Spores biocide resistance

Response to a biocide depends upon the cellular stage of development. Sporulation, a process in which a bacterial spore develops from a vegetative cell, involves seven stages (I—VII Chapter 3) of these, stages IV-VII (cortex and coat development) are the most important in relation to the development of biocide resistance. Resistance to biocidal agents develops during sporulation and may be an early, intermediate or late/very late event. For example, resistance to chlorhexidine occurs at an intermediate stage, at about the same time as heat... 

Mycobacteria are more resistant than other non-sporulating bacteria to a wide range of biocides. Examples of such organisms axe Mycobacterium tuberculosis, theM avium-intracellulare (MAI) group andM. chelonae (M. chelonei). Of the bacteria, however, the most resistant of all to biocides are bacterial spores, e.g. Bacillus subtilis, B. cereus. 

Bacterial resistance to biocides (Table 13.2) is usually considered as being of two types (a) intrinsic (innate, natural), a natural property of an organism, or (b) acquired, either by chromosomal mutation or by the acquisition of plasmids or transposons. Intrinsic resistance to biocides is usually demonstrated by Gram-negative bacteria, mycobacteria and bacterial spores whereas acquired resistance can result by mutation or, more frequently, by the acquisition of genetic elements, e.g. plasmid- (or transposon-) mediated resistance to mercury compounds. Intrinsic resistance may also be exemplified by physiological (phenotypic) adaptation, a classical example of which is biofilm production. 

Other hand, mycobacteria and espeeially bacterial spores are much more resistant. A major reason for this variation in response is associated with the chemical composition and stmcture of the outer cell layers such that there is restricted uptake of a biocide, hi... 

SASPs comprise about 10-20% of the protein in the dormant spore, exist in two forms alfi and y) d are degraded during germination. They are essential for expression of spore resistance to ultraviolet radiation and also appear to be involved in resistance to some biocides, e.g. hydrogen peroxide. Spores (a /3 ) deficient in a//3-type SASPs are much more peroxide-sensitive than are wild-type (normal) spores. It has been proposed that in wild-type spores DNA is saturated with a/j3-type SASPs and is thus protected from free radical damage. 

The first stage in the action of an antibiotic or biocide on a bacterial cell involves interaction between the chemical and the biological entity. Adsorption of a variety of biocides into bacterial cells has been described [33] but this, per se, does not necessarily provide information about the mechanism or site of action of the antibacterial compound. However, resistant cells would usually (but not necessarily) be expected to adsorb less of a chemical than sensitive cells. In non-sporulating bacteria, changes to the outer layers of cells may follow the initial binding to the cell surface or there may be diffusion across the cell envelope in either case, an antibiotic or biocide will penetrate the cell to reach the primary site of action at the cytoplasmic membrane or within the cytoplasm. Little is known about the penetration of antibacterial agents into bacterial spores. 

The response of a developing spore to a biocide depends upon its stage of development [30, 128-132]. Resistance increases during sporulation and may be an early, intermediate or late event [128-132], Resistance to chlor-hexidine takes place at an intermediate stage and appears to be associated with the development of the cortex [133]. In contrast, decreasing susceptibility to glutaraldehyde is a very late event [131, 132] and is linked to the biosynthesis of the spore coats. 

Table 4.7. MECHANISMS OF SPORE RESISTANCE TO BIOCIDAL AGENTS...

SASPs and is thus protected from free radical damage. The a ff mutants do not appear to have been tested against other biocides. In view of their postulated role in protecting DNA, it is interesting to speculate that a///-type SASPs would not be associated with spore resistance to those biocides that have little or no effect on DNA. 

Bacterial resistance to antibiotics and biocides is essentially of two types, intrinsic and acquired. Whilst the latter is of greater significance clinically with antibiotics, specific examples of intrinsic resistance to both antibiotics, e.g. mycobacteria, and biocides (e.g. mycobacteria, Gram-negative bacteria, spores) are also of importance. 

As already pointed out, staphylococci and streptococci are generally more sensitive to biocides than Gram-negative bacteria examples are provided in Table 18.4. On the other hand, mycobacteria and especially bacterial spores are much more resistant. A major reason for this variation in response is associated with the chemical composition and structure of the outer cell layers such that there is restricted uptake of a biocide. In consequence of this cellular impermeability, a reduced concentration of the antimicrobial compound is available at the target site(s) so that the cell may escape severe injury. Another, less frequently observed, mechanism is the presence of constitutive, biocidedegrading enzymes. 

Bacterial spores of the genera Bacillus and Clostridium are invariably the most resistant of all types of bacteria to biocides. Many biocides, e.g. biguanides and QACs, will kill (or at low concentrations be bacteriostatic to) non-sporulating bacteria but not bacterial spores. Other biocides such as alkaline glutaraldehyde are sporicidal, although higher concentrations for longer contact periods may be necessary than for a bactericidal effect. 

Spore coatless forms, produced by treatment of spores under alkaline conditions with UDS (urea plus dithiothreitol plus sodium lauryl sulphate), have been of value in estimating the role of the coats in limiting access of biocides to their target sites. However, this treatment removes a certain amount of spore cortex also. The amount of cortex remaining can be further reduced by subsequent use of lysozyme. The spore coats have an undoubted role to play in conferring resistance of spores to biocides. The cortex also acts as a barrier to some extent. 

Mycobacteria are generally considered to be of intermediate resistance to biocides, including aldehydes, between bacterial spores and other vegetative cells [90], Formalin, aqueous formaldehyde solution and glutaraldehyde are mycobactericidal, although some mycobacterial strains show increased resistance [90, 101, 236]. 

Bacterial spores are invariably the most resistant forms of bacteria to bio-Table 4.3. RESISTANCE OF BIOFILMS TO BIOCIDES... 

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