*2.4. The Diversity of Virulence and Resistance Genes across Microbiomes*

We have argued above that non-housekeeping genes are somewhat free to spread within and between microbiomes (namely between people). Some people may use antibiotics, which select virulence genes that are present in resistant cells and resistance genes but counter-select other genes, including those that confer resistance to unrelated antibiotics and virulence genes encoded in bacterial cells susceptible to the used drug.

The use of antibiotics by sick people may select antibiotic-resistant pathogens, which, by definition, encode virulence genes. Perhaps this is why several studies have shown antibiotic and virulence genes co-occurrence in bacterial genomes [73–75]. In general, one could predict that the continuing use of tons of antibiotics through the decades would co-select virulence and resistance genes. However, it is also manifest that the administration of antibiotics hits several other bacteria, namely commensal and mutualistic bacteria, in the human microbiome [76–78]. Moreover, the use of antibiotics as growth promoters in livestock and agriculture or with prophylactic purposes may select resistant cells independently on whether or not they encode virulence genes [79].

Paradoxically, however, the diversity of resistance genes correlates positively with the diversity of virulence genes across human and environmental microbiomes [72]: microbiomes with a high diversity of one class of genes tend to have a high diversity of the other class. However, Darmancier et al. studied 16,632 bacterial genomes and concluded that no such positive correlation exists at the level of genomes, neither across chromosomes nor across plasmids [80]. The results of these two studies (references [72,80]) are compatible if, and only if, metagenomes with a high diversity of both gene types have those genes mostly located in different genomes. Why should there be a positive correlation at the level of metagenomes but not at the genome level?

In the previous two sections, we have also argued that virulence and resistance genes have conditions to remain in metagenomes for a long time. Each person receives diverse genes of both types during this period, accumulating them in their microbiomes, although not necessarily in the same bacterial cells. Meanwhile, pathogenic bacteria circulate through the human population. Some of those ill people take an antibiotic, which selects cells resistant to it but kills susceptible cells, including those resistant to other drugs. Therefore, a consequence of taking the antibiotic is a decrease in the diversity of drug-resistance genes [81]. Moreover, virulence genes are present in dead cells, decreasing their diversity. This process implies that people who took antibiotics recently are those with the lowest diversity of both resistant and virulence cells [82]. On the other hand, those that took antibiotics a long time ago have the highest diversity of both gene types [82]. The overall result is a positive correlation between the diversities of both gene types across human microbiomes [72,82].

What if there is a misuse (and overuse) of antibiotics, where some people use them even if they are not infected by a bacterial pathogen (or are randomly in contact with antibiotics from environmental contamination)? The process is very similar to the one described above, and the overall result is the same: a positive correlation of both genes' diversities. These are the predictions if the probability that a metagenome loses resistance determinants is lower than the transmission probability between people [82]. We have seen above how unlikely it is that metagenomes lose resistance determinants, so the transmission probability between people must prevail or even the contamination of people from the environment or, for example, non-cooked vegetables [83].

A computer model enabled the analysis and corroboration of the above predictions [82]. The model simulated the transfer of pathogenic bacteria and one hundred categories of resistance and virulence genes. Simulations ran with different network types (regular, smallworld, and random), and the results were always similar (the only difference being that the simulations reached stable results much sooner in small-world and random networks than in the regular network as expected, and according to explanations given above). Control simulations included changing the number of interacting people, the number of virulence and resistance genes, the relative probabilities of losing genes and acquiring them through contacts, or even changing the initial number of people already containing virulence and resistant genes, providing similar results [82].

Until this point, we have shown why the arguments (i) to (iii) presented in the Introduction section are wrong. About the fourth one, our discussion above does not predict a co-location of virulence and resistance genes in bacterial genomes; moreover, the computer model did not need to postulate their co-location to explain the positive correlation between the resistance and virulence genes' diversities in microbiomes observed over human microbiomes. However, one must ask whether the last eighty years of intensive antibiotic use have put virulence and resistance genes together. As mentioned above, there is no correlation between resistance and virulence genes' diversities throughout genomes [80]. Nevertheless, there are indirect signs of the selection of antibiotic-resistant pathogens, given that some categories of resistance and virulence genes preferentially occur in the same genome [80]. For example, Darmancier et al. observed the co-occurrence between type VII secretion systems and fusidic acid resistance genes [80]: fusidic acid is used, e.g., to treat methicillin-resistant *S. aureus* infections, and is also active against tuberculosis [84,85].
