Potential impact of antimicrobial resistance in wildlife, environment and human health

1Microorganisms play an important role in the cycling of elements at a global scale, thus profoundly and directly affecting the environments, in which all of life evolves. While microorganisms affect the environment, the environment in turn also engenders evolutionary pressures on the microorganisms themselves.

2Recent advances in DNA sequencing, high-throughput technologies, and genetic manipulation systems have permitted empirical studies that directly characterize the molecular and genomic bases of evolution. This launched the challenge of unraveling the genotype-phenotype connection, with implications not only for the investigation of evolution, but also physiology, disease risk, development, and biodiversity.

3In microbial populations, evolutionary change is supplied by two sources of new variation: horizontal transfer of genetic material from other, sometimes distantly related species, and mutation. Though, in the short run, mutation is the primary source of new genetic variation driving evolutionary change in microbial populations. In an asexual microbial species, evolutionary changes will happen by the repeated selection of new clones carrying adaptively favorable mutations.

4In fact, firstly, genomic sequencing can determine the complete set of mutations responsible for an advanced phenotype, and has led to the discovery that interactions between these mutations are very usual. Secondly, adaptive mutations commonly target regulatory mechanisms. Thirdly, principles of systems-level optimization cause the genetic changes seen in adaptive evolution, and with a systems-level understanding, these optimization principles can be harnessed for the purposes of metabolic engineering. Fourthly, mutant sub-populations of enhanced fitness invariably arise in growing populations, but their dynamics in the population are complicated due to factors such as natural selection, clonal interference, drift, and frequency-dependent selection.

5Antibiotic-resistant bacteria are extremely important to human health, but the wild reservoirs of resistance determinants are poorly understood. The origins of antimicrobial resistance in the wildlife is important to human health because of the increasing importance of zoonotic diseases as well as the need for predicting emerging resistant pathogens. Wild animals provide a biological mechanism for the spread of antibiotic resistance genes. Antimicrobial-resistant Escherichia coli and Enterococcusspp. isolates originating from wildlife species were reported for the first time from Japanese wild birds. With the new millennium several studies, in different continents, have described the occurrence of antimicrobial-resistant in these bacteria species in wildlife. On the other hand, antimicrobial resistance have been described in others important pathogens, such as Salmonella spp. and methicillin-resistant Staphylococcus aureus (MRSA; ) in wild animals.

6Proximity to human activities influences the antibiotic resistance profiles of the gut bacteria of wild mammals, which live in densely populated microbial habitats in which antibiotics select for resistance. While various bacterial species are important in terms of multiresistance and nosocomial infections in human and veterinary medicine, we consider the Gram-positive vancomycin-resistant enterococci (VRE) and Extended-spectrum Beta-Lactamases producing Gram-negative bacteria like E. coli (ESBL-E. coli) as being key indicator pathogens to trace the evolution of multiresistant bacteria in the environment and wildlife.

7Enterococcus spp. are Gram-positive facultative anaerobic bacteria, spherical, which occur singly, in pairs or short chains and fit within the general definition of lactic acid bacteria. Most enterococci are not virulent and are considered relatively harmless, with little potential for human infection. However, they have also been identified as nosocomial opportunistic pathogens with increased resistance to antimicrobial approved agents. Incidence of VRE among wild animals has been reported in several countries, including in remote areas.

8Escherichia coli is the head of the large bacterial family, Enterobacteriaceae, the enteric bacteria, which are facultatively anaerobic Gram-negative. This intestinal bacterium can be easily disseminated in different ecosystems. For this reason, fecal E. coli is considered to be an important indicator for the selective pressure exerted by the use of antimicrobials on intestinal populations of bacteria. The production of ESBLs by Enterobacteriaceae, specifically by E. coli, has caused a major concern in several countries, being frequently implicated in human infections. Previous reports have described ESBL-containing E. coli strains in healthy wild animals.

