Abstract
Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover colistin resistance mechanisms in ten colistin-resistant Escherichia strains out of 1140 bloodstream isolates, originating from patients hospitalised in a tertiary hospital over a ten-year period (2006 - 2015). Core genome phylogenetic analysis showed that each patient was colonised by a unique strain, suggesting that colistin-resistant strains were acquired independently in each case. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. One strain carried the mobile colistin resistance gene mcr-1.1. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, led to colistin resistance in four strains. While colistin resistance in E. coli can be acquired through mcr-1.1, sequence variation in basRS is another, potentially more prevalent but underexplored, cause of colistin resistance.
Introduction
Escherichia coli is a Gram-negative opportunistic pathogen that is a common cause of bloodstream, urinary tract, and enteric infections1. The rising prevalence of antibiotic resistance in E. coli, in part due to the increasing global spread of the successful multidrug-resistant clade C lineage of ST131, may limit options for future treatments of infections2,3. Due to the emergence and spread of multidrug-resistant clones of E. coli and other Enterobacteriaceae, and the lack of new antibiotics targeting Gram-negative bacteria, colistin (polymyxin E) is increasingly used, despite its neuro- and nephrotoxic side effects, in the treatment of clinical infections with multidrug-resistant and carbapenem-resistant E. coli and other Enterobacteriaceae4–6.
Colistin is a cationic, amphipathic molecule consisting of a non-ribosomal synthesized decapeptide and a lipid tail7,8. Colistin specifically targets Gram-negative bacteria by binding to the anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPS) through electrostatic interactions7–9. Colistin destabilizes the outer membrane, but the subsequent disruption of the inner membrane ultimately leads to cell death9,10. Acquired colistin resistance has been reported in various Gram-negative bacteria that were isolated from clinical, veterinary, and environmental sources11–13. The best-documented mechanism of colistin resistance involves the modification of lipid A with cationic groups to counteract the electrostatic interactions between colistin and lipid A9. Lipid A modifications in Enterobacteriaceae may be mediated by the acquisition of mutations in chromosomally located genes or the acquisition of a mobile genetic element carrying one of the mobile colistin resistance (mcr)-genes, which encode phosphoethanolamine transferases that catalyse the addition of a cationic phosphoethanolamine group to lipid A14–16.
Among Enterobacteriaceae, colistin resistance has been most intensively studied in Salmonella and Klebsiella pneumoniae in which mutations in the regulatory genes mgrB, phoPQ and pmrAB are important mechanisms leading to resistance15,17–19. In E. coli however, mutations in mgrB and phoPQ have not been reported to lead to colistin resistance. This may be caused by the increased rate of dephosphorylation of PmrA (BasR in E. coli) by PmrB (BasS in E. coli) in E. coli compared to other Enterobacteriaceae, which effectively negates the possible activating effects of mutations in phoPQ or mgrB, through PmrD, on the levels of phosphorylated BasR. This may explain why not all of the previously described mutations reported to confer colistin resistance in Salmonella and Klebsiella confer resistance in E. coli14,20–22. In addition, phoPQ expression in E. coli is not only controlled by MgrB but also by the sRNA MicA, adding to the mechanisms controlling PhoPQ activation and making it less likely that the deletion or inactivation of mgrB can contribute to colistin resistance in E. coli14,23. This may explain why colistin resistance in clinical E. coli strains has only been linked to mutations in basRS24–28, although experimental validation of the role of these mutations in colistin resistance is currently mostly lacking.
The PmrAB (BasRS) two-component system plays a crucial role in mediating the modification of LPS that lead to colistin resistance in Gram-negative bacteria14,17. Normally, this two-component system is activated by environmental stimuli, such as the presence of antimicrobial peptides or a low pH. Activation can increase virulence and survival through evasion of the host immune system by upregulating genes associated with modification of LPS, which is the predominant immunogenic molecule of Gram-negative bacteria29,30. In E. coli, the activation of BasRS leads to increased expression of various operons, including its own. This operon also includes eptA, which encodes a lipid A-specific phosphoethanolamine transferase11,14,31.
