Antimicrobial Resistance: Implications for the Food System Microorganisms have an inherent ability to evolve—to mutate and adapt to environmental stressors─allowing them to survive otherwise lethal conditions.

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Classification of antimicrobials

For the purposes of this report, "antimicrobial" is a general term used broadly to refer to any compound, including antibiotics, food antimicrobial agents, sanitizers, disinfectants, and other substances, that acts against microorganisms. The definitions and use of each of these terms differ among various groups. Legal definitions exist for use in a regulatory context.

The term antibiotic is used in this report to refer to drugs used to treat infectious disease in humans, animals, or plants, by inhibiting the growth of or destroying microorganisms; such substances may be naturally occurring, semisynthetic, or synthetic. Antibiotics are also used in food animals to prevent infectious disease and improve the efficiency of feed utilization. Within the antibiotic classification are synthetic antimicrobials such as quinolones, that differ from other substances such as streptomycin, which are natural products or fermentation derived antibiotics. Antibiotics are legally classified as such only when used in humans. They are classified as "veterinary antimicrobial drugs" when used in animals and as "pesticides" when used in plants.

"Biocide" is a general term that refers to chemical agents, such as disinfectants and sanitizers, which are usually broad spectrum. Because biocides vary in antimicrobial activity, other terms may be used to more specifically describe the nature of the antimicrobial activity. For example, terms ending in the suffix "-static," such as "bacteriostatic," are used for agents that inhibit microbial growth without killing the microbes, and terms with the suffix "-cidal," such as "fungicidal," refer to agents that kill the target microbe (McDonnell and Russell 1999). "Disinfectants" destroy or irreversibly inactivate infectious fungi and vegetative bacteria (growing or nonsporeforming), and are used in hospitals, food processing facilities, restaurants, and elsewhere for general purposes (EPA 2005). In the legal connotation, disinfectants include "any oxidant, including but not limited to chlorine, chlorine dioxide, chloramines, and ozone, added to water in any part of the treatment or distribution process, that is intended to kill or inactivate pathogenic microorganisms" (40 CFR §141.2). "Sanitizers," comprised of 2 categories–no-rinse food contact surface sanitizers and nonfood contact surface sanitizers–refer to substances that reduce microbial contamination and destroy vegetative pathogens of public health significance on treated inanimate surfaces. "Sterilants," such as peroxyacetic acid, refer to substances that eliminate all forms of microbial life, including bacterial spores, fungi, and viruses. IFT uses the legal connotation "food antimicrobial agent" to refer to antimicrobial substances, such as nisin and other bacteriocins, including mold inhibitors, which are used to preserve food by preventing microbial growth and subsequent spoilage. Antibiotics cannot legally be used as food additives; thus, they are specifically excluded from this classification.

Classification of resistance

Discussion of antimicrobial resistance, by necessity, must include defining what is meant by resistance. While it would seem that defining resistance would be a simple matter, many definitions exist (Davison and others 2000). Resistance to most traditional, regulatory-approved, or naturally occurring food antimicrobial agents is difficult to characterize because of the lack of a precise definition for such resistance. From a functional perspective, resistance correlates with failure of a given antimicrobial treatment, whereas from a laboratory perspective, resistance is denoted through a "Minimal Inhibitory Concentration" (MIC)2 value that exceeds a threshold value, which may or may not be associated with a clinical outcome. Chapman (1998) stated that a microorganism is resistant if it exhibits "significantly reduced susceptibility" when compared with that of the "original isolate" or a group of sensitive strains. In this report, resistance means "temporary or permanent ability of a microorganism and its progeny to remain viable and/or multiply under conditions that would destroy or inhibit other members of the strain" (Cloete 2003). These terms and the different types of resistance are described below.

As Courvalin (2005) describes, resistance can result from mutations in housekeeping structural or regulatory genes, or alternatively, horizontal acquisition of foreign genetic information. In some cases, resistance may manifest through multiple mechanisms. For example, three different strategies are thought to be involved in resistance to tetracycline (Schnappinger and Hillen 1996). Resistance can also be intrinsic, that is microorganisms without known exposure to antimicrobial agents may be resistant to some agents (see below).

If a resistant strain is isolated from an environment containing an antimicrobial or prepared in the laboratory by exposure to increasing concentrations of an antimicrobial, resistance may be due to a genetic alteration or a temporary adaptation. It may be that temporary adaptation to an antimicrobial through some type of homeostatic mechanism plays a much larger role than true genetic mutation among food-related antimicrobials. To date, research on resistance to food antimicrobials has focused almost exclusively on innate or intrinsic mechanisms of the target microorganisms.

