Mechanisms of Tolerance and Resistance to Chlorhexidine in Clinical Strains of Klebsiella pneumoniae Producers of Carbapenemase: Role of New Type II Toxin-Antitoxin System, PemIK

toxins Article Mechanisms of Tolerance and Resistance to Chlorhexidine in Clinical Strains of Klebsiella pneumoniae Producers of Carbapenemase: Role of New Type II Toxin-Antitoxin System, PemIK Ines Bleriot 1,2 , Lucia Blasco 1,2 , Mercedes Delgado-Valverde 3, Ana Gual-de-Torrella 3, Anton Ambroa 1, Laura Fernandez-Garcia 1,2 , Maria Lopez 1,4, Jesus Oteo-Iglesias 2,4,5 , Thomas K. Wood 6, Alvaro Pascual 2,3,4, German Bou 1,2,4, Felipe Fernandez-Cuenca 2,3,4,† and Maria Tomas 1,2,4,*,† 1 Microbiology Department-Research Institute Biomedical A Coruña (INIBIC), Hospital A Coruña (CHUAC), University of A Coruña (UDC), 15006 A Coruña, Spain; [email protected] (I.B.); [email protected] (L.B.); [email protected] (A.A.); [email protected] (L.F.-G.); [email protected] (M.L.); German.Bou.Ar[email protected] (G.B.) 2Study Group on Mechanisms of Action and Resistance to Antimicrobials (GEMARA) the Behalf of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), 28003 Madrid, Spain; [email protected](J.-O.I.); [email protected] (A.P.); [email protected] (F.F.-C.) 3Clinical Unit for Infectious Diseases, Department of Microbiology and Medicine, Microbiology and Preventive Medicine, Hospital Universitario Virgen Macarena, University of Seville, Biomedicine Insititute of Seville (IBIS), 41009 Seville, Spain; [email protected] (M.D.-V.); [email protected] (A.G.-d.-T.) 4Spanish Network for Research in Infectious Diseases (REIPI), 41071 Seville, Spain 5 Reference and Research Laboratory for Antibiotic Resistance and Health Care Infections, National Centre for Microbiology, Institute of Health Carlos III, 28222 Majadahonda, Spain 6Department of Chemical Engineering, Pennsylvania State University, University Park, PA 16801, USA; [email protected] *Correspondence: [email protected]; Tel.: +34-981-176-399; Fax: +34-981-178-273 †These authors equally contributed to this work. Received: 23 July 2020; Accepted: 29 August 2020; Published: 2 September 2020   Abstract: Although the failure of antibiotic treatment is normally attributed to resistance, tolerance and persistence display a significant role in the lack of response to antibiotics. Due to the fact that several nosocomial pathogens show a high level of tolerance and/or resistance to chlorhexidine, in this study we analyzed the molecular mechanisms associated with chlorhexidine adaptation in two clinical strains of Klebsiella pneumoniae by phenotypic and transcriptomic studies. These two strains belong to ST258-KPC3 (high-risk clone carrying β -lactamase KPC3) and ST846-OXA48 (low-risk clone carrying β -lactamase OXA48). Our results showed that the K. pneumoniae ST258-KPC3CA and ST846-OXA48CA strains exhibited a different behavior under chlorhexidine (CHLX) pressure, adapting to this biocide through resistance and tolerance mechanisms, respectively. Furthermore, the appearance of cross-resistance to colistin was observed in the ST846-OXA48CA strain (tolerant to CHLX), using the broth microdilution method. Interestingly, this ST846-OXA48CA isolate contained a plasmid that encodes a novel type II toxin/antitoxin (TA) system, PemI/PemK. We characterized this PemI/PemK TA system by cloning both genes into the IPTG-inducible pCA24N plasmid, and found their role in persistence and biofilm formation. Accordingly, the ST846-OXA48CA strain showed a persistence biphasic curve in the presence of a chlorhexidine-imipenem combination, and these results were confirmed by the enzymatic assay (WST-1). Keywords: tolerance; persistence; cross-resistance; toxin-antitoxin system; PemI/PemK; Klebsiella pneumoniae Toxins 2020,12, 566; doi:10.3390/toxins12090566 www.mdpi.com/journal/toxins Toxins 2020,12, 566 2 of 17 Key Contribution: Combination of resistance, tolerance and persistence mechanisms seen in clinical isolates under biocide and antimicrobial stress. PemI/PemK system TA system discovered in the plasmid carrying β-lactamase OXA48. 