9The common occurrence of antimicrobial resistance in wildlife has several implications such as: the potential to serve as an environmental reservoir and melting pot of bacterial resistance; the zoonotic potential of enteric bacteria; and the potencial problems of the medical treatment of wildlife. This review aims to summarize the current knowledge on ESBL-E. coli and VRE in wildlife, in turn underlining the need for more large scale research, in particular sentinel studies to monitor the impact of multiresistant bacteria on wildlife.

10The combination of mutation and horizontal transfer has created the overall phylogenetic structure of E. coli and enterococci, resulted to currently recognized four main phylogenetic groups for E. coli and different species for enterococci; and their respective lineages. Thus, an appreciation of mutation and horizontal transfer as important evolutionary processes within bacteria, in general, contributes in understanding of their roles, distribution and mechanistic modes of behavior.

11A multilocus enzyme electrophoresis (MLEE)-based phenogram using 38 enzymes identified four main phylogenetic groups (A, B1, B2, and D) and two accessory groups (C and E) in E. coli. These phylogenetic groups were recovered using the 1,878 genes of the Escherichia spp. core genome and the 2.6 million nucleotides of the E. coli chromosomal backbone, which permitted a robust phylogeny to be developed; the first split in the E. coli phylogenetic history leads to one branch including the strains of group B2 and a subgroup within D that we called group F75 and another branch containing the rest of the species. The remaining strains of group D then appeared from this second branch, followed by group E. Finally, groups A and B1 are sister groups whereas group B2 is included in an ancestral branch. The B2 group reveals the highest diversity at both the nucleotide and the gene content level, supporting its early occurrence in the species lineage and suggesting that it has subspecies status.

12described a triplex polymerase chain reaction (PCR) strategy to assign E. coli isolates quickly to one of these phylogroups. It sought three phylogenetic group markers, the chuA and yjaA genes encoding hypothetical proteins and the TSPE4.C2 DNA sequences situated within a gene encoding putative lipase esterase, and groups were assigned based on different combinations of presence and/or absence of the three amplicons.

13found that strains from phylogenetic groups B2 and D contained more virulence factors than strains from the phylogenetic groups A and B1. Usually, the extraintestinal pathogenic strains belong to groups B2 and D, the commensal strains to groups A and B1, whereas the intestinal pathogenic strains belong to groups A, B1, and D.

14Nowadays, these phylogenetic groups differ in their ecological niches, life-history and some characteristics, such as their ability to exploit different sugar sources, their antimicrobial resistance profiles and their growth rate. A recent survey demonstrated that the majority of the E. coli strains that are able to persist in the environment belong to the B1 phylogenetic group.

15Various researchers analyzed the distribution of the main phylogenetic groups among E. coli strains isolated from human and animal faces; it was found that the relative abundance of phylogenetic groups among mammals is dependent on the host diet, body mass, and climate. A study analyzing fecal strains isolated from birds, non-human mammals, and humans, observed the prevalence of groups D and B1 in birds, A and B1 in non-human mammals, and A and B2 in humans. These different reports concluded that one of the main forces that shapes the genetic structure of E. coli populations among the hosts is domestication. Furthermore, other study in the south of French Guiana, human strains very rarely were observed as belonging to B2 phylogroup (3.7%) whereas wild animal strains were characterized by 46.1% belonging to B2 phylogroup. Moreover, feces from zoo animals were analyzed and a prevalence of group B1 in herbivorous animals and a prevalence of group A in carnivorous and omnivorous animals were found. Furthermore, domesticated animals have a decreased proportion of B2 strains than wild animals (from 30% in wild animals to 14 and 11% in farm and zoo animals, respectively) and an increased proportion of A strains (from 14% in wild animals to 27 and 26% in farm and zoo animals, respectively; ).

16In Portugal, the prevalence of E. coli of groups A and B1 was observed in wild birds as seagulls, birds of prey, and Passeriformes, but also in wild mammals as Iberian lynxes, red foxes, and in Iberian wolf (; Figure 1). In addition, a report conducted by shows that 37% of all ESBL-E. coli isolated from seagulls belong to B2 or D phylogroup, a higher rate than previously reported (27% of all E. coli; ).

