific Reports, 2020Co-Authors: Elizabeth D Lester, Pun To Yung, Adrian PonceAbstract:A fast Endospore Germinability Assay (EGA) was validated with traditional plate counts to enumerate single endospore germination events for monitoring surface sterilization. The assay is based on a time-gated luminescence microscopy technique enabling visualization and enumeration of individual germinating Endospores. Germinating Endospores release calcium dipicolinate to form highly luminescent terbium dipicolinate complexes surrounding each germinating endospore. EGA and heterotrophic plate counting (HPC) were used to evaluate the swab/rinse recovery efficiency of Endospores from stainless steel surfaces. EGA and HPC results were highly correlated for endospore recovery from stainless steel coupons inoculated with range of 1,000 Endospores per coupon down to sterility. Dosage-dependent decrease of surface endospore germinability were observed in dry heat, UV irradiation, oxygen plasma and vaporized hydrogen peroxide treatments, measured with EGA and HPC. EGA is a fast and complementary method to traditional HPC for quantitative sterility assurance testing of surfaces. This work introduces and validates a 15-minute or faster assay for germinable Endospores to complement the conventional lengthy, culture-based surface sterility validation, which is critical in hospitals, food and pharmaceutical industries to help minimize nosocomial infection, food spoilage, and pharmaceutical contamination.
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validation of a clostridium endospore viability assay and analysis of greenland ices and atacama desert soils
Applied and Environmental Microbiology, 2011Co-Authors: Wanwan Yang, Adrian PonceAbstract:Bacterial Endospores are dormant microbial structures that are highly resistant to chemical, physical, and radiation sterilization processes (20, 31). They represent one of the most successful survival strategies of microorganisms and are formed when members of spore-forming genera (e.g., Bacillus and Clostridium) face unfavorable conditions, such as environmental extremes or starvation (19, 34). Once they are formed, Endospores can stay dormant for extended periods of time, from thousands (8, 13, 20, 25, 33) to millions (3, 35) of years, although for the more extreme claims of longevity it is difficult to rule out modern contamination (37). Anaerobic spore-forming clostridia include numerous pathogenic species that are dangerous contaminants. For example, Clostridium botulinum and C. perfringens are common food-poisoning agents that produce toxins which cause diseases such as botulism and human necrotic enteritis (9, 17). C. perfringens, C. difficile, and C. tetani are causative agents of gas gangrene, pseudomembranous colitis, and tetanus (9, 30). Some psychrotrophic clostridia are also responsible for the spoilage of chilled vacuum-packed meat (9). In addition, C. perfringens has been used as an indicator of fecal contamination, because it is present in large numbers in human and animal wastes (4, 6). Due to their resistance to various extreme conditions, Clostridium Endospores are also employed as biological indicators to monitor the effectiveness of various sterilization processes (11, 12). Currently, the standard method for quantifying viable Clostridium Endospores is measuring CFU after heat shock killing of vegetative cells. This method requires several days of incubation and a tedious anaerobic culturing technique, and it is amenable for culturing only fewer than 1% of environmental species (24). Other molecular endospore viability assays include ATP assay (26) and quantitative PCR (qPCR) coupled with propidium monoazide (PMA) (28), which, unlike microscopy-based endospore viability assay (micro-EVA), require extensive sample preparation and are labor-intensive. Previously, we described a spectroscopy-based endospore viability assay (spectro-EVA) to quantify dipicolinic acid (DPA) released from germinating Clostridium spores in liquid suspension (39). Germination was triggered by various germinants, such as l-alanine/NaHCO3, l-lactate, or d-alanine, which cause the release of approximately 108 molecules of a unique biomarker, DPA, from the spore core. Spectro-EVA is based on the detection of DPA in bulk solution via Tb3+-DPA luminescence spectroscopy, with a limit of detection (LOD) of 1,000 spores/ml. Unfortunately, spectro-EVA has even lower detection limits when environmental extracts are analyzed due to sensitivity to interference from contaminants. Previously, this limitation was overcome for the case of Bacillus Endospores by employing a microscopy-based EVA (41), where individual spores are enumerated as they germinate in a microscope field of view. Here we report details of a rapid microscopy-based endospore viability assay (micro-EVA) that enables enumeration of single germinating Clostridium Endospores on Tb3+- and d-alanine-doped agarose. d-Alanine was used as a germinant, which serves to trigger Clostridium spore germination while inhibiting Bacillus spore germination (2, 14, 38). Germination releases DPA from Endospores, and subsequent Tb3+-DPA binding results in green luminescent spots under pulsed UV excitation in a field of view of a time-gated microscope. These were enumerated as germinable Clostridium Endospores (GCEs) using time-gated Tb3+-DPA luminescence microscopy (i.e., micro-EVA). A parallel comparison of micro-EVA data with culturing data validated this method. Finally, we compared micro-EVA to culturing methods to quantify GCEs from two Mars analog environments, Greenland ice core and Atacama Desert.
