Nanoscale Structural and Mechanical Analysis of Bacillus anthracis Spores Inactivated with Rapid Dry Heating

Abstract

Effective killing of Bacillus anthracis spores is of paramount importance to antibioterrorism, food safety, environmental protection, and the medical device industry. Thus, a deeper understanding of the mechanisms of spore resistance and inactivation is highly desired for developing new strategies or improving the known methods for spore destruction. Previous studies have shown that spore inactivation mechanisms differ considerably depending upon the killing agents, such as heat (wet heat, dry heat), UV, ionizing radiation, and chemicals. It is believed that wet heat kills spores by inactivating critical enzymes, while dry heat kills spores by damaging their DNA. Many studies have focused on the biochemical aspects of spore inactivation by dry heat; few have investigated structural damages and changes in spore mechanical properties. In this study, we have inactivated Bacillus anthracis spores with rapid dry heating and performed nanoscale topographical and mechanical analysis of inactivated spores using atomic force microscopy (AFM). Our results revealed significant changes in spore morphology and nanomechanical properties after heat inactivation. In addition, we also found that these changes were different under different heating conditions that produced similar inactivation probabilities (high temperature for short exposure time versus low temperature for long exposure time). We attributed the differences to the differential thermal and mechanical stresses in the spore. The buildup of internal thermal and mechanical stresses may become prominent only in ultrafast, high-temperature heat inactivation when the experimental timescale is too short for heat-generated vapor to efficiently escape from the spore. Our results thus provide direct, visual evidences of the importance of thermal stresses and heat and mass transfer to spore inactivation by very rapid dry heating. Bacterial spores are metabolically dormant cells formed in a process called sporulation, which is generally induced by reduced levels of nutrients in the environment (1-3). Efficient inactivation of spores is of critical importance for a wide range of applications, including biodefense, food safety, environmental protection, and medical device sterilization (4-6). Spores are known to be more resistant to inactivation by heating, radiation exposure, and chemical decontamination than their corresponding vegetative cells. While various methods, including heating, chemical treatment, radiation, and UV treatment, have been used to inactivate spores (4, 6, 7), thermal inactivation is often the method of choice for many applications (8). Thermal inactivation of Bacillus spores in laboratory studies is most often achieved by wet heat in which spores are fully hydrated during heating (9-11) or dry heat in which dry spores are heated on a solid substrate, in an ampoule heated by an oil bath, in a hot air plume, or by infrared heating (5,8,(12)(13)(14)(15)(16)(17)(18)(19)(20). It has long been observed that spores are much less resistant to heat in a well-hydr...

Inactivation of bacterial spores subjected to sub-second thermal stress

“…All these studies show clear correlations between temperature, heating time and spore inactivation efficiency. Although the temperature-kill curves from previous studies [18][19][20]59] are similar to our results (Fig. 2), the spore inactivation mechanisms are different.…”

“…Considering that the water diffusion rate (2 MPa/ms) is closer to the pressurization rate (8 MPa/ms) at $10 4°C /s, but much smaller than the pressurization rate (154 MPa/ms) at $10 5°C /s, it is easy to understand the potentiation of thermal inactivation at higher heating rates by internal pressurization. A similar explanation for spore inactivation was reported by Xing et al [59]. They found that the spore surface was ruptured after rapid heating to 300°C for 10 s, suggesting the vaporized content inside spores cannot promptly diffuse out and therefore the pressure-induced mechanical stresses exceed the yield stress of spores.…”

“…Kumar et al [58] showed that the spore took $0.1 ms to reach the surrounding temperature of $400°C. Xing et al [59] showed that the temperature could reach steady state in less than 0.5 ms. Since our spores are directly in contact with Results are an average of at least 6 replicates and standard deviation is shown.…”

“…From 250°C to 370°C, the viability dropped exponentially by 5 logs. Recently, Xing et al [59] utilized a conductive heating system to rapidly heat Bacillus anthracis spores placed on a gold foil in 0.1 s. They found that the viability decreased exponentially with temperature, with a reduction of 5 logs from 300°C to $700°C. All these studies show clear correlations between temperature, heating time and spore inactivation efficiency.…”

Nanomechanical Characterization of Bacillus anthracis Spores by Atomic Force Microscopy

“…We previously showed that four closely related species of Bacillus spores can be distinguished by surface morphology analysis (54). Using thermal atomic force microscopy (AFM), we recently characterized thermal effects on the topographic morphology and nanomechanical properties of Bacillus anthracis spores at elevated temperatures (55,56). We observed striking changes resembling phase transitions in the physical properties of heated spores on the nanometer scale.…”