## 2 Results
### Results Subsection
#### 2.1.3 Antimicrobial Susceptibility Testing  
The antimicrobial susceptibility test was done for wild-type ST258-KPC3 and ST846-OXA48 wild-type and the two CA strains. According to adaptation to CHLX, an increase in MICs of CHLX was observed in both CA strains. However, no differences in the minimum inhibitory concentration (MIC) values were observed for the other antibiotics tested, except for the colistin, in that in the ST846-OXA48 CA strain showed an increase in the MIC value of 32-fold, that corresponds to a resistance value (Table 2).  
1.4 Characterization of the New TA System, PemI/PemK, Present in a Plasmid in the Strain ST846-OXA48 CA  
A PemI/PemK TA system, whose closest relative is the type II TA toxin-antitoxin system PemK/MazF family toxin belonging to Enterobacteriaceae (Query: 78%; Identity: 99.35%; Code number: WP_077688581.1) and which have not been previously described in _K. pneumoniae_, it was identified by transcriptomic analysis (Table 1) as encoded by a plasmid of _K. pneumoniae_ ST846-OXA48 CA (Figure S1). This plasmid harbors several genes, such as _repA_, _dsbC_, _trpA_, _trbC_, _lusR_, CPBP metalloproteinase, _umuC_, restriction endonuclease, _IS1_, _ssb_, _mobC_, _nikA_, _dotD_/TraH family lipoprotein, secretion systems type IV, _traO_, _traP_, _traQ_, _traW_, _traX_, _dotA/traY_, and _repC_, in addition to another TA system, the RelE/RelB TA system. This PemI/PemK TA system is composed of a 258 bp antitoxin gene (_pemI_) and a 333 bp toxin gene (_pemK_). To confirm that this system is a TA system, _pemI/pemK_ and _pemK_ genes alone were cloned into the overexpression vector pCA24N, widely used in the literature to overexpress TA systems [28; 29], and transformed into the cured plasmid strain ST846-OXA48 CA CP (i.e., lacking plasmids and therefore the plasmid that encodes the PemI/PemK TA system). The toxicity of this TA system was tested by growth curves overexpressing both _pemI/pemK_ and _pemK_ (Figure 2). Overexpression of _pemK_ in ST846-OXA48 CA CP/pCA24N (_pemK_) inhibited bacterial growth, while overexpression of the _pemI/pemK_ system (ST846-OXA48 CA CP/pCA24N (_pemIK_)) in the strain led to normal bacterial growth, which was slightly impaired compared to the empty plasmid. Therefore, the plasmid-based PemI/PemK TA system found in _K. pneumoniae_ ST846-OXA48 CA is functional.  
\begin{table}
\begin{tabular}{c c c c c c c c c c c c c c c} \hline \hline  & & & & & & & & & & & & & & & & \\ \hline
**St258-KPC3** & 9.8 & \(>\)32 & 2 & 64 & 4 & 8 & 4 & \(>\)32 & \(>\)32 & 1024 & 128 & 2 & 16 & 4 & 0.25 \\ \hline
**ST258-KPC3 CA** & 39.1 & \(>\)32 & 2 & 64 & 4 & 8 & 4 & \(>\)32 & \(>\)32 & 1024 & 128 & 2 & 16 & 4 & 0.25 \\ \hline
**ST846-OXA48** & 19.5 & 8 & 8 & 32 & 16 & \(>\)32 & 32 & \(>\)32 & \(>\)32 & 128 & 16 & 8 & 2 & 8 & 0.5 \\ \hline
**ST846-OXA48 CA** & 78.2 & 8 & 8 & 32 & 16 & \(>\)32 & 32 & \(>\)32 & \(>\)32 & 128 & 16 & 8 & 2 & 8 & 16 \\ \hline CHLX, Chlorkeidine; CIP, Ciprofloxacin; TGC, Tigecycline; TOB, Tobramycin; IMP, Imipenem; MRP, Merropenem; GEN, Gentamycin; CAZ, Ceffazidime; TZP, Piperacillin-tazobactam; SAM, Sulbactam; NET, Netilmicin; AMK, Amikacin; MIN, Minococilin; CST, Colistin. & & & & & & & & & & & & \\ \hline \hline \end{tabular} CHLX, Chlorkeidine; CIP, Ciprofloxacin; TGC, Tigecycline; TOB, Tobramycin; IMP, Imipenem; MRP, Merropenem; GEN, Gentamycin; CAZ, Ceffazidime; TZP, Piperacillin-tazobactam; SAM, Sulbactam; NET, Netilmicin; AMK, Amikacin; MIN, Minococilin; CST, Colistin.  
\end{table}
Table 2: MIC values (\(\mu\)g/mL) of different antibiotics for ST258-KPC3, ST258-KPC3 CA, ST846-OXA48, and ST846-OXA48 CA.