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Fast Sterility Assessment by Germinable-Endospore Biodosimetry†
Applied and Environmental Microbiology, 2008Co-Authors: Pun To Yung, Adrian PonceAbstract:The era of modern microbiology began in the 1870s when the life cycle of an endospore-forming pathogen, Bacillus anthracis, was elucidated using new methods for isolating pure cultures from single-cell clones on solid growth media. Bacterial Endospores are dormant microbial structures that are highly resistant to chemical, physical, and radiation sterilization processes (2, 6, 19, 26). In fact, Bacillus subtilis Endospores have survived for 6 years in space while exposed to high-vacuum conditions, temperature extremes, and intense solar and galactic radiation (12, 18). Bacterial Endospores are routinely employed as biological indicators (i.e., biodosimeters) to validate the effectiveness of sterilization methods (e.g., autoclaves) used in the medical device (15, 16), pharmaceutical, health care (5), food preparation, wastewater remediation (23), and biodefense (29) industries. The effectiveness of sterilization processes is measured and reported in terms of sterility assurance levels (SALs), which are defined as the expected probability that a product remains contaminated with viable microorganisms after exposure to a validated sterilization process. A sterilization process that yields predictable SALs is considered to be validated. Confidence in achieving a required SAL is obtained by the use of biological indicators that present a considerably greater population and resistance challenge than the expected bioburden (21), and the most effective way to test the efficiency of a sterilization process is to place biological indicators within and on test products of interest. Currently, endospore inactivation is quantified by measuring the log reduction in CFU. This method, however, requires several days of incubation, during which 20 cycles of cell replication ultimately yield visible colonies that can then be enumerated. In contrast, endospore germination can be initiated and monitored on a timescale of minutes. Germination of Bacillus Endospores can be triggered by simple biomolecules, such as l-alanine, l-asparagine, or glucose (8, 24, 27), which cause the release of approximately 108 molecules of dipicolinic acid (DPA) from the core of the endospore during the first stage of germination. DPA exists in all bacterial Endospores as 5 to 15% of the cellular dry weight and is a unique, defining constituent of the cellular dry weight of Endospores (10, 14, 22). Here we report details of a rapid endospore viability assay (EVA) in which Bacillus atrophaeus Endospores were immobilized on terbium ion (Tb3+)/l-alanine-doped agarose. The l-alanine serves to trigger germination, during which DPA is released from Endospores. The Tb3+ subsequently binds DPA, resulting in green luminescent spots under UV excitation in a microscope field of view, which were enumerated as germinable Endospores using time-gated Tb-DPA luminescence microscopy (i.e., μEVA). Here we validate μEVA against culturing as a method for rapid endospore viability assessment and evaluate its application for monitoring endospore inactivation by thermal and UV sterilization regimens.
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an automated front end monitor for anthrax surveillance systems based on the rapid detection of airborne Endospores
Biotechnology and Bioengineering, 2007Co-Authors: Pun To Yung, Adrian Ponce, Elizabeth D Lester, Greg BearmanAbstract:A fully automated anthrax smoke detector (ASD) has been developed and tested. The ASD is intended to serve as a cost effective front-end monitor for anthrax surveillance systems. The principle of operation is based on measuring airborne endospore concentrations, where a sharp concentration increase signals an anthrax attack. The ASD features an air sampler, a thermal lysis unit, a syringe pump, a time-gated spectrometer, and endospore detection chemistry comprised of dipicolinic acid (DPA)-triggered terbium ion (Tb3+) luminescence. Anthrax attacks were simulated using aerosolized Bacillus atrophaeus spores in fumed silica, and corresponding Tb-DPA intensities were monitored as a function of time and correlated to the number of airborne Endospores collected. A concentration dependence of 102–106 spores/mg of fumed silica yielded a dynamic range of 4 orders of magnitude and a limit of detection of 16 spores/L when 250 L of air were sampled. Simulated attacks were detected in less than 15 min. Biotechnol. Bioeng. 2007; 98: 864–871. © 2007 Wiley Periodicals, Inc.
Pun To Yung - One of the best experts on this subject based on the ideXlab platform.
