Chapter 8 : Mechanisms of Microbial Resistance

General microbial resistance to biocides and biocidal processes.

Initial sequence of events on biocide-microorganism interaction.

A typical bacterial growth curve, showing the four phases of population growth.

The activation and deactivation of the OxyR protein, as an activator in the peroxide-induced oxidative stress response.

The function of various enzymes induced during oxidative stress.

Transport systems in bacteria across a typical cell membrane.

Establishment of the proton motive force.

Summary of the various types of efflux pumps associated with antimicrobial resistance identified in bacteria. A typical Gram-negative bacteria cell wall is shown with associated cytoplasmic and outer membrane (see chapter 1, section 1.3.4.1) proteins. Note that similar cytoplasmic-membrane-associated efflux pumps (MFS and ABC) have been identified in Gram-positive bacteria (which do not have an outer membrane; section 1.3.4.1). Efflux is energy-dependent, derived from the proton motive force (antiporter efflux with H+) or ATP hydrolysis (to ADP and inorganic phosphate). Abbreviations: MFS, major facilitator superfamily; RND, resistance-nodulation-division family; MFP, membrane fusion protein (periplasmic); OMF, outer membrane factor; ABC, ATP-binding cassette family.

Intrinsic mechanisms of microbial resistance to heavy metals. The heavy metal ions can pass through the cell wall and membrane with a subsequent intracellular increase in concentration and inhibitory/biocidal effects (shown on the far left). Mechanisms of resistance are (1) exclusion, due to the presence of various surface structure or more subtle changes in porin or pump specificity to allow the uptake of essential but not biocidal ions; (2) active efflux out of the cell, against a concentration gradient; (3) enzymatic conversion of the ion to a different form, which is less toxic and can be released from the cell; (4) sequestration, in which macromolecules can absorb the biocide, thereby reducing the available concentration; and (5) changes in the structure of target molecules, which reduces their susceptibility to biocidal action.

A P. aeruginosa biofilm on a surface. Individual rod-shaped bacteria can be seen developed in a polysaccharide matrix. Image courtesy of ASM MicrobeLibrary, ©Fett & Cooke, with permission.

The development of a biofilm. Initial attachment (adsorption) of a microorganism may be reversible or permanent, leading to proliferation and extracellular polysaccharide production. This matrix develops over time, allowing the entrapment of nutrients and other microorganisms, which can also proliferate to produce a mature biofilm. Sections of the biofilm can slough off over time, bind to other surfaces, and subsequently develop further biofilms.

An example of Deinococcus radiodurans survival of radiation. The survival of D. radiodurans (solid line) is compared to a typical radiation-sensitive bacterial strain (dotted line) when exposed to increasing doses of γ irradiation (measured in kilograys; chapter 5, section 5.4).

Micrograph of D. radiodurans cells, in a typical tetrad formation. Image courtesy of Max-Planck-Institute for Molecular Genetics.

Microbial growth and optimum temperature conditions. Examples of various vegetative microorganisms are shown, although the sensitivities of specific species to heat vary. Geobacillus spores may be considered hyperthermophilic but are dormant in comparison to their vegetative forms (see section 8.3.11 ).

Microbial growth and optimum pH conditions. Examples of various microorganisms are shown, although specific species vary in their sensitivity to pH.

Microbial growth and optimum salt conditions. Examples of various microorganisms are given, although specific species vary in their sensitivity to salt concentrations.

The basic life cycle of Gram-positive endospore-forming rods. The vegetative growth of the bacteria is limited due to the reduction of essential nutrients (for example, carbon or nitrogen sources) or other environmental factors, causing the initiation of the sporulation cascade, death of the mother cell, and release of the dormant spore. Under the right environmental conditions, conducive to bacterial growth, the spore becomes activated, germinates, and outgrows to produce a viable vegetative bacterial cell over time, which resumes metabolism and multiplication.

Typical bacterial endospore structure. A micrograph of endospores on a surface is shown, with a representation of the various spore layers shown below (not to scale). The shapes of the spores vary depending on the bacterial genus and species.