Relatively little is known about colistin resistance mechanisms in E. coli, other than the acquisition of mcr-genes32. Therefore, we studied a collection of colistin-resistant E. coli strains from bloodstream infections by a combination of whole genome sequencing and matrix-assisted laser desorption-ionisation time-of-flight (MALDI-TOF) analysis of their lipid A, to identify colistin resistance mechanisms in E. coli. The role of mutations in basRS was investigated through the construction of chromosomal integration mutants of different basRS alleles.
Material and methods
Ethical statement
Approval to obtain data from patient records was granted by the Medical Ethics Review Committee of the University Medical Center Utrecht, in Utrecht, The Netherlands (project numbers 16/641 and 18/472).
Colistin-resistant E. coli strains were isolated as part of routine diagnostic procedures. This aspect of the study did not require consent or ethical approval by an institutional review board.
Bacterial strains, growth conditions, and chemicals
Colistin-resistant E. coli strains from bloodstream infections were obtained retrospectively from the microbiologic diagnostics laboratory of the University Medical Center Utrecht in Utrecht, The Netherlands. In initial routine diagnostic procedures, blood cultures were plated on TSA plates with 5% sheep blood. Strains collected up to 2011 were identified and their antibiogram was determined using the BD Phoenix automated identification and susceptibility testing system (Becton Dickinson, Vianen, The Netherlands). From 2011 onwards, species determination was performed by MALDI-TOF on a Bruker microflex system (Leiderdorp, The Netherlands). E. coli strain BW25113 and the BW25113-derived ∆basRS strain BW27848 from the Keio collection were obtained from the Coli Genetic Stock Center33,34. Strains were grown in Lysogeny Broth (LB; Oxoid, Landsmeer, The Netherlands) at 37°C with agitation at 300 rpm unless otherwise noted, with exception of strains containing pGRG36, which were grown at 30°C35. When appropriate, kanamycin (50 mg/L; Sigma-Aldrich, Zwijndrecht, The Netherlands), and ampicillin (100 mg/L; Sigma-Aldrich) were used. Colistin sulphate was obtained from Duchefa Biochemie (Haarlem, The Netherlands). L-(+)-arabinose was obtained from Sigma-Aldrich. Plasmids were purified using the GeneJET Plasmid Miniprep kit (Thermo Fisher Scientific, Landsmeer, The Netherlands). PCR products were purified from gel using GeneJET Gel Extraction and DNA Cleanup Micro Kit (Thermo Fisher Scientific).
Determination of minimal inhibitory concentration
Minimal inhibitory concentrations (MICs) to colistin were determined as previously described36 in line with the recommendations of a joint working group of the Clinical & Laboratory Standards Institute and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/General_documents/Recommendations_for_MIC_determination_of_colistin_March_2016.pdf), using BBL™ Mueller Hinton II (cation-adjusted) broth (MHCAB; Becton Dickinson), untreated Nunc 96-wells round bottom polystyrene plates (Thermo Scientific), and Breathe-Easy sealing membranes (Sigma-Aldrich). The breakpoint value of an MIC > 2 µg/ml for colistin resistance in E. coli was obtained from EUCAST (http://www.eucast.org/clinical_breakpoints/). E. coli MG1655 served as a colistin-susceptible control.
Genomic DNA isolation and whole-genome sequencing
Genomic DNA was isolated using the Wizard Genomic DNA purification kit (Promega, Leiden, The Netherlands) according to the manufacturer’s instructions. DNA concentrations were measured with the Qubit dsDNA Broad Range Assay kit and the Qubit 2.0 fluorometer (Life Technologies, Bleiswijk, The Netherlands).
Sequence libraries for Illumina sequencing were prepared using the Nextera XT kit (Illumina, San Diego, CA) according to the manufacturer’s instructions. Libraries were sequenced on an Illumina MiSeq system with a 500-cycle (2 × 250 bp) MiSeq reagent kit v2. MinION library preparation for barcoded 2D long-read sequencing was performed using the SQK-LSK208 kit (Oxford Nanopore Technologies, Oxford, England, United Kingdom), according to the manufacturer’s instructions, with G-tube (Covaris, Woburn, Massachusetts, United States of America) shearing of chromosomal DNA for 2 x 120 seconds at 1500 g. Sequencing was performed on the MinION sequencer (Oxford Nanopore Technologies) using 2D barcoded sequencing through a SpotON Flow Cell Mk I (R9.4; Oxford Nanopore Technologies).
