https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4432163/
Polymorphic Variation in Susceptibility and Metabolism of Triclosan-Resistant Mutants of Escherichia coli and Klebsiella pneumoniae Clinical Strains Obtained after Exposure to Biocides and Antibiotics
a,b,cABSTRACT
Exposure to biocides may result in cross-resistance to other antimicrobials. Changes in biocide and antibiotic susceptibilities, metabolism, and fitness costs were studied here in biocide-selected Escherichia coli and Klebsiella pneumoniae mutants. E. coli and K. pneumoniae mutants with various degrees of triclosan susceptibility were obtained after exposure to triclosan (TRI), benzalkonium chloride (BKC), chlorhexidine (CHX) or sodium hypochlorite (SHC), and ampicillin or ciprofloxacin. Alterations in antimicrobial susceptibility and metabolism in mutants were tested using Phenotype MicroArrays. The expression of AcrAB pump and global regulators (SoxR, MarA, and RamA) was measured by quantitative reverse transcription-PCR (qRT-PCR), and the central part of the fabI gene was sequenced. The fitness costs of resistance were assessed by a comparison of relative growth rates. Triclosan-resistant (TRIr) and triclosan-hypersusceptible (TRIhs) mutants of E. coli and K. pneumoniae were obtained after selection with biocides and/or antibiotics. E. coli TRIr mutants, including those with mutations in the fabI gene or in the expression of acrB, acrF, and marA, exhibited changes in susceptibility to TRI, CHX, and antibiotics. TRIr mutants for which the TRI MIC was high presented improved metabolism of carboxylic acids, amino acids, and carbohydrates. In TRIr mutants, resistance to one antimicrobial provoked hypersusceptibility to another one(s). TRIr mutants had fitness costs, particularly marA-overexpressing (E. coli) or ramA-overexpressing (K. pneumoniae) mutants. TRI, BKC, and CIP exposure frequently yielded TRIr mutants exhibiting alterations in AraC-like global regulators (MarA, SoxR, and RamA), AcrAB-TolC, and/or FabI, and influencing antimicrobial susceptibility, fitness, and metabolism. These various phenotypes suggest a trade-off of different selective processes shaping the evolution toward antibiotic/biocide resistance and influencing other adaptive traits.
RESULTS
Selection of biocide mutants presenting different phenotypes.
Considering only variants with susceptibility changes involving two or more double dilutions, the only biotypes consistently obtained were triclosan resistance or triclosan hypersusceptibility (Table 2). In E. coli, triclosan-resistant (TRIr) variants were recovered from plates that contained TRI, BKC, or CIP after preconditioning broth exposure with TRI, BKC, or CIP and without preexposure to antimicrobials. Similarly, in K. pneumoniae, TRIr variants were obtained not only from plates containing TRI, BKC, or CIP, after being preexposed to subinhibitory concentrations of TRI, BKC, or CIP, but also to SHC, CHX, and AMP. In K. pneumoniae, TRI-hypersusceptible (TRIhs) variants were recovered from plates containing CHX as a selective agent. Of note, half of the TRIr E. coli variants showed slight increases (less than two double dilutions) in susceptibility to CHX (hypersusceptibility to CHX [CHXhs]), which suggests negative epistasis between the mechanisms of resistance to these two compounds. In K. pneumoniae, TRIr variants appeared to be slightly more resistant to both BKC and CHX than E. coli mutants, but the difference with the wild-type strain MIC was not sufficient to ascertain the robustness of these trends under our experimental conditions.
A large number of colonies of E. coli and K. pneumoniae mutants was obtained in plates supplemented with CIP (either the culture was previously exposed to antibiotics or not), in cases when the same compound was applied during preexposure and plate selection, and when low concentrations of TRI or BKC were used (Table 2).
Antibiotic susceptibility of triclosan mutants.
As described above, TRI resistance was achieved after selection with TRI but also under selection in plates containing BKC or CIP. Table 2 shows the antibiotic susceptibility profile of the TRIr mutants obtained. It is noteworthy that one of the E. coli TRIr variants showing hypersusceptibility to CHX (CHXhs) also presented slightly increased susceptibility to CAZ, GEN, and CLO (Table 2). Some K. pneumoniae TRIr variants showed a remarkable decrease in susceptibility to AMP (1.5- to ≥8-fold MIC increases), CIP (2- to 16-fold), CLO (32- to ≥128-fold), TET (2.6- to ≥16-fold), and CAZ (1.3- to 4-fold). One of them (Kpn_CIP/BKC2) also presented a loss of susceptibility to ERY (≥16-fold) but increased GEN susceptibility (5-fold).
