Characterization of Wet-Heat Inactivation of Single Spores of Bacillus Species by Dual-Trap Raman Spectroscopy and Elastic Light Scattering

Abstract

Dual-trap laser tweezers Raman spectroscopy (LTRS) and elastic light scattering (ELSBacterial spores of Bacillus species are formed in sporulation and are metabolically dormant and extremely resistant to a variety of harsh conditions, including heat, radiation, and many toxic chemicals (37). Since spores of these species are generally present in foodstuffs and cause food spoilage and food-borne disease (37, 38), there has long been interest in the mechanisms of both spore resistance and spore killing, especially for wet heat, the agent most commonly used to kill spores. The killing of dormant spores by wet heat generally requires temperatures about 40°C higher than those for the killing of growing cells of the same strain (37, 43). A number of factors influence spore wet-heat resistance, with a major factor being the spore core's water content, as spores with higher core water content are less wet-heat resistant than are spores with lower core water (15,25). The high level of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) and the types of its associated divalent cations, predominantly Ca 2ϩ , that comprise ϳ25% of the dry weight of the core also contribute to spore wet-heat resistance, although how low core water content and CaDPA protect spores against wet heat is not known. The protection of spore DNA against depurination by its saturation with a group of ␣/␤-type small, acid-soluble spore proteins also contributes to spore wet-heat resistance (14,23,33,37).Despite knowledge of a number of factors important in spore wet-heat resistance, the mechanism for wet-heat killing of spores is not known. Wet heat does not kill spores by DNA damage or oxidative damage (35, 37). Instead, spore killing by this agent is associated with protein denaturation and enzyme inactivation (2, 7, 44), although specific proteins for which damage causes spore death have not been identified. Wet-heat treatment also often results in the release of the spore core's large depot of CaDPA. The mechanism for this CaDPA release is not known but is presumably associated with the rupture of the spore's inner membrane (7). In addition, the relationship between protein denaturation and CaDPA release is not clear, although recent work suggests that significant protein denaturation can occur prior to CaDPA release (7). Almost all information on spore killing by moist heat has been obtained with spore populations, and essentially nothing is known about the behavior of individual spores exposed to potentially lethal temperatures in water. Given the likely heterogeneity of spores in populations, in particular in their wetheat resistances (16,18,39,40), it could be most informative to analyze the behavior of individual spores exposed to high temperatures in water.Raman spectroscopy is widely used in biochemical studies, as this technique has high sensitivity and responds rapidly to subtle changes in molecule structure (1, 22, 31). In addition,

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

“…3c and d). The phenomenon was not observed in previous work on spore inactivation by a number of different agents (28)(29)(30).…”

Effects of High-Pressure Treatment on Spores of Clostridium Species

“…To determine the DPA concentration in the spores that retained DPA, Raman spectra of individual spores were acquired using a laser power of 20 mW at 780 nm with an integration time of 20 s. The DPA concentration in these spores was determined from the intensity of the major CaDPA Raman peak at 1,017 cm Ϫ1 relative to the peak intensity of this same band from a CaDPA solution of known concentration. The Raman spectra of 30 treated spores that did or did not retain CaDPA were averaged as described previously (37)(38)(39)(40).…”

Factors Affecting Variability in Time between Addition of Nutrient Germinants and Rapid Dipicolinic Acid Release during Germination of Spores of Bacillus Species

“…Raman spectra of individual spores trapped by laser tweezers in an optical trap were measured during the germination of these spores under various conditions as described previously (5,35). A number of prominent bands in a dormant spore's Raman spectrum are due almost exclusively to DPA, in particular a very prominent band at 1,017 cm Ϫ1 (5,19,26,35).…”

“…Raman spectra of individual spores trapped by laser tweezers in an optical trap were measured during the germination of these spores under various conditions as described previously (5,35). A number of prominent bands in a dormant spore's Raman spectrum are due almost exclusively to DPA, in particular a very prominent band at 1,017 cm Ϫ1 (5,19,26,35). During spore germination, the DPA-specific Raman bands disappear, thus making it possible to determine the kinetics of DPA release during germination of individual spores (5,19,26).…”

“…More recently, confocal microscopy and then surface adsorption and optical tweezers have been used to capture single spores, and germination events have been followed by methods such as Raman spectroscopy to directly measure DPA release, as well as phase-contrast microscopy and elastic light scattering (3,5,9,10,19,26). While the latter recent advances have allowed accumulation of much information about germination, collection of this type of data for large numbers of individual spores is still labor-intensive, although use of dual optical traps (35) and perhaps multiple traps in the future may alleviate this problem. However, phase-contrast microscopy plus appropriate computer software has recently allowed the monitoring of many hundreds of individual spores for several hours, with automated assessment of various changes in the cells during the period of observation (19).…”