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Detection and Viability Assessment of Endospore-Forming Pathogens
Principles of Bacterial Detection: Biosensors Recognition Receptors and Microsystems, 2020Co-Authors: Adrian Ponce, Stephanie A Connon, Pun To YungAbstract:In this chapter, we explore technology developments for the rapid detection, identification, and viability assessment of endospore-forming pathogens with a focus on Bacillus anthracis. First, we introduce various toxin-producing species and their role as bioinsecticides, probiotics, and bioweapons. We also review the role of Endospores as biological indicators (i.e., dosimeters) for evaluating sterilization regimens, such as autoclaving and wastewater remediation. Monitoring the effectiveness of cleaning and sterilization regimens to maintain good hygiene is required in several major industries, including health care, food, and pharmaceutical industries. In the next section, we review recent developments in DNA-, immuno-, and dipicolinic acid assays, and their applications for detection and monitoring of Bacillus anthracis and other endospore-forming pathogens. Finally, we review viability assays capable of rapid validation of endospore inactivation after sterilization, including assays based on ATP synthesis during stage II germination, and DPA release during stage I germination.
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quantitative and fast sterility assurance testing of surfaces by enumeration of germinable Endospores
Scientific Reports, 2020Co-Authors: Elizabeth D Lester, Pun To Yung, Adrian PonceAbstract:A fast Endospore Germinability Assay (EGA) was validated with traditional plate counts to enumerate single endospore germination events for monitoring surface sterilization. The assay is based on a time-gated luminescence microscopy technique enabling visualization and enumeration of individual germinating Endospores. Germinating Endospores release calcium dipicolinate to form highly luminescent terbium dipicolinate complexes surrounding each germinating endospore. EGA and heterotrophic plate counting (HPC) were used to evaluate the swab/rinse recovery efficiency of Endospores from stainless steel surfaces. EGA and HPC results were highly correlated for endospore recovery from stainless steel coupons inoculated with range of 1,000 Endospores per coupon down to sterility. Dosage-dependent decrease of surface endospore germinability were observed in dry heat, UV irradiation, oxygen plasma and vaporized hydrogen peroxide treatments, measured with EGA and HPC. EGA is a fast and complementary method to traditional HPC for quantitative sterility assurance testing of surfaces. This work introduces and validates a 15-minute or faster assay for germinable Endospores to complement the conventional lengthy, culture-based surface sterility validation, which is critical in hospitals, food and pharmaceutical industries to help minimize nosocomial infection, food spoilage, and pharmaceutical contamination.
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Effect of sonic stimulation on Bacillus endospore germination
Fems Microbiology Letters, 2015Co-Authors: Wen Jie Wu, Pun To YungAbstract:This study investigates the effect of sonic stimulation on Bacillus endospore germination . Germinating Endospores in a microtiter plate were exposed to audible sound wave generated by an array of piezoelectric transducers. In situ germination kinetics was measured by terbium-dipicolinate fluorescence assay, optical density measurement and phase contrast microscopy. Fluorescence results revealed that sonic stimulation (5 kHz at 90 dB) promoted the germination speed by 43.7% ± 11.3% and final germination level by 61.7% ± 11.9% of Bacillus atrophaeus . This acoustic energy absorbed by Endospores is postulated to change membrane permeability and increase enzyme activities; thereby, expediting the germination process. This also raises the likelihood of dormant Endospores undergoing germination because of a rapid release of unidentified chemical mediators for quorum sensing. On the other hand, acoustic effect was not observed in B. subtilis Endospores. This may be attributed to the different spore aspect ratio, 1.43 ± 0.05 for B. atrophaeus and 2.02 ± 0.08 for B. subtilis , which results in a difference in specific absorption rates towards audible sound waves. Our results demonstrate the modulation of endospore germination by an external field to shed light on germination mechanism and cell–wave interaction.
