Mechanisms of Resistance to Biocides

MECHANISMS OF RESISTANCE TO BIOCIDES

Bacterial resistance to biocides has been reported since the 1950s, notably with QACs, biguanides and phenolics. Overall there has been more documented evidence of bacterial resistance to antiseptics than to disinfectants (Table 20.3). It is worth mentioning that some bacteria surviving in biocidal formulations have been associated with outbreaks and pseudo-outbreaks of infection (Weber et al., 2007). Bacterial resistance to all known preservatives has also been reported (Chapman et al., 1998). Recently, much interest has focused on bacteria surviving highlevel disinfection, which is usually employed for the disinfection of medical devices. Thus bacteria surviving exposure to the in-use (high) concentration of highly reactive biocides (e.g. glutaraldehyde, chlorine dioxide, hydrogen peroxide) have been isolated and studied. In 2009, a large outbreak of atypical mycobacteria in at least 38 hospitals in Brazil was reported. These mycobacteria were traced to endoscope contamination and were resistant to 2% w/v glutaraldehyde but also to the clinical concentration of front-line antibiotics against mycobacteria (Duarte et al., 2009). This was the first time that biocide resistance was linked to antibiotic resistance, nosocomial infection and a large infection outbreak.

Over the last 10 years, much progress has been made in understanding the mechanisms conferring resistance to biocides in bacteria. Interestingly, some mechanisms that were thought to occur only with antibiotics have now been described with biocides.

a)   General Mechanisms

A number of mechanisms conferring some level of resistance to biocide exposure have been documented. Traditionally mechanisms of resistance have been divided into intrinsic and acquired resistance. Intrinsic (or innate) resistance is a natural property of the bacteria and provides some explanation as to why some bacteria are less susceptible than others. Intrinsic mechanisms often involve a structural difference, for example, a difference in the permeability of the bacterial membrane to biocides, but also the expression of chromosomal genes such as those encoding for an efflux pump or a degradative enzyme. Acquired resistance refers to the acquisition of a new property by the bacteria through mutation and genetic transfer; such a property can be the mutation of a target site or the transfer of a gene encoding for an efflux pump or a degradative enzyme. It should be noted that when gene transfer occurs, often several genes present on the same conjugative plasmid or transposon can be transferred to a recipient cell at the same time. In this case the term co-resistance is often used to denote the simultaneous acquisition of a number of genes conferring resistance to a number of antimicrobials, both biocides and antibiotics.

Bacterial resistance to a biocide arises often from the presence of several mechanisms that work together to decrease the detrimental concentration of the biocide to a level that is no longer harmful for the bacterium (Table 20.4). The expression of only one mechanism confers low-level resistance often measured as an increase in minimum inhibitory concentration (MIC), but rarely high-level resistance, which can be measured as an increase in minimum bactericidal concentration. Finally, a distinction can be made between mechanisms expressed by a single bacterium and the mechanisms of resistance that arise from a community of bacteria such as in bacterial biofilms.

       i)  Changes in cell permeability

The decrease in biocide penetration arising from changes in cell permeability is well established and has been described with different bacterial genera, notably with Gram-negative bacteria and mycobacteria. It is also the case with bacterial endospores, which are discussed later in this chapter. In Gram-negative bacteria, the outer membrane, and notably the composition of LPS, offers some protection to the cell, by reducing biocide penetration. The role of LPS has been exemplified by researchers with the use of permeabilizing agents and notably ion chelators such as EDTA. EDTA contributes to the removal (loss) of LPS from the outer membrane by scavenging dications involved in the stabilization of LPS in the membrane (section 4.4.1). By losing LPS, the outer membrane becomes more permeable and biocides can penetrate better resulting in enhanced activity. A change in the structure of the outer membrane following a change in protein, fatty acid or phospholipid composition has been associated with a decrease in the efficacy of cationic biocides. In particular, a decrease in the number of porin proteins as a result of biocide exposure has been associated with high-level resistance to QACs in pseudomonads. Recently, a change in surface charge in Pseudomonas aeruginosa has been associated with a decrease in susceptibility to QACs.

In mycobacteria, the lipid-rich outer cell wall (responsible for the waxy appearance of the colonies), and particularly the presence of a mycoyl-acyl-arabinogalactan layer and the composition of the arabinogalactan/ arabinomannan within the cell wall, account for a reduction in biocide penetration; increasing the permeability of the mycobacterial outer cell wall, for example with ethambutol, enhances the activity of biocides and antibiotics.

       ii) Efflux

Efflux pumps are cross-membrane proteins which pump out various substrates including biocides and antibiotics. A large number of efflux pumps in bacteria have been identified and have been divided into five main classes depending on their structure and activity: the small multidrug resistance (SMR) family; the major facilitator superfamily (MFS); the ATP-binding cassette (ABC) family; the resistance-nodulation-division (RND) family; and the multidrug and toxic compound extrusion (MATE) family (Figure 20.3).