Genome assembly, MLST typing, and identification of antibiotic resistance genes
The quality of sequence data was assessed using FastQC v0.11.5 (https://github.com/s-andrews/FastQC). Sequencing reads were trimmed for quality using nesoni v0.115 (https://github.com/Victorian-Bioinformatics-Consortium/nesoni) using standard settings with the exception of a minimum read length of 100 nucleotides. De novo genome assembly using Illumina data was performed using SPAdes v3.6.2 with the following settings: kmers used: 21, 33, 55, 77, 99, or 127, “careful” option turned on and cut-offs for final assemblies: minimum contig/scaffold size = 500 bp, minimum contig/scaffold average Nt coverage = 10-fold37. MinION reads in FastQ format were extracted from Metrichor base-called FAST5-files using Poretools38. Hybrid assemblies were generated of short- and long-read data using SPAdes by specifying Nanopore data with the --nanopore flag. Gene prediction and annotation was performed using Prokka39. MLST typing was performed using the mlst package v2.10 (https://github.com/tseemann/mlst). Assembled contigs were assessed for antibiotic resistance genes using ResFinder 3.240.
Core genome phylogenetic analysis and determination of mutations in candidate colistin resistance determinants
Genome assemblies generated in this study with Illumina data were aligned with 178 complete E. coli genomes and 32 E. albertii genomes that were available from NCBI databases on 24 June 2016 (Supplemental Table 1) using ParSNP v1.241. MEGA6 was used to midpoint root and visualize the phylogenetic tree42. We identified whether non-synonymous mutations were present in basRS by pairwise comparison of the gene sequences of colistin-resistant isolates to their closest matching publicly available genome from the phylogenetic tree using BLAST43. Mutations that were identified in the genome sequences were confirmed through PCR (oligonucleotide primer sequences are provided in Supplemental Table 2) and subsequent Sanger sequencing of the PCR product by Macrogen (Amsterdam, The Netherlands).
Isolation and analysis of lipid A
Isolation of lipid A molecules and subsequent analysis by negative-ion MALDI-TOF mass spectrometry was performed as previously described19,44,45. Briefly, Escherichia strains were grown in LB (Oxoid) and the lipid A was purified from stationary cultures using the ammonium hydroxide/isobutyric acid method described earlier46. Mass spectrometry analyses were performed on a Bruker autoflex™ speed TOF/TOF mass spectrometer in negative reflective mode with delayed extraction using as matrix an equal volume of dihydroxybenzoic acid matrix (Sigma-Aldrich) dissolved in (1:2) acetonitrile-0.1% trifluoroacetic acid. The ion-accelerating voltage was set at 20 kV. Each spectrum was an average of 300 shots. A peptide calibration standard (Bruker) was used to calibrate the MALDI-TOF. Further calibration for lipid A analysis was performed externally using lipid A extracted from E. coli strain MG1655 grown in LB medium at 37°C.
Prediction of functional impact of sequence variation in basRS
We aimed to predict the functional impact of sequence variation in basR and basS using ConSurf47, Scorecons48, and PROVEAN49. Multiple sequence alignments (MSAs) for BasR and BasS amino acid sequences were generated using ConSurf with standard settings. Using the BasR and BasS MSAs, ConSurf was used to determine the rate of conservation of each amino acid residue using the Bayesian calculation method, and JTT as evolutionary substitution model47,50. Scorecons was used to quantify residue conservation, with consideration of the characteristics of each residue, using standard settings48. These methods were used to score conservation of the specific residues, with highly conserved residues being more likely to importantly contribute to protein function. In addition to ConSurf and Scorecons, PROVEAN49 was used to predict the impact of the different BasR and BasS alleles on protein function compared to the publicly available Escherichia reference genome sequences.