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Fast Sterility Assessment by Germinable-Endospore Biodosimetry†
Applied and Environmental Microbiology, 2008Co-Authors: Pun To Yung, Adrian PonceAbstract:The era of modern microbiology began in the 1870s when the life cycle of an endospore-forming pathogen, Bacillus anthracis, was elucidated using new methods for isolating pure cultures from single-cell clones on solid growth media. Bacterial Endospores are dormant microbial structures that are highly resistant to chemical, physical, and radiation sterilization processes (2, 6, 19, 26). In fact, Bacillus subtilis Endospores have survived for 6 years in space while exposed to high-vacuum conditions, temperature extremes, and intense solar and galactic radiation (12, 18). Bacterial Endospores are routinely employed as biological indicators (i.e., biodosimeters) to validate the effectiveness of sterilization methods (e.g., autoclaves) used in the medical device (15, 16), pharmaceutical, health care (5), food preparation, wastewater remediation (23), and biodefense (29) industries. The effectiveness of sterilization processes is measured and reported in terms of sterility assurance levels (SALs), which are defined as the expected probability that a product remains contaminated with viable microorganisms after exposure to a validated sterilization process. A sterilization process that yields predictable SALs is considered to be validated. Confidence in achieving a required SAL is obtained by the use of biological indicators that present a considerably greater population and resistance challenge than the expected bioburden (21), and the most effective way to test the efficiency of a sterilization process is to place biological indicators within and on test products of interest. Currently, endospore inactivation is quantified by measuring the log reduction in CFU. This method, however, requires several days of incubation, during which 20 cycles of cell replication ultimately yield visible colonies that can then be enumerated. In contrast, endospore germination can be initiated and monitored on a timescale of minutes. Germination of Bacillus Endospores can be triggered by simple biomolecules, such as l-alanine, l-asparagine, or glucose (8, 24, 27), which cause the release of approximately 108 molecules of dipicolinic acid (DPA) from the core of the endospore during the first stage of germination. DPA exists in all bacterial Endospores as 5 to 15% of the cellular dry weight and is a unique, defining constituent of the cellular dry weight of Endospores (10, 14, 22). Here we report details of a rapid endospore viability assay (EVA) in which Bacillus atrophaeus Endospores were immobilized on terbium ion (Tb3+)/l-alanine-doped agarose. The l-alanine serves to trigger germination, during which DPA is released from Endospores. The Tb3+ subsequently binds DPA, resulting in green luminescent spots under UV excitation in a microscope field of view, which were enumerated as germinable Endospores using time-gated Tb-DPA luminescence microscopy (i.e., μEVA). Here we validate μEVA against culturing as a method for rapid endospore viability assessment and evaluate its application for monitoring endospore inactivation by thermal and UV sterilization regimens.
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an automated front end monitor for anthrax surveillance systems based on the rapid detection of airborne Endospores
Biotechnology and Bioengineering, 2007Co-Authors: Pun To Yung, Adrian Ponce, Elizabeth D Lester, Greg BearmanAbstract:A fully automated anthrax smoke detector (ASD) has been developed and tested. The ASD is intended to serve as a cost effective front-end monitor for anthrax surveillance systems. The principle of operation is based on measuring airborne endospore concentrations, where a sharp concentration increase signals an anthrax attack. The ASD features an air sampler, a thermal lysis unit, a syringe pump, a time-gated spectrometer, and endospore detection chemistry comprised of dipicolinic acid (DPA)-triggered terbium ion (Tb3+) luminescence. Anthrax attacks were simulated using aerosolized Bacillus atrophaeus spores in fumed silica, and corresponding Tb-DPA intensities were monitored as a function of time and correlated to the number of airborne Endospores collected. A concentration dependence of 102–106 spores/mg of fumed silica yielded a dynamic range of 4 orders of magnitude and a limit of detection of 16 spores/L when 250 L of air were sampled. Simulated attacks were detected in less than 15 min. Biotechnol. Bioeng. 2007; 98: 864–871. © 2007 Wiley Periodicals, Inc.
Kasper Urup Kjeldsen - One of the best experts on this subject based on the ideXlab platform.
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contrasting community composition of Endospores and vegetative firmicutes in a marine sediment suggests both endogenous and exogenous sources of endospore accumulation
Environmental Microbiology Reports, 2019Co-Authors: Carina Cupit, Bente Aagaard Lomstein, Kasper Urup KjeldsenAbstract:: Bacterial Endospores are highly abundant in marine sediments, but their taxonomic identity and ecology is largely unknown. We selectively extracted DNA from Endospores and vegetative cells and sequenced 16S rRNA genes to characterize the composition of the endospore and vegetative Firmicutes communities in the sediment and water column of Aarhus Bay (Denmark). The endospore community in the sediment was dominated by the families Bacillaceae, Lachnospiraceae, Clostridiaceae and Ruminoccocaceae. These families were also represented in the vegetative community in the sediment and the endospore community in the water column. OTUs of high relative abundance in the endospore community were also represented in the vegetative Firmicutes community. Other OTUs were exclusively found in the endospore communities. This suggests that Endospores accumulate in marine sediments due to passive deposition from the water column and sporulation of vegetative cells in the sediment. Some OTUs were detected in the endospore community of the water column and the vegetative community the sediment indicating that Endospores deposited from the water column may germinate upon burial/deposition in the sediment. We provide novel insight into the composition of endospore communities in marine sediments and highlight their role in microbial dispersal and as a seed bank in subsurface sediments.
