Shiwei Wang

Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy

UNLABELLED The Gram-positive spore-forming anaerobe Clostridium difficile is a leading cause of nosocomial diarrhea. Spores of C. difficile initiate infection when triggered to germinate by bile salts in the gastrointestinal tract. We analyzed germination kinetics of individual C. difficile spores using Raman spectroscopy and differential interference contrast (DIC) microscopy. Similar to Bacillus spores, individual C. difficile spores germinating with taurocholate plus glycine began slow leakage of a ∼15% concentration of a chelate of Ca(2+) and dipicolinic acid (CaDPA) at a heterogeneous time T1, rapidly released CaDPA at Tlag, completed CaDPA release at Trelease, and finished peptidoglycan cortex hydrolysis at Tlysis. T1 and Tlag values for individual spores were heterogeneous, but ΔTrelease periods (Trelease - Tlag) were relatively constant. In contrast to Bacillus spores, heat treatment did not stimulate spore germination in the two C. difficile strains tested. C. difficile spores did not germinate with taurocholate or glycine alone, and different bile salts differentially promoted spore germination, with taurocholate and taurodeoxycholate being best. Transient exposure of spores to taurocholate plus glycine was sufficient to commit individual spores to germinate. C. difficile spores did not germinate with CaDPA, in contrast to B. subtilis and C. perfringens spores. However, the detergent dodecylamine induced C. difficile spore germination, and rates were increased by spore coat removal although cortex hydrolysis did not follow Trelease, in contrast with B. subtilis. C. difficile spores lacking the cortex-lytic enzyme, SleC, germinated extremely poorly, and cortex hydrolysis was not observed in the few sleC spores that partially germinated. Overall, these findings indicate that C. difficile and B. subtilis spore germination exhibit key differences. IMPORTANCE Spores of the Gram-positive anaerobe Clostridium difficile are responsible for initiating infection by this important nosocomial pathogen. When exposed to germinants such as bile salts, C. difficile spores return to life through germination in the gastrointestinal tract and cause disease, but their germination has been studied only with population-wide measurements. In this work we used Raman spectroscopy and DIC microscopy to monitor the kinetics of germination of individual C. difficile spores, the commitment of spores to germination, and the effect of germinant type and concentration, sublethal heat shock, and spore decoating on germination. Our data suggest that the order of germination events in C. difficile spores differs from that in Bacillus spores and provide new insights into C. difficile spore germination.

Slow leakage of Ca-dipicolinic acid from individual Bacillus spores during initiation of spore germination

When exposed to nutrient or nonnutrient germinants, individual Bacillus spores can return to life through germination followed by outgrowth. Laser tweezers, Raman spectroscopy, and either differential interference contrast or phase-contrast microscopy were used to analyze the slow dipicolinic acid (DPA) leakage (normally ∼20% of spore DPA) from individual spores that takes place prior to the lag time, Tlag, when spores begin rapid release of remaining DPA. Major conclusions from this work with Bacillus subtilis spores were as follows: (i) slow DPA leakage from wild-type spores germinating with nutrients did not begin immediately after nutrient exposure but only at a later heterogeneous time T1; (ii) the period of slow DPA leakage (ΔTleakage = Tlag - T1) was heterogeneous among individual spores, although the amount of DPA released in this period was relatively constant; (iii) increases in germination temperature significantly decreased T1 times but increased values of ΔTleakage; (iv) upon germination with l-valine for 10 min followed by addition of d-alanine to block further germination, all germinated spores had T1 times of less than 10 min, suggesting that T1 is the time when spores become committed to germinate; (v) elevated levels of SpoVA proteins involved in DPA movement in spore germination decreased T1 and Tlag times but not the amount of DPA released in ΔTleakage; (vi) lack of the cortex-lytic enzyme CwlJ increased DPA leakage during germination due to longer ΔTleakage times in which more DPA was released; and (vii) there was slow DPA leakage early in germination of B. subtilis spores by the nonnutrients CaDPA and dodecylamine and in nutrient germination of Bacillus cereus and Bacillus megaterium spores. Overall, these findings have identified and characterized a new early event in Bacillus spore germination.