1. Introduction The increase in antimicrobial resistance due to the emergence of multi-drug resistant (MDR) pathogens is one of the world’s greatest public health challenges, as it can lead to an era without effective antibiotics [ 1 ]. Recently, the World Health Organization (WHO) published a list of “priority pathogens”, which includes those microorganisms considered a serious threat to human health. Some members of this list are carbapenem-resistant pathogens and are known under the acronym of ESKAPE, including among other species, Klebsiella pneumoniae [ 1 – 3 ]. K. pneumoniae is a Gram-negative, opportunistic bacteria pathogen associated with a wide range of diseases such as urinary tract infections, pneumoniae, septicemia, wounds, and soft tissue infections [ 4 ]. Carbapenem resistance is increasing rapidly worldwide, particularly among K. pneumoniae. The main carbapenem-resistance mechanism is acquisition of plasmid-encoded carbapenemases, which may belong to the molecular class A (i.e., KPCtype), B (i.e., imipenem (IMP)-type, VIM-type, NDM-type) and D (i.e., OXA-48-type). The high-risk clones of K. pneumoniae, in contrast to low-risk clones, have an extraordinary ability to persist and spread in the nosocomial environment, disseminating these carbapenemases and therefore being involved in nosocomial outbreaks [4]. Nevertheless, much less attention has been paid to the presence or occurrence of resistance to antiseptics and biocides, such as chlorhexidine (CHLX) [ 5 ], widely used in hospital settings. CHLX is a symmetric bis-biguanide molecule comprising two chloroguanide chains that are connected by a central hexamethylene chain, and carry two positive charges at physiological pH. CHLX is sparingly soluble in water, and thereby normally formulated with either acetate or gluconate to form water-soluble salts [ 6 ]. The antimicrobial effect of this compound is based on damaging the bacterial membrane, leading to the subsequent leakage of cytoplasmatic material. Therefore, mechanisms conferring resistance toward CHLX include multidrug efflux pumps and cell membrane changes [ 5 ]. Moreover, CHLX adaptation has been associated with the emergence of stable resistance to the last-resort antibiotic colistin (polymyxin E) [7–9]. In general, the failure of antibiotic treatments has been associated with resistance mechanisms. However, it has recently been noted that other mechanisms such as tolerance and persistence were also involved [ 10 ]. The recovery of persistent cells is one of the main causes of prolonged and recurrent infections, that can lead to the complete failure of antibiotic treatments [ 11 ]. In this context, it is important to distinguish between resistant, tolerant, and persistent bacteria [ 12 ]. The term resistance is generally used to describe the inherited ability of a bacterial population to grow in the presence of high concentrations of antibiotics, regardless of the duration of treatment [ 12 ], due to active defense mechanisms associated with mutations [ 10 ]. Whereas, the term tolerance is used to describe the ability, inherited or not, of a bacterial population to survive the transient exposure of high concentrations of antibiotics without causing changes in minimum inhibitory concentrations (MICs), due to the deceleration of essential biological processes [ 10 – 12 ]. It is important to emphasize that despite the slow-growth rate, tolerant bacteria keep a metabolically active state. In contrast to resistance and tolerance, persistence is characterized by the ability, not inherited, of a bacterial subpopulation (around 0.001–1%) [ 10 ] to resist antibiotics by growth arrest due to the inactivation of their metabolism and their non-replicative state, thus it is due to their dormant state. Persistent bacteria exhibit transient levels of tolerance to antibiotics that do not affect their MICs, so once the drug pressure is removed and their metabolism is reactivated, they can rapidly re-grow. Nowadays, it is known that multiple molecular mechanisms are involved in the formation of persistent bacteria such as the stringent response molecule Toxins 2020,12, 566 3 of 17 (p)ppGpp, stress response, SOS response, quorum sensing, toxin-antitoxin (TA) systems, efflux pumps, the ROS response and energy metabolism, among others [11]. The involvement of TA systems in cell