Construction of chromosomal basRS transgene insertions
Chromosomal transgene insertions of basRS were constructed in BW27848 by utilizing the Tn7 transposon system on the pGRG36 plasmid35. The promoter of the eptA-basRS operon was fused to the basRS coding sequence by separate PCRs for the promotor region and the basRS amplicon, with high fidelity Phusion Green Hot Start II DNA Polymerase (Thermo Fisher Scientific) using strain-specific primers (Supplemental Table 2; oligonucleotides were obtained from Integrated DNA Technologies, Leuven, Belgium). The promoter and the basRS amplicon were subsequently fused by overlap PCR. Fused PCR products were cloned into pCR-Blunt II-TOPO using the Zero Blunt TOPO PCR Cloning kit (Thermo Fisher Scientific), and subsequently subcloned into pGRG3635. Electrocompetent BW25113 and BW27848 E. coli cells were prepared as described previously51 and transformed using the following settings: voltage 1800V, capacitance 25 µF, resistance 200Ω, with a 0.2 cm cuvette using the Gene Pulser Xcell electroporation system (Bio-Rad Laboratories, Veenendaal, The Netherlands). Transformants were grown at 30°C. After confirming integration of the Tn7 transposon at the attTn7 site by PCR (primers listed in Supplemental Table 2) and Sanger sequencing (Macrogen), the pGRG36 plasmid was cleared by culturing at 37°C.
Inverse PCR site-directed mutagenesis was performed on amplicons cloned in pCR-Blunt II-TOPO to reverse the mutations that were identified in colistin-resistant strains to the sequences of basR or basS in the closest matching publicly available genome52. After gel purification of the amplified fragments, (hemi)methylated fragments were digested using DpnI (New England Biolabs (NEB), Ipswich, Massachusetts, United States of America). Subsequently the vector was recircularized using the Rapid DNA Ligation kit (Thermo Fisher Scientific) after phosphorylation using T4 Polynucleotide kinase (NEB). The constructs were then transformed into chemically competent DH5α E. coli cells (Invitrogen, Landsmeer, The Netherlands). Mutated sequences were subsequently subcloned to pGRG36 as described above.
Data availability
Sequence data has been deposited in the European Nucleotide Archive (accession number PRJEB27030).
Statistical analysis
Statistical significance was determined using the non-parametric Kruskal-Wallis one-way, two-tailed ANOVA test. Correction for multiple comparison testing was performed using Dunn’s correction. Family-wise significance was defined as a p-value < 0.05.
Results
Low prevalence of colistin resistance in invasive Escherichia bloodstream isolates
A total of 1140 bloodstream isolates (collected from January 2006 to December 2015), for which automated antibiotic susceptibility profiles were available, and for which species identification had been performed, were available for this study. Twelve isolates were deemed resistant to colistin through routine diagnostic procedures. Two of those isolates were isolated from the same patient, on the same day, and were thus considered duplicates, and only one of these was included in this study. In ten of the eleven remaining isolates, colistin resistance, defined as an MIC > 2 µg/ml colistin, was confirmed through broth microdilution (Fig. 1). Strain A783 was a false positive for colistin resistance during automated susceptibility testing in routine diagnostic procedures, and was excluded from further analysis, leaving ten isolates for further investigation (Table 1). The estimated prevalence of colistin resistance in E. coli strains causing bloodstream infections isolated from January 2006 to December 2015 was thus determined to be 0.88%. Three patients had received colistin in the three months before isolation of the colistin-resistant strain, for varying indications (Table 1). Two of these patients received colistin to treat infections, but all three patients were also administered colistin as part of selective digestive or oropharyngeal decontamination (SDD/SOD), a prophylactic antibiotic treatment widely used in Dutch intensive care units53. The ten colistin-resistant strains were analyzed further in this study to determine their relatedness and mechanism through which they had developed colistin resistance.
Colistin-resistant bloodstream E. coli isolates are not clonal
To assess the phylogenetic relationships between the colistin-resistant strains, a phylogenetic tree was generated based on the assembled contigs of the colistin-resistant strains and 210 publicly available complete genome sequences (Supplemental Table 1). Based on a core genome alignment of 874 kbp, we did not observe direct transmission of colistin-resistant strains between patients (Fig. 2a). Three colistin-resistant strains (strains I1121, H2129, and G821) belonged to the globally disseminated ST131 clone (Fig. 2a), and all three were dispersed throughout the multidrug-resistant clade C of ST1313,54. This indicates that these ST131 strains have independently acquired colistin resistance (Fig. 2b). Strain A2361 clustered among E. albertii, although it had been typed as E. coli in routine diagnostic procedures.
