a literature search using relevant terms such as  zn and animal feed , bacteria , ( antibiotic resistance or antimicrobial resistance ) , requirement , bioavailability using the advanced search builder provided by pubmed ( www.ncbi.nlm.nih.gov/pubmed ) or web of science was performed . a similar search using the same terms , but the reference lists in the selected citation were scrutinized to identify additional articles or reports , overlooked by the searches . the titles of all hits were scanned , and for those that were of potential relevance , the abstracts were also scanned . of these , for those of potential relevance , the full text was obtained and assessed whether it was of relevance to this paper . original and review articles , and textbook content were included as references in this paper . a list of the articles on zn / cu driven co - selection of antibiotic resistance , which fulfilled the inclusion criteria with summary of the findings and main conclusion , is presented in table 1 . mrsa : methicillin resistant staphylococcus aureus ; mssa : methicillin sensitive staphylococcus aureus , arg : antimicrobial resistance gene the microbiota associated with animals represents a complex assemblage of microorganisms covering all three domains of life ( bacteria , achaea and eukarya ) ( 5 ) . all body sites are colonized , with the lower gastrointestinal tract being the most densely populated . in the number of cells , the microbiota generally outnumbers the host by a factor of 10 , while with respect to the number of genes the microbiota contains 100 times more genes than the host . thus , the gut microbiota can be considered an organ in itself ( 6 ) . the function of the microbiota is to protect the host from pathogen invasion , to train the immune system , to extract energy from low accessible nutrients , and in addition to produce essential vitamins and metabolites needed by the host ( 7 ) . generally , lower gut microbes are strictly anaerobic due to the anaerobic conditions in that area . the anaerobic conditions are mainly created by microbial respiration ( 8) . the porcine gut microbiota shows a dominance of the phyla firmicutes and bacteroidetes , which is similar to that of humans ( 9 , 10 ) , while the chicken microbiota is lower in bacteroidetes , and higher in lactobacillus and proteobacteria 
 ( 11 ) . it has been proposed that diet is the main driver for the composition and functioning ( nutrient breakdown and metabolite production ) of the gut microbiota since microorganisms in the gut can utilize nutrient compounds that the host can not break down ( 12 ) . mainly due to the complexity , we have very limited knowledge of what shapes the host associated microbiota composition ( 10 ) . the main unresolved questions are if the host - associated microbiota is shaped by bacterial  bacterial competition , or if the host can shape the composition ( 13 ) . in microbial ecosystems there is a delicate balance between trace metals such as zn and cu as limiting factors , and the toxic effect of these ( 14 ) . zn and cu are common co - factors in enzymes , while the effects of elevated exposure are more diverse , ranging from the replacement of other trace elements , binding to enzymes , and oxidation . it has been hypothesized that the antimicrobial effect of zn and cu leads to growth promotion in a similar manner as for the effect of antibiotic - based growth promoters ( 15 ) . for cu , although it has been reported that there is a shift in the microbiota associated with exposure ( 16 ) , it could be that its main pathogen related effect is through increased host tolerance for lipopolysaccharides ( 17 ) . a range of pathogens can exploit host responses through inflammation induction ( 18 , 19 ) . for instance , upon inflammation the host will produce chelating agents such as calprotectin that will limit the availability of the trace elements and thereby bacterial growth ( 20 ) . for zn , however , recent evidence suggests that pathogens can have a competitive advantage over the commensal microbiota under zn limiting conditions , thereby being promoted under an inflamed state ( 14 , 21 ) . since diarrhea in itself can lead to zn depletion and this could also promote the pathogen survival . the potential mechanisms of growth promotional effects of zn / cu are attributed to their antimicrobial activities , similar to that of antibiotics , in that gut microbiota are altered to reduce fermentation loss of nutrients and to suppress gut pathogens ( 22 ) . the data from ( 22 ) illustrated reduced sizes of major groups of bacteria among the porcine gastrointestinal commensals , namely , the lactobacilli and streptococci by elevated doses of dietary zno and cuso4 . the reduced level of these commensals in the proximal part of the gastrointestinal tract may benefit the host animal by allocating more feed components for its growth performance . furthermore , feeding the animals high dietary zno doses resulted in an altered pattern of organic acid accumulation , with lower levels of lactate and succinate in the stomach and small intestine and an accumulation of these compounds in the cecum and colon . how this influences the physiology of the animals needs further elucidation in detail , since lactic acid produced in the stomach is normally considered a part of the natural defense mechanism of the host , whereas lactate accumulation in the large intestine has mainly been observed in connection with various disorders . cuso4 reduced the number of coliforms in the large intestine , which may be a part of other mechanisms , such as the suppression of specific pathogens and induced resistance of the animal to pathogen adhesion and invasion as well as pathogen - produced toxins ( 23 ) . trace elements like zn and cu may be toxic to bacteria and this microbial toxicity may be due to their chemical affinity to the thiol groups of macro - biomolecules but also depends on the solubility of the metal compounds under physiological conditions . to avoid cellular toxicity to elevated trace element exposure , bacteria have evolved mechanisms of metal tolerance . both the mechanisms of toxicity and tolerance to trace elements in bacteria are discussed extensively in the review article of seiler and berendock ( 24 ) . the authors concluded that in addition to antibiotic agents , heavy metals used in animal farming and aquaculture might promote the spread of antibiotic resistance via co - selection . it has been proved that antimicrobial agents other than antibiotics have the ability to promote a co - selection process , indirectly selecting for antibiotic resistance ( 25 ) . the trace elements like zn and cu seems to