Individual Spores Research Articles

Visualization of Germination Proteins in Putative Bacillus cereus Germinosomes.

Bacillus cereus can survive in the form of spores for prolonged periods posing a serious problem for the manufacture of safe shelf-stable foods of optimal quality. Our study aims at increasing knowledge of B. cereus spores focusing primarily on germination mechanisms to develop novel milder food preservation strategies. Major features of B. cereus spores are a core with the genetic material encased by multiple protective layers, an important one being the spores′ inner membrane (IM), the location of many important germination proteins. To study mechanisms involved in germination of B. cereus spores, we have examined the organization of germinant receptors (GRs) in spores′ IM. Previous studies have indicated that in spores of B. cereus ATCC 14579 the L-alanine responsive GR, GerR, plays a major role in the germination process. In our study, the location of the GerR GR subunit, GerRB, in spores was examined as a C-terminal SGFP2 fusion protein expressed under the control of the gerR operon′s promoter. Our results showed that: (i) the fluorescence maxima and integrated intensity in spores with plasmid-borne expression of GerRB-SGFP2 were significantly higher than in wild-type spores; (ii) western blot analysis confirmed the expression of the GerRB-SGFP2 fusion protein in spores; and (iii) fluorescence microscopy visualized GerRB-SGFP2 specific bright foci in ~30% of individual dormant spores if only GerRB-SGFP2 was expressed, but, noticeably, in ~85% of spores upon co-expression with GerRA and GerRC. Our data corroborates the notion that co-expression of GR subunits improves their stability. Finally, all spores displayed bright fluorescent foci upon expression of GerD-mScarlet-I under the control of the gerD promoter. We termed all fluorescent foci observed germinosomes, the term used for the IM foci of GRs in Bacillus subtilis spores. Our data are the first evidence for the existence of germinosomes in B. cereus spores.

The impact of high power ultrasound for controlling spoilage by Alicyclobacillus acidoterrestris: A population and a single spore assessment

Consumer demand for healthier, microbiologically safe and stable, higher quality and minimally processed foods has increased in the last decades, promoting the application of non-thermal process technologies. Ultrasound processing is gaining attention because of its potential for improving quality and safety while retaining product flavor, texture, color and nutrient composition. Recently, Alicyclobacillus acidoterrestris has been recognized by manufacturers and processors in the fruit industry as a new type of thermoacidophilic, endospore-forming spoilage bacterium for commercialized fruit juices that can withstand the high temperatures applied in pasteurization process and spoil them producing taint compounds. Based on the above, the present study was undertaken to investigate the separate and combined effect of ultrasound treatment operating at 26 kHz, 90 μm, 200 W for 5, 10, 15 min and heat stress (at 80 °C for 10 min) on the recovery of A. acidoterrestris spores in K broth adjusted to pH = 4.5 with 25% (w/v) citric acid at temperatures between 35 and 45 °C at population and individual spore level. At the population level, statistically significant differences in average lag time of A. acidoterrestris spores from different treatments at 35 and 45 °C have been found. After the applied ultrasound (at 100% Amplitude for 10 min) and heat shock (at 80 °C for 10 min), the average lag time of A. acidoterrestris spores at 35 °C (7.24 ± 0.34 h) and 45 °C (6.38 ± 0.18 h) has been shown to be longer compared to the control at 35 °C (5.68 ± 0.36 h) and at 45 °C (2.87 ± 0.19 h), while for this combination, the maximum specific growth rate was not significantly different from the control samples. Additionally, the findings of this study have shown that λ among individual spores was greatly variable when they were treated by ultrasound (at 100% Amplitude for 10 min) and/or thermal treatment (at 80 °C for 10 min). The application of ultrasound and thermal treatment resulted in a more pronounced effect on median lag phase duration (25.74 h) than the heat shock (11.63 h) and affected both the position and the spread of the λ distributions of A. acidoterrestris spores. This work would help to better assess and optimize the proposed combined treatments, since ultrasound and thermal treatments could work in a synergistic way on delaying the lag time of the tested bacterial spores.