Use of Raman Spectroscopy and Phase-Contrast Microscopy To Characterize Cold Atmospheric Plasma Inactivation of Individual Bacterial Spores

ABSTRACT Raman spectroscopy and phase-contrast microscopy were used to examine calcium dipicolinate (CaDPA) levels and rates of nutrient and nonnutrient germination of multiple individual Bacillus subtilis spores treated with cold atmospheric plasma (CAP). Major results for this work include the following: (i) >5 logs of spores deposited on glass surfaces were inactivated by CAP treatment for 3 min, while deposited spores placed inside an impermeable plastic bag were inactivated only ∼2 logs in 30 min; (ii) >80% of the spores treated for 1 to 3 min with CAP were nonculturable and retained CaDPA in their core, while >95% of spores treated with CAP for 5 to 10 min lost all CaDPA; (iii) Raman measurements of individual CAP-treated spores without CaDPA showed differences from spores that germinated with l-valine in terms of nucleic acids, lipids, and proteins; and (iv) 1 to 2 min of CAP treatment killed 99% of spores, but these spores still germinated with nutrients or exogenous CaDPA, albeit more slowly and to a lesser extent than untreated spores, while spores CAP treated for >3 min that retained CaDPA did not germinate via nutrients or CaDPA. However, even after 1 to 3 min of CAP treatment, spores germinated normally with dodecylamine. These results suggest that exposure to the present CAP configuration severely damages a spores inner membrane and key germination proteins, such that the treated spores either lose CaDPA or can neither initiate nor complete germination with nutrients or CaDPA. Analysis of the various CAP components indicated that UV photons contributed minimally to spore inactivation, while charged particles and reactive oxygen species contributed significantly. IMPORTANCE Much research has shown that cold atmospheric plasma (CAP) is a promising tool for the inactivation of spores in the medical and food industries. However, knowledge about the effects of plasma treatment on spore properties is limited, especially at the single-cell level. In this study, Raman spectroscopy and phase-contrast microscopy were used to analyze CaDPA levels and kinetics of nutrient- and non-nutrient-germinant-induced germination of multiple individual spores of Bacillus subtilis that were treated by a planar CAP device. The roles of different plasma species involved in spore inactivation were also investigated. The knowledge obtained in this study will aid in understanding the mechanism(s) of spore inactivation by CAP and potentially facilitate the development of more effective and efficient plasma sterilization techniques in various applications.

Memory of Germinant Stimuli in Bacterial Spores

ABSTRACT Bacterial spores, despite being metabolically dormant, possess the remarkable capacity to detect nutrients and other molecules in their environment through a biochemical sensory apparatus that can trigger spore germination, allowing the return to vegetative growth within minutes of exposure of germinants. We demonstrate here that bacterial spores of multiple species retain memory of transient exposures to germinant stimuli that can result in altered responses to subsequent exposure. The magnitude and decay of these memory effects depend on the pulse duration as well as on the separation time, incubation temperature, and pH values between the pulses. Spores of Bacillus species germinate in response to nutrients that interact with germinant receptors (GRs) in the spores inner membrane, with different nutrient types acting on different receptors. In our experiments, B. subtilis spores display memory when the first and second germinant pulses target different receptors, suggesting that some components of spore memory are downstream of GRs. Furthermore, nonnutrient germinants, which do not require GRs, exhibit memory either alone or in combination with nutrient germinants, and memory of nonnutrient stimulation is found to be more persistent than that induced by GR-dependent stimuli. Spores of B. cereus and Clostridium difficile also exhibit germination memory, suggesting that memory may be a general property of bacterial spores. These observations along with experiments involving strains with mutations in various germination proteins suggest a model in which memory is stored primarily in the metastable states of SpoVA proteins, which comprise a channel for release of dipicolinic acid, a major early event in spore germination. IMPORTANCE Cellular memory is defined as a sustained response to a transient environmental stimulus, and yet its generation and storage have not been described in bacterial spores. We demonstrate here that bacterial spores of multiple species retain memory of transient exposures to germinant stimuli that can result in altered responses to subsequent exposure. Memory was induced by activation of germinant receptors (GRs) or by GR-independent germinants and was accessed by both GR-dependent and GR-independent germinants. Analysis of effects on memory of exposure to GR-dependent and GR-independent germinants as well as in spores lacking various germination proteins suggests a model in which memory is stored primarily in metastable states of SpoVA proteins which comprise a channel for release of spore dipicolinic acid. Spore memory can also significantly reduce the concentration of nutrient germinants necessary to trigger germination, and this may be used to respond to low levels of nutrient germinants. Cellular memory is defined as a sustained response to a transient environmental stimulus, and yet its generation and storage have not been described in bacterial spores. We demonstrate here that bacterial spores of multiple species retain memory of transient exposures to germinant stimuli that can result in altered responses to subsequent exposure. Memory was induced by activation of germinant receptors (GRs) or by GR-independent germinants and was accessed by both GR-dependent and GR-independent germinants. Analysis of effects on memory of exposure to GR-dependent and GR-independent germinants as well as in spores lacking various germination proteins suggests a model in which memory is stored primarily in metastable states of SpoVA proteins which comprise a channel for release of spore dipicolinic acid. Spore memory can also significantly reduce the concentration of nutrient germinants necessary to trigger germination, and this may be used to respond to low levels of nutrient germinants.