have potential to act as a selective pressure that forces the proliferation and evolution of zn / cu and antibiotic resistance not only at the farm level , but also in the environment . the total amounts and concentrations used of zn and cu in feed may differ among countries , due to f. ex . restrictions imposed by national legislation . as a consequence different selective pressure might be exerted within different countries . data regarding development of resistance against zn and cu in bacteria of human origin is deficient . the average exposure of zn and cu in humans , from food , is probably usually far lower than the exposure in animals fed diets supplemented with these trace elements . in bacterial isolates found in animals ( table 1 ) , elevated minimum inhibitory concentration ( mic ) values to zn and cu were detected in several opportunistic bacterial species compared to background isolates . the data presented in these studies indicate that such elevation in mic - values may be due to elevated exposure of these trace elements in animal feed . mic is defined as the lowest concentration of a given agent that inhibits growth of a microorganism under standard laboratory conditions . testing for susceptibility against zn / cu in various bacterial species was performed using either a micro - dilution technique or an agar - dilution technique and under different methodological conditions ( studies listed in table 1 ) . there is currently no standardized and approved method to determine the mic values for zn / cu . among the examined bacterial species , the development of resistance to cu in enterococci is associated with the presence of a cu resistance gene ( tcrb ) , which is often located on a plasmid ( 3 , 2628 ) . in enterococci , the cu resistance gene tcrb was shown to be associated with resistance to the macrolide antibiotic erythromycin ( ermb ) . a conjugation study demonstrated co - transfer of tcrb and ermb genes between e. faecium and e. faecalis 
 ( 3 ) . transferable cu resistance tcrb has been reported in these enterococci isolated from piglets , calves , poultry , as well as humans in denmark ( 29 ) . several studies performed in denmark show a link between resistance to cu and resistance to macrolides and also to glycopeptides ( vancomycin ) in enterococcal isolates of pig origin ( 30 , 31 ) . the authors concluded that there is a frequent occurrence of cu resistance gene in these isolates , where cu sulphate is being used in large amounts as feed additive . the glycopeptide antibiotic avoparcin has been used as a growth promoter in animal production by adding to feed , in many european countries , including norway , in the past ( from mid-1970s ) . however , it has been prohibited since the 1990s because of the development of vancomycin resistance in bacteria , in particular in enterococci . the discontinued use of avoparcin in animal feed has resulted in a reduction in the number of vancomycin - resistant organisms isolated from animals ( 32 , 33 ) . because avoparcin and vancomycin are similar in structure , bacteria resistant to avoparcin are resistant to vancomycin as well . vancomycin - resistant enterococci ( vre ) spread rapidly and have become a major problem in many countries . the possibility of transfer of vancomycin resistance genes from enterococci to other gram - positive bacteria , like staphylococci , raises significant concerns about the emergence of vancomycin - resistant s. aureus 
 ( 34 ) . nowadays , vancomycin constitutes one of the last resort antibiotics for treatment of mrsa infection in humans . several studies in table 1 have demonstrated an association between resistance to zn and resistance to methicillin in staphylococci ( 4 , 35 , 38 ) . ( 38 ) found that mrsa strains from pigs from european countries , canada , and china had a high prevalence of zn resistance ( mainly associated with czrc gene ) , whereas the corresponding mssa were susceptible . similar association between resistance to zn and resistance to methicillin was also observed in samples from veal farms from the netherlands . methicillin is not the drug of choice for treatment of infection in veterinary medicine , neither in norway nor in any countries within eu . there is a lack of knowledge regarding the source of methicillin  zn - resistant staphylococci in animals . it is not clear whether the methicillin - resistant staphylococci in animals are of human origin and have been resistant to zn after exposure to feed or the zn - resistant staphylococci have been resistant to methicillin , due to exposure to antibiotic(s ) . a recent publication from germany ( 37 ) showed a higher diversity of e. coli clones in piglets fed with diets supplemented with zn compared to the background control group . the proportion of multiresistant e. coli was significantly increased in the zn group compared to the control group . the authors suggested two possible mechanisms for their results : 1 ) co - selection via zn resistance as some of the isolates were both zn and antimicrobial resistant ; 2 ) enhanced plasmid uptake under the influence of zn , as the authors detected several resistance plasmids in isolates of the zn feeding group . there is a lack of data which can demonstrate whether cu / zn - resistant bacteria may acquire antibiotic resistance genes / be antibiotic resistant or antibiotic - resistant bacteria are more capable to be cu / zn - resistant than antibiotic susceptible bacteria . the resistance genes to these trace elements are identified in some bacterial species from animals . resistance to zn / cu and its link to antibiotics resistance in bacterial species originated from animal resistance genes to zn / cu are often located on plasmids , which may be transferable to other bacteria , intra- and inter - species . although overuse of antibiotics in agriculture and medicine is partially responsible for the increased level of antibiotic resistance in bacteria , exposure to trace metals may also contribute to antibiotic resistance , even in the absence of antibiotics themselves . a resistance link between zn and methicillin resistance to cu is often linked to resistance to macrolides ( e.g. erythromycin ) or glycopeptides ( e.g. vancomycin ) in enterococci . zn supplementation to animal feed may increase the proportion of multiresistant e. coli in gut microbiota . the transmission of zn / cu - resistant bacteria with resistance to antimicrobial agents to human microbiota can not be discounted . our knowledge regarding mechanisms of induction of zn / cu resistance in bacteria is limited and knowledge on dose - response relations is lacking . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .