Resistance is not so futile: Endospores and resistance mechanisms

Endospores present a concern in controlled environments due to their resistance and indefinite survivability. The production of a spore is part of a sophisticated stress response. Here, the bacterial genome is copied and transferred into the safety of a spore (sporulation).

The spore remains dormant until environmental conditions improve. When conditions are favorable, the spore will germinate (generally rapidly) and become a functioning, vegetative cell.

This week’s article looks at what endospores are, how they are formed, and their relative resistance as part of improving our understanding of contamination control.

What is an endospore?

Endospores were first described by Robert Koch in 1876. A bacterial endospore is a structure designed to protect the genome. The term endospore is used because the spore emerges from within the mother cell. The spore does not emerge until it is fully formed. The release process requires the mother cell to undergo lysis. Sporulation involves:

? A cell replicates its DNA.

? The DNA divides asymmetrically.

? The two copies of the genome are separated into two ‘compartments’ (sporangium).

? The smaller compartment (called the pre-spore) develops into a spore.

? The spore is released into the surrounding environment.

The resultant spore is metabolically dormant and partially dehydrated. Consequently, they are able to survive in nutrient-free and harsh environments (1). The spore will either persist or germinate (where a vegetative cell is formed). Germination is triggered by specific germinants, which are environment and species dependent (2).

The limit of viability is unknown. There have been cases of spores being held in flasks of alcohol in laboratories for decades. In addition, viable spores have been recovered from dried plant samples dating from 1640 (3) (and some viable spores of Bacillus marismortui isolated from salt crystals are thought to be 25 million years old) (4). Certainly, spores can survive for thousands of years (5).

Spore structure

Each layer of the spore structure protects against DNA damage. Working from inside out, the spore consists of (6):

1. A dehydrated central core containing the genome (where water has been replaced with Ca2+-dipicolinic acid).

2. An inner spore membrane.

3. Germ cell wall (peptidoglycan).

4. Cortex.

5. Outer spore membrane.

6. Spore coat: comprising of a series of thin, concentric layers.

Some bacteria possess a different type of outermost glycoprotein layer called the exosporium (in particular those of the B. cereus group). This has hair-like projections (7) and plays a role in enabling the spore to sense the external environment and potentially favorable conditions for germination. The exosporium is composed of approximately 50% protein, 15% lipids and 20% carbohydrates.

There are many differences with the spore coat between bacterial species, including differences in thickness and with the number of folds. The folds assist with the expansion in the volume of the spore core that occurs when the relative humidity changes as a result of different environments (8).

Which bacteria form endospores?

The bacteria capable of forming endospores, of most interest, are those of the phylum Firmicutes: the aerobic Bacillaceae (like Bacillus species) and the anaerobic Clostridia. Bacillus subtilis is often studied as a model organism for sporulation.

Where are endospores found?

Endospores are common to soil and in environments considered ‘extreme’ from hot springs to arctic sediments. Spores are also found on a variety of packaging and potentially within harsher environments, like cleanrooms.

Spores and resistance

Endospores are of interest to those seeking to achieve contamination control. This is because endospores display remarkable resistance properties. Spores are resistant to ultraviolet radiation; several chemicals (including disinfectants like quaternary ammonium compounds, oxidizing agents, aldehydes, acid, alcohols, and alkali, plus genotoxic agents); heat, as well as other stress factors. Spores are much more resistant to mechanical disruption than growing cells, such as sound waves (sonication) and shaking with glass beads (9).

With chemicals, spores are also resistant to low and high pH values, such as with 1 M acid or base (10). Spores are very resistant to desiccation and can survive multiple cycles of hydration and desiccation at moderate vacuum (~10?Pa). This makes them resistant to freeze-drying (11).

Resistance is species and strain dependent. Taking ultraviolet light, researchers established that a wild-type B. pumilus coded SAFR-032 is more resistant to 254 nm UV radiation than the B. pumilus type strain, ATCC 7061 (the most highly UV-resistant spores yet to be discovered) (12).

What confers protection?

Protection and resistance of the spore is a product of its structure, with various parts of the structure either conferring resistances to varied factors or reinforcing another component. If we strip down the spore components, we can observe the following types of resistance as related to structure:

1. Essential to spore protection and survival is the proteinaceous spore coat (13). (B. subtilis has over 80 different proteins in its spore coat, for example). The coat architecture varies according to the bacterial species; however, the spore coat is complex and formed of many different proteins (sometimes it is referred to as a ‘mineralized gel-like protoplast’). This outer coat provides chemical and enzymatic resistance. Part of this is due to a barrier effect due to coat morphogenetic proteins (14).

