## 3 Discussion  
Due to the emergence of MDR pathogens over the past few decades, public health officials faces new challenges, such as the alarming increase in antimicrobial resistance, as well as the emerging link between resistance strategies used by bacteria against antibiotics and biocides [18]. This last problem is even more worrisome due to the routinely and uncontrolled use of antiseptics and biocides in clinical practice [9]. One example of this is CHLX, a bis-biguanide antiseptic of cationic nature that has  
Figure 5: Enzymatic activity by the colorimetric assay (WST-1 based) of the strain _K. pneumoniae_ ST846-OXA48 CA and ST846-OXA48 CA CP in the presence of the combination of IMP (50 \(\times\) MIC) and CHLX (10 \(\times\) MIC). The growth control is ST846-OXA48 CA strain without antibiotic pressure. ***, \(p\)-value \(<\) 0.001 and ***, \(p\)-value \(<\) 0.0001. The errors bars represent the standard deviation of the three experiment replicates. bactericidal activity through membrane disruption [31]. For these reasons it of great interest to decipher the molecular mechanisms involved in the adaptation to CHLX in clinical strains of _K. pneumoniae_, producers of carbapenemases. In order to determine at the molecular level the effect of the adaptation to CHLX in a strains of _K. pneumoniae_, we performed a phenotypic study in the presence of CHLX (\(10\times\) MIC), which showed that the adaptation to CHLX led to the activation of two different molecular mechanisms in the clinical strains of _K. pneumoniae_ ST258-KPC3 CA and ST846-OXA48 CA. In effect, the ST258-KPC3 CA strain presented a growth curve typical of resistant bacteria, where a slight reduction in the bacterial population occurs during the time of activation of defense mechanisms [32] (e.g., efflux pumps, TA systems, quorum network), followed by a regrowth period similar to the control. In contrast, ST846-OXA48 CA had a characteristic growth curve of tolerant bacteria, where the strain undergoes a drastic reduction or arrest of growth during the first four hours of exposure to the bactericide [31]. These results were corroborated by the transcriptomic study where the transcriptomic profile of the ST258-KPC3 CA strain revealed the overexpression of a larger number of genes compared to ST846-OXA48 CA strain, especially those related to the overexpression of efflux pumps, which are generally considered as a basic molecular mechanism associated with resistance [33]. In our study, the efflux pump gene that was overexpressed in ST258-KPC3 CA was the methyl viologen resistance gene _smvA_. SmvA is responsible for resistance to CHLX in _K. pneumoniae_ strains previously adapted to CHLX, due to the interruption of the _smvR_ gene (_smvA_ repressor) [9]. The antimicrobial susceptibility test revealed the appearance of cross-resistance to colistin (polymycin E), in the ST846-OXA48 CA strain tolerant to CHLX, increasing its MIC value 32-fold. These results were also corroborated by the transcriptomic studies, in which a high level of expression of the colistin resistance genes, _pmrD_ and _pmrK_ could be observed. This phenomenon of cross-resistance was also described previously, where five out of six strains of _K. pneumoniae_ adapted to CHLX also presented resistance to colistin, increasing their MIC values from 2-4 mg/L to 64 mg/L [9]. This phenotype is due to the common biochemical characteristics of CHLX and colistin: in fact, both are cationic compounds with hydrophobic functions [6]. As for the other antibiotics tested, no change in the MIC value was observed in the strain adapted to CHLX compared to the wild-types. In the previous study of [PERSON] et al. (2018), a combination of \(\beta\)-lactam antibiotic IMP with the CHLX biocide caused in some strains of _Acinetobacter baumantii_ the formation of a subpopulation of persistent bacteria [12]. This phenomenon could be observed in the ST846-OXA48 CA strain in the presence of the same combination.