physiology, specifically in: (i) biofilm formation by regulating fimbriae [ 13 , 14 ], (ii) bacterial persistence, by generating slowly-growing cells tolerant to antibiotics and environmental changes [ 15 – 19 ], (iii) plasmid maintenance [ 20 , 21 ], (iv) general stress response [ 22 ], and (v) phage inhibition [ 23 – 25 ] is becoming clearer [ 18 ]. A TA system is a module of two genes encoding a stable toxin and an unstable antitoxin. Under normal growth conditions the antitoxin inhibits the toxin, but under stress conditions the antitoxin is degraded, leaving the toxin free to inhibit the basic cellular processes like DNA replication or protein synthesis, and also promoting plasmid maintenance, slow growth and latency [ 18 ]. These systems are widely distributed and found in the bacterial chromosome, plasmids, and bacteriophages [2]. In this context, this study provides a better comprehension of the molecular mechanisms associated with chlorhexidine adaptation (CA) in two clinical strains of K. pneumoniae, both of which produce carbapenemase: ST258-KPC3 (high-risk clone carrying β -lactamase KPC3) and ST846-OXA48 (low-risk clone carrying β -lactamase OXA48), from a phenotypic and transcriptomic point of view. It should be noted that international high-risk clones of K. pneumoniae are among the most common nosocomial pathogens. The success of these clones is due to their facility to spread their plasmids, which carry a considerable variety of antimicrobial resistance genes [ 26 , 27 ]. Thus, the study of this type of clones is of great clinical relevance. Moreover, this study aims to characterize a new toxin-antitoxin system (PemIK) located in a plasmid inside the ST846-OXA48CA strain, and to examine the possible role of this system in persistence and in biofilm formation. 2. Results 2.1. Results Subsection 2.1.1. Time-Killing Curve in the Presence of CHLX (10 ×MIC) The time-killing curves of the strains ST258-KPC3CA and ST846-OXA48CA in the presence of CHLX (10 × MIC) showed two different growth patterns (Figure 1). The strain ST258-KPC3CA showed a slight reduction in its bacterial population, occurring in the first two hours of CHLX exposure, decreasing from 6 LogCFU/mL (1.47 × 10 6 CFU/mL) to 4 LogCFU/mL (7.75 × 10 4 CFU/mL) at 2 h (Figure 1A). This slight reduction in the bacterial population occurs during the activation of defense mechanisms, such as efflux pumps, which can reduce the effective concentration of the drug in the cell. In contrast, the ST846-OXA48CA strain, dramatically reduced its bacterial population during the first four hours of CHLX exposure, decreasing from of a bacterial population of 6 LogCFU/mL (2.43 × 10 6 CFU/mL) to 2 LogCFU/mL (1.05 × 10 2 CFU/mL) at 4 h (Figure 1B). After this period, both bacterial strains grew again reaching respectively a bacterial population of 6 LogCFU/mL (6.63 × 10 6 CFU/mL) and 5 LogCFU/mL (2.75 × 10 5 CFU/mL) at 48 h. The curves have the characteristics of a resistant strain in the first case (ST258-KPC3CA) and a tolerant strain in the second one (ST846-OXA48CA), according to the definition of each mechanisms. Resistance is the ability of bacterial population to grow at a similar rate in the presence of an environmental stress, while the tolerance is the ability of a bacterial population to withstand the stress. Toxins 2020,12, 566 4 of 17 Toxins 2020, 12, x FOR PEER REVIEW 4 of 17 Figure 1. Time-killing curve in the presence of chlorhexidine (CHLX) (10 × minimum inhibitory concentration (MIC)) in K. pneumoniae chlorhexidine adaptation (CA) strains ST258-KPC3CA (A) and ST846-OXA48CA (B). The same strains without being exposed to biocide pressure are used as controls. The errors bar represents the standard deviation of the three replicates experiment. 