The role of efflux pumps is to remove harmful substances from the bacterial cytoplasm, including biocides (e.g. QACs and phenolics), to levels that are not damaging for the cell. The quantity of antimicrobial pumped out depends upon the number of pumps present, their expression and efficacy. Some studies have shown that high-level resistance can be achieved by efflux, for instance against the bisphenol triclosan, but usually, the effect is an increase in MIC.

      iii)  Enzymatic inactivation

Enzymatic degradation plays a role in reducing the harmful concentration of a biocide and has been observed with aldehydes (e.g. aldehyde dehydrogenase), oxidizing agents (e.g. catalases, superoxide dismutase, hydroxyperoxidases), phenolics and parabens. In the case of metallic salts such as silver, the ionic form is reduced to the inactive metal. The role of enzymatic inactivation has not been widely studied, but it is unlikely that a bacterial enzyme will contribute to high-level resistance to a biocide.

iv)       Modification of target site

To date, resistance conferred by the modification of a biocide target site has only been observed with triclosan. At a low concentration this bisphenol interacts specifically with a bacterial enoyl-acyl reductase carrier protein, which is involved in the synthesis of fatty acid. Triclosan has been shown to interact with a number of structurally related enzymes in many bacterial genera. A modification of the enzyme confers a low-level resistance to triclosan, although some studies claim that a high-level resistance has been observed. This is unlikely, since at a high concentration triclosan interacts with multiple target sites to bring about a bactericidal effect.

v)     Change in metabolic pathway

A change in metabolic pathway that confers resistance to a biocide is a relatively new concept that was thought to occur only with sulphonamides. However, bacterial adaptation to triclosan, as measured by an extended lag phase of growth followed by a normal exponential phase, has been observed in several bacterial genera. The recent use of a microarray in Salmonella enterica serovar Typhimurium enabled the identification of a ‘triclosan resistance network’ including an alternative pathway to the production of pyruvate and fatty acid. In Staphylococcus aureus showing reduced sensitivity to triclosan, a change in lipid composition of the cell membrane was associated with altered expression of various genes involved in fatty acid metabolism. Low-level QAC resistance in Serratia marcescens may arise from a change in synthetic or metabolic pathways.

b) Induction Of Resistance

The induction of antimicrobial resistance in bacteria following biocide exposure is a relatively recent concern which is particularly pertinent to the increasing number of commercially available products containing a low concentration of a biocide. This low, often subinhibitory, concentration can induce the expression of resistance mechanisms that can confer bacterial survival to biocide and/or antibiotic exposure. Low-level resistance as measured by an increase in MIC has often been observed. Prior to the use of genomics, proteomics and metabolomics, induction of resistance in bacteria was observed with an increase in lag phase of growth and a decrease in growth rate, an overexpression of efflux pumps and the production of guanosine 5′-diphosphate 3′-diphosphate (ppGpp). An increase in DNA repair was also associated with an increase in bacterial survival following biocide exposure.

More recently, a change in expression of regulons commanding a number of responses, such as a stress type of response (and repair mechanism), increased efflux, change in membrane composition, and a change in metabolic and synthetic pathways, has been recorded following exposure to a low concentration of biocides. Such a global response is of concern as it also confers a decreased susceptibility to antibiotics, and recent evidence suggests it might also lead to overexpression of virulence determinants.

It can be noted that the concentration of a biocide that promotes mutation in bacteria might be low. Indeed, a number of investigations have observed that the mutation rate increases in the presence of an active efflux system. The effect of bacterial mutation in the development of resistance has not been widely investigated, with the exception of triclosan. It is also possible that biocides that interact with the bacterial genome (e.g. dyes, oxidizing agents) might produce a higher mutation rate.

c)  Dissemination Of Resistance

Surprisingly, the dissemination of biocide resistance mechanisms between bacteria has been little studied. The acquisition of new genetic determinants, notably by the process of conjugation, is of concern as it is often dependent on the presence of large transferable plasmids, and transposons which encode for many genes including bacterial resistance to antibiotics and virulence factors. When several resistance genes are transferred at the same time, the term co-resistance is used. For example, the resistance to the QAC benzalkonium chloride in Staphylococcus aureus has been associated with the presence of plasmids containing qac, bla and tet resistant genes (encoding for efflux pumps and a β-lactamase). A number of mechanisms of resistance such as efflux and degradative enzymes have been documented to be transferred between bacteria. The extent of such dissemination is difficult to measure, although it is thought to readily occur in bacterial communities such as a biofilm.