A representation of a typical sporulation process, with the key (seven) stages identified.

The development of resistance of bacterial endospores to biocides and biocidal processes. The defined stages of sporulation are given from stage III (engulfment of the forespore) to stage VII (release of the mature spore), as described in Fig. 8.19 . The point at which the developing spore demonstrates resistance to each biocide or biocidal process is indicated.

Loss of resistance to various biocides and heat during bacterial endospore germination and outgrowth. Many biocides inhibit the activation of endospores (sporistatic) and are bactericidal to vegetative cells. Others (marked by asterisks) are also sporicidal at higher concentrations and temperatures and longer exposure times. Biocides, at low concentrations and temperatures, have also been shown to specifically inhibit germination or outgrowth following activation of the spore.

Typical life cycle of Streptomyces. A desiccated spore, under the right environmental conditions, will germinate and initiate hyphal growth. Under conditions of environmental stress and/or nutrient limitation, aerial hyphae develop in parallel with the production of secondary metabolites, including antibiotics and hydrolytic enzymes to assist in survival. The aerial filaments separate by simple cross-wall division to form prespore compartments and the development of desiccated spores, which can be released into the environment.

The potential revival of microorganisms after biocidal treatment. On exposure, microorganisms may survive due to lack of a contact or intrinsic resistance to the biocide. In other cases, the damaged microorganisms can be revived by active repair mechanisms and undergo subsequent growth or infectivity. Damaged microorganisms may therefore be initially uncultivable by normal laboratory methods but remain viable over time. Biocidal effects may also be sufficient to render the microorganism unviable (cell death or loss of infectivity).

A representation of the mycobacterial cell wall structure.

A representation of a typical Gram-positive bacterial cell wall structure.

A representation of a typical Gram-negative bacterial cell wall structure.

The primary mechanisms of action of penicillin and of bacterial resistance to it. The antibiotic normally penetrates through the cell wall to the cytoplasmic membrane-associated penicillin-binding proteins (PBPs) and disrupts their role in the synthesis of the cell wall peptidoglycan (shown on the left). The first mechanism of resistance (1) is exclusion from the cell, which can be intrinsic or acquired. In the second (2) the target PBPs have mutated to become less sensitive to penicillin binding. The third (3) is the presence (naturally, induced, or otherwise acquired) of β-lactamases, which hydrolyze the antibiotic.

The modes of bacterial tolerance to triclosan due to acquired mutations. Exclusion (1) may be due to the loss of outer membrane porin proteins (reduced influx) or changes in the outer/inner membrane lipid structure. Efflux mechanisms (2) include overproduction of cell membrane/wall-associated efflux pumps due to mutation in regulator proteins. Enoyl reductases are specific targets for triclosan in the inhibition of fatty acid biosynthesis, with mutations identified that are associated with less affinity of triclosan for these enzymes (3). Finally, the overproduction of enoyl reductases or other proteins provides greater tolerance of the biocide (4).

Demonstration of the resistance of M. chelonae glutaraldehyde-resistant strains. The aldehyde antimicrobial activity of a wild-type, glutaraldehyde-sensitive strain of M. chelonae was compared to a glutaraldehyde-resistant strain. A formulation of 2% glutaraldehyde demonstrated little to no effect against the resistant strain; another aldehyde (0.55% OPA) demonstrated efficacy but required greater exposure time than for the wild-type strain.

The simplified structure of a typical mercury-resistance operon in Gram-negative bacteria. The numbers, types, and control of expression of proteins from the operon can vary from isolate to isolate. In all cases a mercuric reductase (MerA) is expressed; however, only broad-spectrum resistance-associated plasmids and transposons are found to have MerB homologues (which are enzymes that hydrolyze organomercurial compounds to release the mercuric ion for subsequent reduction). The other proteins expressed are involved in mercury transport and control of the expression of the operon.

Mechanisms of resistance to mercury.