Innate (Intrinsic) resistance. As is the case for a natural property of a microorganism, innate resistance is chromosomally controlled (Russell 1991). Innate resistance is related to the general physiology or anatomy of a microorganism and stems from pre-existing mechanisms or properties. This type of resistance is most likely responsible for differences in resistance observed among different types, genera, species, and strains of microorganisms in identical environmental conditions and concentrations. Innate resistance may stem from the complexity of the cell wall, efflux mechanisms (means by which microbes pump antimicrobials out of the cell [Gilbert and McBain 2003]), or enzymatic inactivation of the antimicrobial (Russell 2001). For example, because of the complexity of their cell walls, Gram-negative bacteria generally have a higher level of resistance to antibacterial agents than typical Gram-positive bacteria (Russell and Chopra 1996). More specifically, Gram-negative bacteria are innately resistant to penicillin G by virtue of their double membrane structure, which prevents the antibiotic from accessing the cell wall target. Similarly, Mycobacterium species are more resistant than other nonsporeforming bacteria due to high lipid content in their cell walls and comparatively high hydrophobicity. Other bacteria–Bacillus, Pseudomonas, Corynebacterium, Micrococcus, and the fungus Aspergillus have innate resistance to benzoate because they are capable of metabolizing the compound to succinic acid and acetyl coenzyme A (Chipley 1993). Innate resistance is not considered an important clinical problem because antibiotics were never intended for use against intrinsically resistant bacteria.

There are certain circumstances in which antimicrobial agents do not adversely affect bacteria that are generally susceptible to the particular agent. Because the efficacy of most food antimicrobials and sanitizers is dependent upon and influenced by the conditions of the application, some situations may permit bacterial resistance that would not have occurred otherwise (IFT 2002a). Exposure conditions, such as the environmental conditions (temperature, pH, and food composition) of the antimicrobial application, or interaction of the antimicrobial with components of the suspension medium or food product can influence the efficacy of the antimicrobial agent (Davidson 2001). For example, organic matter can quench the hypochlorite ion and therefore eliminate its efficacy at killing generally susceptible bacterial populations (Kotula and others 1997). However, microorganisms that are generally susceptible to antibiotics can themselves also become temporarily resistant to an antimicrobial through activation of silent, resident gene(s) that confer this resistance. A good example of this occurring is observed with the survivability of biofilm-associated cells versus planktonic (free floating/living) cells (Frank and Koffi 1990; Mosteller and Bishop 1993; Mustapha and Liewen 1989). Microbial cells in biofilms exhibit resistance primarily through the protection provided by extracellular materials such as exopolysaccharides. Also, nongrowing bacterial cells are resistant to many antibiotics that target cell wall synthesis. Once conditions again become favorable for growth, these bacterial cells become susceptible again to these cell wall inhibitors. Also, the few reports of resistance to food antimicrobial preservatives and sanitizers are attributed to microbial stress responses to sublethal stressors, such as low or high temperatures, acidity, osmolarity, low moisture, high atmospheric pressure, low oxygen or anaerobic conditions, gas atmospheres, competing bacteria, and low nutrient environments that trigger physiological changes and subsequently confer resistance to these compounds (Abee and Wouters 1999; Archer 1996; Samelis and Sofos 2003a; Sheridan and McDowell 1998; Sofos 2002a).

Acquired (Extrinsic) resistance. Acquired resistance results from genetic changes that occur through mutation of the antimicrobial's target site within the bacterium or acquisition of genetic material encoding resistance via plasmids3 or transposons4 containing integron sequences5 (Roe and Pillai 2003; Russell 1991, 1996; Russell and Chopra 1996). Acquired resistance, the most common type of antibiotic resistance, has been well studied for antibiotics, but has not been well studied for food antimicrobial agents and sanitizers. Acquisition of genes for β-lactamase (an enzyme capable of breaking down and inactivating β-lactam antibiotics [penicillins and cephalosporins]) and mutation of one of the subunits of DNA gyrase (the target of fluoroquinolones) are examples of this type of resistance. Another example includes resistance of some microorganisms to sanitizing compounds, such as quaternary ammonium compounds (QACs), as a result of the presence of plasmid-encoded efflux pumps that remove the QACs (Russell 1997).

Although acquired resistance is of concern in the use of food antimicrobial agents and sanitizers, occurrence of such resistance appears to be rare. Unlike antibiotics, which generally have specific target sites, biocides (that is, disinfectants, sanitizers, antimicrobials) typically act nondiscriminately against multiple nonspecific targets (Bower and Daeschel 1999); thus, single mutations or biochemical alterations of cellular targets seldom confer resistance to biocides.

Adaptation. For certain types of antimicrobials, adaptation may be demonstrated by exposing a microorganism to a stepwise increase in concentration of the substance. This type of resistance, however, is often unstable; the microorganism may revert back to the sensitive phenotype when grown in an antimicrobial-free medium, termed "back-mutation" (Russell 1991). In the absence of selection pressure, the mutations associated with resistance may actually reduce fitness of the bacterial strain compared to the wild type, parental strain. Stabilizing, secondary, compensatory mutations are sometimes needed to maintain resistance and reduce fitness cost associated with the original "resistance" mutation.