Describing the Individual Spore Variability and the Parameter Uncertainty in Bacterial Survival Kinetics Model by Using Second-Order Monte Carlo Simulation.

The objective of this study was to separately describe the fitting uncertainty and the variability of individual cell in bacterial survival kinetics during isothermal and non-isothermal thermal processing. The model describing bacterial survival behavior and its uncertainties and variabilities during non-isothermal inactivation was developed from survival kinetic data for Bacillus simplex spores under fifteen isothermal conditions. The fitting uncertainties in the parameters used in the primary Weibull model was described by using the bootstrap method. The variability of individual cells in thermotolerance and the true randomness in the number of dead cells were described by using the Markov chain Monte Carlo (MCMC) method. A second-order Monte Carlo (2DMC) model was developed by combining both the uncertainties and variabilities. The 2DMC model was compared with reduction behavior under three non-isothermal profiles for model validation. The bacterial death estimations were validated using experimentally observed surviving bacterial count data. The fitting uncertainties in the primary Weibull model parameters, the individual thermotolerance heterogeneity, and the true randomness of inactivated spore counts were successfully described under all the iso-thermal conditions. Furthermore, the 2DMC model successfully described the variances in the surviving bacterial counts during thermal inactivation for all three non-isothermal profiles. As a template for risk-based process designs, the proposed 2DMC simulation approach, which considers both uncertainty and variability, can facilitate the selection of appropriate thermal processing conditions ensuring both food safety and quality.

Timing of fungal spore release dictates survival during atmospheric transport

Fungi disperse spores to move across landscapes and spore liberation takes different patterns. Many species release spores intermittently; others release spores at specific times of day. Despite intriguing evidence of periodicity, why (and if) the timing of spore release would matter to a fungus remains an open question. Here we use state-of-the-art numerical simulations of atmospheric transport and meteorological data to follow the trajectory of many spores in the atmosphere at different times of day, seasons, and locations across North America. While individual spores follow unpredictable trajectories due to turbulence, in the aggregate patterns emerge: Statistically, spores released during the day fly for several days, whereas spores released at night return to ground within a few hours. Differences are caused by intense turbulence during the day and weak turbulence at night. The pattern is widespread but its reliability varies; for example, day/night patterns are stronger in southern regions. Results provide testable hypotheses explaining both intermittent and regular patterns of spore release as strategies to maximize spore survival in the air. Species with short-lived spores reproducing where there is strong turbulence during the day, for example in Mexico, maximize survival by releasing spores at night. Where cycles are weak, for example in Canada during fall, there is no benefit to releasing spores at the same time every day. Our data challenge the perception of fungal dispersal as risky, wasteful, and beyond control of individuals; our data suggest the timing of spore liberation may be finely tuned to maximize fitness during atmospheric transport.

Pseudocercospora exilis Causing Leaf Spot Disease on Brassica rapa subsp. parachinensis in China

