## 2 Results
### Results Subsection
#### 2.1.5 Biofilm Formation Assay  
Since TA systems have been associated with the arrest of bacterial growth and the formation of biofilms, we studied the effect of the PemI/PemK TA system and the PemK toxin on biofilm formation (Figure 3). Production of PemK toxin resulted in a significant decrease in biofilm formation compared to the control (ST846-OXA48 CA CP/pCA24N) (\(p\)-value \(<\) 0.001). Moreover, production of PemI/PemK restored a similar phenotype as the control, lacking a significant difference in biofilm formation (\(p\)-value \(<\) 0.05). Therefore, the PemI/PemK TA system influences _K. pneumoniae_ biofilm formation. Figure 3: Biofilm formation assay. (**A**) Biofilm strained with 10% crystal violet was dissolved in 30% acetic acid. (**B**) Box and whisker plot of the optical density of biofilm produced by the strains ST846-OXA48 CA CP/pCA24N, ST846-OXA48 CA CP/pCA24N (PemIK), and ST846-OXA48 CA CP/pCA24N (PemK). The biofilm formation was expressed as the ratio between OD580/600 nm, in order to normalize the data. Boxes indicate the lower and upper quartile. Horizontal lines in each box represents the median value of biofilm formation. The mean biofilm formation for each strain is indicated by a +. Vertical lines extending from each box represent the minimum and maximum biofilm formation. *, \(p\)-value \(<\) 0.05. All experiments were performed in triplicates. Figure 2: Growth curves of ST846-OXA48 CA CP containing pCA24N plasmids with _pemIK_ (dark grey, dashed line) and _pemK_ (light grey, dotted line) in the presence of 1 mM IPTG. The strain ST846-OXA48 CA CP is used as a control as it carries the empty plasmid pCA24N (black). The errors bar represents the standard deviation of the three experimental replicates. 1.6 Time-Killing Curve in the Presence of Imipenem or in Combination with Chlorhexidine for ST846-OXA48 CA and ST846-OXA48 CA CP  
The time-killing curves of _K. pneumoniae_ ST846-OXA48 CA tolerant to CHLX (Figure 4A) were performed in the presence of imipenem (IMP) (\(50\times\) MIC) alone or in combination with CHLX (\(10\times\) MIC). A drastic reduction in the number of CFU was observed during the first four hours in the presence of IMP (\(50\times\) MIC), decreasing from \(7\) LogCFU/mL (\(9.35\times 10^{7}\) CFU/mL) to \(3\) LogCFU/mL (\(1.50\times 10^{3}\) CFU/mL). However, bacterial regrowth occurred after four hours, reaching similar levels of CFU/mL as the control (\(8\) LogCFU/mL (\(6.83\times 10^{8}\) CFU/mL) vs. \(9\) LogCFU/mL (\(1.73\times 10^{9}\) CFU/mL)) at \(28\) h and exceeding it at \(48\) h (\(9\) LogCFU/mL (\(1.25\times 10^{9}\) CFU/mL) vs. \(9\) LogCFU/mL (\(1.07\times 10^{9}\) CFU/mL)). In the case of the combination of IMP and CHLX, a greater CFU reduction than IMP alone was observed, with no CFU detected after \(4\) h. Nevertheless, a regrowth of the bacterial population was observed after \(28\) h. Thus, ST846-OXA48 CA in presence of the combination of IMP and CHLX showed a characteristic behavior of the persistent subpopulation. Figure 4: Time-killing curve in the presence of IMP (\(50\times\) MIC) and in presence of the combination of IMP (\(50\times\) MIC) and CHLX (\(10\times\) MIC) for the strains of _K. pneumoniae_ ST846-OXA48 CA (**A**) and ST846-OXA48 CA CP (**B**). The controls are the strains without exposure to any stress (IMP, IMP + CHLX). The error bars represent the standard deviation of the three replicates of the experiment. In the case of the ST846-OXA48 CA CP strain (Figure 4B), in which the plasmid was removed by means of a curing agent, 3% sodium dodecyl sulfate (SDS) (10% _w/v_ pH = 7.4), a drastic reduction in the number of CFU, was observed both with IMP alone as for in the combination of IMP and CHLX. In fact, in the case of IMP alone no CFUs were recovered at 20 h while, in the case of the combination, no CFUs were recovered at 4 h (Figure 4). Finally, the culture was considered dead as no regrowth was observed throughout the rest of the assay.