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identity abundance and reactivation kinetics of thermophilic fermentative Endospores in cold marine sediment and seawater
Frontiers in Microbiology, 2017Co-Authors: Marta Volpi, Bente Aagaard Lomstein, Bo Barker Jørgensen, Andreas Sichert, Kasper Urup KjeldsenAbstract:Cold marine sediments harbor Endospores of fermentative and sulfate-reducing, thermophilic bacteria. These dormant populations of Endospores are believed to accumulate in the seabed via passive dispersal by ocean currents followed by sedimentation from the water column. However the magnitude of this process is poorly understood because the Endospores present in seawater were so far not identified, and only the abundance of thermophilic sulfate-reducing Endospores in the seabed has been quantified. We investigated the distribution of thermophilic fermentative Endospores (TFEs) in water column and sediment of Aarhus Bay, Denmark, to test the role of suspended dispersal and determine the rate of endospore deposition and abundance in the sediment. We furthermore aimed to determine the time course of reactivation of the germinating TFEs. TFEs were induced to germinate and grow by incubating pasteurized sediment and water samples anaerobically at 50°C. We observed a sudden release of the endospore component dipicolinic acid immediately upon incubation suggesting fast endospore reactivation in response to heating. Volatile fatty acids (VFAs) and H2 began to accumulate exponentially after 3.5 h of incubation showing that reactivation was followed by a short phase of outgrowth before germinated cells began to divide. Thermophilic fermenters were mainly present in the sediment as Endospores because the rate of VFA accumulation was identical in pasteurized and non-pasteurized samples. Germinating TFEs were identified taxonomically by reverse transcription, PCR amplification and sequencing of 16S rRNA. The water column and sediment shared the same phylotypes, thereby confirming the potential for seawater dispersal. The abundance of TFEs was estimated by most probable number enumeration, rates of VFA production, and released amounts of dipicolinic acid during germination. The surface sediment contained ~105-106 inducible TFEs cm-3. TFEs thus outnumber thermophilic sulfate-reducing Endospores by an order of magnitude. The abundance of cultivable TFEs decreased exponentially with sediment depth with a half-life of 350 years. We estimate that 6 x109 anaerobic thermophilic Endospores are deposited on the seafloor per m2 per year in Aarhus Bay, and that these thermophiles represent >10% of the total endospore community in the surface sediment.
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Endospores of thermophilic bacteria as tracers of microbial dispersal by ocean currents
The ISME Journal, 2014Co-Authors: Albert Leopold Müller, Ilias Lagkouvardos, Casey R J Hubert, Júlia Rosa De Rezende, Kasper Urup Kjeldsen, Bo Barker Jørgensen, David Berry, Alexander LoyAbstract:Microbial biogeography is influenced by the combined effects of passive dispersal and environmental selection, but the contribution of either factor can be difficult to discern. As thermophilic bacteria cannot grow in the cold seabed, their inactive spores are not subject to environmental selection. We therefore conducted a global experimental survey using thermophilic Endospores that are passively deposited by sedimentation to the cold seafloor as tracers to study the effect of dispersal by ocean currents on the biogeography of marine microorganisms. Our analysis of 81 different marine sediments from around the world identified 146 species-level 16S rRNA phylotypes of endospore-forming, thermophilic Firmicutes . Phylotypes showed various patterns of spatial distribution in the world oceans and were dispersal-limited to different degrees. Co-occurrence of several phylotypes in locations separated by great distances (west of Svalbard, the Baltic Sea and the Gulf of California) demonstrated a widespread but not ubiquitous distribution. In contrast, Arctic regions with water masses that are relatively isolated from global ocean circulation (Baffin Bay and east of Svalbard) were characterized by low phylotype richness and different compositions of phylotypes. The observed distribution pattern of thermophilic Endospores in marine sediments suggests that the impact of passive dispersal on marine microbial biogeography is controlled by the connectivity of local water masses to ocean circulation.
Bo Barker Jørgensen - One of the best experts on this subject based on the ideXlab platform.
