Heat resistance of bacterial spores: Are we ‘straining’ for answers?

Bacterial spores are extremely tolerant to heat, chemicals, and to harsh physical conditions (1). As an example, with chemical resistance both the outer and inner spore coats limit the uptake of biocidal agents by preventing penetration of the agent to its site of action. The effect of this leads to spores being up to 100,000 times more resistant to disinfectants than vegetative bacterial cells (2).

?

(Tim Sandle is a pharmaceutical microbiologist, his website is Pharmaceutical Microbiology Resources).

?

Hence, spores are of concern to pharmaceutical and healthcare facilities due to their resistance and dormancy. Of particular concern are Bacillus species and in this LinkedIn article, the topic is heat-resistance.

?

Spores will be killed at a given level of heat under controlled conditions. Heat denatures certain proteins that are required for germination and structural material (dipicolinic acid) is released from the spore core (3).

?

Heat resistance

?

Different bacteria have different levels of resistance to heat.

?

Vegetative cells

?

Where vegetative cells have degrees of heat resistance, these are generally destroyed as pasteurization temperatures. Cells are destroyed in a variety of ways as heat damages the cytoplasmic membrane, ribosomal RNA, and causes the denaturation of proteins. Relative resistance of vegetative cells depends on how they respond to the external stressor (heat) and to the extent they can repair the effects of heat damage.

?

As to why some species are more resistant to heat than others and why some strains within the same species are more resistant, this relates to differences with heat-shock proteins, proteases, and transport proteins. Between strains these molecular factors are affected by the presence or absence of genes (or gene mutations).

?

Heat resistance is also influenced by environmental conditions, including pH, water activity and osmoprotectants (molecules that protect cells against osmotic stress). Environmental conditions influence the expression of proteins within the bacterial cell.

?

Spores

?

To destroy bacterial spores, sterilization temperatures are required. Typical bacterial spores are resistant to approximately 40–45°C higher temperatures than their corresponding vegetative cells (4). Beyond this general rule, the heat resistance of microorganisms varies greatly between different species and also between different strains of the same species (5).

?

Of the types of bacteria that produce spores, there is considerable variation between Bacillus and Clostridium species when it comes to heat resistance. Greater thermoresistances occur with the genera Geobacillus, Anoxybacillus, and Moorellaas (the most resistant of these are isolated from geothermal springs and would not be found within the typical pharmaceutical environment; although some thermopiles, like Heyndrickxia sporothermodurans are found in rural environments and this organism has a D-value only slightly below that of G. stearothermophilus (6).

?

Generally (although not always) the heat resistance of the bacterium correlates with the temperature at which spores will germinate, assuming optimal conditions in terms of available nutrients and water etc. This explains why the Geobacillus stearothermophilus spores used to evaluate moist heat are cultivated at 55-600C and Bacillus atrophaeus spores used to evaluate dry heat are cultivated at 30-35oC.

?

Strain differences

?

Different strains of the same species can show differences in heat resistance, even under the same cultural conditions. For example, research has shown, at a 95% prediction interval and of log10D120 (the D-value at 120?C) for B. cereus spores was as large as 2.2 log10. (7) One metareview asserts that strain variability in spore heat resistance accounted for approximately two-thirds of the variability found in the literature.

?

In many cases, the different heat resistance of bacterial strains correlates to operons (a cluster of genes that are transcribed together to give a single messenger RNA (mRNA) molecule, which therefore encodes multiple proteins). With many species of Bacillus, the presence of the spoVA2 operon on a mobile genetic element designated spoVA2mob is the key determinant for increasing heat resistance. The transfer of genetic material is often facilitated by bacteriophages (viruses that infect bacteria).

?

As well as the presence of this operon, the copy number of the spoVA2mob operon is also an important determinant (8). Copy numbers can be determined using a droplet digital PCR assay. For example, studies have shown that strains of B. amyloliquefaciens and B. subtilis can contain up to three copies of the spoVA2mob operons, making them more heat resistant than other members of the genera. It follows that those Bacillus strains carrying multiple copies of the spoVA2mob operon exhibit decimal reduction times (D values) that can be up to 100-fold higher than strains of the same species that do not carry this operon. Other operons impart the same effects on other organisms, such as the SASP ssp4 gene that seems to elevate heat resistance with some species of Clostridium (9).

?

The exact molecular functions of the proteins encoded in the spoVA2mob operon during sporulation and germination are not clear; however, multiple studies have demonstrated its influence. One possible connection is with altering mechanosensitive channels that are known to sense changes in the membrane tension and osmotic changes.

?

Sporulation processes also influence heat resistance. Studies have shown that the medium matters (particularly the presence of cations like potassium, magnesium, calcium and manganese) and the maturation period as the vegetative cell forms (longer maturation times can lead to increased cross-linkage of proteins, as shown in time-lapse phase-contrast and fluorescence microscopy). These are interesting characteristics for the manufacturers of biological indicators, should there be concerns about increasing D-values (or so-termed ‘resistance creep’).