Acquired silver resistance in Salmonella associated with the pMG101 plasmid. pMG101 is a 180-kb plasmid that encodes the sil tolerance genes in an ∼14-kb section (above). The genes are encoded in three operons: silCFBAGP, silRS, and silE. Central to the mechanism of resistance are the SilR and SilS proteins (below). SilS is a sensor protein that detects the increased levels of silver ions and causes the derepression of SilR that represses the expression of silCFBAGP. SilA, B, and C are components of an RND-type efflux system that pumps silver ions out of the cell. SilF is an accessory protein that transports ions to the efflux mechanism, and SilE is a silver-binding protein that sequesters the heavy metal availability. The action of SilP remains to be further defined.

pSK41, an example of a multidrug resistance plasmid in staphylococci (not drawn to scale). pSK41 is a 46.4-kB plasmid encoding various genes for its transfer by conjugation (tra), resistance to antibiotics (gentamycin, tobramycin, and kanamycin by aacA-aphD; neomycin by aadD; and bleomycin by ble), and a biocide efflux pump (qacC). The plasmid contains sequences from a transposon (Tn4001) and an integrated plasmid (pUB110).

Mechanisms of viral resistance to biocides. The typical structure of an enveloped virus is shown as an example (chapter 1, section 1.3.5). Resistance can be due to indirect factors such as (A) viral clumping and protection (e.g., due to the presence of associated soils) or (B) directly to the structure of the virus particles, including the presence of an envelope, capsid structure, and access to damage the internal nucleic acid.

Mechanisms of resistance and modes of action of biocides against prions. Prions (as hydrophobic protein fibrils) are present in associated macromolecules (including lipids, carbohydrates, and proteins), through which the biocidal process needs to penetrate. Following penetration, ineffective processes include those that have no effect on proteins, that fix proteins, or that cause prion protein dispersal (potentially leading to cross-contamination). Biocidal processes that denature and/or hydrolyze (or degrade) proteins have been shown to be effective against prions; however, with denaturation alone, renaturation of the protein could occur to give the reassociated infectious protein form. Further, protein hydrolysis may be partial or complete. In some cases, partial hydrolysis of the protein is ineffective, because smaller components of the protein retain an infectious nature.

Scanning electron micrograph of an encapsulated C. neoformans strain. The capsule appears as a loose fibrillar network. (Reprinted from A. Casadevall and J.R. Perfect, Cryptococcus neoformans [ASM Press, Washington, DC, 1998].)

Example of a mass of fungal mycelial growth. Image courtesy of CDC-PHIL/Dr. Lucile K. Georg, 1971 (ID#15693), with permission.

Fungal (ascomycota) life cycle. The life cycle shown is typical of ascomycota such as Neurospora. In the case of yeasts such as Saccharomycetes, the single cells reproduce asexually by binary fission or budding and sexually by two cells uniting to lead to the development of ascospores. Asexual and sexual spores (which are different in structure) are formed in other fungi, although in some cases only asexual conidiospores have been described ( Table 8.34 ).

Examples of various types of fungal spores and spore-bearing structures. Zygomycota: a, aseptate hypha; b, zygospore; c, sporangiophore (with spores within sporangia); d, sporangiospores. Basidiomycota: e, basidiomata; f, basidium; g, naked basidiospores; h, hypha with clamp connections. Ascomycota: i, ascomata; j, ascus, containing spores; k, ascospores; l, septate hypha. Deuteromycetes: m, pycnidium; n, conidiophore; o, conidiogenous cells; p, conidia. Oomycota: q, zoospore (motile); r, gametangia; s, oospores. (Reprinted from J. Guarro et al., Clin Microbiol Rev 12:454-500, 1999.)

C. parvum oocysts and sporozoites.

Acanthamoeba cyst.

Micrograph examples of helminth eggs from Enterobius (left) and Fasciola (right). Enterobius image: Image courtesy of CDC-PHIL/B.G. Partin; Dr. Moore, 1978 (ID#14617), with permission. Fasciola image: Image courtesy of CDC-PHIL/Dr. Mae Melvin, 1973, with permission.

Representation of the structure of a helminth (Ascaris) egg.