Chinese flowering cabbage (Brassica rapa L. subsp. parachinensis) is an important vegetable crop of China and widely cultivated in the southern provinces. During February of 2017 to 2019, a severe leaf spot disease was seen on Chinese flowering cabbage plants in different agricultural fields around Lianzhou and Guangzhou cities of Guangdong province, China. Disease incidence reached up to 70% in severely infested fields. Early disease symptoms observed were small circular or irregular spots of brown color, 3 to 11 mm in diameter and often coalesced. Ten symptomatic leaves were cut into small pieces (5 × 5 mm), surface sterilized with 1% NaOCl, plated onto potato dextrose agar (PDA) medium, and incubated at room temperature for 7 days under continuous fluorescent light. A total of 11 pathogen isolates were purified by successively subculturing on new PDA medium plates using the single-spore technique. A cercosporoid fungus was consistently isolated from diseased leaves. The colonies were raised and greenish black in color. Conidiophores were brown, subcylindrical, multiseptate, straight or slightly curved, unbranched, and smooth. Conidiogenous cells were pale brown, smooth, and arranged terminally. Conidia were pale brown, smooth, obclavate or fusoid, straight to slightly curved, with obtuse apex, one- to seven-septate, and 44.3 to 69.12 × 4.8 to 6.2 μm (n = 20). The morphological and cultural characteristics of all isolates were consistent with Pseudocercospora exilis Hern.-Gut. & Dianese (Hernandez-Gutierrez and Dianese 2009; Silva et al. 2016). One monosporic isolate (PE-1) was obtained by plating individual spores on V8 agar medium. To further confirm the species, genomic DNA was extracted from the hyphal mass of the isolate PE-1 using the DZup fungal DNA extraction kit (Sangon Biotech, Shanghai, China). The internal transcribed spacer (ITS) and translational elongation factor (EF-1α) regions of the isolate PE-1 were amplified with primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999) and sequenced by Sangon Biotech. The obtained DNA sequences were deposited in GenBank (accessions MN971630 for the ITS sequence and MN991198 for the EF-1α). BLAST analysis showed that the ITS and EF-1α sequences shared 99% nucleotide identity with the previously submitted P. exilis sequences (GenBank accessions NR147302 and KT290193 for ITS and EF-1α, respectively). Pathogenicity testing was performed by spraying conidial suspension (1 × 10⁶ conidia/ml) of isolate PE-1 on the leaves of 40-day-old Chinese flowering cabbage plants. Five plants were used for inoculation. Another five plants were sprayed with distilled sterilized water to serve as a control. Plants were kept in transparent boxes for 2 days to retain moisture and subsequently incubated in a growth room at 28 ± 2°C with a 12-h photoperiod. Similar leaf spots were observed on the leaves of all inoculated plants after 1 week of inoculation. No symptoms were seen on control plants. The same pathogen was reisolated consistently from inoculated leaves. The species identity was confirmed by both morphological and molecular characteristics, thus fulfilling Koch’s postulates. Previously, P. exilis has been reported to cause leaf spot disease on Chamaecrista orbiculata in Brazil (Hernandez-Gutierrez and Dianese 2009). To our knowledge, this is the first report of leaf spot on Chinese flowering cabbage caused by P. exilis. This disease can cause severe economic losses, because Chinese flowering cabbage is mainly sold in the market and used in salads or cooked dishes. Timely measures must be taken to manage this disease.

Cyclic di-GMP in Streptomycetes: A New Conformation, New Binding Mode, New Receptor, and a New Mechanism to Control Cell Development.

A recent paper by Gallagher etal. (2020) demonstrates that c-di-GMP controls spore formation in Streptomyces venezuelae through sequestering the sporulation sigma factor σWhiG and presents the crystal structure of a ternary complex between c-di-GMP, σWhiG, and its anti-sigma factor, RsiG.

Understanding the Effects of High Pressure on Bacterial Spores Using Synchrotron Infrared Spectroscopy.

Bacterial spores are extremely resistant life-forms that play an important role in food spoilage and foodborne disease. The return of spores to a vegetative cell state is a three-step process, these being activation, germination, and emergence. High-pressure (HP) processing is known to induce germination in part of the spore population and even to inactivate a high number of Bacillus spores when combined with other mild treatments such as the addition of nisin. The aim of the present work was to investigate the mechanisms involved in the sensitization of spores to nisin following HP treatment at ambient temperature or with moderate heating leading to a heterogeneous spore response. Bacillus subtilis spores were subjected to HP treatment at 500 MPa at 20 and 50°C. The physiological state of different subpopulations was characterized. Then Fourier transform infrared (FTIR) microspectroscopy coupled to a synchrotron infrared source was used to explore the heterogeneity of the biochemical signatures of the spores after the same HP treatments. Our results confirm that HP at 50°C induces the germination of a large proportion of the spore population. HP treatment at 20°C generated a subpopulation of ungerminated spores reversibly sensitized to the presence of nisin in their growth medium. Regarding infrared spectra of individual spores, spores treated by HP at 50°C and germinated spores had similar spectral signatures involving the same structural properties. However, after HP was performed at 20°C, two groups of spores were distinguished; one of these groups was clearly identified as germinated spores. The second group displayed a unique spectral signature, with shifts in the spectral bands corresponding to changes in membrane fluidity. Besides, spores spectra in the amide region could be divided into several groups close to spectral properties of dormant, germinated, or inactivated spores. The part of the spectra corresponding to α-helix and β-sheet-structures contribute mainly to the spectral variation between spores treated by HP at 20°C and other populations. These changes in the lipid and amide regions could be the signature of reversible changes linked to spore activation.

