*5.4. Virulence*

The premise plumbing environment exhibits several features that could possibly contribute to the virulence of resident OPs. Wargo [38] describes features of drinking water plumbing that could prime OPs to infect cystic fibrosis patients, although the interactions described in this review could also pose risk to otherwise immunocompromised individuals. Such features that are relevant to pipe material include [38]:


Some studies sugges<sup>t</sup> that the above types of interaction may increase the pathogenic potential of premise plumbing-associated OPs specifically. Copper resistance is important to mammalian host infection for *P. aeruginosa* [311] and *A. baumannii* [312,313], and other evidence suggests that exposure to copper in aquatic environments selects for greater copper resistance among certain OPs [129,303,304]. Copper and other divalent metals may also play a role in nutrient acquisition and pathogenesis even after infecting hosts [314].

The effects of iron exposure on OPs are not as apparent. *L. pneumophila* serogroup 1 grown in medium that was iron limited (0.017–0.056 mg/L) has been shown to lose its virulence [315], indicating that limiting adequate concentrations of iron could not only decrease the presence of *Legionella* but also the likelihood of human infection. Iron also plays a role in modulating various behaviors, including modulating virulence factor production in *P. aeruginosa* and *A. baumannii* [316–321], but it is unclear what e ffects exposure to iron have on virulence in the premise plumbing environment. This subject is largely unexplored and more research is needed to determine the overall e ffects of the premise plumbing environment on OP virulence.

#### *5.5. Antibiotic Resistance and Tolerance*

Copper, among other heavy metals has been shown to exert selection pressure, leading to enhanced survival of antibiotic resistant bacteria. In fact, heavy-metal-associated co-selection and cross-selection has been proposed to be as much of a concern for environmental propagation of antibiotic resistance as antibiotics themselves [322]. Increases in antibiotic resistance genes at the community scale have been identified after long-term copper exposure in soil [323–326], sediment [327], and drinking water [327]. Bench-scale tests using bacterial isolates from biofilters [328] and wastewater [329] inoculated into growth media have shown that a selective or inductive e ffect of copper can take places within hours. However, these studies were performed with copper concentrations 5–77 times greater than the 1.3 mg/<sup>L</sup> US EPA copper action level and similarly in exceedance of the Chinese Standard for Drinking Water Quality of 1 mg/<sup>L</sup> [209] and WHO Guideline for Drinking-Water Quality of 2 mg/<sup>L</sup> [82]. Thus, these concentrations may not be representative of potable water systems. One study examining antibiotic resistant and sensitive strains of *Staphylococcus aureus* showed that the more antibiotic resistant strain survived longer in a copper container [90]. As discussed above, copper may also better support *Acanthamoeba* than other materials, while in one study *L. pneumophila* grown within *A. polyphaga* demonstrated increased tolerance to all antibiotics tested (rifampin, ciprofloxacin, and erythromycin) compared to those grown in culture media [330]. The role of copper plumbing and other pipe materials in these emerging areas of research is worthy of further investigation.

There is more limited evidence that the presence of iron may also induce or select for antibiotic resistance, as observed for *P. aeruginosa* using iron-amended growth media [330] and the gu<sup>t</sup> microbiomes of mice supplied with iron-amended water [331]. The latter case, while using an iron concentration more than 25 times the EU drinking water standard of 0.2 mg/<sup>L</sup> [332] and 16 times both the US EPA National Secondary Drinking Water Standard and Chinese Standard for Drinking Water Quality of 0.3 mg/L, may be of particular concern, as it suggests that pipe corrosion products have the potential to select for antibiotic resistance inside the infected host organism.