In particular, the thick proteinaceous coat detoxifies reactive chemicals. Superoxide dismutase – a protein involved in spore coat formation – helps to create the thick, striated outer layer. It is thought that superoxide dismutase may serve to detoxify potentially damaging chemicals in contact with the spore surface (15).

Furthermore, protection against oxidizing agents is provided by the catalases and other enzymes that the coat possesses.

2. Beneath the coat resides a very thick layer of specialized peptidoglycan called the cortex. Cortex formation involves the dehydration of the spore core, which aids in resistance to elevated temperature.

3. A germ cell wall resides under the cortex. This layer of peptidoglycan will become the cell wall of the bacterium if the endospore germinates. The cross-linked peptidoglycan structure surrounding the core is implicated in heat resistance of the spore.

4. The inner membrane, under the germ cell wall, is a major permeability barrier against several potentially damaging chemicals and ultraviolet light.

5. The center of the endospore, the core, exists in a very dehydrated state and houses the cell's DNA, ribosomes and copious quantities of dipicolinic acid (up to 10% of the spore's dry weight). The calcium-dipicolinic acid chelate protects the DNA from UV damage.

Small acid-soluble proteins are also only found in endospores. These proteins bind and condense DNA and they are responsible for resistance to DNA-damaging chemicals.

Protection can appear in other ways, such as through cellular agglomeration (‘clumping’ of spores) (16).

Limitations of resistance

Spores demonstrate high resistance to both heat and radiation; however, there are limits. Spores can be destroyed by temperatures exceeding the boiling point of water (100^C). However, different organisms show variations of resistance above this temperature and even those killed at 100^C can survive for many hours (as temperatures increase above this point the time required for destruction tends to decrease). Mutations impacting both the spore coat and the inner membrane can increase the resistance of a spore to moist heat. For example, with B. subtilis an acquired transposon with an operon, termed spoVA2mob has been shown to increase resistance (17). This same mutation also confers increased resistance to hydrogen peroxide.

Moist heat is more effective than dry heat and it can kill spores by exerting DNA damage, plus the inactivation of one or more essential germination components. With the difference between moist heat and dry heat, spores are resistant to ~20^C higher temperatures when dry heat is applied compared to when they are heated in water. However, with sufficient heat and time dry heat can be an effective form of sterilization (18). One concern is if dry heat does not achieve total kill the possibility of mutations increases – mutations equipped better for DNA repair following dry heat exposure (19).

Resistance to microwave heating is possible; this is an under-researched area. A 2.0 kW microwave has sufficient irradiation to rupture the spore coat and inner membrane (this is considerably above the common domestic microwave, which operates at 0.5 kW).

Spores are resistant to radiation to a certain degree and if the damage to the spore DNA is not complete then many spores can repair the radiation damage to their DNA when they germinate and the vegetative cell emerges (20) (and spore resistance to ionizing radiation resistance is increased by the presence of a number of DNA repair enzymes in spores). Bacterial spores can tolerate distinct levels of gamma radiation, depending on the species and the specific conditions. For example, B. anthracis spores require a dose of 1.5 × 10^6 rads to be inactivated, while B. megaterium vial-1 spores show influence from a wide range of spore loads and gamma radiation doses.

Further, in terms of radiation, the type of radiation is significant. Spores are generally resistant to ultraviolet light; however, prolonged exposure to ionizing radiation (X-rays and gamma rays) will kill endospores. In some cases, high hydrostatic pressure can also be effective at spore destruction (>1000 MPa) (21).

With sporicidal disinfection, ethylene oxide, chlorine-based compounds, practice acid and hydrogen peroxide (22) at an appropriate concentration and over a suitable time period (typically 10 minutes or greater), prove to be effective (23).

Another method of destruction is to encourage spores to germinate, such as through gentle heat, and then to increase the heat to kill the resultant vegetative cells (as with Tyndallization) (24).

Transfer of spores

Spores can be spread over considerable distances e.g., transfer via trolley wheels, cardboard etc. For controlled environments, many of the risk factors relate to the transfer of materials and associated disinfection (as discussed in previous LinkedIn articles).

Summary

Bacterial spores show remarkable resistance to many of the environmental factors within controlled environments and the standard decontamination methodologies. Factors important in spore resistance include the spore coat proteins, such as those able to detoxify chemical agents; the relative impermeability of the spore's inner membrane; the protection of spore DNA by its saturation with alpha/beta-type small, acid-soluble spore proteins; and the ability to undertake DNA repair (25).

Sterilization methods and sporicidal disinfectants, provided they can hit their targets, are effective. However, an important emphasis should be given to preventing spores from being present in critical areas in the first place.

References

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