An extended analysis of susceptibility to a wide variety of antimicrobial substances using Phenotype MicroArrays (Biolog, Inc.) was performed not only for TRIr mutants (4 E. coli and 2 K. pneumoniae) but also for mutants for which the MIC of TRI (MICTRI) showed either a slight increase (only a one-dilution difference) or a slight decrease in comparison with that of the wild parental strain (2 E. coli and 3 K. pneumoniae strains, respectively). The parental strains Ec_HEC30 and Kpn_39.11 were also included. The results are shown in Tables S1 and S2 in the supplemental material. Some common patterns were observed, even though the phenotypes of susceptibility were diverse in the different mutants.
All six E. coli TRIr mutants analyzed were more resistant to cefoxitin, cefuroxime, tetracycline, and dodine (a fungicide) but showed enhanced susceptibility to highly diverse antimicrobials, remarkably, those that increase membrane permeability (such as poly-l-lysine, polymyxin B, and metaborate, a herbicide), and to many others. The five K. pneumoniae mutants analyzed (2 TRIr and 3 triclosan susceptible [TRIs]) were more tolerant to penicillin, the tetracycline derivative penimepicycline, and novobiocin and more susceptible to compounds acting on membrane permeability, such as colistin (a polypeptide polycationic antibiotic), methyl viologen or paraquat (oxidant herbicide), and cobalt (a toxic cation). The two TRIr K. pneumoniae mutants (Kpn_CIP/BKC2 and Kpn_TRI/TRI2) exhibited decreased antimicrobial susceptibility to a wide panel of agents (to 57 and 58 chemicals, respectively, with 52 compounds being commonly affected), which comprised several antibiotic classes (see Table S2 in the supplemental material). On the contrary, TRIhs K. pneumoniae variants were mainly susceptible to several more antimicrobials than their parental strain. These results indicate that the acquisition of resistance to biocides can either decrease or increase susceptibility to a wide variety of different cell growth-inhibiting agents.
Acquisition of resistance to biocides alters the physiology of E. coli and K. pneumoniae.
One interesting issue derived from testing some of the analyzed mutants was the possible alteration of their normal physiology. Alterations in colony morphology were observed for some of the TRIr mutants (Ec_CIP/CIP, Kpn_CIP/BKC2, Kpn_AMP/TRI2, and Kpn_TRI/CIP), as reflected in the smaller size exhibited by their colonies in comparison with that of the corresponding parental strains, whereas other TRIr and TRIhs mutants (Kpn_AMP/Tri2, Kpn_BKC/BKC1, and Kpn_SHC/CHX2) formed mucous colonies (see Fig. S1 in the supplemental material) with enhanced biofilm formation (not shown). All of them exhibited the same XbaI-digested genomic DNA profile as their respective parental ancestor, suggesting that the alterations in physiological behavior are not due to large genomic reorganizations (see Fig. S2 in the supplemental material).
To further understand the effect of acquiring biocide resistance on the physiology of K. pneumoniae and E. coli, the use of carbon sources in each mutant was compared to that in the parental strains. The clustering of metabolic profiles of both the mutants and their respective wild-type strains is shown in Fig. 1 and in Tables S3 and S4 in the supplemental material. Among E. coli TRIr mutants, the most remarkable finding was the enhanced use of d-malic acid by all strains analyzed and the more efficient growth of most mutants in arbutin (β-d-glucopyranoside). Some TRIr mutants (Ec_TRI/TRI2 and Ec_CIP/CIP), characterized by a similar antibiotic susceptibility profile, were able to use some amino acids (hydroxyl-l-proline), carboxylic acids, carbohydrates, and alcohols more efficiently than the parental strain. However, they differ in their ability to metabolize different substrates (adonitol, d-glucosaminic acid, m-inositol, d-psicose, l-lyxose, d-allose, salicin, α-hydroxyglutaric acid γ-lactone, and dihydroxyacetone).
