Antimicrobial agents of various types have been in use for well over 100 years. Examples include antibiotics for animals, plants and/or people, sanitizers and disinfectants used for hard surfaces, and sanitizers used for hands and skin. Microbial isolates have shown a level of resistance to some of these antimicrobial agents, creating legitimate concern over the development of such resistance to biocides through their normal use. While resistance to antibiotics is well described, tolerance to sanitizers and disinfectants is less well understood. In this article, we will explain the difference between antibiotic and disinfectant resistance and their impacts on their performance.
Differences between antibiotics and disinfectants resistance
In the media, and to a lesser extent in other literature, little distinction is made between resistances to different classes of antimicrobial agents, contributing to ambiguity on whether resistance to one agent is linked to resistance to other agents. There are many who think that resistance to antibiotics is the same as resistance to sanitizers and disinfectants and that a solution that addresses resistance to one antimicrobial will work for the others. In contrast to antibiotics, resistance problems with disinfectants occur much less frequently, as the two operate by different mechanisms. While antibiotics act specifically on certain biological processes in the metabolism of the bacteria, disinfectants act non-specifically on the entire biological structures of the cell. So it is not surprising that the bacteria can modify these specific sites of attack (by antibiotics) by undergoing mutations. However, the bacterial cells are much less able to adapt and protect themselves against the attack of disinfectants.
What causes antibacterial tolerance and how does it occur
Even though the adaptations of microbes to disinfectants are less well described, some mechanisms are known that allow a certain tolerance. However, the distinction between resistance and tolerance must first be made. Tolerance is often a lower level of insensitivity and is rarely complete. This low level of insensitivity is differentiated from resistance, which is a high level of insensitivity and can be near immunity to an antimicrobial.
- Acquired resistance is often gained via genetic mutation or the acquisition of genes encoding tolerance from other organisms through horizontal gene transfer. And it may also be a result of a stress response, such as through the production of biofilms.
- A biofilm is a community of microorganisms that are associated with a surface and are encased within extracellular polymeric substances (EPS). Biofilms can form in diverse environments and as a result, noncellular materials such as soil and mineral crystals can be found within a biofilm matrix. Bacteria in biofilms are phenotypically and genetically different and are harder to kill than their planktonic counterparts because the EPS limits direct contact between microbial cells in biofilms and antimicrobials. Persister cells are a phenotypically different sub-population of cells in biofilms which are more tolerant to antimicrobials. Once removed from a biofilm and the protecting EPS, most bacteria are as susceptible as other organisms that had not been part of the biofilm.
- Efflux pumps are capable of moving a variety of different toxic compounds out of cells and directly accord bacteria additional tolerance to antimicrobials through the extrusion into the external environment of sanitizers or disinfectants that have diffused into the cell. Typically they also improve tolerance to antimicrobials by enhancing biofilm formation.
- Genotypic resistance is a form of resistance that is encoded by specific genes. Such resistance genes may arise via mutation during mitosis (a process of cell duplication) and can encode a system that can reduce the efficacy of an antimicrobial (e.g. efflux pumps).
The different tolerance of microbes to disinfectants depends on different factors. For example, the different structure of the bacterial cell can lead to different susceptibilities to antimicrobials. In addition, there are bacteria that have the ability to form spores (e.g. Clostridioides difficile). This spore state gives the microorganisms increased resistance to certain chemicals or environmental influences (such as high temperatures).
Tolerance to disinfectants and sanitizers – does it impact the performance of such products?
For all antimicrobials, in-use concentration plays an important role in the risk of developing resistance or tolerance to the antimicrobial. Antimicrobials represent a selective pressure on microorganisms. When microorganisms are exposed to a high dose of antimicrobials (acute exposure), they tend to have a much lower chance to develop resistance or tolerance. In contrast, chronic exposure to low levels of antimicrobials represents a higher risk of resistance because such low but prolonged selective pressure allows microorganisms to develop specific mechanisms for resistance and tolerance (Donaghy, 2019) (Kampf, 2019) (Weber, 2006).
Are there differences in the active disinfectant ingredients?
Quaternary Ammonium Chloride (Quat): Tolerance to quat has been relatively well characterized. At levels above the Minimum Inhibitory Concentration (MIC – minimum level of biocide needed for antimicrobial activity), quats disrupt cell membranes. When quats are used at sub-MICs, the mode of action is complicated and always includes multiple processes like modification of the cell’s membranes, hyper expression of efflux pumps, or acquisition of quat specific efflux genes. Although many efflux mechanisms that can provide tolerance to quat can also provide resistance to antibiotics, it is not clear that there is a causal relationship where the use of quat leads to antibiotic resistance or vice versa. Unsurprisingly, there are a variety of quat tolerance genes and those genes can be widely spread and relatively common. However, the impact of such genes on phenotypic tolerance is not clear. It is also not clear that there is an impact of quat tolerance genes on sublethal levels of quat. In general, tolerance genes appear to have no impact on tolerance to recommended use levels of quat sanitizers and disinfectants.