2.1.2. Transcriptomic Study All the transcriptomic results are deposited in the NCBI database as a GenBank BioProject (Code number: PRJNA609262) and GEO series (Code number: GSE147316). The transcriptomic profile from the ST258-KPC3CA isolate indicated a probable CHLX resistant profile. Indeed, this strain has a higher number of overexpressed genes (Log2fold change >1.5), especially for transporters and efflux pumps such as the methyl viologen resistance gene smvA (Log2fold change: 3.635), which is involved in the cationic biocide resistance. However, strain ST846-OXA48CA showed what it was as a CHLX tolerant profile, with repressed genes (Log2FoldChange <1.5) for efflux pumps, TA systems, SOS response, and ppGpp mechanisms (Table 1). This strain also showed high levels of expression of genes, pmrD, and pmrK (Log2fold change 2.360 and 1.570, respectively), characteristics of the colistin resistance. Therefore, these transcriptomic results corroborated the results obtained by the timekilling curves, showing activation of molecular mechanisms of resistance and tolerance molecular mechanisms in response to CHLX in ST258-KPC3CA and ST846-OXA48CA strains, respectively. Figure 1. Time-killing curve in the presence of chlorhexidine (CHLX) (10 × minimum inhibitory concentration (MIC)) in K. pneumoniae chlorhexidine adaptation (CA) strains ST258-KPC3CA ( A ) and ST846-OXA48CA ( B ). The same strains without being exposed to biocide pressure are used as controls. The errors bar represents the standard deviation of the three replicates experiment. 2.1.2. Transcriptomic Study All the transcriptomic results are deposited in the NCBI database as a GenBank BioProject (Code number: PRJNA609262) and GEO series (Code number: GSE147316). The transcriptomic profile from the ST258-KPC3CA isolate indicated a probable CHLX resistant profile. Indeed, this strain has a higher number of overexpressed genes (Log2fold change >1.5), especially for transporters and efflux pumps such as the methyl viologen resistance gene smvA (Log2fold change: 3.635), which is involved in the cationic biocide resistance. However, strain ST846-OXA48CA showed what it was as a CHLX tolerant profile, with repressed genes (Log2FoldChange <1.5) for efflux pumps, TA systems, SOS response, and ppGpp mechanisms (Table 1). This strain also showed high levels of expression of genes, pmrD, and pmrK (Log2fold change 2.360 and 1.570, respectively), characteristics of the colistin resistance. Therefore, these transcriptomic results corroborated the results obtained by the time-killing curves, showing activation of molecular mechanisms of resistance and tolerance molecular mechanisms in response to CHLX in ST258-KPC3CA and ST846-OXA48CA strains, respectively. Toxins 2020,12, 566 5 of 17 Table 1. Gene expression in response to CHLX in the strain ST258-KPC3CA and ST846-OXA48CA. Mechanism Gene aDescription ST258-KPC3CA ST846-OXA48CA Log2 FoldChange ID Gene Log2 FoldChange ID Gene Transporter smvA Methyl viologen protein (cationic biocide resistance) 3.635 HGAILKPD_00917 1.209 EMNICGIE_00134 actP Acetate permease ActP (cation/acetate symporter) 2.724 HGAILKPD_00571 0.649 EMNICGIE_02128 csbX MFS superfamily 2.549 HGAILKPD_04325 0.618 EMNICGIE_04796 lldP L-lactate permease 2.486 HGAILKPD_04496 0.085 EMNICGIE_00243 cysW Ferric iron ABC transporter 2.181 HGAILKPD_02877 −0.266 EMNICGIE_02673 potA ABC transporter 1.749 HGAILKPD_02785 −0.351 EMNICGIE_03352 pmrD Signal transduction protein PmrD (colistin resistance) - - 2.360 EMNICGIE_04427 pmrK Polymyxin resistance protein PmrK (colistin resistance) - - 1.570 EMNICGIE_02839 ATP metabolism atpD ATP synthase beta chain −0.209 HGAILKPD_02375 −0.232 EMNICGIE_00435 TA systems ortT Orphan toxin OrtT 0.731 HGAILKPD_02791 0.727 EMNICGIE_00095 pemI Programmed cell death antitoxin PemI - - −0.100 EMNICGIE_05097 pemK Programmed cell death toxin PemK - - −0.302 EMNICGIE_05098 (p)ppGpp gppA Guanosine-5 0 -triphosphate,3 0 -diphosphate pyrophosphatase −0.765 HGAILKPD_02586 −0.399 EMNICGIE_03280 ROS response cydA Cytochrome d ubiquinol oxidase subunit I 1.318 HGAILKPD_03209 0.441 EMNICGIE_03423 cybB Cytochrome b561 0.456 HGAILKPD_02756 0.196 EMNICGIE_00060 SOS system yedK Putative SOS response-associated peptidase YedK 1.117 HGAILKPD_04848 0.003 EMNICGIE_04152 yebG DNA damage-inducible gene in SOS regulon 0.722 HGAILKPD_02193 0.455 EMNICGIE_04716 a All gene expression showed have a p-value <0.05, and overexpression and repression were considered from as a Log2fold change of at least 1.5 and 0.5, respectively. (-) Not detected. The rows shaded differently reveal those genes of high relevance in this study, the cationic biocide resistance