Seven out of 9 TRIr K. pneumoniae mutants showed variable FC (4 to 66%). The Kpn_CIP/BKC2 mutant with striking reduced susceptibility to several antibiotics (CAZ, AMP, CIP, ERY, CLO, and TET) showed the highest FC value and the longest lag phase (Fig. 2). This variant was unique in presenting such an increase in the susceptibility to GEN. Together with other two mutants that demonstrated appreciable FCs (Kpn_AMP/TRI2 and Kpn_SHC/CIP, with FC values of 11 and 15%, respectively), an impaired ability to metabolize a high number of carboxylic acids, amino acids, sugars, and amino sugars involved in peptidoglycan synthesis was observed in comparison with parental strains. Finally, K. pneumoniae TRIhs variants (Kpn_NE/CHX2 and Kpn_CIP/CHX2), with phenotypes of increased susceptibility, showed low FC.
Growth kinetics of E. coli and K. pneumoniae mutants and their parental strains. The parental strains are HEC30 (E. coli) and 39.11 (K. pneumoniae). The average data are represented in the graphics. Growth rates were determined in plain LB broth at 37°C for 24 h. O.D.600, optical density at 600 nm.
A comparison of the mutants reveals that decreased susceptibility to TRI accompanied by a remarkable decreased susceptibility to CIP or ERY is associated with higher physiological costs in both E. coli and K. pneumoniae. The finding that mutants with high FC show increased metabolism of some carbon sources, in the case of E. coli, or decreased metabolic ability for other substrates, in the case of K. pneumoniae, suggests that FC may depend on the environment and available nutrients.
Gene expression of acrAB and marA, and soxS and ramA.
Gene expression of acrAB, acrEF, and acrCD and marA, soxS, and ramA was evaluated in different TRIr and TRIhs mutants exhibiting different FC values (Table 3). Increased expression of both acrF and marA was detected for one E. coli mutant (Ec_CIP/CIP), with notable changes in the susceptibility to TRI or CIP (10-fold increase in MIC) and an increased metabolism of some carbon sources, as well as a high FC value (26%). The mutant Ec_TRI/TRI2 presented the highest FC and increased metabolism of certain carbon sources, although the expression of the tested genes was not modified. Conversely, decreased expression of acrB, acrF, marA, and soxS (fold changes and standard deviations: 0.26 ± 0.17, 0.18 ± 0.15, 0.32 ± 0.18, and 0.21 ± 0.18, respectively) was observed in a TRIr mutant (4-fold increase in MIC for Ec_BKC/BKC) that showed 6% FC and a 2-fold increase in the MIC of CAZ (MICCAZ).
Mutants with a low fitness cost, corresponding to phenotypes of decreased (4-fold increase in MICTRI for Kpn_TRI/TRI2) and increased susceptibility to TRI (4-fold decrease in MICTRI for Kpn_NE/CHX2 and Kpn_CIP/CHX2), showed increased expression of acrB and acrF and either down- or overexpression of acrB and marA, respectively. The mutant Kpn_SHC/CIP, with a 15% FC, showed increased acrF expression. However, the variant with the highest FC had enhanced expression of the transcriptional regulator ramA (fold change, 45.01 ± 40.81). Overexpression of ramA was previously involved in ciprofloxacin resistance of S. enterica obtained after increasing passages in CIP (35).
Analysis of FabI amino acid changes.
Different amino acid changes were identified in TRIr E. coli mutants with MICs of 4 (Gly93Val in Ec_TRI/TRI2 mutant) and 1 mg/liter (Leu94Phe in Ec_NE/TRI1 mutant), both located in the binding site of FabI and the second reported here for the first time. Another isolate showing up to five mutations did not show any TRI resistance phenotype. Mutations in the fabI gene were not observed in K. pneumoniae mutants.
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In summary, this work reports the effects of exposure to biocides and antibiotics in selected mutants showing polymorphic changes, including resistance to antimicrobial compounds, differential fitness costs, and changes in the metabolic profiles in E. coli and K. pneumoniae. The complex variety of antimicrobial mechanisms of action for biocides affecting different basic networks of bacterial physiology is consequently mirrored by the complex variety of resistance mechanisms, determining secondary effects on susceptibility to other compounds, metabolism, and ultimately environment-dependent influences on fitness and colonization/virulence.
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