Oxidizers (Chlorine, Peracetic Acid, Hydrogen Peroxide) act as denaturants of proteins by reacting with thiol and amino groups and as a result damage the cellular structures, including the cell wall, membranes and nucleic acids.
As oxidizers target almost all cellular structures indiscriminately, resistance through specific genes and cellular processes are not expected and have not been shown in the literature. However, bacteria can have reduced susceptibility to oxidative stress by forming a biofilm or other phenotypic tolerance. Biofilm-forming microorganisms produce extracellular polymeric substances which quench the effects of oxidizers. Therefore, a higher concentration of oxidizing agent is usually needed to kill microorganisms in biofilms. Some microorganisms produce the enzyme catalase which converts hydrogen peroxide to oxygen and water and can provide intrinsic resistance to very low levels of H202. However, most disinfectants and sanitizers use peroxide at a level that can overcome inactivation by catalase.
Alcohol’s efficacy in killing vegetative bacteria is primarily due to denaturing of proteins with solutions of 60-80% alcohol being typically recommended (WHO, 2009). However, alcohol solutions are not effective antimicrobial agents against bacterial spores (WHO, 2009) (Boyce, 2018). A recent study by Pidot (2018) demonstrated a varying range of efficacy (10-fold range) of alcohol against different isolates of Enterococcus faecium, suggesting the potential that tolerance or resistance mechanisms exist. However this study was done using a 23% solution of isopropanol, which is significantly below the level of alcohol that we’ve just discussed, thus the clinical significance of the finding is uncertain. When the standard 70% alcohol solutions were used instead the efficacy met expectations and was consistent across the various isolates. A study by Tinajero (2019) found no difference in susceptibility to alcohol among Enterococcus faecium isolates after hospital-wide adoption of alcohol-based hand rubs.
Surface disinfectants containing alcohol are typically formulated with other ingredients in order to enhance efficacy and decrease adsorption to soils (Boyce, 2018). Alcohol-based disinfectants are poor cleaners, evaporate rapidly, and are often flammable making them an inferior choice for general surface disinfection (Boyce, 2018). Their biocidal efficacy is known to be compromised by the presence of soils, especially protein-based soils (Boyce, 2018) (WHO, 2009). Using an alcohol-based disinfectant it would be expected that a pre-cleaning of surfaces, which itself would have an impact on the level of microorganisms remaining on the surface. Thus it is uncertain that the results in Pidot (2018) have practical significance for either hand hygiene or surface disinfection.
Conclusion - Based on current literature:
In recent years, several published studies have shown that bacteria can develop resistance not only to antibiotics but also tolerances to disinfectants and sanitisers. However, the publications mainly link this to QUAT as an active ingredient and to long-term sublethal exposure, which is not used in practice. In addition, biofilms must also be specifically considered for the reasons outlined in this article. In any case, there is a need for further research in these areas.
References:
Boyce JM. Alcohols as surface disinfectants in healthcare settings. Infect Cont and Hosp Epidemiol, 2018; 39 (3): 323-328.
Donaghy JA, Balamurugan J, Goodburn K, Grunwald L, Jensen AJ, Kanagachandran K, LaFforgue H, Seefelder W, Quentin MC. Relationship of sanitizers, disinfectants, and cleaning agents with antimicrobial resistance. J Food Prot. 2019; 82(5): 889–902.
Kampf G. Adaptive bacterial response to low level chlorhexidine exposure and its implications for hand hygiene. Microbial Cell. 2019; 6(7): 307-320.
Pidot SJ, Gao W, Buultjens AH, Monk IR, Guerillot R, Carter GP, et. al. Increasing tolerance of hospital Enterococcus faecium to handwash alcohols. Science Translational Medicine. 2018; 10: eaar6115.
Tinajero CG, Bobadilla-Del Valle M, Alvarez JA, Mosqueda JL, De Leon AP, Macias AE. Vancomycin-resistant Enterococcus faecium sensitivity to isopropyl alcohol before and after implementing alcohol hand rubbing in a hospital. Am J of Infect Cont. 2019; 47: e-27-29.
Weber DJ, Rutala WA. Use of germicides in the home and the healthcare setting: Is there a relationship between germicide use and antibiotic resistance? Infect Cont and Hosp Epidemiol. 2006; 27(10): 1107-1119.
World Health Organization. WHO guidelines on hand hygiene in health care. WHO Press. 2009; Geneva, Switzerland.
The content of this piece is excerpted from a larger article published by Diversey, Peter Teska
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