By screening for acquired antibiotic resistance genes through ResFinder 3.2, we found that only strain E3090 carried the mcr gene mcr-1.1 (0.086% of all bloodstream isolates; Supplemental Fig. 1). After long-read sequencing and hybrid assembly, the mcr-1.1-gene in this strain appeared to be located as the sole antibiotic resistance gene on a 32.7 kbp IncX4-type plasmid. This mcr-1.1 carrying IncX4-type plasmid from E3090 shares 99% identity to the previously reported mcr-1.1 carrying IncX4-type plasmid pMCR-1_Msc (GenBank accession MK172815.1) harboured by E. coli isolated from patients in Russia55, confirming the global dissemination of this plasmid56. In all strains studied here, a variety of acquired resistance genes was observed (Supplemental Fig. 1), reflecting the non-clonal nature of the colistin-resistant strains. The three colistin-resistant ST131 strains possessed different repertoires of acquired resistance genes, further excluding recent transmission between patients of the ST131 strains studied here. Strain F2745 and E2372 carried only one, and two resistance genes respectively, while strain A2361 did not possess any acquired resistance genes.
Escherichia isolates exclusively acquire colistin resistance by modification of phosphate groups of lipid A
To determine which modifications to lipid A are affecting colistin resistance in E. coli we extracted lipid A from the clinical strains and the colistin-susceptible control E. coli strain MG1655, and subjected them to MALDI-TOF mass spectrometry. The lipid A produced by all E. coli strains showed lipid A species with a mass-to-charge ratio (m/z) of 1797, corresponding to the canonical unmodified E. coli hexa-acylated lipid A (Fig. 3). Colistin-resistant strains showed additional lipid A species at m/z 1921, consistent with the addition of phosphoethanolamine (m/z 124) to the hexa-acylated species. Additional species were detected in the lipid A produced by strains E650 and Z821. Species m/z 2036 indicated the addition of palmitate (m/z 239) to the hexa-acylated species m/z 1797, whereas species m/z 2160 was consistent with the addition of palmitate to the hexa-acylated lipid A species containing phosphoethanolamine (m/z 1910).
The E. alberti strain A2361 produced lipid A distinct from E. coli. Species m/z 1825 is likely to represent a hexa-acylated species corresponding to two glucosamines, two phosphates, four 3-OH-C14, and two C14. Species m/z 1948 is consistent with the addition of phosphoethanolamine to the hexa-acylated species, with a further addition of palmitate to produce lipid A species m/z 2187. Species m/z 1868 and m/z 2107 correspond to the loss of the second phosphate group, compared to m/z 1948, and m/z 2187.
Identification of candidate mutations involved in colistin resistance
As mutations in basRS have been suggested to cause to colistin resistance in E. coli24–28, we next aimed to establish the contribution of the basRS alleles in the colistin-resistant phenotype of these bloodstream isolates. Due to the multidrug-resistant nature of the clinical isolates (Supplemental Fig. 1), we were unable to generate targeted mutations in these strains. Therefore, we made chromosomal transgene insertion mutants of the different basRS alleles in the attTn7 site in the BW25113-derived ∆basRS strain BW27848 using the Tn7 transposon system. By making chromosomal transgenes insertions, rather than using an in trans complementation method, we excluded copy number effects by plasmids, and the need to use antibiotics to select for the presence of a plasmid. Since BW27848 still possesses the gene encoding for the phosphoethanolamine transferase EptA, we constructed sequences that consisted of the fused sequences of the promotor region of the eptA-basR-basS operon and the basRS coding sequences in order to prevent etpA gene dose-dependent effects. We were unable to generate the construct for strain E650, presumably due to the toxicity of the insert.