Prediction of Aerosolized Fungal Spores by Determining Respiratory Intensity of the Growing Colony

ABSTRACT Aerosolization of fungal spores from moldy surfaces can cause adverse health effects. The spores of an older colony are more easily detached than those of a younger colony. When the growth age of a colony is known, the aerosolization of spores can be more accurately predicted. This study proposed the adoption of respiratory intensity as an indicator of the growth age of Aspergillus niger colonies. Respiratory intensity is the mass of carbon dioxide produced by the respiration of a colony per unit quantity of spores within a certain time. Two types of colonies, with homogeneous and heterogeneous age distributions, were aerosolized in a wind tunnel to determine the spore release proportion and validate the method. The results revealed that the respiratory intensity of the homogeneous colony is closely correlated with the growth age when the age of the colony is less than 14 days under standard growth conditions. The spore release proportion increases with the growth age and the blowing speed. The predicted spore release proportion for a heterogeneous colony is more precise when the colony is divided into multiple sub-colonies to characterize the growth age for each individual spore release proportion. The relative errors of the predicted spore release proportions as compared with the test values were less than 10%.

Method for RNA extraction and transcriptomic analysis of single fungal spores

Transcriptomic analysis of single cells has been increasingly in demand in recent years, thanks to technological and methodological advances as well as growing recognition of the importance of individuals in biological systems. However, the majority of these studies have been performed in mammalian cells, due to their ease of lysis and high RNA content. No single cell transcriptomic analysis has yet been described in microbial spores, even though it is known that heterogeneity at the phenotype level exists among individual spores. Transcriptomic analysis of single spores is challenging, in part due to the physically robust nature of the spore wall. This precludes the use of methods commonly used for mammalian cells. Here, we describe a simple method for extraction and amplification of transcripts from single fungal conidia (asexual spores), and its application in single-cell transcriptomics studies. The method can also be used for studies of small numbers of fungal conidia, which may be necessary in the case of limited sample availability, low-abundance transcripts or interest in small subpopulations of conidia.•The method allows detection of transcripts from single conidia of Aspergillus niger•The method allows detection of genomic DNA from single conidia of Aspergillus niger

A late Holocene record of human impacts on tropical environments from non-pollen palynomorphs, Albertine Rift, western Uganda

AbstractNon-pollen palynomorphs and elemental geochemistry data from Lake Kifuruka in western Uganda provide evidence of environmental change in the tropical African region since the beginning of the Holocene. The multi-proxy record presented here shows that dry conditions dominated the end of the Pleistocene evidenced by calcium enriched sediments and suppressed fungal taxa activity. Moist conditions dominated the early Holocene and persisted until just after 1960 cal yr BP. Elevated frequencies of individual fungal spore taxa associated with herbivory and soil erosion, includingSordaria-type,Sporormiella-type,Chaetomium-type, andGlomus-type, about 4300 cal yr BP suggests a significant environmental change that could be linked to human activities. A convergence of multiple proxy data, including microscopic charcoal, elemental geochemistry, and fungal spores, strongly support the occurrence of anthropogenic forest disturbance in the Albertine Rift about 4300 cal yr BP.

