A biocide is defined as an active chemical molecule that controls the growth of, or kills, bacteria and other microorganisms in a biocidal product [3,6,7,8]. Biocidal substances act in different ways and sometimes several biocides are combined within a single product to increase the overall antimicrobial efficacy [8]. The mechanisms of action and tolerance to a wide range of biocides on bacteria have been reviewed and described widely in the literature [5,9,10,11]. Biocides generally act on multiple targets unless present at sub-inhibitory or low concentration [8], although exact mechanisms of action are not fully understood, and are also organism specific. Reported actions include effects on multiple structural and functional components of the bacteria, thereby disrupting cell walls, cell membranes, cross-linking of proteins, and nucleic acids. Reported mechanisms that bacteria use to reduce the impact of biocides include changes in the permeability of the cell membrane to block the biocide; pumps to reduce the intracellular concentration of the biocide; and enzymes to degrade certain biocides. Due to differences in their membrane, Gram-negative bacteria are generally less susceptible to many biocides than Gram-positive bacteria [3].

Biocides, such as Quaternary Ammonium Compounds (QACs), chlorine-releasing agents, and biguanides are widely used in food animal production. Examples of biocide use include: the cleaning and disinfecting of buildings and equipment; decontaminating ponds and equipment in fish farming; in footbaths for operators outside animal housing; in livestock footbaths to treat and prevent the spread of foot infections such as digital dermatitis; to clean the udders of animals used for milk production; and for preserving specific products such as eggs or semen [3,6,8,12]. They may also be used in anti-fouling paints used in aquaculture to reduce the growth of attached organisms on fish cages and nets [13,14]. Biocides are generally not used within body tissues, but some such as organic acids and essential oils (EOs) are added to animal feed and water as antimicrobial controls [3,15].

While increased AMR to triclosan (5-Chloro-2-(2,4-dichlorophenoxy)phenol) is often discussed in the literature, it is of limited relevance to food animal production [16], as this product was used almost exclusively in human-related products. Due to health concerns and the potential impact on the environment, it has been banned in many countries. 

Some metals (such as cobalt, copper, iron, manganese, molybdenum, selenium, and zinc) are essential in the diet of living things to maintain various physiological functions and are usually added as nutritional supplements in animal feed [17]. They also have antimicrobial properties and may be used for this purpose in food animal production. The antimicrobial modes/mechanisms of action of metals on bacteria have been reviewed and described in the literature [5,18,19]. As with biocides, the exact mechanisms of action still remain unclear and are also organism specific, but reported actions include effects on the cell wall or membrane; interactions with DNA; binding or inhibition of enzymes and membrane proteins. Reported mechanisms that bacteria use to reduce the impact of metals are similar to those used to reduce the impact of biocides and include enzymes to modify the metal; changes in the permeability of the cell membrane to block the metal; and efflux pumps to reduce the intracellular concentration of the metal. Again, as with biocides, due to differences in their membrane Gram-negative bacteria are generally less susceptible to metals than are Gram-positive bacteria [3].

Copper and zinc are widely used in the pig and poultry sectors as in-feed growth promotors and for enteric disease control [3]. Zinc is also used in aquaculture as a supplement in feed [13,20]. Metals are often used in higher concentrations than needed to ensure adequate nutrition [21,22]. Since the bioavailability of metals in feed is usually quite low, unabsorbed metals are excreted in feces and may accumulate in soil, water, and sediments from food animal production practices. One study in the USA found 90% of in-feed copper and zinc fed to pigs was shed in feces [21]. Although the use of forms of these metals with higher bioavailability (organic forms rather than inorganic) allow for substantial reductions of dietary inclusion rates and consequentially less environmental impact [23,24].

The total amounts and concentrations used of copper and zinc in food animal production may differ among countries, due to restrictions imposed by national legislation. For example, the permitted maximum zinc content in animal feed in the EU (Regulation 2016/1095) is 180 mg zinc/kg for salmonids and in milk replacers for calves; 150 mg zinc/kg for piglets, sows, and all fish species other than salmonids; and 120 mg zinc/kg for other species. The permitted maximum copper content in animal feed in the EU (Regulation 2018/1039) is 15 mg copper/kg for immature bovines (cattle) before the start of rumination; 30 mg copper/kg for other bovines (cattle); 15 mg copper/kg for ovines (sheep); 15 mg copper/kg for caprines (goats); 150 mg copper/kg for piglets suckling and weaned up to 4 weeks after weaning; 100 mg copper/kg for piglets from 5th week after weaning up to 8 weeks after weaning; 50 mg copper/kg for crustaceans; and 25 mg copper/kg for other species.