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freezing tolerance of thermophilic bacterial Endospores in marine sediments
Frontiers in Microbiology, 2019Co-Authors: Margaret Cramm, Bo Barker Jørgensen, Anirban Chakraborty, Carmen Li, Emil S Ruff, Casey R J HubertAbstract:: Dormant Endospores of anaerobic, thermophilic bacteria found in cold marine sediments offer a useful model for studying microbial biogeography, dispersal, and survival. The dormant endospore phenotype confers resistance to unfavorable environmental conditions, allowing dispersal to be isolated and studied independently of other factors such as environmental selection. To study the resilience of thermospores to conditions relevant for survival in extreme cold conditions, their viability following different freezing treatments was tested. Marine sediment was frozen at either -80°C or -20°C for 10 days prior to pasteurization and incubation at +50°C for 21 days to assess thermospore viability. Sulfate reduction commenced at +50°C following both freezing pretreatments indicating persistence of thermophilic Endospores of sulfate-reducing bacteria. The onset of sulfate reduction at +50°C was delayed in -80°C pretreated microcosms, which exhibited more variability between triplicates, compared to -20°C pretreated microcosms and parallel controls that were not frozen in advance. Microbial communities were evaluated by 16S rRNA gene amplicon sequencing, revealing an increase in the relative sequence abundance of thermophilic endospore-forming Firmicutes in all microcosms. Different freezing pretreatments (-80°C and -20°C) did not appreciably influence the shift in overall bacterial community composition that occurred during the +50°C incubations. Communities that had been frozen prior to +50°C incubation showed an increase in the relative sequence abundance of operational taxonomic units (OTUs) affiliated with the class Bacilli, relative to unfrozen controls. These results show that freezing impacts but does not obliterate thermospore populations and their ability to germinate and grow under appropriate conditions. Indeed the majority of the thermospore OTUs detected in this study (21 of 22) could be observed following one or both freezing treatments. These results are important for assessing thermospore viability in frozen samples and following cold exposure such as the very low temperatures that would be encountered during panspermia.
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identity abundance and reactivation kinetics of thermophilic fermentative Endospores in cold marine sediment and seawater
Frontiers in Microbiology, 2017Co-Authors: Marta Volpi, Bente Aagaard Lomstein, Bo Barker Jørgensen, Andreas Sichert, Kasper Urup KjeldsenAbstract:Cold marine sediments harbor Endospores of fermentative and sulfate-reducing, thermophilic bacteria. These dormant populations of Endospores are believed to accumulate in the seabed via passive dispersal by ocean currents followed by sedimentation from the water column. However the magnitude of this process is poorly understood because the Endospores present in seawater were so far not identified, and only the abundance of thermophilic sulfate-reducing Endospores in the seabed has been quantified. We investigated the distribution of thermophilic fermentative Endospores (TFEs) in water column and sediment of Aarhus Bay, Denmark, to test the role of suspended dispersal and determine the rate of endospore deposition and abundance in the sediment. We furthermore aimed to determine the time course of reactivation of the germinating TFEs. TFEs were induced to germinate and grow by incubating pasteurized sediment and water samples anaerobically at 50°C. We observed a sudden release of the endospore component dipicolinic acid immediately upon incubation suggesting fast endospore reactivation in response to heating. Volatile fatty acids (VFAs) and H2 began to accumulate exponentially after 3.5 h of incubation showing that reactivation was followed by a short phase of outgrowth before germinated cells began to divide. Thermophilic fermenters were mainly present in the sediment as Endospores because the rate of VFA accumulation was identical in pasteurized and non-pasteurized samples. Germinating TFEs were identified taxonomically by reverse transcription, PCR amplification and sequencing of 16S rRNA. The water column and sediment shared the same phylotypes, thereby confirming the potential for seawater dispersal. The abundance of TFEs was estimated by most probable number enumeration, rates of VFA production, and released amounts of dipicolinic acid during germination. The surface sediment contained ~105-106 inducible TFEs cm-3. TFEs thus outnumber thermophilic sulfate-reducing Endospores by an order of magnitude. The abundance of cultivable TFEs decreased exponentially with sediment depth with a half-life of 350 years. We estimate that 6 x109 anaerobic thermophilic Endospores are deposited on the seafloor per m2 per year in Aarhus Bay, and that these thermophiles represent >10% of the total endospore community in the surface sediment.
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Endospores of thermophilic bacteria as tracers of microbial dispersal by ocean currents
The ISME Journal, 2014Co-Authors: Albert Leopold Müller, Ilias Lagkouvardos, Casey R J Hubert, Júlia Rosa De Rezende, Kasper Urup Kjeldsen, Bo Barker Jørgensen, David Berry, Alexander LoyAbstract:Microbial biogeography is influenced by the combined effects of passive dispersal and environmental selection, but the contribution of either factor can be difficult to discern. As thermophilic bacteria cannot grow in the cold seabed, their inactive spores are not subject to environmental selection. We therefore conducted a global experimental survey using thermophilic Endospores that are passively deposited by sedimentation to the cold seafloor as tracers to study the effect of dispersal by ocean currents on the biogeography of marine microorganisms. Our analysis of 81 different marine sediments from around the world identified 146 species-level 16S rRNA phylotypes of endospore-forming, thermophilic Firmicutes . Phylotypes showed various patterns of spatial distribution in the world oceans and were dispersal-limited to different degrees. Co-occurrence of several phylotypes in locations separated by great distances (west of Svalbard, the Baltic Sea and the Gulf of California) demonstrated a widespread but not ubiquitous distribution. In contrast, Arctic regions with water masses that are relatively isolated from global ocean circulation (Baffin Bay and east of Svalbard) were characterized by low phylotype richness and different compositions of phylotypes. The observed distribution pattern of thermophilic Endospores in marine sediments suggests that the impact of passive dispersal on marine microbial biogeography is controlled by the connectivity of local water masses to ocean circulation.