First Report of Leaf Spot Caused byPseudocercospora vitisonBidens pilosain Brazil

Bidens pilosa L. (Asteraceae) is an aggressive weed in annual and perennial crops with high potential to reduce crop yields in several countries (Dias et al. 2017). It also may host pathogens such as Alternaria dauci (J.G. Kuhn) J.W. Groves & Skolko (Lin et al. 2015) and Podosphaera xanthii (Castagne) U. Braun & N. Shishkoff (Cho et al. 2013). In Brazil, this weed is distributed across all grape-producing areas. Leaf blight on grape, caused by Pseudocercospora vitis (Lev.) Speg., is an important disease that causes premature defoliation. In March 2018, plants of B. pilosa showing leaf spots were found in a 1-ha organic orchard of fox grape cultivar Bordo in the metropolitan region of Curitiba in Parana state, Brazil. The orchard had high leaf blight incidence (above 80%). The disease normally started in early summer; in December, and around March, complete defoliation occurred caused by leaf blight. B. pilosa was distributed throughout the orchard, and around 50% of leaves were infected. Leaf spots initially appeared slightly chlorotic, becoming pale brown to dark brown. Lesions were circular or irregular, 2 to 8 mm in diameter, and often coalesced. Microscopic examination revealed that conidia were hyaline to light brown, cylindrical, straight or slightly curved, with a rounded apex. They had 4 to 10 transverse septa, 24 to 82 μm in length and 5 to 8 μm in width, with conidiophores grouped in synnema. Isolates were recovered from lesions on tissue surfaces that were disinfested by immersion in 70% alcohol for 30 s and then in 0.5% NaClO for 30 s. Pieces were washed three times with sterile distilled water and cultured on potato dextrose agar (PDA) at room temperature for 9 days under continuous fluorescent light. Hyphal tips of the obtained colony were transferred to V8 agar medium and cultured at room temperature for 4 weeks under continuous fluorescent light. Individual spores were collected and deposited in V8 agar medium to obtain one monosporic isolate (PCLEMID02). The colony presented slow growth, with 15-mm diameter after 1 week of PDA cultivation at 25°C under a 12-h photoperiod, and was dark gray on top, black on reverse. The morphological and cultural characteristics of the isolate were consistent with those described for Pseudocercospora sp. (Ellis 1971). To confirm the species, the nuclear gene regions ITS, ACT, and EF-1α of isolate PCLEMID02 were amplified and sequenced for multigene phylogenetic analysis. The DNA sequences generated were deposited in GenBank (MH330685, MH891492, and MH891491). Phylogenetic analysis was performed with the concatenated sequence alignment of three genes using MEGA 6.0 software. The neighbor-joining phylogenetic tree showed that isolate PCLEMID02 from B. pilosa clustered in one separate clade with 100% bootstrap support with sequences of P. vitis (GenBank GU269829.1, GU320533.1, and GU384541.1). To perform the pathogenicity test, a suspension containing 1 × 10⁴ conidia/ml was prepared from one isolate (PCLEMID02) with 3-week-old cultures grown on V8 agar. The conidial suspension was sprayed on the leaf surface of B. pilosa and grapes. It used 20 leaves ranging in age from 30 to 45 days. Another 20 leaves received water instead of inoculum and served as controls. All leaves were left in a moist chamber for 24 h and subsequently stored in a greenhouse at 28 ± 2°C with a 12-h photoperiod. Only the inoculated leaves showed symptoms. Leaf blight on grape and leaf spots on B. pilosa were observed 12 and 25 days after inoculation, respectively. Both the inoculated leaves and the controls were submitted to the reisolation process. The pathogen was effectively reisolated only on symptomatic leaves and presented the same cultural, morphological, and molecular characteristics as the colony used for inoculation. Thus, B. pilosa is a host of P. vitis and may allow the survival of the pathogen in the dormancy period of the grapevine and, consequently, contribute primary inoculum for the development of leaf blight on grape.

Laser Tweezers Raman Spectroscopy (LTRS) to Detect Effects of Chlorine Dioxide on Individual Nosema bombycis Spores.