17It is interesting to note that fecal samples from red foxes showed that the E. coli isolates from phylogenetic groups A and D were predominant. Similar results were observed in chickens and swine, in wild boars and in wild birds in the same geographical area.

18With the exception of E. faecium and E. faecalis, the enterococci are infrequently described to be involved in human pathogenesis. Though, in some countries association of strains of E. faecalis and E. faecium with human disease has reached proportions of serious concern. However, these enterococci species can show significant differences in the incidence of virulence factors and antimicrobial resistance. Generally, E. faecalis appears to harbor more virulence traits while E. faecium strains were generally free of virulence factors. In addition, considering the distribution of the antimicrobial resistance according to the species, the E. faecium possessed a higher level of resistance than E. faecalis. Actually, the majority of hospital-derived isolates of E. faecalis cluster in three clonal complexes, CC2, CC9, and CC87, and included the highest proportion of multiresistant isolates., while the CC17 is a major group of genetic lineage of E. faecium that has widely spread worldwide and it is associated with hospital outbreaks. Although E. faecalis CC2 and CC9 strains has been detected outside hospitals in farm animals and environment, to our knowledge has not been identified in wild animals. On the other hand, E. faecium CC17 was recovered from seagulls in Portugal.

19Recent comparisons of available genome sequences support the concept of a hospital-associated clade that is genetically distinct from most commensal isolates from animals and humans. In Portugal, concerning wild animals, E. faecium were found to be the main species in seagulls; birds of prey, partridges, Iberian wolf, red foxes, wild boars, gilthead seabream, and Echinoderms, while E. faecalis was dominant in Passeriformes and wild rabbits. On the other hand, E. hirae was the predominant specie isolated from Iberian lynx (,; Figure 2).

20Along with seagulls and birds of prey in Portugal, migratory Canada geese report showed also the occurrence of E. faecium and E. faecalis species. Furthermore, different reports found that all enterococci isolates detected in wild mammals and also in glaucous gulls were E. faecium species. Another study performed in Brazil, showed the occurrence of E. faecalis in non-human primate (capuchin monkeys and common marmoset) fecal samples. In American bison fecal samples, E. casseliflavus was the predominant species recovered with 62.4%, followed by E. faecalis (16%; ).

21With the arrival of next-generation sequencing, it was quickly possible to investigate hundreds of strains to get a better knowledge at the whole-genome level the evolutionary processes acting in populations, opening the era of “population genomics”. Certainly further studies using new high throughput technologies are mandatory to completely understand the evolution of predominant clones and species in different hosts and environments.

22Large-scale epidemiological studies provide insight into the diversity and complexity of E. coli and enterococci niches. Thus, genome plasticity has contributed to the emergence of new virulence traits: some clusters of genes or genomic islands, including pathogenicity islands (PAIs) should be discovered only in a subset of strains and favored in some specific environments. Furthermore, several alternative combinations of genes could promote similar adaptations to a given environment. Gene gain and loss make fundamental contributions to new habitat adaptation and the emergence of new lineages. Strains related to hospital infections were found to have significantly larger overall average genome size than strains from non-hospitalized humans or animals groups. The prevalence of virulence factors genes is variable among commensal populations. On a global scale, the human microbiota is characterized by a higher prevalence of virulence genes than the microbiota of other organisms. In animals, the presence of virulence genes increases with body mass, which reveals the gut complexity of larger animal. Thus, virulence factors and their change in prevalence among hosts may reflect some local adaptation to commensal habitats rather than virulence per se.