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Pre‐column liquid chromatographic determination of dipicolinic acid from bacterial Endospores
Limnology and Oceanography-methods, 2012Co-Authors: Bente Aa Lomstein, Bo Barker JørgensenAbstract:A high performance liquid chromatographic (HPLC) method for fluorescence detection of dipicolinic acid (DPA), a unique component of bacterial Endospores, was developed for rapid and highly sensitive quantification of Endospores. DPA was pre-column complexed to the lanthanide metal terbium (Tb3+). Surplus free Tb3+ and the Tb3+-DPA complex were separated by reverse-phase gradient chromatography and the fluorescence emission was read at 545 nm after exitation at 270 nm. The limits of quantification (LOQ) and detection (LOD) were 0.26 and 0.08 nM, respectively. The method was used to detect DPA extracted from Bacillus subtilis Endospores, DPA extracted from B. subtilis Endospores added to more complex substrates such as marine sediment and canned tuna and DPA extracted from indigenous Endospores in a coastal marine sediment. Endospores represented 4% of the microbial community (bacterial cells + Endospores) in active surface sediment from the Aarhus Bay.
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pre column liquid chromatographic determination of dipicolinic acid from bacterial Endospores
Limnology and Oceanography-methods, 2012Co-Authors: Bente Aa Lomstein, Bo Barker JørgensenAbstract:A high performance liquid chromatographic (HPLC) method for fluorescence detection of dipicolinic acid (DPA), a unique component of bacterial Endospores, was developed for rapid and highly sensitive quantification of Endospores. DPA was pre-column complexed to the lanthanide metal terbium (Tb3+). Surplus free Tb3+ and the Tb3+-DPA complex were separated by reverse-phase gradient chromatography and the fluorescence emission was read at 545 nm after exitation at 270 nm. The limits of quantification (LOQ) and detection (LOD) were 0.26 and 0.08 nM, respectively. The method was used to detect DPA extracted from Bacillus subtilis Endospores, DPA extracted from B. subtilis Endospores added to more complex substrates such as marine sediment and canned tuna and DPA extracted from indigenous Endospores in a coastal marine sediment. Endospores represented 4% of the microbial community (bacterial cells + Endospores) in active surface sediment from the Aarhus Bay.
Michael G Ganzle - One of the best experts on this subject based on the ideXlab platform.
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the copy number of the spova2mob operon determines pressure resistance of bacillus Endospores
Applied and Environmental Microbiology, 2019Co-Authors: Zhen Li, Felix Schottroff, David J Simpson, Michael G GanzleAbstract:The spoVA2mob operon confers heat resistance to Bacillus spp., and the resistance correlates to the copy number of the operon. Bacillus Endospores also exhibit a strong variation in resistance to pressure, but the underlying mechanisms of endospore resistance to pressure are not fully understood. We determined the effects of multiple spoVA2mob operons on high-pressure resistance in Bacillus Endospores. The copy numbers of the spoVA2mob operon in 17 strains of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus velezensis, and Bacillus pumilus were determined via droplet digital PCR (ddPCR) and genome sequencing. These strains contained between 0 and 3 copies of the spoVA2mob operon; the quantification of the gene copy number by ddPCR was as accurate as whole-genome sequencing. We further tested the pressure resistance of 17 Bacillus Endospores at 600 MPa and 80°C. Strains with one or no spoVA2mob operon had significantly lower pressure resistance than strains with two or three copies of the operons (P < 0.001), indicating that redundant spoVA2mob operons in Bacillus contributed to higher pressure resistance of Endospores. The copy number of the spoVA2mob operon was not related to the dipicolinic acid (DPA) content of Endospores. Overall, the copy number of the spoVA2mob operon contributes to pressure resistance of Bacillus Endospores. This improves our understanding of the pressure resistance mechanisms in Bacillus spp. and may inform the development of high-pressure sterilization in food processing.IMPORTANCEBacillus spp. are considered pressure-resistant microorganisms, but the resistance mechanisms remain unknown. The spoVA2mob operon is a mobile genetic element, and it can transfer to pathogenic or spoilage organisms by horizontal gene transfer. Results in this study indicate that multiple copies of the spoVA2mob operon mediate high-pressure resistance of Bacillus Endospores, and it might contribute to the identification of the source of pressure-resistant pathogens and spoilage organisms that may contaminate the food supply. The droplet digital PCR (ddPCR) system is well suited for analysis in some human diseases due to its high efficiency and capability to provide high precision; however, no relevant studies in food microbiology have been reported so far. This study demonstrates a novel application of ddPCR in food microbiology.