The microsporidium Nosema bombycis (Nb) causes pebrine, a fatal disease in sericulture. Nb is effectively killed by chlorine dioxide (ClO2); however, the precise killing mechanism remains unclear. We used laser tweezers Raman spectroscopy (LTRS) to monitor the action of ClO2 on individual Nb spores in real time. Raman peaks of ClO2 appeared in Nb spores, corresponding to decreased peaks of trehalose that gradually disappeared. A peak (1658 cm-1) corresponding to the protein α-helix significantly weakened while that (1668 cm-1) corresponding to irregular protein structures was enhanced; their intensities were negatively correlated in a certain time range and dependent on ClO2 concentration. The intensities of peaks at 782 cm-1 (nucleic acids) and 1004 cm-1 (phenylalanine of protein) did not change evidently even under extremely high ClO2 concentrations. Thus, ClO2 rapidly permeates the Nb spore wall, changing the protein secondary structure to lose biological function and destroy permeability, causing trehalose to leak out. These effects are ClO2 concentration-dependent, but no other obvious changes to biomacromolecules were detected. Single-cell analysis using LTRS is an effective method to monitor the action of chemical sporicides on microbes in real time, providing insight into the heterogeneity of cell stress resistance.

Single-cell analysis reveals individual spore responses to simulated space vacuum

Outer space is a challenging environment for all forms of life, and dormant spores of bacteria have been frequently used to study the survival of terrestrial life in a space journey. Previous work showed that outer space vacuum alone can kill bacterial spores. However, the responses and mechanisms of resistance of individual spores to space vacuum are unclear. Here, we examined spores’ molecular changes under simulated space vacuum (~10−5 Pa) using micro-Raman spectroscopy and found that this vacuum did not cause significant denaturation of spore protein. Then, live-cell microscopy was developed to investigate the temporal events during germination, outgrowth, and growth of individual Bacillus spores. The results showed that after exposure to simulated space vacuum for 10 days, viability of spores of two Bacillus species was reduced up to 35%, but all spores retained their large Ca2+-dipicolinic acid depot. Some of the killed spores did not germinate, and the remaining germinated but did not proceed to vegetative growth. The vacuum treatment slowed spore germination, and changed average times of all major germination events. In addition, viable vacuum-treated spores exhibited much greater sensitivity than untreated spores to dry heat and hyperosmotic stress. Among spores’ resistance mechanisms to high vacuum, DNA-protective α/β−type small acid-soluble proteins, and non-homologous end joining and base excision repair of DNA played the most important roles, especially against multiple cycles of vacuum treatment. Overall, these results give new insight into individual spore’s responses to space vacuum and provide new techniques for microorganism analysis at the single-cell level.

Enhanced method for High Spatial Resolution surface imaging and analysis of fungal spores using Scanning Electron Microscopy

Efficient, fast and new micro-analytical methods for characterization of ultrastructures of fungal spores with electron microscopy are very much required and essential. SEM analysis of biological materials, especially fungi, requires optimal preparation of the specimen and often requires the usage of dried samples which demands a challenging sample preparation. In the present investigation, we described a fast and improved method for the preparation of fungal specimen for scanning electron microscopy (SEM). The fungus, Curvularia lunata was grown on the surface of sterile Whatman No.1 filter paper which was overlaid on Potato Dextrose Agar (PDA) medium, gold coated immediately after removal from the growth medium and subjected to imaging. Generally, SEM imaging is done with samples that were fixed with chemical fixatives, dehydrated and gold coated specimens, but here we describe an easy and more efficient sample preparation for SEM which enabled enhanced image quality and precision visualization of fungal cells, especially the spores. The developed method has enabled the analysis of even the robust samples like fungal spores that to eliminating special temperature requirement. The ultimate goal was to develop an improved protocol/method applied to analysis of fungal spores with greater coverage about fungal specimen preparation. This method permits the use of rapid sample preparation and will allow us to imaging of individual spore or conidia structures in the context of fungal cell architecture which clarifies our understanding in fungal taxonomy/biology.

