## 3 Results
### X. citri Persister Cells Are Killed by Cisplatin, Mitomycin C, and High Copper Concentrations  
Even with the suppression of metabolic routes (usually targeted by conventional antimicrobial molecules), persister cells can be killed by chemicals that do not interfere directly with metabolism and whose actions are independent of growth. At least two compounds are reported to kill persister cells: cisplatin [48] and mitomycin C [49; 50]. In addition, since the copper killing mechanism for microorganisms is possibly independent of growth [51], we investigated the behavior of _X. citri_ persister cells when exposed to these three chemicals: cisplatin, mitomycin C, and copper. _X. citri_ persister cells were obtained using tetracycline as the inducer (45 min, as shown in Figure 1B), and after this, the cultures were submitted to treatments with the different molecules, using a 5 \(\times\) MIC concentration. Cisplatin killed persisters and exponentially growing cells at very similar rates, without any colonies observed after 1 h (Figure 3A). Mitomycin C also killed exponentially growing and persister cells, but complete eradication was observed only after 2 h (Figure 3B). In contrast, copper took longer to exterminate persister cells compared to exponentially growing cells, since persisters cells were killed after 2 h and exponential cells after 1 h of (Figure 3C). Noteworthy, for all cases, persisters were undetectable within 2 h (Figure 3A-C). This is a very different scenario than observed for ciprofloxacin treatment, where it was necessary to use a much higher concentration (\(\sim\)100 \(\times\) MIC) and even after 6 h, persister cells were still detectable, although exponential cells were no longer present (Figure 1B). Therefore, cisplatin, mitomycin C, and copper are effective for killing _X. citri_ persister cells. Figure 2: Induction of _X. citri_ persisters after pretreatment with copper, zinc, and high temperature. (**A**) _X. citri_ cultures pre-treated with copper 1 \(\times\) minimal inhibitory concentration (MIC) (160 \(\upmu\)g/mL) for 15, 30, 45 and 60 min or with zinc 1 \(\times\) MIC (322 \(\upmu\)g/mL) for 60 min, before ciprofloxacin treatment (50 \(\upmu\)g/mL, 3 h). Fold changes in CFU in relation to control (without copper or zinc pretreatment) are shown as the mean of biological replicates and the symbols *** (\(p<0.001\)), **(\(p<0.01\)) indicate significant differences using Student’s _t_-test. (**B**) Cultures were grown at 30 \({}^{\circ}\)C, and after reaching the mid-log phase, flasks were transferred to 37 \({}^{\circ}\)C for 3 h and 6 h before ciprofloxacin treatment (50 \(\upmu\)g/mL, 3 h, to kill non-persisters). Fold changes in CFU counting are shown (relative to control without heat-treatment). Results are shown as the mean of biological replicates and symbol * (\(p<0.05\)) and **(\(p<0.01\)) indicate significant differences using Student’s _t_-test. To compare the killing potential of copper as a persister killing agent without the interference of growth media, we obtained persister cells by pretreating _X. citri_ with tetracycline (Figure 1A), but, this time, the cells were washed and suspended in 0.85% NaCl buffer. To this bacterial suspension of persisters, copper was added at 32 \(\upmu\)g/mL and 160 \(\upmu\)g/mL (which corresponds to 0.2 \(\times\) MIC and 1 \(\times\) MIC, respectively). For comparison, the same experiment was performed with the addition of cisplatin instead of copper. We observed that after only 30 min, the lowest copper concentration (0.5 \(\times\) MIC) eradicated _X. citri_ persisters, while for cisplatin treatments, even after 60 min of incubation and using a higher concentration (1 \(\times\) MIC), cisplatin could not eliminate these cells (Figure 3D). Overall, these results show that copper, depending on the environmental condition, can act as a persister inducer but can also kill persisters.