gene, the colistin resistance genes, and the novel TA system PemI/PemK. Background colour indicates the genes of most interest from this study. Toxins 2020,12, 566 6 of 17 2.1.3. Antimicrobial Susceptibly 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-OXA48CA strain showed an increase in the MIC value of 32-fold, that corresponds to a resistance value (Table 2). Table 2. MIC values ( µ g/mL) of different antibiotics for ST258-KPC3, ST258-KPC3CA, ST846-OXA48, and ST846-OXA48CA. MIC (µg/mL) Strain CHLX CIP TGC TOB IMP MRP GEN CAZ TZP SAM NET DOX AMK MIN CST ST258-KPC3 9.8 >32 2 64 4 8 4 >32 >32 1024 128 2 16 4 0.25 ST258-KPC3CA 39.1 >32 2 64 4 8 4 >32 >32 1024 128 2 16 4 0.25 ST846-OXA48 19.5 8 8 32 16 >32 32 >32 >32 128 16 8 2 8 0.5 ST846-OXA48CA 78.2 8 8 32 16 >32 32 >32 >32 128 16 8 2 8 16 CHLX, Chlorhexidine; CIP, Ciprofloxacin; TGC, Tigecycline; TOB, Tobramycin; IMP, Imipenem; MRP, Meropenem; GEN, Gentamycin; CAZ, Ceftazidime; TZP, Piperacillin-tazobactam; SAM, Sulbactam; NET, Netilmicin; AMK, Amikacin; MIN, Minociclin; CST, Colistin. 2.1.4. Characterization of the New TA System, PemI/PemK, Present in a Plasmid in the Strain ST846-OXA48CA A PemI/PemK TA system, whose closest relative is the type II TA toxin-antitoxin system PemK/MazF family toxin belonging to Enterobacteriacea (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-OXA48CA (Figure S1). This plasmid harbors several genes, such as repA,dsbC,trbA,trbC,lusR, CPBP metalloprotease, umuD, 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-OXA48CA 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-OXA48CA CP/pCA24N (pemK) inhibited bacterial growth, while overexpression of the pemI/pemK system (ST846-OXA48CA 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-OXA48CA is functional. Toxins 2020,12, 566 7 of 17 Toxins 2020, 12, x FOR PEER REVIEW 3 of 17 Figure 2. Growth curves of ST846-OXA48CA 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 ST846OXA48CA 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. 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-OXA48CA 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 (pvalue < 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 ST846OXA48CA CP/pCA24N, ST846-OXA48CA CP/pCA24N (PemIK), and ST846-OXA48CA CP/pCA24N Figure 2. Growth curves of ST846-OXA48CA 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-OXA48CA 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. 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-OXA48CA 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. Toxins 2020, 12, x FOR PEER REVIEW 3 of 17 Figure 2. Growth curves of ST846-OXA48CA 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 ST846OXA48CA 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. 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-OXA48CA 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 (pvalue < 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 ST846OXA48CA CP/pCA24N, ST846-OXA48CA CP/pCA24N (PemIK), and ST846-OXA48CA CP/pCA24N 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-OXA48CA CP/pCA24N, ST846-OXA48CA CP/pCA24N (PemIK), and ST846-OXA48CA 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. Toxins 2020,12, 566 8 of 17 2.1.6. Time-Killing Curve in the Presence of Imipenem or in Combination with Chlorhexidine for ST846-OXA48CA and ST846-OXA48CA CP The time-killing curves of K. pneumoniae ST846-OXA48CA tolerant to CHLX (Figure 4A) were performed in the presence of imipenem (IMP) (50 × MIC) alone or in combination with CHLX (10 × MIC). A drastic reduction in the number of CFU was observed during the first four hours in the presence of IMP (50 × MIC), decreasing from 7 LogCFU/mL (9.35 × 10 7 CFU/mL) to 3 LogCFU/mL (1.50 × 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 × 10 8 CFU/mL) vs. 9 LogCFU/mL (1.73 × 10 9 CFU/mL)) at 28 h and exceeding it at 48 h (9 LogCFU/mL (1.25 × 10 9 CFU/mL) vs. 9 LogCFU/mL (1.07 × 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-OXA48CA in presence of the combination of IMP and CHLX showed a characteristic behavior of the persistent subpopulation. Toxins 2020, 12, x FOR PEER REVIEW 5 of 17 Figure 4. Time-killing curve in the presence of IMP (50 × MIC) and