The mechanisms by which bacteria become antibiotic-resistant are diverse, complex and not completely understood. In this regard, commensal E. coli strains, especially those inhabiting the intestines of humans and warm-blooded animals, have been shown to play a pivotal role in the onset and diffusion of antibiotic-resistance genes [21,22]. The high intestinal bacterial density, together with antibiotic exposure, due to global indiscriminate and, in many cases, inappropriate use of antibiotics, have likely led to the selection and spread of antibiotic-resistance mutants among commensal E. coli strains, which are considered a primary reservoir of antibiotic-resistance genes. Moreover, the remarkable E. coli genome plasticity may have favored the acquisition of virulence-associated determinants that may be transmitted to bacteria of the same and/or of related species [8,23].

The results of epidemiological surveys indicate that 20–45% of ExPEC isolates were resistant to first-line antibiotics including beta-lactams, fluoroquinolones and trimethoprim–sulfamethoxazole [24,25,26,27], so that the management of patients suffering from MAR ExPEC infections has become more complicated.

The fluoroquinolones are a family of broad-spectrum antibiotics used since the late 1980s for the treatment of severe infections, especially UTIs, although long-term therapy with fluoroquinolones is linked to serious adverse effects. Consequently, the U.S. Food and Drug Administration recommends the restricted use of these antibiotics to UTIs untreatable with commonly used antibiotics. Nevertheless, fluoroquinolones continue to be widely prescribed in several countries [16,17,18,19,20] and the incidence of resistant E. coli is continuously increasing. To resist to the action of fluoroquinolones (the inhibition of topoisomerase enzymes, DNA gyrase and topoisomerase IV), E. coli may become resistant by mutations that alter the target enzymes, by reducing the entrance of the antibiotic into the cell or by activating efflux systems, which extrude the drug from the cell. Aminoglycosides are a family of bactericidal broad-spectrum antibiotics that may also act in synergy with other antibiotics. Despite the adverse side effects due to the prolonged use of these antibiotics, several members of this family were intensively used in the past, until they were replaced in the 1980s with cephalosporins, carbapenems and fluoroquinolones. The mechanism of action of aminoglycosides targets the binding to the aminoacyl-tRNA recognition site (A-site) of the 16S rRNA of 30S ribosome, leading to the inhibition of polypeptide synthesis and subsequent cell death. Resistance to aminoglycosides may occur based on several mechanisms, namely (i) enzymatic modification and inactivation of the aminoglycosides, mediated by aminoglycoside acetyltransferases, nucleotidyltransferases or phosphotransferases, both in Gram-positive and -negative bacteria; (ii) increased efflux of the antibiotic outside the cell; (iii) decreased permeability; and iv) modifications of the 30S ribosomal subunit that alter the binding of the aminoglycosides [28,29,30,31,32,33].

In the pre-antibiotic era, heavy metals were largely used as chemotherapeutics and/or as antimicrobials in human and animal healthcare, and some heavy metals are still in use today in medicine and agriculture settings [34]. As with antibiotics, resistance to heavy metals has recently increased, and ExPECs presenting combined resistance to both antibiotics and heavy metals are commonly isolated [9,13,16,17,18,19,35,36]. This finding is of particular concern considering that heavy metals have been associated with the co-selection of antibiotic resistance genes and that, thanks to mobile genetic elements, heavy metal resistance genes are transmitted to microorganisms of the same or of related species [37,38]. Although the efficacy of metal preparations is controversial, nevertheless, several heavy metals are still in use in agriculture and aquaculture (i) as organic/inorganic fertilizers; (ii) as preservatives in animal food production and (iii) as biocides and antimicrobials in cases of antibiotic failure [39]. Due to their elevated toxicity, some of the treatments with metals such as mercury and arsenic/antimony have now been eliminated or greatly limited in several countries [40,41]. Since they are chemically stable elements, heavy metals accumulate in soil and water and exercise a strong selective pressure that may trigger the onset of metal-resistant mutants. Thus, decontamination of metal-polluted zones appears to be an inevitable step to limit the development and diffusion of both antibiotic and metal resistance genes [42]. This is of particular concern also considering that the environmental metal pollution is remarkably more diffused than antibiotic pollution. According to what is reported above, the strong association between metal exposure, metal- and antibiotic-resistance genes led the European Scientific Committee for Emerging and Newly Identified Health Risks to warn about bacterial exposure to heavy metals, which may boost the onset and the spread of antibiotic-resistance genes [35]. Apart from their importance in co-selection, heavy metals also play a role in the regulatory mechanisms governing complex aspects of the bacterial physiology, such as the two-component CpxA/CpxR system of Gram-negative bacteria, which senses and responds to stress stimuli, such as those caused by cell-envelope-targeting drugs and heavy metals. Once activated, the CpxA/CpxR system activates the transcription of 11 out of 28 Cu-inducible genes whose expression is also required to transform Cu-susceptible microorganisms into Cu-tolerant derivatives [43]. Furthermore, as well as counteracting antibiotic activity, bacteria have to face the potential lethal effects of poisonous oxygen derivatives (molecular oxygen does not react with cell components, but its derivatives are toxic) such as hydrogen peroxide and superoxide anions, which directly undermine the metabolic activities of enzymes co-factored with iron and flavins. Consequently, when bacteria accumulate substantial reactive oxygen insults, coming from environmental or cellular sources, growth is inhibited. Although hydrogen peroxide and superoxide anions do not oxidize DNA directly, these molecules feed directly or indirectly the generation of the highly reactive hydroxyl radicals, which damages the bacterial chromosome [44]. Characteristically, three reactive oxygen species (ROS) are considered, i.e., superoxide anions (O²⁻), hydrogen peroxide (H₂O₂) and hydroxyl radicals (HO•), though other reactive and abundant molecules may exist. Several common oxidants derived from molecular oxygen are found in the environment or even produced by the cellular metabolism. To meet this challenge, E. coli activates complex mechanisms to resist the accumulation of these radicals. The activity of the sigma factor protein family is linked to bacterial stress responses. In E. coli, activation of RpoE is required for maintaining the integrity of periplasmic and outer membrane compartments; it is regulated by Zn and also responsible for the transition to Zn- and Cu-tolerance phenotypes [45]. Again, Cu has also been shown to regulate the expression of the oxidative stress-responsive regulatory gene soxR, whose encoded SoxS protein regulates the expression of the AcrAB efflux pump [46]. Lastly, it has been recently reported that exposure to sub-inhibitory concentrations of heavy metals, such as Cu, Ag, Cr and Zn, promotes the conjugative transfer of antibiotic-resistance genes. This finding highlights even more the urgent need to develop new, effective strategies to control the environmental diffusion of these metal pollutants [9,47,48,49,50].