The colistin MIC determination of the generated basRS chromosomal transgene insertion mutants from strains I1121, H2129, G821, and Z821 had significantly higher colistin MIC values than the BW27848::Tn7-empty strain (adjusted p-values of 0.0195, 0.0094, 0.0008, and 0.008 respectively), with observed MIC values higher than the 2 µg/ml cut-off for resistance as set by EUCAST (Fig. 4). As expected, the basRS allele of the mcr-1.1 positive strain E3090 did not lead to colistin resistance. We were unable to show the contribution of basRS to colistin resistance in the additional four colistin-resistant strains (F2745, E2372, D2373, A2361) that lacked mcr-1.1.
Mutations in the basRS genes contribute to colistin resistance in E. coli
By construction of the chromosomal transgene insertion mutants, we identified the ability of the basRS sequences of four strains (I1121, H212, G821, and Z821) to cause colistin resistance in BW27848. To identify the mutations in the basRS alleles of these strains that cause the found resistance, we compared the basRS encoding sequences of those strains causing resistance to the phylogenetically most closely related publicly available E. coli genome sequences used in the construction of Fig. 2. None of these reference strains were reported to be colistin-resistant, or carried any of the mcr-genes. This comparison revealed four distinct mutations: a L10R substitution in BasS in I1121, a G53S substitution in BasR in H2192, the duplication of the HAMP-domain in BasS in G821, and a A159P substitution in BasS in Z821 (Fig. 5). As expected, in the mcr-1.1 positive strain E3090 no mutations in basRS were identified.
To assess the possible role of observed variations in basRS in colistin resistance, we used ConSurf47 and Scorecons48 to score conservation of the specific residues. In addition, we used PROVEAN49 to predict the impact of the mutations on protein function. As input sequences, we used the BasR and BasS amino acid sequences of the genomes that most closely matched to the colistin-resistant strains (Fig. 2), as this allowed the scoring of the identified substituted residues. The rate of conservation of an amino acid indicates its importance in the protein, with higher conserved residues being more likely to be important for correct protein functioning. ConSurf results indicated that one of the three substituted residues, A159 in BasS, was highly conserved (Fig. 5). In contrast, Scorecons shows that the specific characteristics of all three affected residues were conserved. The PROVEAN results suggested that the G53S substitution in BasR, and the duplication of the HAMP-domain and A159P substitution in BasS could impact protein function. ConSurf or Scorecons could not be used for the analysis of the observed duplication in strain G821. Thus, for all observed mutations, an impact of the substitution of the residues on protein functioning was supported by at least one method.
Using these in silico prediction methods, we hypothesised that the observed mutations were impacting the normal functioning of the BasRS two-component system. To assess whether the mutations in basRS identified by comparing the basRS sequences of the clinical strains I1121, H2129, G821, and Z821, and their closest match in the set of 178 publicly available E. coli genome sequences (Fig. 5) were causal to the development of colistin resistance, the identified mutations were reversed through site-directed inverse PCR mutagenesis to match the publicly available genome sequence. The MIC values of these mutants returned to levels similar to that of the colistin-susceptible BW27848::Tn7-empty strain (Fig. 6). These experiments support the in silico predictions on the functional impact of the basRS sequence variations observed in the colistin-resistant E. coli strains.
Discussion
In the present study, we identified the mechanisms through which E. coli bloodstream isolates can develop colistin resistance. We did not find evidence for transfer of colistin-resistant strains between patients, suggesting that colistin resistance has been acquired independently in all cases. In seven patients colistin-resistant strains were isolated without the patients being previously exposed to the drug. All colistin-resistant strains had LPS that was modified by the addition of phosphoethanolamine to the lipid A moiety of LPS. Resistance in one of the bloodstream isolates could be explained by the acquisition of mcr-1.1. In four other strains, we identified mutations in basRS that contribute to colistin resistance. Although colistin-susceptible strains that were isogenic to the resistant strains were not available, we were able to pinpoint the mutations in basRS leading to resistance in these strains by matching the genomic sequences of our nosocomial isolates with publicly available genomes, none of which were reported to be colistin-resistant, and subsequent construction of chromosomally integrated basRS transgene alleles in the ∆basRS strain BW27848. The mechanisms of colistin resistance in the remaining five strains remain to be characterized.