23Interactions between community members are required for community development and maintenance; and can also drive some diversification. Many genes that are carried by mobile elements code for traits that are expressed outside of the cell. Such traits are involved in bacterial sociality, such as the production of public goods, which benefit a cell’s neighbors, or the production of bacteriocins, which harm a cell’s neighbors. To out-compete other clones, the production of colicins can represent a useful strategy in a structured environment. Colicins are the most expansively studied bacteriocins produced by E. coli. This could permit unadapted strains to colonize the gut and, hence, allow numerous clones to coexist in the long term. It may also promote diversification in a clone, as some strains may try to benefit from the production of the colicin but avoid paying the associated cost. However, the secretion of bacteriocins can be defined as a “spiteful” behavior in which are costly for the producer and cause harm to other members of the population.

24Horizontal gene transfer is a key step in the evolution of bacterial pathogens. Chromosomal structures such as PAIs have been shown to extensively contribute to the evolution of bacterial pathogens by providing dynamic changes of the bacterial genome composition leading to a bacterial evolution in quantum leaps. Different mechanisms have been proposed for transfer of PAIs across the species border including phage transfer, mobilization by conjugative transposons and plasmids. The fact that these microorganisms are less virulent than others is not reassuring, since the acquirement of virulence genes by bacteria is possible. Further research is required to characterize molecular and cellular interactions between the host and enterococci which lead to intra-species genetic transfer and virulence factors in enterococci species.

25Antimicrobial resistance is a worldwide problem in human and veterinary medicine. Commonly, it is usual that the major risk factor for the increase of this situation is an extensive use of antimicrobials that leads to the dissemination of resistant bacteria and resistance genes in animals and humans. The appearance of multiresistant bacteria of human and veterinary origin is probably accompanied by co-contamination of the environment apparently leading to a great health concern. As in the hospital environment, the agricultural use of antimicrobials agents selects for antibiotic resistance. These antimicrobial drugs from both hospital and agricultural sources can persist in soil and aquatic environments, and the selective pressure imposed by these compounds may affect the treatment of human diseases. In addition, the prophylactic use of antibiotics in fish farms has led to a rise in the number of resistant bacteria. However, the use of antibiotics by humans is not the only selective pressure for antibiotic resistance in natural microbial communities: compounds and conditions that occur in these communities may provide additional selection pressures. In fact, most antimicrobial agents are produced by strains of fungi and bacteria that occur naturally in all environments, including soil.

26Bacteria may also acquire resistance determinants without direct exposure to an antimicrobial through horizontally mobile elements including conjugative plasmids, integrons, and transposons. These mobile elements can simply transfer antimicrobial resistance genes from one bacterium to another. Antimicrobial agents exert a selective pressure not only on pathogenic, but also on commensal bacteria of the intestinal tract of humans and animals. In a broader view, increasing evidence suggests that components such as integrons and their gene cassettes played significant roles in genome evolution and fluidity within the bacterial kingdom.

27In E. coli, the phylogenetic group A strains and some group D strains are especially permissive to the development of resistance to third-generation cephalosporins. Conversely, phylogenetic group B2 strains are less resistant than the remaining strains, regardless of the molecular mechanism implicated in the acquisition of resistance, and have a lower prevalence of integrons in commensal E. coli strains from both human and animal hosts. This could reveal the relative decrease of phylogenetic group B2 strains in domesticated animals in which antimicrobials are used considerably.

28The class of beta-lactam antibiotics is among the most important groups of antimicrobial agents in human and veterinary medicine. Besides the first widely used antimicrobial substance penicillin, other members of this family have gained a similar importance over the last decades, namely the first- to fourth- generation cephalosporins and the beta-lactamase-inhibitors. All beta-lactams interfere with the final stage of peptidoglycan synthesis through acting on penicillin-binding proteins, thereby preventing the bacterial cell wall from forming. The most common resistance mechanism of Enterobacteriaceae spp. against beta-lactams is the inactivation of the antibiotic by breaking up the nitrogen-carbonyl bond in the beta-lactam ring. The emergence and wide dissemination of ESBLs among clinical E. coli isolates in hospitals, has caused a major concern in several countries, being frequently implicated in human infections. These infections have a great impact on public health due to an increased incidence of treatment failure and severity of disease. ESBLs mainly include TEM, SHV, and CTX-M enzymes. Among them, the highest number of variants described in the last years corresponds to the CTX-M family. The presence of CTX-M enzymes render E. coli resistant to a variety of beta-lactams, and are transferred via plasmids that can also include resistance genes to several unrelated classes of antimicrobial agents.