Other uses of metals include use in livestock footbaths to treat and prevent the spread foot infections such as digital dermatitis [22,25] and in wound dressings [3]. Following concerns over therapeutic use of zinc at high concentrations in animal production potentially leading to an increased prevalence of livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA) and environmental contamination, zinc is now only permitted in the EU and UK at concentrations up to 150 ppm for nutritional use [26]. Copper is the principal biocidal component of anti-fouling paints used in aquaculture to reduce the growth of attached organisms on fish cages and nets [13,14]. Copper has also been studied as an antimicrobial alternative to stainless steel surfaces in food production and processing environments [27]. The use of silver and zinc nanoparticles as antimicrobial controls and alternatives to antibiotics in food animal production have received attention in recent years [9,28,29].

Co-selection mechanisms for biocides and/or metals and clinically as well as veterinary-relevant antibiotics are described widely in the literature [3,5,12,30,31,32], amongst others. There are two main types of related resistance/tolerance co-selection mechanisms:

Cross-resistance adaptions may be normally present (intrinsic) in the bacteria, or readily acquired by mutation or genetic transfer under appropriate conditions [3,12]. Such adaptions include efflux pumps (transport proteins involved in the extrusion of toxic substrates from within cells into the external environment [33]), biofilm formation, spore formation, nutrient stress responses, and reduced cell envelope permeability [3,12].

Efflux pumps may expel a broad range of unrelated and structurally diverse compounds. Thus, whether intrinsic or acquired, bacteria possessing efflux pumps have substantial potential for cross-resistance to antibiotics, biocides, and/or metals, though this does depend on the nature of the efflux pump [3,33].

Biofilms are complex structures formed by different or single types of bacteria adhering to surfaces which may enhance resistance/tolerance to different antimicrobial agents [34]. Biofilms produce an extracellular matrix that provides a diffusion barrier, plus a potential site for neutralization or binding, of chemical agents, and an enhanced medium for bacterial genetic exchange [3,12]. Bacterial biofilms have been well documented to be highly resistant to antimicrobials [35]. The presence of multiple species of bacteria in biofilms may allow for horizontal gene transfer (HGT) of resistance genes between different bacteria [36]. Biofilms can generate a state of hypermutability (capability for excessive mutation) in bacteria in part due to stress and slower growth that stimulates the development of resistance/tolerance, which may also co-select for AMR [22,34].

Co-resistance/co-transfer may be acquired through the release of resistance genes in MGEs. They may potentially allow some proportion of the bacterial population to survive an otherwise terminal challenge, increasing the risk of selection of organisms permanently adapted to the antimicrobial agent [3]. There can be a genetic link between resistance/tolerance to different agents (co-resistance) through the co-location of different resistance genes on MGEs [3,37,38].

Resistance in many antimicrobial-resistant bacteria (ARB) is encoded by genes that are carried on large conjugative plasmids [39]. These plasmids typically contain multiple antibiotic resistance genes (ARGs) as well as genes that confer reduced susceptibility/tolerance to biocides (BRGs) and/or metals (MRGs), and there are numerous examples reported in the literature [40,41,42,43,44,45]. However, an analysis of the co-occurrence of ARGs, BRGs, and MRGs by Pal et al. [46] concluded that plasmids provide limited opportunities for biocides and metals to promote HGT of AMR through co-selection (though this was more common in bacteria of animal origin), whereas greater possibilities exist for indirect selection (and therefore clonal selection) via chromosomal BRGs and MRGs.

There is evidence that zinc and/or copper may co-select for LA-MRSA due to co-location of the zinc/copper MRG czrC and the methicillin resistance gene mecA (or its homologue mecC) within the staphylococcal cassette chromosome (SCC) SCCmec element [47,48,49,50,51,52,53]. SCCmec is a MGE that can also transfer to other Staphylococcus spp. [54].

There is evidence that some adaptations that enable resistance to antimicrobial agents may result in associated costs to the organism, usually termed “fitness cost”. An example being broad substrate efflux pumps, which consume cell energy resources and indiscriminately remove some useful metabolic substances from the cell [3,16]. Carriage of plasmids (containing resistance genes) have also been cited as another example [39]. However, it has also been reported that compensatory mutations can arise that offset such plasmid fitness costs [55,56].