*2.3. Selection and Weak Counter-Selection of Virulence and Resistance Genes*

Proteins encoded by virulence genes—e.g., toxins and cell surface proteins that enable bacterial attachment to host tissues, among others—can help bacteria colonize hosts [45]. Therefore, newly acquired virulence genes may not affect fitness or confer immediate advantages to bacteria, while recently acquired resistance determinants often impose a fitness cost on the bacterial cell. Therefore, these gene types may have different effects on

their new hosts when newly acquired. However, the claim that resistant determinants are costly is somewhat complex.

The fitness costs of drug resistance determinants may evolve towards lower values or even become zero. For example, compensatory mutations often arise and diminish or even eliminate the deleterious effects of resistance mutations [46–56]. Moreover, resistance determinants (mutations or plasmids) can decrease the fitness cost of other resistant determinants, e.g., through epistatic interactions [57–59]. Furthermore, plasmid–plasmid interactions may facilitate plasmid transfer [60–62], sometimes compensating for plasmid costs [63,64].

Sometimes compensatory mutations arise in the resistance-encoding mobile genetic element, not the bacterial chromosome. In this case, when moving into another bacterial host, this genetic element carries both the resistance gene and the compensatory mutations, therefore imposing no cost on its new host [52,65].

A recent study involved 9275 patients during hospital stays over two years at the Ramon y Cajal University Hospital (Madrid, Spain). Alonso-del Valle et al. measured the cost of *pOXA-48\_K8*: a naturally isolated plasmid. This plasmid was the most successful *pOXA-48*-like plasmid in an extensive collection of extended-spectrum ß-lactamaseand carbapenemase-producing enterobacteria isolated from the gut of 105 out of those 9275 patients (1.13%). The authors introduced this plasmid in 25 isolates of *Klebsiella pneumoniae* and 25 of *Escherichia coli* naïve to the *pOXA-48\_K8* plasmid to determine the distribution of fitness effects on the plasmid in those 50 strains. These strains were isolated from patients coinciding on the hospital ward with others colonized with bacteria harboring *pOXA-48*-like plasmids [66].

As expected, the plasmid imposed a fitness cost to bacterial cells: a mean cost of 2.9%. Although small, this effect is statistically significant. However, individual values varied considerably. Most fitness effects were null, and the authors only observed a fitness cost in 14 strains (28%). Interestingly, the plasmid conferred a fitness advantage to the host in seven strains (14%) [66]. It is crucial to note that, even if the plasmid decreases bacterial fitness in 28% of the strains, the plasmid can succeed and "amplify" in the other 58% of the strains where the plasmid is neutral or among the 14% where the plasmid confers a fitness benefit [7,27,67].

Alonso-del Valle et al. measured the fitness of plasmid-bearing strains through competition assays. This method is appropriate because it mimics the real competition taking place between plasmid-bearing and plasmid-free cells in the gut microbiota. These competition assays were performed in agitated liquid media to avoid plasmid transfer into the competitor [66].

All the accumulated knowledge about the cost of resistant determinants points in the same direction: even if plasmid-encoded resistance determinants impose a fitness cost to naïve recipient cells, soon the cost diminishes or disappears. Therefore, resistant cells may avoid being outcompeted by sensitive cells after a critical adaptation period to a resistance-encoding gene.

One must remember that human tissues and the gut are structured media with minimal or no agitation. In such environments, plasmids can transfer to neighboring naïve cells. Possibly, the plasmid imposes a fitness cost to their neighbors, leaving resources to the donor cells. Therefore, even if plasmids are costly in the donor cells, these may succeed by imposing fitness costs on adjacent competitors, acting as harmful agents [68]. This harming behavior is advantageous to donor cells for the following reasons [68]: (i) donor cells have already adapted to the plasmid presence due, for example, to compensatory mutations [56]; (ii) the plasmid transfers to recipient cells (transconjugant cells); (iii) these cells are not adapted to the plasmid presence because they never harbored it; (iv) these non-adapted transconjugant cells replicate slower and use fewer resources than before the plasmid arrival; (v) donor cells may uptake the unused nutrients and replicate.

Furthermore, it is interesting to note that, just because they are transferable (and not due to the gain of additional genes), conjugative plasmids may have an adaptive value to their hosts, not only as harmful agents, as explained above [68], but also as promoters of bacterial biofilms, which confer protection against antimicrobials [63,69,70].

Therefore, epidemiological or evolutionary studies of antibiotic resistance must assume that resistance genes are widespread worldwide. For over eight decades, tons of antibiotics have been used and thrown into the environment [71]. The prolonged use of these drugs has promoted the clonal expansion of resistant cells and the worldwide spread of resistant clones and putative mobile genetic elements carrying resistance genes. Not surprisingly, nowadays, many metagenomes, including human and environmental ones, contain resistance genes; sometimes, this includes a diverse set of those genes as well as virulence genes [72].