29Carbapenems such as imipenem or meropenem possess the most consistent activity against ESBL-producing Enterobacteriaceae strains. Both antibiotics are considered the agents of choice in the treatment of infections due to ESBL-producing organisms However, Enterobacteriaceae-producing carbapenemases have rapidly emerged and disseminated worldwide, including in the wild. The carbapenemases, such as the New Delhi metallo-beta-lactamase 1 (NDM-1) hydrolyze all β-lactam antibiotics, including carbapenems, and their high potential for rapid, wide dissemination constitutes a major clinical and public health threat.

30The increase in the occurrence of nosocomial infections caused by enterococci in particular E. faecium, is at least partly due to the wide variety of intrinsic and acquired resistances to glycopeptides and aminoglycosides, among others, posing a challenge to therapeutic options. Moreover, infections caused by other Enterococcus species (E. faecalis, E. durans, E. mundtii, E. avium, E. raffinosus, E. gallinarum, and E. casseliflavus) occasionally occur and warrant attention. Enterococci are expert in acquiring and transferring elements that confer resistance to antimicrobials and they are also known to be intrinsically resistant to numerous antimicrobials. As a result, therapeutic alternatives for treatment of enterococcal infections are increasingly limited. Evolution of enterococci toward resistance to multiple antimicrobials is also a major cause of concern.

31The acquisition of vancomycin resistance by enterococci has seriously affected the treatment and infection control of these organisms. Different types of glycopeptide resistance and their biochemical mechanisms have been described in enterococci: acquired type (vanA, vanB, vanD, vanE, vanG, and vanL); and low-level intrinsic type (vanC, associated with the E. gallinarum, E. casseliflavus, and E. flavescens species). VanA-type resistance, which was the first to be elucidated and which is the most common, is characterized by high levels of resistance to glycopeptides, vancomycin, and teicoplanin and is mediated by transposon Tn1546 or closely related elements that are chromosomally or plasmid located. The development of newer antimicrobial drugs, such as linezolid and daptomycin with activity against many VRE strains. has improved this situation; however resistance to these agents has already been described.

32An important difficulty in evaluating the causal relationship between antimicrobial use and resistance is the confounding influence of geography: the co-localization of resistant bacterial species with antimicrobial use does not essentially involve causation and could represent the presence of environmental conditions and factors that have independently contributed to the incidence of resistance.

33The collection of all antimicrobial resistance genes and their precursors in pathogenic and non-pathogenic bacteria and also in antimicrobial producing-organisms is referred as the antimicrobial resistome, a concept that has been advanced to serve as a framework for understanding the ecology of resistance on a global scale.

34Usually, wildlife is not exposed to clinical antimicrobial agents but can acquire antimicrobial-resistant bacteria through contact with humans, animals and the environment, where water polluted with feces appears to be the most significant vector of contamination. The incidence of commensal and pathogenic bacteria in fecal contaminations can be expected to be a connection between the environment and settings with regular or even constant antimicrobial pressure (aquaculture, livestock farming, human, and veterinary clinical settings), resulting in a constant release of antimicrobial-resistant human and animal bacteria into the environment through wastewater or manure. Additionally, the detection of antimicrobial-resistant bacteria in aquatic environments affected by human and animal wastewater and soil provides evidence for this hypothesis. In this context the common use of antimicrobials in aquaculture is also of utmost importance due to possible direct influences on wild animals. As intestinal bacteria like E. coli and enterococci can be easily disseminated in different ecosystems through water, they are intensively used as indicator species for fecal pollution. In this sense, it is essential to interpret the evolutionary and ecological forces that influence in the population structure of the commensal strains to fully understand the antimicrobial resistance and virulence of pathogenic strains. Certainly, the selective pressures in the habitats of commensal strains may coincidentally promote the emergence of antimicrobial resistance and virulence factors, rendering commensal strains reservoirs of virulent and resistant strains.