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effects of nisin and reutericyclin on resistance of Endospores of clostridium spp to heat and high pressure
Food Microbiology, 2013Co-Authors: Simmon Hofstetter, Michael G Ganzle, David Gebhardt, Linda Ho, Lynn M McmullenAbstract:Abstract The effects of high pressure, temperature, and antimicrobial compounds on Endospores of Clostridium spp. were examined. Minimal inhibitory concentrations (MIC) of nisin and reutericyclin were determined for vegetative cells and Endospores of Clostridium sporogenes ATCC 7955, Clostridium beijerinckii ATCC 8260, and Clostridium difficile 3195. Endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 were exposed to 90 °C and 90 °C/600 MPa in the presence of 16 mg L −1 nisin or 6.4 mg L −1 reutericyclin for 0–60 min in a 0.9% saline solution. Dipicolinic acid (DPA) release was measured using a terbium-DPA fluorescence assay, and endospore permeability was assessed using 4′,6-diamidino-2-phenylindole (DAPI) fluorescence. Vegetative cells of C. sporogenes ATCC 7955 exhibited higher sensitivity to nisin relative to Endospores, with MIC values 0.23 ± 0.084 mg L −1 and 1.11 ± 0.48 mg L −1 , respectively. Nisin increased DPA release when Endospores were treated at 90 °C; however, only C. sporogenes ATCC 7955 exhibited higher inactivation, suggesting strain or species specific effects. Reutericyclin did not enhance spore inactivation or DPA release. Use of nisin in combination with high pressure, thermal treatments enhanced inactivation of Endospores of Clostridium spp. and may have application in foods.
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use of the fluorescent probe laurdan to label and measure inner membrane fluidity of Endospores of clostridium spp
Journal of Microbiological Methods, 2012Co-Authors: Simmon Hofstetter, Christian Denter, Roland Winter, Lynn M Mcmullen, Michael G GanzleAbstract:Abstract A method for measuring the fluidity of inner membranes of populations of Endospores of Clostridium spp. with a fluorescent dye was developed. Cells of Clostridium beijerinckii ATCC 8260 and Clostridium sporogenes ATCC 7955 were allowed to sporulate in the presence of 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) on a soil-based media. Labeling of Endospores with LAURDAN did not affect endospore viability. Removal of the outer membranes of Endospores was done using a chemical treatment and confirmed using transmission electron microscopy (TEM). Two-photon confocal laser scanning microscopy (CLSM), and generalized polarization (GP) measurements were used to assess fluorescence of Endospores. Lipid composition analysis of cells and Endospores was done to determine whether differences in GP values are attributable to differences in membrane composition. Removal of the outer membranes of Endospores did not significantly impact GP values. Decoated, labeled Endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 exhibited GP values of 0.77 ± 0.031 and 0.74 ± 0.027 respectively. Differences in ratios of fatty acids between cells and Endospores are unlikely to be responsible for high GP values observed in Endospores. These GP values indicate high levels of lipid order and the exclusion of water from within inner membranes of Endospores.
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comparison of pressure and heat resistance of clostridium botulinum and other Endospores in mashed carrots
Journal of Food Protection, 2004Co-Authors: Dirk Margosch, Michael G Ganzle, Matthias A Ehrmann, Rudi F VogelAbstract:Inactivation of bacterial Endospores in food requires a combination of pressure and moderate heat. Endospore resistance of seven Clostridium botulinum strains was compared with those of Bacillus spp. (B. cereus, B. subtilis, B. licheniformis, B. smithii, B. amyloliquefaciens) and Thermoanaerobacterium thermosaccharolyticum with respect to pressure (600 to 800 MPa) and temperature (80 to 116°C) treatments in mashed carrots. A large variation was observed in the pressure resistance of C. botulinum spores. Their reduction after treatments with 600 MPa at 80°C for 1 s ranged from more than 5.5 log units to no reduction. Spores of the proteolytic C. botulinum TMW 2.357 exhibited a greater resistance to pressure than spores from all other bacteria examined, with the exception of B. amyloliquefaciens. Heat resistance of spores did not correlate with pressure resistance, either within strains of C. botulinum or when C. botulinum spores were compared with spores of T. thermosaccharolyticum. A quantitative release ...