Resistance is the ability of a microorganism to exhibit reduced sensitivity to an antimicrobial treatment that would be effective against other organisms. There are several kinds of resistance, including intrinsic resistance, phenotypically acquired resistance, and genetically acquired resistance.
Intrinsic resistance is the ability of an organism to be insensitive to an antimicrobial condition due to the nature of the microorganism. For instance, some microorganisms can form bacterial spores that enable them to survive conditions like extreme temperatures and drying, as well as exposure to many disinfectants and sanitizers. In particular, non-oxidizing antimicrobials, such as phenolics, alcohol, and quaternary ammonium chloride (QAC), are unable to penetrate a spore coat. And with oxidizing biocides, it generally takes far higher levels and exposure times to inactivate a spore compared to a normal microorganism. For example, it may take 5,000 parts per million (ppm) and several minutes or more to inactivate a spore compared to only 50 ppm of chlorine and 30 seconds.
Another form of intrinsic resistance is displayed by mycobacteria, which have a cell wall that is very hydrophobic and contains a lot of natural wax. This can prevent many biocides, especially non-oxidizing biocides, from penetrating the cell wall. This barrier can be overcome but it requires a higher level of biocide, longer exposure time or the use of other ingredients.
Intrinsic resistance is generally a very stable trait and is closely linked to the basic structure of various microorganisms. In general, the intrinsic resistance of microorganism to biocides is, from most resistant to least resistant: spores>mycobacteria>non-enveloped viruses>gram negative bacteria>gram positive bacteria>enveloped viruses.
Phenotypically Acquired Resistance
The ability of microorganisms to become insensitive to an antimicrobial treatment as a result of how and where the organism grows is considered phenotypically acquired resistance. An example is biofilms, complex communities of microorganisms like bacteria, yeast, molds, protozoa, and viruses. Biofilms attach to surfaces and secrete a material that strengthens and protects the biofilm. Organisms in a biofilm are far more resistant to antimicrobial agents than organisms that are freely in suspension. This increased resistance occurs because antimicrobial agents can’t physically reach the microorganisms through the secreted material or they are inactivated by the material.
Organisms that are on a soiled surface or even in solution with a heavy soil load are also often very resistant to biocides. As with biofilms, this is a result of the biocide being inactivated by the soil or physically prevented from reaching the organism.
Unlike intrinsic resistance, phenotypically acquired resistance is not a stable trait of microorganisms. If the organisms in a biofilm are suspended in solution so that they are no longer protected by the secreted material, the organisms are as sensitive to a biocide as an organism that was not in the biofilm. Or, if the soil is removed, the organisms will become sensitive to biocides. This is one reason why it is important to clean a surface before sanitizers are used.
Acquired Genotypic Resistance
Genetically acquired resistance is insensitivity to a biocide that a microorganism gains either via a mutation or through a transfer of resistance genes from one organism to another. A mutation is a change in an organism’s DNA, and on rare occasions, can make a microorganism resistant to biocides. Exposure to antimicrobials at sub-lethal levels over time can encourage this kind of mutation. Thus, it is critical that all sanitizers and disinfectants are used at the recommended concentrations and in the recommended way. It is also important that biocides drain properly. Pooling or standing solutions of antimicrobials that get diluted to below lethal levels increase the chance that a mutant resistant to that biocide can develop.
Organisms can also acquire a genetic resistance to a biocide via acquisition of a resistance gene from another microorganism. There are different modes of transfer, but the end result is that an organism that was previously sensitive to a biocide can suddenly acquire the genes that cause it to be resistant to the biocide or even multiple antimicrobial agents.
Oftentimes this kind of resistance is not stable. A mutation or resistance gene may only offer a survival advantage as long as the biocide is present. For instance, a mutation in a binding site that makes it less likely that a QAC can bind to a microorganism may also interfere with the binding of nutrients that are important for the cell to survive. While the QAC is present, the mutation acts as a survival advantage but once the QAC is removed, the mutant is still not able to absorb nutrients easily and may disappear from a population once the biocide is gone.
The Impact of Resistance
Many people get concerned about genetically acquired resistance to sanitizers, yet this is one of the least relevant forms. The confusion may be a result of the legitimate concern over genetically acquired resistance to antibiotics. However, antibiotics and the biocides that are used in sanitizers and disinfectants are different compounds used in different ways. Antibiotics often have a single binding site on a target microorganism and a single site at which they are active. They are also used at levels that are very close to the lowest possible level at which the antimicrobial is effective, referred to as the minimum inhibitory concentration (MIC). That means a mutation in a single binding site or active site in a microorganism can make that organism nearly immune to an antibiotic, particularly if the antibiotic is used at levels that are near its MIC.
Biocides have many ways that they can kill microorganisms. In some cases, there may be hundreds or even thousands of binding sites or places in a bacterial cell where the biocide is active. Even if a cell mutates so that a site on its surface no longer binds a biocide, it may have a very limited effect on the effectiveness of the biocide. Another factor to consider is use levels. A sanitizer or disinfectant is often used at many times the MIC for that antimicrobial. For example, the MIC for a typical QAC against many organisms is 0.5-2 ppm. An organism that acquires resistance to a QAC may be able to tolerate 2-5 times that much QAC to survive 1-10 ppm. However, QAC is used at 200-800 ppm in many applications, so this level of resistance really has little or no effect.
Intrinsic resistance is also not a particularly relevant resistance as long as due care is taken when selecting sanitizers and disinfectants. Because this characteristic is stable and is inherent to the nature of target microorganisms, the effectiveness of an antimicrobial can be tested against the microorganism and the antimicrobial’s label will indicate which microorganisms the treatment is effective against.
Often the most serious form of resistance is phenotypically acquired resistance, or organisms growing in biofilms or those protected by soil. The most important step for controlling these kinds of organisms is good cleaning practices. Yet when there is a microbial problem, many people change sanitizers on the assumption that organisms have acquired a genetic resistance or use the sanitizers at higher than recommend concentrations. Unfortunately, because most sanitizers and many disinfectants are poor cleaners and because the chemicals are often prevented from physically contacting microorganisms in biofilms or soil, such responses are ineffective. The correct response to this kind of resistance is to clean better.
Dr. Grinstead, a senior food safety technology fellow with Sealed Air’s Diversey Care division, is a food safety microbiologist with more than 20 years of research and development experience. Reach him at firstname.lastname@example.org.