Effects of Steam Autoclave Treatment on Geobacillus stearothermophilus Spores

To determine the mechanism of autoclave killing of Geobacillus stearothermophilus spores used in biological indicators (BIs) for steam autoclave sterilization, and rates of loss of spore viability and a spore enzyme used in BIs.

Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant, Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy

Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T1) and reached a maximum after the completion of CaDPA release (Trelease) and spore cortex lysis (Tlysis).

Analysis of the Germination of Individual Clostridium sporogenes Spores with and without Germinant Receptors and Cortex-Lytic Enzymes

The Gram-positive spore-forming anaerobe Clostridium sporogenes is a significant cause of food spoilage, and it is also used as a surrogate for C. botulinum spores for testing the efficacy of commercial sterilization. C. sporogenes spores have also been proposed as a vector to deliver drugs to tumor cells for cancer treatments. Such an application of C. sporogenes spores requires their germination and return to life. In this study, Raman spectroscopy and differential interference contrast (DIC) microscopy were used to analyze the germination kinetics of multiple individual C. sporogenes wild-type and germination mutant spores. Most individual C. sporogenes spores germinated with L-alanine began slow leakage of ∼5% of their large Ca-dipicolinic acid (CaDPA) depot at T1, all transitioned to rapid CaDPA release at Tlag1, completed CaDPA release at Trelease, and finished peptidoglycan cortex hydrolysis at Tlys. T1, Tlag1, Trelease, and Tlys times for individual spores were heterogeneous, but ΔTrelease (Trelease – Tlag1) periods were relatively constant. However, variability in T1 (or Tlag1) times appeared to be the major reason for the heterogeneity between individual spores in their germination times. After Trelease, some spores also displayed another lag in rate of change in DIC image intensity before the start of a second obvious DIC image intensity decline of 25–30% at Tlag2 prior to Tlys. This has not been seen with spores of other species. Almost all C. sporogenes spores lacking the cortex-lytic enzyme (CLE) CwlJ spores exhibited a Tlag2 in L-alanine germination. Sublethal heat treatment potentiated C. sporogenes spore germination with L-alanine, primarily by shortening T1 times. Spores without the CLEs SleB or CwlJ exhibited greatly slowed germination with L-alanine, but spores lacking all germinant receptor proteins did not germinate with L-alanine. The absence of these various germination proteins also decreased but did not abolish germination with the non-GR-dependent germinants dodecylamine and CaDPA, but spores without CwlJ exhibited no germination with CaDPA. Finally, C. sporogenes spores displayed commitment in germination, but memory in GR-dependent germination was small, and less than the memory in Bacillus spore germination.

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

ABSTRACT 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.