Some of the mutations we experimentally link to colistin resistance in this study, have previously been associated with colistin resistance or the functioning of the BasRS two-component system. In this study, we demonstrated that the amino acid change L10R in BasS (strain I1121) also confers colistin resistance. An amino acid substitution in the same position of BasS (L10P) was previously experimentally proven to cause colistin resistance in E. coli26. The glycine in position 53 of BasR has previously been reported to be altered in colistin-resistant Enterobacteriaceae57,58 including in E. coli59. The G53S change specifically, as in isolate H2192, has been experimentally proven to contribute to colistin resistance in Klebsiella (previously Enterobacter) aerogenes 60,61 and Salmonella enterica subsp. enterica serovar Typhimurium62 and we extend those findings to E. coli here. The previously unidentified duplication of 162 nucleotides in basS (strain G821) leads to the introduction of a second HAMP domain in BasS and confers colistin resistance in the BW27848 background. The HAMP domain is widespread in bacteria and is commonly involved in signal transduction as part of two-component systems63. We hypothesise that the addition of an extra HAMP domain in BasS may change signal transduction in the protein, leading to the constitutive activation of the histidine kinase domain of BasS, increased phosphorylation of BasR and upregulated expression of eptA, ultimately resulting in the addition of phosphoethanolamine to lipid A. Finally, we demonstrate that the A159P substitution in BasS (observed in strain Z821) contributes to colistin resistance. A mutation leading to a A159V substitution was found in an in vitro evolution study in which E. coli was evolved towards colistin resistance64, and in clinical colistin-resistant E. coli isolates65, but experimental confirmation of the role of alterations in A159 in colistin resistance in E. coli was so far lacking. Our data suggest that the basRS alleles of three E coli strains (F2745, E2372, and D2373), and the E. albertii strain A2361, do not confer resistance in the BW25113 E. coli background. Because E. albertii is phylogenetically distinct from E. coli, its basRS allele may not function optimally in an E. coli background, explaining the inability of the transgene insertion complementation in the basRS deletion of BW25113 E. coli strain to cause colistin resistance66.
The observed modification of lipid A with phosphoethanolamine in all isolates underlines the crucial role of phosphoethanolamine transferases in the ability of Escherichia to become resistant to polymyxins14. The lipid A of three of the colistin-resistant strains was also modified with palmitate, but the contribution of lipid A palmitoylation to colistin resistance in clinical E. coli strains is currently unknown. The reliance of Escherichia on the modification of lipid A by phosphoethanolamine to acquire colistin resistance, suggests that the inhibition of this class of enzymes by blocking the conserved catalytic site31 could be a target for future drug development and opens the possibility of combination therapy with colistin and an inhibitor of phosphoethanolamine transferase67. With the increasing clinical issues posed by infections with multidrug-resistant Gram-negative bacteria, there is an urgent need to better understand resistance mechanisms to last-resort antibiotics like colistin. While the discovery of the mcr genes have generated considerable interest in transferable colistin resistance genes, our data suggest that chromosomal mutations remain an important cause of colistin resistance among clinical isolates in the genus Escherichia.
Funding information
W.v.S. was funded through an NWO-Vidi grant (grant 917.13.357), and a Royal Society Wolfson Research Merit Award. Work in J.A.B. laboratory was supported by Biotechnology and Biological Sciences Research Council (BBSRC, BB/P020194/1) and Queen’s University Belfast start-up. T.L.B. is the recipient of a PhD fellowship funded by the Department for Employment and Learning (Northern Ireland, UK).
Author contributions
A.B.J. conceived and designed experiments, performed experiments, analysed data, and wrote the manuscript. T.B.L. performed experiments, and analysed data. N.P.M. performed experiments, and analysed data. M.J.M.B. wrote the manuscript. R.J.L.W. wrote the manuscript. J.A.B. analysed data, and wrote the manuscript. W.v.S. conceived and designed experiments, wrote the manuscript, and supervised the study. All authors reviewed and approved the final version of the manuscript.
Competing interests
The authors declare no conflicts of interest.
Acknowledgements
We thank Eline A.M. Majoor for technical support and L. Marije Hofstra and Lidewij W. Rümke for their review of patient records. We also thank the Utrecht Sequence Facility and Ivo Renkens for their expertise in MinION Nanopore sequencing.