35Despite the commensal character of E. coli and enterococci, they are commonly involved in animal and human infections that implicate the use of antimicrobials, which increases public health preoccupations to the list of implications that arise from the spread of ESBL-E. coli and VRE into wildlife. Furthermore, the increasing frequency of community-acquired ESBL-E. coli and VRE infections and the occurrence in livestock farming has been observed recently, suggesting a successful transmission as well as persistence of ESBL-E. coli and VRE strains outside hospital settings. An additional parallel global phenomenon is the spread of ESBL-E. coli and VRE into the environment beyond human and domesticated animal populations, and this appears to be directly induced by antimicrobial practice. This might be a significant cause of the community-onset of ESBL-E. coli and VRE infections but can result (i) in an involvement of wildlife in ESBL-E. coli and VRE spread and transmission into fragile environmental niches, (ii) in subsequent colonization of wild animal populations which can turn into an infectious source or even a reservoir of ESBL-E. coli and VRE, (iii) in new putative infection cycles between wildlife, domesticated animals and humans, and (iv) in difficulties of wildlife medical treatment.

36Monitoring the prevalence of resistance in indicator bacteria such as fecal E. coli and enterococci in different populations (animals, patients and healthy humans) makes it possible to compare the prevalence of resistance and to detect transfer of resistant bacteria or resistance genes from animals to humans and vice versa. Just recently, there has been increasing interest in resistant bacteria and resistance genes isolated from wild animals.

37The degree of colonization varies a lot between different animal species. Therefore, ESBL-E. coli and VRE prevalence is clearly influenced by sampling schemes, by geographic regions, by host spectrum of these bacteria and by the degree of synanthropic behavior shown by host species. It is important also to take in consideration the limitations that occur interpreting these results. For instance, ESBL-E. coli prevalence in different Portuguese geographical areas ranged from 0.5% in birds of the remote Azores islands in the Atlantic Ocean to 32% for birds of the Portugal’s Northern Portuguese coast. A lower prevalence of ESBL-E. coli was also observed (0.8%) in glaucous-winged gulls of Kamchatka peninsula in Russia. These findings suggest that wild animals living in urban areas are more susceptible to carry ESBL-E. coli than those living in remote areas.

38Due to their diversity in ecological niches and their ease in picking up human and environmental bacteria, wild birds might act as mirrors of human activities. Within the heterogeneous class of birds, two groups seem to be in the focus of ESBL-E. coli and VRE carriage in wildlife: birds of prey and waterfowl/water related species. However, recent studies in Passeriformes have also described a significant incidence of VRE.

39Although wild birds, such as birds of prey, have only rare contact with antimicrobial agents, in disagreement with the existence of direct selective pressure, they can be contaminated or colonized by resistant bacteria. Water contact and acquisition via food seem to be major aspects of transmission of resistant bacteria of human or veterinary origin to wild animals. In the other hand, wild birds such as seagulls are often opportunistic marine feeders along the shoreline or offshore, but also eat the food sources provided by humans, especially garbage. Migrating birds that travel long distance seem to act as transporters, or as reservoirs, of resistant bacteria and may consequently have a significant epidemiological role in the dissemination of resistance, as well as being mirrors of the spectrum of pathogenic microorganisms present in humans. Reports on marine fish showed the presence of ESBL-E. coli and VRE in gilthead seabream, indicating a dissemination of ESBL-E. coli and VRE into the Atlantic ocean. Moreover, it has previously been demonstrated that seagulls shared strains with isolates cultured from wastewater treatment plants and landfills. This highlights the possibility of bacterial exchange between human sewage and birds.