Equally important to the horizontal transfer of resistance is the maintenance of resistant determinants (plasmids) following the continuous presence of biocides. Although this issue has not been widely studied, it is of interest with the use of biocidal products which are documented to leave a residual concentration of biocide on surfaces, antimicrobial surfaces, and the continuous presence of biocides in certain applications, such as drinking water chlorination.

d)   Bacterial Spores

The formation of a spore is a mechanism of bacterial survival when growth conditions are detrimental for the vegetative form. Such adverse conditions include lack of food but also the presence of biocides and other detrimental physical and chemical conditions. The spore structure is unique and confers upon the spore high level resistance to biocides. Hence only a few biocides, mainly highly reactive ones such as aldehydes, oxidizing agents and chlorine-releasing agents, are sporicidal, while others such as biguanides, QACs and phenolics are sporostatic despite their bactericidal effect on the vegetative bacteria. It should also be noted that sporicides take time to kill spores, usually a minimum of 5 minutes contact, and at a high concentration; aldehydes such as glutaraldehyde and formaldehyde need a much longer contact time and, in the case of ortho-phthalaldehyde, a raised temperature.

             i)  Sporulation and germination

Sporulation, a process in which a bacterial spore develops from a vegetative cell , involves seven stages (I–VII); 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 resistance, whereas decreasing susceptibility to glutaraldehyde is a very late event.

Bacillus spore coatless mutants and chemically induced coatless spores have shown the role of the spore coats in limiting access of biocides to the spore core. The cortex also acts as a barrier to some extent.

During germination and/or outgrowth, metabolism and biosynthetic processes increase and cells regain their sensitivity to antibacterial agents. Some inhibitors act at the germination stage (e.g. phenolics, parabens), whereas others such as chlorhexidine and the QACs do not affect germination but inhibit outgrowth. Glutaraldehyde, at low concentrations, is an effective inhibitor of both stages.

              ii) Spore Structure

The spore structure and the interaction between a biocide and the spore have been particularly well documented in the genus Bacillus. The spore core (protoplast, sometimes referred to as the germ cell) is enclosed within a cell wall which is surrounded by the cortex and several spore coats. Sometimes an exosporium may surround the spore.

The spore core is the target site of sporicides since it is the location of RNA, DNA, dipicolinic acid (DPA) and most of the calcium, potassium, manganese and phosphorus. Also present are substantial amounts of low molecular weight basic proteins, the small acid-soluble spore proteins (SASPs) which are rapidly degraded during germination. SASPs, comprising about 10–20% of the protein in the dormant spore, exist in two forms (α/β and γ) and 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 (α−/β−) deficient in α/β-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 α/β-type SASPs and is thus protected from free radical damage.

e)  Bacterial Biofilms

Bacteria are generally associated with surfaces in a complex community called biofilms. Following attachment to a surface, a bacterium will go through a series of metabolic and phenotypic changes, leading to the formation of microcolonies embedded within a matrix of secreted exopolysaccharides. Bacteria in biofilms have been shown to be less susceptible to antibiotics and biocides than planktonic bacteria. There are several biocide resistance mechanisms, all contributing to a ‘biofilm-associated phenotype’: reduction in biocide penetration, reduced bacterial metabolism, quiescence, enzymatic inactivation and efflux (Table 20.5).

Biocides have been observed to change the composition of a complex biofilm, composed of different bacterial genera or/and species. For example, polyhexamethylene biguanides (PHMB), chlorhexidine and Bardac (a QAC) have been shown to select for pseudmonads to the detriment of Gram-positive bacteria. The bisphenol triclosan was shown to reduce the genera diversity of a complex waste drain biofilm and to decrease the overall susceptibility of the remaining population.

f)    Misuse And Abuse Of Biocides

The indiscriminate use of biocides in an increasing number of applications and, notably, the use of sub-optimal low concentrations has fuelled the debate on emerging bacterial cross-resistance to antibiotics used for human and animal medicine. This is based on in vitro evidence that some mechanisms conferring a decrease in biocide susceptibility can also lead to resistance to therapeutic concentrations of antibiotics. Some of the most common mechanisms involved include expression and over-expression of efflux pumps and changes in cell permeability and metabolism. However, there is no useful rule of thumb to predict cross-resistance between biocide and antibiotic resistance in bacteria. In addition, emerging cross-resistance following biocide exposure in situ has not been widely reported. The most significant study to date was reported in 2009 and concerned an outbreak in 38 hospitals of an isolate of Mycobacterium massiliense resistant to 2% glutaraldehyde and to antimycobacterial therapeutic antibiotics.