?

Another heat resistance feature is ‘super dormant spores’, which generally display elevated heat resistance, which is the product of reduced core water content (10). As well as lower water content, increased muramic acid cross-linking in the cortex also leads to extended dormancy and potential increased heat resistance.

?

Pressure resistance

?

Some methods of heat transfer require alternations to pressure (as with an autoclave) and the application of high-pressure is used as an antimicrobial step in the food industry. The pressure resistance of bacterial spores does not correlate to heat resistance. An organism like Bacillus amyloliquefaciens, as an example, is relatively resistant to pressure (11).

?

It is also the case that other spore properties, like the content of dipicolinic acid (DPA) and water, further determine a spore’s resistance to both pressure and heat. Hence, the resistance of spores to pressure-associated thermal sterilization is also influenced by a spore’s ability to retain DPA.

?

Pressure and temperature, coming together.

?

Studies suggest that the copy number of spoVA2mob operon per genome correlates to the spore DPA content and pressure resistance. This property may not only be strain dependent, for it is a mobile genetic element that can transfer to organisms by horizontal gene transfer meaning that other species may acquire the genes.

?

What is the significance?

?

As well as some spores being more resistant to heat that others, genetic mechanisms see some species of Bacillus being more resistant than others and some strains within the same species being considerably more resistant than others, through mechanisms where organisms readily and reproducibly evolve to acquire much enhanced endospore heat resistance.

?

These factors place considerable emphasis on the importance of overkill cycles when qualifying devices that function to the achieve sterilization by heat.

?

Readers may be interested in the article “Rogue Biological Indicators: Are They A Real Phenomenon?” at: https://www.researchgate.net/publication/340563409_Rogue_Biological_Indicators_Are_They_A_Real_Phenomenon

?

References

?

1.?????Setlow P. 2003. Spore germination. Curr Op in Microbiol 6:550–556

2.?????Morató, J., Ribas, F. Microbial response to disinfectants in Handbook of Water and Wastewater Microbiology, 2003

3.?????Wang G, Zhang P, Setlow P, LiY Q. 2011. Kinetics of germination of wet-heat-treated individual spores of Bacillus species, monitored by Raman spectroscopy and differential interference contrast microscopy. Appl. Environ. Microbiol.77:3368–79

4.?????Wohlgemuth, S., K?mpfer, P. Bacterial Endospores in Encyclopedia of Food Microbiology (Second Edition), 2014

5.?????Reineke, K. and AMathys, A. Endospore Inactivation by Emerging Technologies: A Review of Target Structures and Inactivation Mechanisms,Annual Review of Food Science and Technology 2020 11:1, 255-274

6.?????Huemer, I.A., Klijn, N., et al. Thermal death kinetics of spores of Bacillus sporothermodurans isolated from UHT milk. Int Dairy J 1998, 8, 851–855

7.?????vanAsseltED,ZwieteringMH.2006.Asystematicapproachtodetermineglobalthermalinactivationparametersforvariousfoodpathogens.Int.J.FoodMicrobiol.107:73–82

8.?????Berendsen EMM, Koning RAA, Boekhorst J, et al. 2016. High-level heat resistance of spores of Bacillus amyloliquefaciens and Bacillus licheniformis results from the presence of a spoVA operon in a Tn1546 transposon. Front Microbiol 7:1912

9.?????LiJ, P. Sarker,D. ?MR, McClane, M. 2009 Further characterization of Clostridium perfringens small acid soluble protein-4 (ssp4) properties and expression. PLOS ONE 4: e6249

10.?Ghosh S, Setlow, P. 2009. Isolation and characterization of super dormant spores of Bacillus species. J. Bacteriol. 191:1787–97

11.?den Besten HMW, Wells-Bennik MHJ, Zwietering MH. 2018. Natural diversity in heat resistance of bacteria and bacterial spores: impact on food safety and quality. Annu Rev Food Sci Technol 9:383–410

12.?Berendsen EM, Boekhorst J, Kuipers OP, Wells-Bennik M. 2016. A mobile genetic element profoundly increases heat resistance of bacterial spores. ISME J 10:2633–2642

13.?Dongmin T., Katrien K., et al. Bacillus weihenstephanensis can readily evolve for increased endospore heat resistance without compromising its thermotype, International Journal of Food Microbiology, 2021 341: ?https://doi.org/10.1016/j.ijfoodmicro.2021.109072

14.?Berendsen EM, Boekhorst J, Kuipers OP, Wells-Bennik M. 2016. A mobile genetic element profoundly increases heat resistance of bacterial spores. ISME J 10:2633–2642