40Another important host of these bacteria appears to be in wild rodents. Although these animals have previous been in the focus of research on ESBL in wildlife in different continents, they have only been detected in urban rats. On the other hand, VRE have been earlier described in wild rodents. This synantropic species can easily pickup human waste and frequently interacts with human feces in the sewage system in urban environments and can therefore easily acquire multiresistant bacteria. Remarkably, wild boars have also been described as hosts of these bacteria in Europe, which might expose their omnivorous feeding behavior. Recent studies revealed the presence of ESBL-producing E. coli and VRE isolates in Iberian wolf and/or Iberian lynx. The incidence of ESBL-E. coli and VRE in red foxes may be due their diet as these wild animals usually hunt wild rabbits, small rodents and birds. It is important to point out that some studies reported the presence of antimicrobial-resistant isolates in wild rabbits and wild rodents. Foxes are on top of the food chain, perhaps accumulating multiresistant bacteria from their prey. All these evidences may contribute in the acquisition and spread of antimicrobial-resistant bacteria even in the absence of direct antimicrobial pressure.

41These wild animals act as reservoirs of resistance genes and they could spread resistant bacteria throughout the wild environment. These researchers address the issue of antimicrobial-resistant microorganism proliferation in the environment and the related potential human health and environmental impact.

42The level of resistant bacteria detected in wild animals seems to relate well with the degree of association with human activity. In fact, human density, natural preservation state, livestock or the reserve of an area may be significant criteria for the proliferation of antimicrobial-resistant bacteria. Nevertheless, several studies report the occurrence of multidrug-resistant bacteria in remote places or preservation areas therefore underlining the complexity of the spread of antimicrobial resistance in wild animals. These discoveries propose, on one hand, an influence of migratory behavior of wild birds into remote areas, or on the other hand the omnipresence of human influence in various ecological niches of the planet via human feces. Different reports showed that areas with high livestock and human density and an assumable frequent interaction of wildlife with human influenced habitats of any kind (livestock farms, landfills, sewage systems, or wastewater treatment facilities) result in a higher risk for wildlife to acquire antimicrobial-resistant bacteria.

43The strength of trillions upon trillions of microorganisms, combined with the ancient force of evolution by constant, insistent variation, will inevitably overpower the drugs. Their spectrum is selected to involve pathogenic bacteria and antimicrobials constantly select naturally resistant bacteria. As bacteria quickly evolve to acquire resistance to the available antimicrobials, it is a constant race for scientists to develop effective strategies to combat infection and to reveal new therapeutic targets.

44Moreover, antimicrobial resistance evolving and spreading among bacterial pathogens is a public health problem of increasing magnitude. Since the beginning of the antimicrobial era, the selective pressure caused by the use of antimicrobials in clinical, veterinary, husbandry, and agricultural practices is considered the major factor responsible for the occurrence and spread of antimicrobial-resistant bacteria. The evolution of antimicrobial resistance in bacteria is related to the evolution of antimicrobial production. Though, resistance has also been discovered in the absence of antimicrobial exposure, as in bacteria from wildlife, raising an interest about the mechanisms of emergence and persistence of resistant strains under similar conditions, and the implications for resistance control strategies. Monitoring antimicrobial resistance in wildlife from remote areas could also be a useful tool to evaluate the impact of anthropic pressure.

45support that in ecological studies of antimicrobial resistance, there has possibly been too much focus on resistant organisms and not enough on resistance genes. Due to the capability of bacteria to transfer resistance genes, even among distantly related bacteria, analyses of antimicrobial resistance emergence, dissemination and persistence might be better conducted at the gene level.

46Until now, genomics-based investigation into E. coli and enterococci has focused on the identification of genes directly implicated in virulence. However, the fundamental physiology and response mechanisms to environmental conditions of E. coli and enterococci remained relatively poorly understood. This is a serious oversight because during infection the microbial fitness is an important reason in the success of any microbial pathogen. Further genome-wide reports aiming to define genes that are important during infection and colonization or exposure to antimicrobials will deliver significant data on relevant aspects of E. coli and enterococcal biology. This knowledge can consequently be useful for the development of novel treatment approaches to combat microbial infections.

47The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.