in presence of the combination of IMP (50 × MIC) and CHLX (10 × MIC) for the strains of K. pneumoniae ST846-OXA48CA (A) and ST846OXA48CA 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. Figure 4. Time-killing curve in the presence of IMP (50 × MIC) and in presence of the combination of IMP (50 × MIC) and CHLX (10 × MIC) for the strains of K. pneumoniae ST846-OXA48CA ( A ) and ST846-OXA48CA 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. Toxins 2020,12, 566 9 of 17 In the case of the ST846-OXA48CA CP strain (Figure 4B), in which the plasmid was removed by means of a curing agent, 3% sodium dodecyl sulfate (SDS) (10% w/vpH =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. Thus, the ST846-OXA48CA CP strain, unlike the ST846-OXA48CA strain, showed more sensitive behavior curve pattern in the presence of IMP alone and in the presence of the combination of drug. These results may suggest that the absence of the plasmid containing both the β -lactamase OXA48 and the TA system PemI/PemK could be a factor responsible for the absence of regrowth. Moreover, the lack of the TA system PemI/PemK could be implicated in the non-appearance of a persistent subpopulation in presence of the combination of IMP and CHLX, contrary to what happens in the strain ST846-OXA48 CA. 2.1.7. Enzymatic Analysis Using the Cell Proliferation Reagent WST-1 The results of the time-killing curves in the presence of the IMP and CHLX combination was confirmed by enzymatic analysis using the cell proliferation reagent WST-1 (Figure 5), which measures the omnipresent reducing agents NADH and NADPH as biochemical markers to evaluate the metabolic activity of the cell [ 30 ]. Indeed, ST846-OXA48CA lacks metabolic activity/cell proliferation at 24 h (OD480 nm <0.01), whereas it presents a significant increase at 48 h (OD480 nm >0.4; p-value <0.0001), confirming regrowth in the bacterial culture. In contrast, the ST846-OXA48CA CP strain, despite showing significant differences (p-value <0.002) between 24 and 48 h in terms of metabolic activity/cell proliferation, is considered as a dead culture since its OD480 nm is less than 0.1. Toxins 2020, 12, x FOR PEER REVIEW 6 of 17 2.1.7. Enzymatic Analysis Using the Cell Proliferation Reagent WST-1 The results of the time-killing curves in the presence of the IMP and CHLX combination was confirmed by enzymatic analysis using the cell proliferation reagent WST-1 (Figure 5), which measures the omnipresent reducing agents NADH and NADPH as biochemical markers to evaluate the metabolic activity of the cell [30]. Indeed, ST846-OXA48CA lacks metabolic activity/cell proliferation at 24 h (OD480 nm < 0.01), whereas it presents a significant increase at 48 h (OD480 nm > 0.4; p-value < 0.0001), confirming regrowth in the bacterial culture. In contrast, the ST846-OXA48CA CP strain, despite showing significant differences (p-value < 0.002) between 24 and 48 h in terms of metabolic activity/cell proliferation, is considered as a dead culture since its OD480 nm is less than 0.1. Figure 5. Enzymatic activity by the colorimetric assay (WST-1 based) of the strain K. pneumoniae ST846-OXA48CA and ST846-OXA48CA CP in the presence of the combination of IMP (50 × MIC) and CHLX (10 × MIC). The growth control is ST846-OXA48CA strain without antibiotic pressure. ***, pvalue < 0.001 and ****, p-value < 0.0001. The errors bars represent the standard deviation of the three experiment replicates. 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 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 × 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-KPC3CA and ST846-OXA48CA. In effect, the ST258-KPC3CA 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, ST846OXA48CA 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 ST258KPC3CA strain revealed the overexpression of a larger number of genes compared to ST846Figure 5. Enzymatic activity by the colorimetric assay (WST-1 based) of the strain K. pneumoniae ST846-OXA48CA and ST846-OXA48CA CP in the presence of the combination of IMP (50 × MIC) and CHLX (10 ×MIC). The growth control is ST846-OXA48CA 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. 3. 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