Vertical Transmission of Fusarium circinatum Mitoviruses FcMV1 and FcMV2-2 via Microconidia

Pine Pitch Canker disease, caused by the pathogenic fungus Fusarium circinatum, affects conifer species worldwide. However, the virulence of the pathogen may be affected by the presence of mycoviruses. The aim of this laboratory-based study was to investigate the probability and rate of transmission of F. circinatum mitoviruses FcMV1 and FcMV2-2 via microconidia. Ten isolates of mitovirus-infected F. circinatum were subcultured to produce a total of 100 single-spore colonies (ten replicates per isolate). The total RNA and cDNA obtained from each spore isolate (monosporic culture) were amplified by PCR with specific primers for detection of F. circinatum mitoviruses FcMV1 and FcMV2-2. The mitoviruses were detected in a high percentage of the individual spore isolates (between 60% and 100% depending on the fungal isolate). However, the probability of transmission was not statistically significantly associated with either the F. circinatum isolate or the viral strain. A high proportion of transmission via microconidia is critical for development of a biological control program against Pine Pitch Canker (PPC) disease in forests. However, further studies are needed to establish the effect of these mitoviruses on the virulence of F. circinatum.

Genotyping of individual Ceratonova shasta (Cnidaria: Myxosporea) myxospores reveals intra-spore ITS-1 variation and invalidates the distinction of genotypes II and III.

Genotypes of the myxosporean parasite Ceratonova shasta are defined by the number of ATC repeats in the parasite's ribosomal DNA internal transcribed spacer region 1. These genotypes correlate with specific salmonid fish hosts. We observed coho salmon (Oncorhynchus kisutch) and rainbow trout (Oncorhynchus mykiss) with mixtures of genotypes II and III, and assumed that this was a consequence of fish having an aggregate infection from multiple individual parasites. We hypothesized that although multiple ITS copies are present within a parasite spore, the DNA sequences of these copies are identical, and thus individual C. shasta spores are a single genotype. We tested this by extracting and sequencing DNA from individual myxospores. We trialed three approaches for in-tube DNA extraction; digestion with proteinase K was superior to simply rehydrating spores, or incubation in the buffer. Sequences from 14 myxospores were each a mixture of genotypes II and III. Therefore, intra-genomic ribosomal DNA variants exist within individual parasite spores, and II and III should no longer be regarded as discrete C. shasta genotypes. This single-spore genotyping approach will be a useful tool for testing validity of other C. shasta genotypes, and for correctly matching genotype with phenotype for mixed infections of other myxozoan species.

Germination, Outgrowth, and Vegetative-Growth Kinetics of Dry-Heat-Treated Individual Spores of Bacillus Species.

DNA damage kills dry-heated spores of Bacillus subtilis, but dry-heat-treatment effects on spore germination and outgrowth have not been studied. This is important, since if dry-heat-killed spores germinate and undergo outgrowth, toxic proteins could be synthesized. Here, Raman spectroscopy and differential interference contrast microscopy were used to study germination and outgrowth of individual dry-heat-treated B. subtilis and Bacillus megaterium spores. The major findings in this work were as follows: (i) spores dry-heat-treated at 140°C for 20 min lost nearly all viability but retained their Ca2+-dipicolinic acid (CaDPA) depot; (ii) in most cases, dry-heat treatment increased the average times and variability of all major germination events in B. subtilis spore germination with nutrient germinants or CaDPA, and in one nutrient germination event with B. megaterium spores; (iii) B. subtilis spore germination with dodecylamine, which activates the spore CaDPA release channel, was unaffected by dry-heat treatment; (iv) these results indicate that dry-heat treatment likely damages spore proteins important in nutrient germinant recognition and cortex peptidoglycan hydrolysis, but not CaDPA release itself; and (v) analysis of single spores incubated on nutrient-rich agar showed that while dry-heat-treated spores that are dead can complete germination, they cannot proceed into outgrowth and thus not to vegetative growth. The results of this study provide new information on the effects of dry heat on bacterial spores and indicate that dry-heat sterilization regimens should produce spores that cannot outgrow and thus cannot synthesize potentially dangerous proteins.IMPORTANCE Much research has shown that high-temperature dry heat is a promising means for the inactivation of spores on medical devices and spacecraft decontamination. Dry heat is known to kill Bacillus subtilis spores by DNA damage. However, knowledge about the effects of dry-heat treatment on spore germination and outgrowth is limited, especially at the single spore level. In the current work, Raman spectroscopy and differential interference contrast microscopy were used to analyze CaDPA levels in and kinetics of nutrient- and non-nutrient germination of multiple individual dry-heat-treated B. subtilis and Bacillus megaterium spores that were largely dead. The outgrowth and subsequent cell division of these germinated but dead dry-heat-treated spores were also examined. The knowledge obtained in this study will help understand the effects of dry heat on spores both on Earth and in space, and indicates that dry heat can be safely used for sterilization purposes.

Morphological and molecular characteristics of Kudoa scomberomori (Myxosporea: Kudoidae) firstly foundfrom the muscles of info-pacific king mackerel Scomberomorus guttatus in Quang Binh province, Vietnam

The genus Kudoa includes about 100 described species. In Vietnam, so far, only one species, Kudoa monodactyli, has been reported. During servey for parasites of marine fish in Quang Binh province, Vietnam in 2017, we found Myxozoan samples in 8 out of 15 Indo-Pacific king mackerel (Scomberomorus guttatus). They are identified as K. scomberomori based on the results of morphological and molecular analyses. A great number of individual spores form white pseudocysts with thin membrane within the muscle fibre. The pseudocysts were 0.1-0.3 × 0.4-0.7 mm. The shape of fresh spore like a flower petal. The spore of K. scomberomori was 6.65 ± 0.09 (6.53-6.76) µm in length, 7.42 ± 0.13 (7.29-7.58) µm in width and 6.55 ± 0.11 (6.40-6.65) µm in thickness. Each spore has six shell valves with same size. Each shell valve contained one polar capsule measuring 3.42 ± 0.09 (3.32-3.48) µm in length and 1.30 ± 0.14 (1.14-1.46) µm in width. This is the first report of K. scomberomori in Vietnam and Indo-Pacific king mackerel is recorded as a new host for this species. Citation: Nguyen Ngoc Chinh, Ha Duy Ngo, Nguyen Huu Duc, Nguyen Thuy Linh, Pham Ngoc Doanh, 2017. Morphological and molecular characteristics of Kudoa scomberomori (Myxosporea: Kudoidae) firstly foundfrom the muscles of info-pacific king mackerel Scomberomorus guttatus in Quang Binh province, Vietnam. Tap chi Sinh hoc, 40(1): x-xx. DOI: 10.15625/0866-7160/v40n1.10671. *Corresponding author: chinhnn89@gmail.com Received 13 September 2017, accepted 2 December 2017

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy.

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Effects of lowering water activity by various humectants on germination of spores of Bacillus species with different germinants

The effect of water activity (aw), as lowered by different dietary humectants, on the germination of Bacillus subtilis, Bacillus megaterium and Bacillus cereus spores with germinants that act by different mechanisms has been investigated and compared. Germination of spores of these species by all of the germinants investigated was inhibited as aw decreased, with the general order of efficacy for these non-ionic humectants being sucrose > trehalose > glycerol. The effect of lowering aw on germination by germinant receptor (GR)-dependent germinants was not appreciably altered by varying germinant concentrations, was generally not much more effective with spores lacking coats or an outer membrane, and was less pronounced with heat-activated spores. Analysis of the effect of aw on spore germination via different mechanisms showed that GR-dependent germination was least sensitive to aw, while germination via activation of spore cortex peptidoglycan hydrolysis or dipicolinic acid release was more sensitive. However, germination by high hydrostatic pressure was less sensitive to inhibition by low aw, than was germination by other germinants. Examination of the GR-dependent germination of individual spores indicated that aw acted most strongly in inhibiting the commitment step of germination, while exerting smaller effects on dipicolinic acid release or cortex peptidoglycan hydrolysis.